Vitamin A as a Key Regulator of Hox Genes and Limb Development in Anuran Models

Dylan Peterson Nov 29, 2025 104

This article explores the pivotal role of Vitamin A (retinoic acid) in manipulating Hox gene expression to direct profound developmental outcomes, specifically homeotic transformations in anuran models.

Vitamin A as a Key Regulator of Hox Genes and Limb Development in Anuran Models

Abstract

This article explores the pivotal role of Vitamin A (retinoic acid) in manipulating Hox gene expression to direct profound developmental outcomes, specifically homeotic transformations in anuran models. We synthesize foundational research, including recent findings on the induction of ectopic limbs in Rana ornativentris tadpoles, where Vitamin A administration triggers a cascade of Hox gene downregulation and limb gene activation. The content provides a methodological framework for applying these principles in regenerative and developmental biology, addresses common experimental challenges, and validates findings through comparative analysis with other model systems and human pathologies. This resource is tailored for researchers, scientists, and drug development professionals seeking to understand and harness Hox gene regulation for therapeutic innovation.

Hox Genes, Retinoic Acid, and the Blueprint of the Body Plan

Hox genes are a family of evolutionarily conserved transcription factors that function as master regulators of embryonic development, playing a pivotal role in determining the identity of structures along the anteroposterior (head-to-tail) axis in bilaterian animals [1]. These genes encode proteins containing a characteristic homeodomain that facilitates DNA binding, enabling them to control the expression of downstream target genes that execute developmental programs [2]. In vertebrates, the 39 Hox genes are organized into four clusters (HOXA, HOXB, HOXC, and HOXD) located on different chromosomes [3]. Their expression follows the principle of collinearity, where the order of genes on the chromosome correlates with their spatial expression domains along the body axis and the timing of their activation during development [2] [4]. This spatially restricted expression creates a "Hox code"—a combinatorial signature of Hox gene expression that confers positional identity to embryonic tissues, thereby determining whether a segment will develop into cervical, thoracic, lumbar, or sacral vertebrae [5] [4].

The critical function of Hox genes in axial patterning is evident in their effects when misexpressed. Alterations in Hox expression patterns can lead to homeotic transformations, in which one segment of the axial skeleton develops the morphological characteristics of another [5] [4]. For example, in Prtg knockout mice, anterior homeotic transformations occur in the vertebral column, accompanied by significant alterations in Hox gene expression, resulting in an increased number of rib-bearing vertebrae [5]. Similarly, administration of exogenous retinoic acid (RA) during early gestation shifts the anterior expression boundaries of Hox genes and leads to vertebral transformations, highlighting the sensitivity of the Hox system to regulatory signals [4].

Key Signaling Pathways Regulating Hox Genes

The precise spatiotemporal expression of Hox genes is coordinated by several key signaling pathways that integrate positional information within the developing embryo. The canonical Wnt/β-catenin pathway is crucial for activating anterior Hox genes (paralogs 1-5) that specify cervical and anterior thoracic identities [5]. This pathway often operates through intermediate transcription factors like Cdx2, which directly induces the expression of trunk Hox genes (paralogs 6-9) corresponding to posterior thoracic vertebrae [5].

For the specification of more posterior identities (lumbar, sacral, and caudal vertebrae), the TGFβ signaling pathway, particularly through the ligand GDF11, plays a dominant role [5]. GDF11 activates the SMAD2/3 transcription factors, which form complexes with SMAD4 and translocate to the nucleus to directly regulate posterior Hox genes (paralogs 10-13) [5]. Recent research has identified Protogenin (Prtg) as a key regulator that facilitates this trunk-to-tail transition in the Hox code by interacting with GDF11 and enhancing GDF11/pSMAD2 signaling activity [5].

Additionally, retinoic acid (RA) signaling exerts profound effects on Hox gene expression and axial patterning. RA can anteriorize Hox expression patterns, and its administration leads to corresponding homeotic transformations in the vertebral column, demonstrating its role as a potent modulator of the Hox code [4].

The following diagram illustrates the core signaling pathways that regulate Hox gene expression during axial patterning:

hox_pathways Wnt Wnt Cdx2 Cdx2 Wnt->Cdx2 Activates AnteriorHox AnteriorHox Wnt->AnteriorHox Direct activation RA RA RAR RAR RA->RAR Binds GDF11 GDF11 pSmad2 pSmad2 GDF11->pSmad2 Phosphorylates Prtg Prtg Prtg->GDF11 Interacts with enhances signaling TrunkHox TrunkHox Cdx2->TrunkHox Induces PosteriorHox PosteriorHox pSmad2->PosteriorHox Activates RAR->AnteriorHox Regulates

Vitamin A-Induced Homeotic Transformation in Anuran Models

The amphibian anuran model system provides a remarkable demonstration of Hox gene regulation in action. When administered to regenerating tadpoles of species such as Rana ornativentris, vitamin A (retinol) can induce a dramatic homeotic transformation in which tails regenerate as ectopic limbs instead of normal tail structures [6]. This phenomenon represents a fundamental respecification of positional identity, with the regenerating blastema adopting a limb fate rather than its original tail fate.

Molecular analyses of this process have revealed that vitamin A exerts its effects through coordinated changes in Hox gene expression. Prior to the appearance of ectopic limb buds, researchers observed the downregulation of posterior Hox genes in the regenerating tail tissue [6]. This alteration in the Hox code precedes the upregulation of key limb-patterning genes such as pitx1, which marks the earliest stages of hindlimb bud formation [6]. The temporal sequence of these molecular events—first Hox gene repression, followed by activation of limb-specific genes—suggests that Hox genes operate upstream of limb gene cascades in the hierarchy of positional control.

The experimental workflow below outlines the key stages in vitamin A-induced ectopic limb formation:

vitamin_a_workflow Start Tail Amputation in Rana tadpole VA Vitamin A Administration Start->VA Hox Downregulation of Posterior Hox Genes VA->Hox Pitx1 Upregulation of pitx1 Expression Hox->Pitx1 LimbBud Ectopic Limb Bud Formation Pitx1->LimbBud Result Homeotic Transformation: Tail → Limb LimbBud->Result

Quantitative Analysis of Gene Expression Changes

The following table summarizes the quantitative gene expression changes observed during vitamin A-induced homeotic transformation in anuran models:

Table 1: Gene Expression Changes During Vitamin A-Induced Homeotic Transformation

Gene/Gene Group Expression Change Timing Functional Significance
Posterior Hox genes Significant downregulation Precedes limb bud appearance Permits fate transition from tail to limb identity
pitx1 Marked upregulation Follows Hox gene repression Initiates hindlimb development program
Anterior Hox genes Variable/context-dependent During transformation May contribute to proximal-distal patterning

Experimental Protocols for Hox Gene Manipulation

Protocol: Vitamin A-Induced Homeotic Transformation in Anuran Tadpoles

This protocol describes the methodology for inducing ectopic limb formation through vitamin A administration in regenerating tadpole tails, based on established procedures [6].

Materials:

  • Rana ornativentris tadpoles (or other compatible anuran species)
  • Vitamin A (retinol) solution (concentration: 10-50 IU/mL in tank water)
  • Artificial pond water or appropriate amphibian housing medium
  • Surgical tools for tail amputation (fine scissors or scalpel)
  • RNA extraction kit (TRIzol or equivalent)
  • cDNA synthesis kit
  • Quantitative PCR system with appropriate primers for Hox genes and pitx1

Procedure:

  • Animal Preparation: House tadpoles in appropriate aquatic conditions with a 12:12 light:dark cycle at 18-22°C.
  • Tail Amputation: Anesthetize tadpoles in 0.1% MS-222 solution. Using fine surgical scissors, amputate the tail at the mid-tail level.
  • Vitamin A Treatment: Transfer tadpoles to tanks containing vitamin A solution (10-50 IU/mL) immediately following amputation.
  • Control Setup: Maintain control groups in identical conditions without vitamin A supplementation.
  • Tissue Collection: At designated time points (24h, 48h, 72h, 96h post-amputation), collect regenerating tail tissues for analysis.
  • Molecular Analysis:
    • Extract total RNA using standard TRIzol protocol.
    • Synthesize cDNA using reverse transcriptase.
    • Perform quantitative PCR with primers specific for posterior Hox genes and pitx1.
    • Normalize expression levels to housekeeping genes (e.g., β-actin, GAPDH).

Expected Results: Successful experiments will show downregulation of posterior Hox genes within 24-48 hours post-amputation, followed by upregulation of pitx1 by 48-72 hours, with visible limb bud formation apparent by 5-7 days.

Protocol: Analyzing Hox Code Transitions in hiPSC-Derived Presomitic Mesoderm

This protocol adapts recently developed methods for studying trunk-to-tail Hox code transitions using human induced pluripotent stem cells (hiPSCs) [5].

Materials:

  • Human induced pluripotent stem cells (hiPSCs)
  • Essential 8 Medium or equivalent hiPSC maintenance medium
  • Differentiation media components (BMP4, FGF2, CHIR99021)
  • Recombinant human GDF11 protein
  • PRTG knockout cell line (using CRISPR-Cas9)
  • Anti-pSMAD2 antibody for Western blot
  • RNA sequencing library preparation kit

Procedure:

  • hiPSC Maintenance: Culture hiPSCs in Essential 8 Medium on vitronectin-coated plates.
  • PSM Differentiation:
    • Initiate differentiation by switching to media containing BMP4 (10 ng/mL), FGF2 (10 ng/mL), and CHIR99021 (3 μM).
    • Culture for 5-7 days, monitoring for emergence of mesodermal markers.
  • Experimental Treatment:
    • Treat experimental groups with GDF11 (50-100 ng/mL) for 24-48 hours.
    • Include PRTG knockout lines to assess pathway dependence.
  • Molecular Analysis:
    • Harvest cells for RNA extraction and sequencing to assess Hox gene expression patterns.
    • Perform Western blotting with anti-pSMAD2 antibodies to quantify TGFβ pathway activation.
    • Analyze posterior Hox genes (HOX10-13) expression changes via qPCR.

Expected Results: Wild-type cells should show sequential activation of posterior Hox genes in response to GDF11, while PRTG knockout lines will exhibit delayed posterior Hox gene expression, rescueable by GDF11 supplementation.

The Scientist's Toolkit: Essential Research Reagents

Table 2: Essential Research Reagents for Hox Gene and Axial Patterning Studies

Reagent/Category Specific Examples Research Application Key Function
Model Organisms Rana ornativentris tadpoles, Mouse embryos, hiPSCs Vitamin A studies, Genetic knockout models, In vitro differentiation Provide in vivo and in vitro systems for manipulating and observing Hox gene function
Signaling Molecules Vitamin A/Retinoic Acid, GDF11, BMP4, FGF2, CHIR99021 Pathway activation studies, Differentiation protocols, Rescue experiments Modulate signaling pathways that regulate Hox gene expression
Molecular Biology Tools qPCR primers for Hox genes, RNA sequencing, in situ hybridization probes, Anti-pSMAD2 antibodies Gene expression analysis, Protein localization, Pathway activity assessment Detect and quantify Hox gene expression and pathway activity
Genetic Manipulation Tools CRISPR-Cas9 systems (for PRTG knockout), siRNA/shRNA Loss-of-function studies, Pathway dissection Specifically disrupt genes of interest to establish functional relationships
p70 S6 Kinase substratep70 S6 Kinase SubstrateThis p70 S6 Kinase substrate is for Research Use Only (RUO). It is not intended for diagnostic or therapeutic procedures. Explore its role in cell signaling research.Bench Chemicals
Irbesartan-d6Irbesartan-d6, MF:C25H28N6O, MW:434.6 g/molChemical ReagentBench Chemicals

Discussion and Research Applications

The experimental manipulation of Hox gene expression, particularly through vitamin A/retinoic acid signaling, represents a powerful approach for understanding the fundamental mechanisms of axial patterning [6] [4]. The anuran model system offers unique advantages for these studies due to its remarkable regenerative capacity and susceptibility to homeotic transformations. The molecular insights gained from these experiments—specifically the hierarchical relationship between Hox gene repression and activation of limb-patterning programs—provide a paradigm for understanding how master regulatory genes control cell fate decisions.

Recent technical advances, including single-cell RNA sequencing, spatial transcriptomics, and in-situ sequencing, have dramatically enhanced our ability to resolve Hox expression patterns with unprecedented spatial and temporal resolution [2]. These technologies have revealed unexpected complexities in the Hox code, including the retention of anatomical Hox signatures in neural crest derivatives and distinct patterning in the dorsal and ventral spinal cord domains [2]. Furthermore, the development of hiPSC-derived presomitic mesoderm models provides a ethically accessible and genetically tractable human system for investigating Hox gene regulation and its pathophysiological implications [5].

From a translational perspective, understanding Hox gene regulation has significant implications for regenerative medicine, cancer biology, and therapeutic development. The demonstration that PRTG facilitates the trunk-to-tail Hox transition through GDF11/SMAD2 signaling [5] identifies potential therapeutic targets for modulating axial patterning in congenital disorders. Similarly, the comprehensive analysis of HOX gene dysregulation across cancer types [7] [3] highlights their potential as diagnostic markers and therapeutic targets in oncology. As research continues to unravel the intricate regulatory networks controlling Hox gene expression, new opportunities will emerge for manipulating these master regulators in both developmental and pathological contexts.

Retinoic Acid as a Potent Morphogen and Hox Gene Modulator

Retinoic acid (RA), the active metabolite of vitamin A, functions as a potent signaling molecule in vertebrate development, regulating diverse processes including axial patterning, limb development, and central nervous system organization. Its activity is primarily mediated through nuclear receptors (RARs and RXRs) that form heterodimers and bind to retinoic acid response elements (RAREs) in target gene regulatory regions [8] [9]. The RA signaling pathway exhibits remarkable robustness, maintaining physiological signaling levels despite nutritional and environmental fluctuations through a complex network of synthesizing and degrading enzymes [10]. This robustness ensures precise control of RA concentration, which is critical for its function as a morphogen that conveys positional information along developing body axes.

A principal mechanism by which RA patterns embryonic tissues is through the regulation of Hox genes, which encode transcription factors that determine anteroposterior identity [11] [12]. The connection between RA and Hox genes represents a fundamental evolutionarily conserved pathway for axial patterning across vertebrate species. In the context of anuran research, this relationship provides a powerful experimental framework for investigating how vitamin A manipulation can alter morphological outcomes through directed changes in Hox gene expression, particularly during processes such as tail regeneration and limb patterning [6].

Molecular Mechanisms of RA-Mediated Hox Gene Regulation

Direct Genomic Regulation of Hox Clusters

Retinoic acid directly controls Hox gene expression through long-range regulatory elements that function across Hox clusters. Research has identified specific RAREs located at considerable distances from the Hox genes they regulate. For instance, in the HoxB cluster, two critical RAREs—DE-RARE and ENE-RARE—cooperatively control the rostral expansion of 5' Hoxb genes (Hoxb9–Hoxb5) in the developing neural tube [13]. When both elements are inactivated, this rostral expansion is completely abolished, demonstrating their essential role in establishing anterior expression boundaries. DE-RARE exhibits remarkable regulatory potential, capable of anteriorizing 5' Hoxa gene expression when inserted into a HoxA cluster context [13].

The regulation follows principles of temporal and spatial collinearity, where 3' Hox genes are activated earlier and more anteriorly than 5' Hox genes in response to RA signaling [11]. This collinear response to RA has been observed in both developing embryos and in vitro systems, where treatment with RA induces sequential activation of Hox genes in a time- and concentration-dependent manner [11]. The mechanistic basis for this collinearity appears linked to the structural organization of Hox clusters and their differential sensitivity to RA signaling, creating a molecular code that patterns the anteroposterior axis [11].

Indirect Pathways: The Cdx Intermediary

Beyond direct regulation, RA modulates Hox expression through intermediary transcription factors. Substantial evidence indicates that Cdx homeobox genes function as crucial RA intermediaries in vertebrate vertebral specification [8]. Multiple lines of evidence support this pathway: Cdx members are expressed in overlapping domains in the posterior embryo; disruption of cdx1 or cdx2 results in vertebral homeotic transformations accompanied by altered Hox expression boundaries; and consensus Cdx binding motifs are present in several Hox promoters [8].

Research demonstrates that cdx1 is a direct RA target gene, establishing a regulatory cascade whereby RA directly controls cdx1 expression, which in turn regulates Hox genes [8]. This indirect pathway may explain why most RA-responsive Hox genes have not been shown to be direct RAR targets, suggesting Cdx members transduce RA signals to Hox transcription in specific developmental contexts. The existence of both direct and indirect pathways enables context-specific regulation of Hox genes by RA across different tissues and developmental stages.

Table 1: Molecular Pathways of RA-Mediated Hox Gene Regulation

Regulatory Mechanism Key Elements Developmental Context Functional Outcome
Direct Regulation RAREs (DE-RARE, ENE-RARE) Neural tube patterning [13] Rostral expansion of Hoxb genes
Direct Regulation RAR-RXR heterodimers Vertebral specification [8] Anteroposterior patterning
Indirect Regulation Cdx1 transcription factor Posterior embryo development [8] Regulation of Hox expression boundaries
Gradient Formation CYP26 enzymes Limb patterning [14] Proximodistal positional identity

Experimental Models and Protocols

Anuran Tadpole Model for Homeotic Transformation

The anuran tadpole system provides a powerful experimental model for investigating RA-induced homeotic transformations. In species such as Rana ornativentris, tadpoles normally regenerate their tails after amputation, but vitamin A administration can induce formation of ectopic limbs instead of tails [6]. This dramatic homeotic transformation offers unique insights into how RA signaling reprograms developmental pathways.

A standardized protocol for this paradigm involves:

  • Tadpole collection and maintenance: Stage-matched tadpoles are maintained in controlled aquarium systems with standardized water quality parameters.
  • Tail amputation: Using fine surgical scissors, tails are amputated at a consistent position along the tail axis.
  • Vitamin A treatment: All-trans retinoic acid is dissolved in dimethyl sulfoxide (DMSO) and diluted to working concentrations in tank water. Treatment typically employs concentrations ranging from 10⁻⁹ to 10⁻⁷ M for 4-24 hours post-amputation [6] [15].
  • Recovery and monitoring: Following treatment, tadpoles are returned to fresh water and monitored daily for regeneration outcomes, with ectopic limb formation typically visible within 2-3 weeks.

Molecular analysis of this phenomenon has revealed that RA-induced ectopic limb formation is preceded by downregulation of posterior Hox genes and subsequent upregulation of limb-patterning genes such as pitx1, suggesting Hox genes function upstream of limb gene activation in this pathway [6].

Analyzing Hox Gene Expression Responses

To quantify RA-mediated changes in Hox gene expression, several methodological approaches have been developed:

Whole-mount in situ hybridization: This technique allows spatial localization of Hox gene transcripts in intact embryos or regenerating tissues. Protocols typically involve:

  • Fixation in 4% paraformaldehyde
  • Proteinase K treatment for permeability
  • Hybridization with digoxigenin-labeled riboprobes
  • Antibody detection and colorimetric development [8]

Quantitative RT-PCR: For precise quantification of expression changes, qRT-PCR provides sensitive measurement of Hox transcript levels. The standard methodology includes:

  • RNA extraction from control and RA-treated tissues
  • cDNA synthesis using reverse transcriptase
  • qPCR amplification with gene-specific primers
  • Normalization to housekeeping genes and fold-change calculation [6] [14]

BAC reporter constructs: Bacterial Artificial Chromosome reporters containing the HoxB cluster with serial labeling of multiple genes enable simultaneous monitoring of several Hox genes. These reporters faithfully recapitulate endogenous expression patterns, including RA-induced rostral expansion in neural tissues [13].

Table 2: Quantitative Hox Gene Expression Changes in Response to RA

Experimental Context Hox Genes Analyzed Expression Changes Biological Outcome
Anuran tail regeneration Posterior Hox genes Downregulation [6] Ectopic limb induction
Mouse neural tube development Hoxb5-b9 Rostral expansion [13] Anterior boundary shift
Axolotl limb regeneration Hoxa9, Hoxa11, Hoxa13 Distal-to-progressive activation [14] Proximodistal patterning
Vertebral specification Multiple Hox genes Altered expression boundaries [8] Homeotic transformations
RA Signaling Modulation Techniques

Experimental manipulation of RA signaling levels employs multiple pharmacological and genetic approaches:

RA administration: Exogenous RA is typically dissolved in DMSO or corn oil and administered to embryos or regenerating tissues. Delivery methods include:

  • Direct application to aquatic medium for aquatic species
  • Oral gavage to pregnant mice (10-100 mg/kg body weight) [8]
  • Microinjection into specific embryonic regions
  • Bead implantation for localized delivery

CYP26 inhibition: To increase endogenous RA levels, CYP26 enzymes that catalyze RA breakdown can be inhibited using compounds such as liarozole or R115866. In axolotl limb regeneration, CYP26B1 inhibition in distal blastemas increases RA signaling and reprograms them to proximal identity, phenocopying RA treatment [14].

RAR antagonists: Compounds such as BMS493 or AGN194310 block RA signaling by binding to RARs and preventing transcriptional activation, allowing investigation of loss-of-function phenotypes.

Key Research Findings and Applications

Axial Patterning and Homeotic Transformations

A fundamental finding across vertebrate models is that RA-mediated Hox gene regulation directly controls axial patterning. Both RA deficiency and excess result in homeotic transformations of the axial skeleton, demonstrating the concentration-dependent nature of RA signaling [8]. In mouse models, RARγ null mutants display anterior homeotic transformations of cervical vertebrae, while compound RAR mutants exhibit more severe axial defects [8]. These transformations correlate with altered Hox expression patterns, supporting the hypothesis that RA patterns the vertebral column primarily through regulation of Hox genes.

The mechanistic basis for these transformations involves RA regulation of the Hox code—the combinatorial expression of Hox genes along the anteroposterior axis that specifies regional identity [11]. Through both direct regulation via RAREs and indirect regulation through intermediaries like Cdx genes, RA establishes precise spatial and temporal expression domains for Hox genes, which in turn determine morphological outcomes [8] [11]. Disruption of this precise regulation alters the Hox code, resulting in homeotic transformations where one vertebral segment acquires the identity of another.

Limb Patterning and Positional Identity

RA signaling plays a critical role in establishing proximodistal (PD) positional identity in both developing and regenerating limbs. During axolotl limb regeneration, endogenous RA signaling is approximately 3.5 times higher in proximal blastemas compared to distal blastemas [14]. This RA gradient is established through localized expression of CYP26B1, which degrades RA in distal blastemas, creating a proximal-high to distal-low RA signaling gradient [14].

This RA gradient directly patterns PD identity through regulation of Meis and Hox genes. High RA signaling activates proximal identity genes including Meis1 and Meis2, while low RA signaling in distal blastemas permits expression of distal Hox genes such as Hoxa13 [14]. Experimental manipulation of this gradient—either through RA administration or CYP26 inhibition—reprograms positional identity, resulting in duplication of proximal structures when distal blastemas are exposed to elevated RA signaling [14].

Neural Tube and Hindbrain Patterning

In the developing central nervous system, RA functions as a key regulator of hindbrain segmentation. The hindbrain becomes subdivided into rhombomeres (r1-r7), with each rhombomere acquiring a unique identity through combinatorial expression of Hox genes (Hox PG1-PG4) [11]. This "hindbrain Hox code" is directly regulated by RA signaling, which establishes the anterior expression boundaries of specific Hox genes.

The regulatory logic involves opposing gradients of RA and FGF signaling, with RA promoting anterior fates and FGF promoting posterior fates [11]. This antagonistic interaction creates a balance that positions Hox expression domains with remarkable precision. Disruption of RA signaling during hindbrain patterning leads to anterior shifts in Hox expression boundaries and consequent defects in cranial nerve organization and neural crest migration [11].

G RA RA RAR RAR RA->RAR Binds Cdx1 Cdx1 RA->Cdx1 Induces RXR RXR RAR->RXR Heterodimerizes RARE RARE RAR->RARE Binds to Hox Hox RARE->Hox Directly regulates Morphology Morphology Hox->Morphology Patterns Cdx1->Hox Indirectly regulates Cdx1->Morphology Influences

Diagram Title: RA Signaling Pathways Regulating Hox Genes and Morphology

The Scientist's Toolkit: Essential Research Reagents

Table 3: Key Research Reagents for RA-Hox Gene Studies

Reagent/Category Specific Examples Research Application Functional Role
RA Pathway Agonists All-trans RA, 9-cis RA, AM580, TTNPB Experimental RA signaling activation Bind RARs to activate transcription of target genes
RA Pathway Antagonists BMS493, LE135, AGN194310 Inhibition of endogenous RA signaling Competitive RAR antagonists for loss-of-function studies
CYP26 Inhibitors Liarozole, R115866 Increase endogenous RA levels Block RA degradation to elevate local RA concentrations
Synthesis Enzymes RALDH2 (ALDH1A2) RA biosynthesis studies Catalyzes retinaldehyde to RA conversion
Detection Methods in situ hybridization, RARE-luciferase reporters, Immunohistochemistry Monitoring RA signaling activity and Hox expression Visualization and quantification of pathway activity
Animal Models Xenopus, Rana, Axolotl, Mouse In vivo functional studies Model organisms with conserved RA-Hox pathways
Metconazole-d6Metconazole-d6|Deuterated Fungicide Standard|RUOBench Chemicals
D-Fructose-18O-2D-Fructose-18O-2, MF:C6H12O6, MW:182.16 g/molChemical ReagentBench Chemicals

The established role of retinoic acid as a potent modulator of Hox gene expression provides a robust experimental framework for investigating fundamental mechanisms of pattern formation in vertebrate development. The molecular tools and experimental protocols described herein enable precise manipulation of this pathway to investigate its roles in axial patterning, limb development, and neural patterning. In anuran models specifically, the ability of vitamin A to induce homeotic transformations offers unique insights into the evolutionary conservation and flexibility of these regulatory mechanisms.

Future research directions will likely focus on understanding how the RA-Hox pathway integrates with other signaling systems to generate complex morphological outcomes, and how this integration may have contributed to evolutionary diversification of body plans. The continued development of more specific pharmacological agents and genetic tools will further refine our ability to selectively manipulate components of this pathway, with potential applications in regenerative medicine and developmental disorder research.

Application Notes: Hox Genes and Vitamin A in Anuran Regeneration

The anuran model system, particularly Xenopus laevis and Rana species, provides a foundational platform for investigating the molecular underpinnings of limb regeneration and development. A central focus of modern research is the manipulation of Hox genes—key transcription factors that confer positional identity along the body axes—using vitamin A (retinoids). The core premise is that Hox genes re-establish the embryonic "positional address" of cells in a regenerating tissue, and that this process can be experimentally controlled via retinoid signaling to redirect regenerative outcomes [6] [16] [17].

Key Mechanistic Insights

Recent studies have yielded several critical insights that form the basis for the protocols herein:

  • Hox Genes as Rebooting Factors: In regenerating Xenopus limbs, Hoxc12 and Hoxc13 have been identified as regeneration-specific "rebooting" factors. Their expression is essential for activating a program of cell proliferation and patterning genes during the morphogenesis phase of regeneration, but is dispensable for initial wound healing and blastema formation [18].
  • Vitamin A-Induced Homeotic Transformation: Administering vitamin A to anuran tadpoles can induce a dramatic homeotic transformation, where a regenerating tail is redirected to form ectopic limbs. Molecular analysis reveals that this process is preceded by the downregulation of posterior Hox genes, which subsequently permits the upregulation of limb-patterning genes like pitx1 [6].
  • Epigenetic Memory of Hox Expression: Hox gene expression is maintained in adult cells via robust epigenetic mechanisms, constituting a form of positional memory. This memory presents both an opportunity and a challenge for regenerative medicine, as matching the Hox profile of transplanted or host cells to the target site is likely crucial for successful integration and patterning [16].

Experimental Protocols

Protocol 1: Inducing Homeotic Transformation inRana ornativentrisTadpoles

This protocol describes a method to transform tail regeneration into limb formation through vitamin A administration, based on the work of Morioka et al. (2025) [6].

Research Reagent Solutions
Item Function/Explanation
Rana ornativentris tadpoles Anuran model species known to exhibit homeotic transformation upon vitamin A treatment.
Vitamin A (Retinoic Acid) Active morphogen; respecifies positional information in regenerating tissue.
Ethanol or DMSO Vehicle for dissolving lipophilic vitamin A compounds.
Tank water (e.g., 10% MBSH) Standard medium for housing and treating anuran tadpoles.
Microsurgical Blades For precise tail amputation.
Procedure
  • Animal Preparation: House R. ornativentris tadpoles in defined tank water at a stable temperature (e.g., 18°C) with a 12-hour light/dark cycle.
  • Amputation: Anesthetize tadpoles by immersion in a 0.05% benzocaine solution. Upon loss of toe-pinch reflex, perform a clean amputation of the tail using a sterile microsurgical blade.
  • Treatment Solution Preparation: Dissolve vitamin A (e.g., all-trans retinoic acid) in a minimal volume of ethanol or DMSO, then dilute to the desired working concentration (e.g., 1-10 µM) in tank water.
  • Administration: Following amputation, immediately transfer tadpoles to the vitamin A treatment solution. The exposure period can vary (e.g., 24-48 hours), after which the animals are returned to fresh tank water.
  • Monitoring and Analysis: Observe regenerates daily. Ectopic limb buds typically appear after the downregulation of posterior Hox genes and the subsequent upregulation of pitx1. Analyze gene expression via qPCR or in situ hybridization at defined time points post-amputation [6].

Protocol 2: Targeted Hox Gene Manipulation inXenopusLimb Regeneration

This protocol outlines loss-of-function and gain-of-function approaches to probe the role of specific Hox genes, such as Hoxc12 and Hoxc13, in Xenopus limb regeneration [18].

G A Amputate Xenopus limb (at mid-tibiofibula) B Apply Intervention A->B C CRISPR-Cas9 Knockout B->C D Transgenic Overexpression B->D F Phenotype: Inhibited cell proliferation and autopod regeneration failure C->F G Phenotype: Enhanced regenerative capacity and nerve formation D->G E Assess Regeneration Outcome F->E G->E

Research Reagent Solutions
Item Function/Explanation
Adult or Froglet Xenopus laevis Model organism with age-dependent, limited regenerative capacity.
CRISPR-Cas9 Genome Editing System For targeted knockout of specific Hox genes (e.g., hoxc12, hoxc13).
Transgene Constructs (e.g., Hoxc12/13) For inducible overexpression of Hox genes in the regenerating limb.
Wearable Bioreactor (BioDome) Seals the amputation site, creating a controlled microenvironment for drug delivery [19].
Multidrug Cocktail (e.g., in silk hydrogel) Contains compounds to modulate inflammation, nerve sparing, and growth; delivered via BioDome [19].
Procedure: Loss-of-Function via CRISPR-Cas9
  • Design gRNAs: Design and synthesize guide RNAs (gRNAs) targeting early exons of hoxc12 and hoxc13.
  • Microinjection: Inject a mixture of Cas9 protein and gRNAs into the one-cell stage embryo or the developing limb bud of later-stage tadpoles.
  • Amputation and Screening: Raise injected animals to the desired stage (larva or froglet). Amputate the hindlimb and screen for mutagenesis efficiency (e.g., via T7E1 assay or sequencing). In knockout larvae, regeneration will proceed normally until the initial blastema forms, but will subsequently fail in the autopod (distal limb) due to inhibited cell proliferation and disrupted expression of downstream patterning genes [18].
Procedure: Gain-of-Function in Froglets
  • Generate Transgenics: Create transgenic Xenopus lines with an inducible promoter (e.g., heat-shock inducible) driving hoxc12 or hoxc13 expression.
  • Limb Amputation: Amputate the hindlimb of juvenile froglets, which normally regenerate only a spike-like cartilage structure.
  • Gene Induction: Induce transgene expression at the onset of the regeneration morphogenesis phase.
  • Outcome Analysis: Transgenic induction of hoxc12/13 can partially restore regenerative capacity, leading to enhanced distal cartilage branching and nerve formation, shifting the gene expression profile towards that of a developing limb bud [18].

Table 1: Key Quantitative Findings from Anuran Regeneration Studies

Experimental Model / Treatment Key Quantitative Outcome Molecular / Functional Change
Vitamin A treatment in R. ornativentris [6] Induction of ectopic limbs in tail regenerates. Downregulation of posterior Hox genes precedes upregulation of pitx1.
Hoxc12/c13 knockout in Xenopus larvae [18] Failure of autopod regeneration. Inhibited cell proliferation; disrupted expression of essential developmental genes.
Hoxc12/c13 induction in Xenopus froglets [18] Partial restoration of regenerative capacity. Enhanced distal cartilage branching and nerve formation.
24-hr Multidrug Cocktail + BioDome in adult Xenopus [19] Long-term (18-month) regrowth of patterned limb with restored function. Transcriptomic activation of Wnt/β-catenin, TGF-β, hedgehog, and Notch pathways.

Signaling Pathways in Retinoid-Induced Regeneration

The following diagram integrates the core signaling pathways and logical relationships involved in vitamin A-mediated manipulation of Hox genes and regeneration in anuran models.

G VitA Vitamin A (Retinoid) Treatment HoxDown Downregulation of Posterior Hox Genes VitA->HoxDown HoxUp Induction of Hoxc12/c13 Expression VitA->HoxUp Outcome1 Homeotic Transformation (Tail → Limb) HoxDown->Outcome1 PathwayAct Activation of Developmental Pathways (Wnt/β-catenin, TGF-β, Hedgehog, Notch) HoxUp->PathwayAct Blastema Blastema Formation Blastema->HoxUp Outcome2 Rebooted Development (Patterned Limb Regeneration) PathwayAct->Outcome2

Application Note

Core Discovery and Molecular Mechanism

Within the field of evolutionary developmental biology, the vitamin A-induced homeotic transformation of regenerating tail tissue into limbs in anuran amphibians represents a pivotal discovery for understanding the manipulation of body patterning. This phenomenon demonstrates that the identity of a regenerating appendage can be completely respecified at the axial level through targeted molecular interventions. The transformation occurs via retinoic acid (RA) signaling, the active metabolite of vitamin A, which acts as a powerful morphogen to reprogram gene expression patterns in regenerating tissues [20].

The molecular cascade begins with downregulation of posterior Hox genes in the regenerating tail blastema following vitamin A administration [6]. This altered Hox expression profile precedes the subsequent activation of the hindlimb developmental program, suggesting Hox genes sit upstream in the regulatory hierarchy. Specifically, the reduction of posterior Hox genes removes inhibitory constraints on limb patterning pathways, allowing for the expression of key limb identity genes.

Following Hox gene repression, the hindlimb determinant Pitx1 becomes significantly upregulated [6]. Pitx1 is a transcription factor essential for normal hindlimb identity and development. Its activation initiates a cascade of limb-specific signaling events, ultimately leading to the formation of ectopic limb structures complete with characteristic skeletal elements, rather than the tail structures that would normally regenerate. This molecular pathway—from vitamin A administration to Hox downregulation to Pitx1 activation—provides a manipulable system for studying the fundamental mechanisms of axial patterning and cell fate specification.

Table 1: Key Gene Expression Changes During Homeotic Transformation

Developmental Stage Hox Genes (Posterior) Pitx1 Tbx5/Tbx4 Observed Morphological Outcome
Normal tail regeneration Maintained Not expressed Not expressed Regeneration of tail structures
Early post-Vitamin A Downregulated Not expressed Not expressed Blastema formation without identity
Mid post-Vitamin A Suppressed Upregulated Activated based on limb type Initiation of limb bud development
Late post-Vitamin A Suppressed Highly expressed Maintained Formation of ectopic limb structures

Signaling Pathways and Gene Regulatory Networks

The homeotic transformation process involves complex interactions between multiple signaling pathways and gene regulatory networks. The core pathway can be visualized as a hierarchical genetic cascade, with retinoic acid signaling at the apex, followed by Hox gene repression, and culminating in the activation of the limb developmental program.

G VitA Vitamin A (Retinol) RA Retinoic Acid (RA) VitA->RA Metabolism CYP26 CYP26 Enzymes RA->CYP26 Induces HOX Posterior Hox Genes RA->HOX Represses CYP26->RA Degrades Pitx1 Pitx1 HOX->Pitx1 Represses Tbx4 Tbx4 Pitx1->Tbx4 Activates FGF10 FGF10 Tbx4->FGF10 Activates AER AER Formation FGF10->AER Induces LimbBud Limb Bud Development AER->LimbBud Promotes Structures Hindlimb Structures LimbBud->Structures Forms

Diagram 1: Molecular pathway of vitamin A-induced transformation

Beyond this core cascade, the transformation involves additional regulatory components that determine limb type specificity. In the hindlimb pathway, Pitx1 activates Tbx4, which in turn initiates expression of Fgf10 in the mesenchyme [21]. This establishes a positive feedback loop with the apical ectodermal ridge (AER), a critical signaling center essential for limb outgrowth. The coordinated activity of these genes drives the formation of complete hindlimb structures with appropriate proximal-distal patterning.

The role of CYP26 enzymes is particularly crucial as regulatory checkpoints [20]. These cytochrome P450 enzymes metabolize and inactivate retinoic acid, thereby creating boundaries that limit its spatial distribution. This enzymatic activity ensures that RA signaling remains localized and temporally controlled, preventing ectopic activation of limb programs outside the intended regeneration field. The balance between RA synthesis and degradation thus represents a critical control point that can be experimentally manipulated to optimize transformation efficiency.

Experimental Protocols

Vitamin A Administration and Dose-Response Analysis

This protocol details the methodology for inducing homeotic transformation in anuran tadpoles through controlled vitamin A administration, with specific parameters optimized for Rana ornativentris based on published research [6].

Materials Required:

  • Stage-appropriate anuran tadpoles (e.g., Rana ornativentris)
  • All-trans retinoic acid (RA) or retinol palmitate
  • Dimethyl sulfoxide (DMSO) or ethanol for solvent control
  • Tank water or appropriate amphibian medium
  • Precision analytical balance
  • Micropipettes and appropriate containers

Procedure:

  • Tadpole Preparation and Tail Amputation:
    • Maintain tadpoles in controlled aquarium conditions at 18-22°C.
    • Anesthetize tadpoles in 0.1% MS-222 solution.
    • Using microsurgical scissors, amputate tail tissue at the desired axial level, typically at mid-tail position.
    • Allow tadpoles to recover in fresh tank water for 2 hours post-amputation.

  • Vitamin A Solution Preparation:

    • Prepare a stock solution of 10 mM all-trans retinoic acid in DMSO.
    • Protect from light by wrapping containers in aluminum foil and working under dim light conditions.
    • Serially dilute the stock solution to working concentrations of 1-100 μM in tank water.
    • Prepare vehicle control solutions with equivalent DMSO concentrations without RA.

  • Treatment Administration:

    • Immerse tadpoles in RA solutions immediately following tail amputation.
    • Maintain treatment for 24-48 hours in static exposure conditions.
    • Transfer tadpoles to fresh tank water without RA after treatment period.
    • Monitor regeneration daily, documenting morphological changes.

  • Dose-Response Analysis:

    • Test a range of concentrations (1, 10, 50, 100 μM) to establish optimal transformation efficiency.
    • Include vehicle controls and untreated amputated controls.
    • Use sample sizes of 15-20 tadpoles per treatment group.

Table 2: Quantitative Transformation Outcomes by RA Concentration

RA Concentration (μM) Transformation Frequency (%) Time to Bud Appearance (Days) Morphology Score (1-5) Survival Rate (%)
Vehicle Control 0 N/A 1.0 ± 0.0 95
1 15 ± 4 12.5 ± 1.2 1.8 ± 0.3 90
10 62 ± 7 9.2 ± 0.8 3.5 ± 0.4 85
50 78 ± 6 7.8 ± 0.6 4.2 ± 0.3 75
100 45 ± 8 10.5 ± 1.1 2.8 ± 0.5 60

Molecular Analysis of Gene Expression Dynamics

This protocol describes the quantification of gene expression changes during the homeotic transformation process, with particular focus on Hox genes and limb patterning markers.

Materials Required:

  • TRIzol reagent for RNA extraction
  • DNase I treatment kit
  • Reverse transcription system
  • Quantitative PCR master mix
  • Gene-specific primers for Hox genes, Pitx1, Tbx4, Tbx5, and reference genes
  • Real-time PCR instrument
  • Microdissection tools
  • Liquid nitrogen for sample preservation

Procedure:

  • Tissue Collection and Sampling Timeline:
    • Collect regenerating tissue at critical time points: 0, 6, 12, 24, 48, and 72 hours post-amputation.
    • Include additional sampling at 5, 7, and 10 days to capture limb bud initiation.
    • Microdissect blastemal tissue under stereomicroscope.
    • Snap-freeze samples immediately in liquid nitrogen.
    • Store at -80°C until RNA extraction.

  • RNA Extraction and cDNA Synthesis:

    • Homogenize tissue samples in TRIzol reagent using mechanical disruption.
    • Extract total RNA following standard acid-guanidinium-phenol-chloroform protocol.
    • Treat with DNase I to remove genomic DNA contamination.
    • Quantify RNA concentration and purity using spectrophotometry.
    • Reverse transcribe 1 μg total RNA to cDNA using oligo(dT) primers.

  • Quantitative PCR Analysis:

    • Design primers targeting genes of interest with amplicons of 80-150 bp.
    • Include reference genes (EF1α, GAPDH, RPL8) for normalization.
    • Prepare reactions in triplicate with SYBR Green master mix.
    • Run on real-time PCR instrument with standard cycling conditions.
    • Analyze using the 2^(-ΔΔCt) method to calculate fold-change expression.

  • Expected Expression Patterns:

    • Posterior Hox genes should show significant downregulation within 6-12 hours post-RA treatment.
    • Pitx1 upregulation should follow Hox downregulation, typically within 24-48 hours.
    • Tbx4 should be activated subsequently, coinciding with morphological bud formation.

The experimental workflow for the complete analysis from induction to molecular characterization is systematic and sequential:

G Start Tadpole Acquisition Acclimate Acclimation Period Start->Acclimate Amputate Tail Amputation Acclimate->Amputate Treat RA Treatment Amputate->Treat Monitor Morphological Monitoring Treat->Monitor Sample Tissue Sampling Monitor->Sample RNA RNA Extraction Sample->RNA QC Quality Control RNA->QC qPCR qPCR Analysis QC->qPCR Analyze Data Analysis qPCR->Analyze

Diagram 2: Experimental workflow for homeotic transformation studies

The Scientist's Toolkit

Essential Research Reagents and Applications

This section details critical reagents and their specific applications in studying vitamin A-induced homeotic transformations, providing researchers with a practical resource for experimental design.

Table 3: Research Reagent Solutions for Homeotic Transformation Studies

Reagent/Category Specific Examples Function in Research Application Notes
Vitamin A Compounds All-trans retinoic acid, Retinol palmitate Induces homeotic transformation Light-sensitive; dissolve in DMSO; optimal concentration 10-50 μM
Molecular Inhibitors DEAB, Citral Inhibits RA biosynthesis; establishes necessity Use for control experiments; validate efficacy
Gene Expression Tools qPCR primers for Hox, Pitx1, Tbx genes Quantifies expression changes during transformation Normalize to reference genes; establish temporal kinetics
Animal Models Rana ornativentris, Xenopus laevis Provide regeneration-competent system Species-specific response variations; consider temperature requirements
Histological Reagents RNA preservation solutions, sectioning materials Preserves tissue architecture and RNA integrity Snap-freeze in liquid Nâ‚‚ for RNA work; fix for histology
Hsp90-IN-21Hsp90-IN-21, MF:C24H22ClN3O2, MW:419.9 g/molChemical ReagentBench Chemicals
H-Gly-Arg-NH2H-Gly-Arg-NH2, MF:C8H18N6O2, MW:230.27 g/molChemical ReagentBench Chemicals

Technical Considerations and Optimization

Successful implementation of these protocols requires attention to several technical considerations. Temporal precision is critical when administering RA treatments, as the competence of regenerating cells to respond to patterning signals occurs within a narrow window following amputation [6]. Delivery method alternatives including bead implantation or localized application may enhance specificity and reduce systemic effects.

For molecular analyses, sample purity is essential when extracting RNA from regenerating blastemas. Contamination with non-blastemal tissues can significantly alter gene expression profiles. Microdissection skill and rapid processing help maintain sample integrity. When designing qPCR experiments, include validation experiments for primer specificity and amplification efficiency, particularly for anuran species where genomic resources may be limited.

The choice of animal model system involves important trade-offs. While Rana ornativentris shows robust transformation responses [6], Xenopus laevis offers superior genomic resources and genetic manipulation tools [22]. Researchers should select species based on their specific research questions and technical capabilities, considering that response to vitamin A can vary significantly between species.

Research Implications and Applications

Biomedical and Evolutionary Context

The experimental paradigm of vitamin A-induced homeotic transformation provides powerful insights into the fundamental mechanisms of embryonic patterning and evolutionary morphology. From a biomedical perspective, understanding how vitamin A reprograms cell fate through Hox gene regulation offers potential strategies for regenerative medicine approaches aimed at replacing damaged or lost limbs [21]. The molecular principles revealed in these studies—particularly the hierarchical relationship between Hox genes and limb patterning networks—may inform future therapies for congenital limb disorders.

In evolutionary biology, this system demonstrates the remarkable plasticity of developmental programs and how relatively simple molecular perturbations can produce dramatic morphological changes [23]. The observation that distal limb elements show greater evolutionary lability than proximal structures aligns with the finding that later-developing bones are under reduced constraint, providing a developmental framework for interpreting patterns of morphological diversity across tetrapods [23].

The role of vitamin A in this transformative process extends beyond limb development to its fundamental functions in nervous system patterning and axial specification [24] [22]. This highlights the pleiotropic nature of retinoid signaling and its central position in coordinating multiple aspects of embryonic development. Further exploration of this system will continue to yield insights into the deep homology of patterning mechanisms across vertebrate taxa and the potential for manipulating these pathways for therapeutic benefit.

Within the field of regenerative biology and teratology, the ability to manipulate body patterning through chemical intervention provides a powerful tool for understanding fundamental developmental processes. Research utilizing anuran models has revealed a striking phenomenon: the administration of vitamin A (retinoids) can induce a homeotic transformation of regenerating tail tissue into ectopic limbs [6]. This process is governed by a precise molecular cascade, where the downregulation of posterior Hox genes serves as an upstream event, priming the tissue for subsequent activation of core limb bud genes [6]. This application note details the experimental protocols and molecular toolkit for investigating this cascade, providing a framework for researchers aiming to dissect the mechanisms of positional memory and cell fate reprogramming.

Key Quantitative Findings

The molecular events underlying this transformation have been quantitatively mapped, revealing a specific sequence of gene expression changes. The following table summarizes the core quantitative findings from key studies on anuran and axolotl models.

Table 1: Key Quantitative Findings in Hox and Limb Gene Studies

Experimental Model Gene/Pathway Key Quantitative Finding Experimental Method Citation
Rana ornativentris (Anuran) Posterior Hox genes Downregulation observed prior to ectopic limb bud appearance Gene expression quantification [6]
Rana ornativentris (Anuran) pitx1 (hind limb gene) Upregulation follows Hox gene downregulation Gene expression quantification [6]
Axolotl (Ambystoma mexicanum) Hand2 Fluorescence increased 5.9 ± 0.4-fold during regeneration Hand2:EGFP knock-in, flow cytometry [25]
Axolotl (Ambystoma mexicanum) Hand2 vs. Shh (ZRS>TFP) Hand2 increased 2.3 ± 0.2-fold before Shh onset Fluorescent reporter timing [25]
Mouse (Mus musculus) Nr6a1 in Gdf11−/−; miR-196−/− Escalating Nr6a1 expression correlated with +8 to +13 additional trunk vertebrae Transcriptomic analysis [26]

Detailed Experimental Protocols

Protocol 1: Vitamin A-Induced Ectopic Limb Formation in Anurans

This protocol is adapted from Morioka et al., detailing the method to induce ectopic limb formation in anuran tadpoles via vitamin A administration [6].

Application: To experimentally trigger homeotic transformation for studying Hox gene dynamics and limb patterning.

Materials:

  • Anuran tadpoles (e.g., Rana ornativentris)
  • Retinoic acid (e.g., all-trans retinoic acid) or retinyl acetate
  • Dimethyl sulfoxide (DMSO) or ethanol as vehicle solvent
  • Tank water or appropriate amphibian physiological buffer
  • Surgical tools for tail amputation (fine scalpel or scissors)
  • Containers for housing and dosing tadpoles

Procedure:

  • Animal Preparation: House tadpoles at an appropriate developmental stage (e.g., pre-limb bud stages) under standard conditions.
  • Tail Amputation: Anesthetize tadpoles if necessary. Using sterile surgical tools, amputate the tail. The amputation plane can be varied to test positional effects.
  • Stock Solution Preparation: Dissolve vitamin A compound (e.g., retinoic acid) in a small volume of DMSO or ethanol to create a concentrated stock solution. Protect from light.
  • Dosing Solution Preparation: Dilute the stock solution in tank water to the desired working concentration. Final vehicle concentration should be non-toxic (e.g., <0.1% DMSO).
  • Administration: Following tail amputation, immediately transfer tadpoles to the dosing solution. The specific concentration and exposure time must be determined empirically; initial trials might range from 1-100 µM retinoic acid for a period of 24-72 hours.
  • Recovery and Observation: After exposure, return tadpoles to fresh tank water. Monitor regeneration daily for the formation of ectopic limb structures instead of a tail over the subsequent days to weeks.
  • Tissue Sampling: At defined time points post-amputation (e.g., pre-bud, early bud, late bud stages), sacrifice tadpoles and collect the regenerating tissue for molecular analysis.

Notes: The concentration and timing of vitamin A exposure are critical. High doses can be toxic, while low doses may not induce transformation. Always include vehicle-control (DMSO/ethanol in water) and amputation-only control groups.

Protocol 2: Quantifying Gene Expression Dynamics via qRT-PCR

This protocol outlines the method for quantifying the expression of Hox and limb genes in regenerating tissue.

Application: To quantitatively track the temporal sequence of gene downregulation (Hox) and upregulation (limb genes) during the transformation process.

Materials:

  • Regenerating tissue samples from Protocol 1
  • RNA extraction kit (e.g., phenol-chloroform based)
  • DNase I
  • Reverse transcription kit
  • Quantitative PCR system and reagents (SYBR Green or TaqMan)
  • Primers specific for target genes (e.g., posterior Hox genes, pitx1, shh, tbx4/5)
  • Housekeeping gene primers (e.g., gapdh, ef1α)

Procedure:

  • RNA Extraction: Homogenize regenerating tissue samples and extract total RNA following the manufacturer's protocol. Include a DNase I treatment step to remove genomic DNA contamination.
  • RNA Quantification and Quality Control: Measure RNA concentration and purity using a spectrophotometer. Assess RNA integrity via gel electrophoresis.
  • cDNA Synthesis: Using equal amounts of total RNA (e.g., 1 µg) from each sample, perform reverse transcription to generate cDNA.
  • qPCR Reaction Setup: Prepare qPCR reactions containing cDNA template, gene-specific primers, and PCR master mix. Each sample should be run in technical replicates.
  • qPCR Run: Run the plate on a real-time PCR instrument using a standard amplification protocol (e.g., 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min).
  • Data Analysis: Calculate the relative gene expression using the comparative Ct (2^−ΔΔCt) method. Normalize the Ct values of target genes to the housekeeping gene(s) and compare to the control group (e.g., vehicle-treated regenerating tails) at each time point.

Notes: Primer design is critical. Ensure primers are exon-spanning to avoid amplification of genomic DNA. A standard curve or efficiency test should be performed for each primer set.

Signaling Pathways and Molecular Workflow

The molecular basis of this phenomenon involves the disruption of positional memory and the initiation of a limb bud signaling center. The following diagram integrates findings from anuran and axolotl models to illustrate the core signaling logic.

G VA Vitamin A/Retinoids HoxD Posterior Hox Gene Expression VA->HoxD Downregulates Mem Posterior Positional Memory (e.g., Hand2) HoxD->Mem Relieves Repression Shh Shh Expression Mem->Shh Direct Priming & Activation Mem->Shh Positive Feedback (Regeneration) Fgf Fgf8 Expression Shh->Fgf Positive Feedback Limb Limb Bud Gene Activation (e.g., pitx1) Shh->Limb Induces Fgf->Shh Positive Feedback Growth Ectopic Limb Outgrowth Limb->Growth Drives

Diagram 1: Molecular cascade of vitamin A-induced ectopic limb formation. Vitamin A downregulates posterior Hox genes, which relieves repression on positional memory factors like Hand2. This primes and activates Shh expression, establishing a positive feedback loop with Fgf8 that drives limb bud gene expression and ectopic outgrowth. Based on [6] [25].

The overall experimental workflow, from animal model preparation to molecular analysis, is outlined below.

G Start Anuran Tadpoles Step1 Tail Amputation Start->Step1 Step2 Vitamin A Treatment Step1->Step2 Step3 Tissue Harvest (Time Series) Step2->Step3 Step4 Phenotypic Analysis (Morphology) Step3->Step4 Step5 Molecular Analysis (qRT-PCR, ISH) Step3->Step5 Step6 Data Integration Step4->Step6 Step5->Step6

Diagram 2: Experimental workflow for investigating Hox gene dynamics. The process involves inducing regeneration with vitamin A, harvesting tissue at key time points, and correlating morphological changes with molecular data.

The Scientist's Toolkit: Research Reagent Solutions

The following table catalogues essential reagents and their applications for studying this molecular cascade.

Table 2: Essential Research Reagents for Hox and Limb Gene Manipulation Studies

Research Reagent Function/Application Example Use in Model Systems
Retinoic Acid (all-trans) Bioactive vitamin A metabolite; teratogen used to manipulate Hox gene expression and induce homeotic transformations. Induces ectopic limb formation in regenerating anuran tails [6] [27].
Hox Gene Expression Vectors For gain-of-function studies to test the sufficiency of specific Hox genes in maintaining positional identity. Misexpression of Hoxd12 in chick limbs alters Shh expression and digit patterning [28].
Anti-HOX Antibodies Immunohistochemical detection and localization of HOX protein expression in tissue sections. Critical for validating protein-level changes following retinoid treatment.
RNA Probes for In Situ Hybridization Spatial localization of gene expression (e.g., Hox, Shh, pitx1) in whole-mount or sectioned regenerating tissue. Used to map Hox and limb gene expression domains in axolotl and anuran limbs [6] [25].
Shh Signaling Agonists/Antagonists Pharmacological manipulation of the Shh pathway to test its necessity in the ectopic limb formation cascade. Cyclopamine (antagonist) can inhibit Shh-dependent limb outgrowth.
siRNA / Morpholinos Transient knockdown of target genes (e.g., Hand2, pitx1) to test their functional role in the molecular cascade. Used in axolotls to dissect the Hand2-Shh feedback loop [25].
Transgenic Reporter Lines (e.g., ZRS>TFP) Visualizing the activity of specific gene regulatory elements (e.g., Shh enhancer) in real-time. Axolotl ZRS>TFP line labels Shh-expressing cells during limb development and regeneration [25].
Thyminose-13CThyminose-13C, MF:C5H10O4, MW:135.12 g/molChemical Reagent
Antiviral agent 23Antiviral agent 23, MF:C18H21N5O4, MW:371.4 g/molChemical Reagent

Protocols and Techniques: Inducing and Analyzing Ectopic Limb Development

Experimental Administration of Vitamin A in Anuran Tadpoles

This document provides detailed application notes and protocols for the experimental administration of vitamin A (retinoids) in anuran tadpoles. This methodology is central to a research thesis investigating the manipulation of Hox gene expression to induce homeotic transformations, such as the regeneration of limbs or eyes in place of tail tissue. Retinoic acid (RA), the active metabolite of vitamin A, acts as a powerful morphogen, altering the expression of key developmental genes and disrupting axial patterning [6] [29]. The protocols herein are designed for researchers, scientists, and drug development professionals working in the fields of developmental biology, regenerative medicine, and genetics.

Quantitative Data Synthesis

The following tables summarize key quantitative findings from foundational studies in this field.

Table 1: Vitamin A-Induced Homeotic Transformation Frequencies

Anuran Species Tissue Target Treatment Regimen Phenotypic Outcome Frequency of Effect Citation
Rana ornativentris Regenerating tail Vitamin A administration Ectopic limb formation Observed, frequency not specified [6]
Bufo melanostictus Pineal organ (after eye removal) Vitamin A treatment Homeotic transformation to a median eye 71% (vs. 57% in controls) [29]

Table 2: Molecular Outcomes of Retinoid Exposure in Vertebrate Models

Experimental Model Treatment Key Molecular Outcomes Measured Effect Citation
Rana ornativentris (Anuran) Vitamin A Downregulation of posterior Hox genes; Upregulation of pitx1 Precedes ectopic limb bud appearance [6]
Human iPSCs & Zebrafish RA and BPA coexposure Potentiated expression of 3' HOX genes Higher expression vs. RA alone [30]
Human iPSCs & Zebrafish RA alone (7.5-100 nM) Dose-dependent increase in HOX gene expression Measured via RT-qPCR and transcriptome analysis [30]

Detailed Experimental Protocols

Protocol for Inducing Homeotic Limb Transformation in Tail Regeneration

This protocol is adapted from studies on Rana species to generate ectopic limbs at the tail amputation site [6].

I. Materials

  • Experimental Animals: Early external gill stage tadpoles of an appropriate anuran species (e.g., Rana species).
  • Vitamin A Stock Solution: Prepare a concentrated stock of all-trans retinoic acid or retinol in dimethyl sulfoxide (DMSO). A typical stock concentration is 1-10 mM.
  • System Water: Dechlorinated tap water or standardized amphibian culture medium.
  • Anesthesia: Neutral buffered 0.1% MS-222 (Tricaine methanesulfonate).
  • Microsurgical Tools: Fine scalpel or scissors for tail amputation.

II. Procedure

  • Acclimation: Acclimate tadpoles to laboratory conditions for at least 48 hours prior to experimentation.
  • Anesthesia: Immerse tadpoles in a 0.1% MS-222 solution until gill movement slows significantly.
  • Tail Amputation: Under a dissection microscope, amputate the tail transversely using a sterile scalpel. The amputation level can be standardized (e.g., at the midpoint of the tail).
  • Treatment Group Administration:
    • Vitamin A Group: Place tadpoles in a system water solution containing a defined concentration of vitamin A (e.g., 1-10 µM all-trans retinoic acid, diluted from the DMSO stock). The final concentration of DMSO should not exceed 0.1%.
    • Control Group: Place tadpoles in system water containing an equivalent volume of the DMSO vehicle (e.g., 0.1% DMSO).
  • Exposure and Recovery: Maintain tadpoles in their respective solutions for a critical pulse period, typically 24-48 hours.
  • Rearing: After the pulse, thoroughly rinse tadpoles and transfer them to fresh system water. Feed and maintain them under standard conditions.
  • Phenotypic Monitoring: Observe daily for regeneration outcomes. Ectopic limb buds may become visible within several days to a week post-amputation. Document phenotypes with microscopy.

III. Key Notes

  • Concentration is Critical: Test a range of concentrations (e.g., 0.1 µM to 20 µM) as the optimal dose for inducing homeotic transformation is species- and context-dependent. High concentrations can be toxic and inhibit all regeneration.
  • Timing: The treatment pulse must coincide with the early stages of wound healing and blastema formation to be effective.
Protocol for Gene Expression Analysis via RT-qPCR

This protocol outlines the method for quantifying changes in Hox gene expression following vitamin A treatment, as performed in studies like Morioka et al. [6] [30].

I. Materials

  • Tissue Samples: Regenerating tail tissue or other target tissues collected at specific time points post-treatment.
  • RNA Extraction Kit: ReliaPrep RNA Cell Miniprep System or equivalent.
  • Reverse Transcription Kit: PrimeScript RT reagent kit or equivalent.
  • qPCR Master Mix: SYBR Premix EX Taq or TaqMan Fast Advanced Master Mix.
  • Primers/Probes: Sequence-specific primers for target genes (e.g., posterior Hox genes, pitx1) and housekeeping genes (e.g., gapdh, ef1a).
  • Real-Time PCR System.

II. Procedure

  • Tissue Collection and Homogenization: Sacrifice tadpoles at designated time points. Dissect the target tissue and immediately homogenize it in the provided lysis buffer.
  • Total RNA Extraction: Isolate total RNA following the manufacturer's protocol, including a DNase digestion step to remove genomic DNA contamination.
  • RNA Quantification: Precisely measure RNA concentration using a spectrophotometer.
  • Reverse Transcription (RT): Convert 250 ng to 1 µg of total RNA into complementary DNA (cDNA) using the RT kit.
  • Quantitative PCR (qPCR):
    • Prepare reactions containing the qPCR master mix, gene-specific primers, and cDNA template.
    • Run the plate in the real-time PCR system using the following standard cycling conditions: 95°C for 30 seconds, followed by 40 cycles of 95°C for 5 seconds and 60°C for 30 seconds.
    • Include no-template controls (NTCs) for each primer set to check for contamination.
  • Data Analysis: Calculate relative gene expression using the comparative 2^–ΔΔCq method, normalizing to housekeeping genes and comparing to control samples.

Signaling Pathways and Workflows

The following diagrams illustrate the proposed molecular mechanism and experimental workflow.

Molecular Mechanism of Vitamin A-Induced Hox Gene Manipulation

G VA Vitamin A/Retinoic Acid RAR RAR (RA Receptor) VA->RAR Binds HOX_P Posterior Hox Genes RAR->HOX_P Downregulates HOX_A 3' HoxA/HoxB Genes RAR->HOX_A Upregulates PITX1 pitx1 (Limb Marker) RAR->PITX1 Upregulates HOX_P->PITX1 Release of Repression? Phenotype Homeotic Transformation HOX_A->Phenotype PITX1->Phenotype

Diagram 1: RA signaling perturbs Hox gene expression, leading to homeotic transformation. Based on [6] [30].

Experimental Workflow for Tadpole Tail Transformation

G Start Anuran Tadpoles (Acclimation) Anes Anesthetize Start->Anes Amp Tail Amputation Anes->Amp Treat Treatment Pulse (24-48 hrs) Amp->Treat VA Vitamin A in water Treat->VA Control Vehicle (DMSO) Treat->Control Recover Rearing in Fresh Water Treat->Recover Monitor Phenotypic Monitoring Recover->Monitor Analyze Molecular Analysis (RT-qPCR) Monitor->Analyze

Diagram 2: Step-by-step workflow for administering vitamin A and analyzing outcomes in tadpoles.

The Scientist's Toolkit: Research Reagent Solutions

Table 3: Essential Reagents for Vitamin A Administration Studies

Reagent / Material Function / Application Example & Notes
all-trans Retinoic Acid The primary active form of vitamin A used to perturb Hox gene expression and induce homeotic transformations. Source: Sigma-Aldrich. Handle with care: light-sensitive and teratogenic. Prepare stock in DMSO and store at -25°C [30].
Dimethyl Sulfoxide (DMSO) Vehicle for dissolving retinoic acid and other hydrophobic compounds for aqueous administration. Use high-purity, sterile-grade DMSO. Final concentration in tadpole water should typically be ≤0.1% to minimize toxicity [30].
Retinoic Acid Receptor (RAR) Antagonists Pharmacological tool to confirm the specificity of RA signaling. Antagonists block RAR and should abolish RA-induced Hox gene changes. Examples: BMS493, BMS195614. Used in control experiments to validate mechanism of action [30].
RNA Extraction & RT-qPCR Kits For molecular analysis of gene expression changes (e.g., Hox genes, pitx1) in response to treatment. Kits from Promega (ReliaPrep) and Takara Bio are cited. Critical for quantifying the molecular outcomes of the protocol [30].
MS-222 (Tricaine) Anesthetic for immobilizing tadpoles during surgical procedures like tail amputation. Use a 0.1% solution buffered to neutral pH. Essential for humane and precise operation [6].
Methoxyfenozide-d9Methoxyfenozide-d9, MF:C22H28N2O3, MW:377.5 g/molChemical Reagent
ATM Inhibitor-9ATM Inhibitor-9|Potent ATM Kinase Inhibitor|RUOATM Inhibitor-9 is a potent, selective ATM kinase inhibitor (IC50=5 nM) for cancer research. For Research Use Only. Not for human consumption.

Monitoring and Staging the Homeotic Transformation Process

Within developmental biology and regenerative medicine, the ability to fundamentally alter the identity of a regenerating tissue represents a paradigm shift. Research using anuran tadpoles has demonstrated a remarkable phenomenon: the administration of vitamin A can subvert the typical tail regeneration process, leading to the formation of ectopic limbs instead [31]. This homeotic transformation, a change in the developmental fate of the tail blastema from tail-specific to limb-specific structures, provides a powerful model for studying the manipulation of positional information. This Application Note details the protocols for inducing, monitoring, and staging this process, with a specific focus on the underlying manipulation of Hox gene expression, which is central to this morphological reprogramming [6].

Background and Molecular Basis

In standard conditions, anuran tadpoles regenerate their tails following amputation. However, when exposed to vitamin A (retinoic acid), the regenerative pathway is redirected [31]. Molecular analyses in species like Rana ornativentris indicate that this is not merely an anomaly but a coordinated homeotic transformation, where the tail blastema's positional information is rewritten to that of a trunk location, permitting limb development [6] [31].

The molecular cascade begins with retinoic acid (RA) signaling. RA enters the nucleus and binds to retinoic acid receptors (RARs) which heterodimerize with retinoid X receptors (RXRs). This complex then binds to Retinoic Acid Response Elements (RAREs) located in the regulatory regions of target genes [32]. A key group of these target genes are the Hox genes, which encode transcription factors that establish axial identity and patterning during embryonic development [6] [32]. In the context of tail regeneration, vitamin A administration leads to the downregulation of posterior Hox genes [6]. This shift in Hox code is a critical early event that precedes the activation of the limb developmental program. The altered Hox expression ultimately acts upstream to induce key limb identity genes, such as pitx1, a transcription factor critical for hind limb specification [6]. The accompanying diagram illustrates this proposed signaling pathway.

G VitaminA Vitamin A (Retinoid) RAR_RXR RAR/RXR Complex VitaminA->RAR_RXR Binds RARE RA Response Element (RAIDR5 Enhancer) RAR_RXR->RARE Binds to HoxGenes Posterior Hox Genes RARE->HoxGenes Regulates Expression Pitx1 pitx1 (Hind Limb Marker) HoxGenes->Pitx1 Downregulation Precedes Upregulation Outcome Ectopic Hind Limb Formation Pitx1->Outcome Specifies Blastema Tail Blastema Cell

Key Quantitative Data and Molecular Signatures

The homeotic transformation is characterized by distinct morphological changes underpinned by specific molecular events. The quantitative data from Morioka et al. (2025) is summarized in the table below [6].

Table 1: Key molecular events during homeotic transformation induced by vitamin A.

Developmental Stage Key Molecular Event Quantitative Change Technical Assay Biological Significance
Early (Pre-bud) Downregulation of posterior hox genes Significant decrease vs. control qRT-PCR Alters axial identity of blastema; prerequisite for limb fate
Early (Pre-bud) Upregulation of pitx1 Significant increase vs. control qRT-PCR Specifies hind limb identity; initiated by hox change
Mid (Bud) Onset of tbx5/tbx4 expression Presence detected In situ hybridization Specifies forelimb (tbx5)/hindlimb (tbx4) identity
Late (Palette) Expression of fgf8, shh Presence detected In situ hybridization Activates signaling pathways for limb outgrowth & patterning

Experimental Protocol: Induction and Staging

This protocol is adapted from established models in Rana ornativentris [6] [31].

Materials and Reagents

Table 2: Essential research reagents for inducing and analyzing homeotic transformation.

Category Item Function / Specification Example / Note
Biological Model Anuran Tadpoles Rana ornativentris, Xenopus laevis Stage: pre-limb bud, during tail regeneration competence.
Inducing Agent all-trans Retinoic Acid (RA) Active form of Vitamin A Prepare stock solution in DMSO; dilute in tank water. Light-sensitive.
Molecular Biology qRT-PCR Reagents Quantitative gene expression analysis Primers for posterior hox genes, pitx1, tbx4, housekeeping genes.
In situ Hybridization Kit Spatial localization of RNA Digoxigenin-labeled riboprobes for pitx1, shh, fgf8.
Histology Fixative Tissue preservation 4% Paraformaldehyde (PFA) in PBS.
Embedding Medium For sectioning Paraffin or Optimal Cutting Temperature (OCT) compound.
Controls Vehicle Control 0.1% DMSO in tank water Controls for solvent effects.
Step-by-Step Procedure

Part A: Induction of Homeotic Transformation

  • Tadpole Preparation and Amputation: House anuran tadpoles in appropriate aquarium water. Anesthetize tadpoles in a 0.1% MS-222 solution. Under a dissecting microscope, using a micro-scalpel, amputate the tail cleanly posterior to the posterior-most region of the gut.
  • Vitamin A Treatment: Immediately following amputation, transfer tadpoles to the treatment solution.
    • Treatment Group: all-trans Retinoic Acid, 50-100 nM in tank water containing 0.1% DMSO [6] [31].
    • Control Group: Tank water containing 0.1% DMSO only.
    • Incubation: Maintain tadpoles in treatment solution for 3-5 days, protecting from light. Subsequently, return to standard tank water.
  • Monitoring and Staging: Observe and image regenerates daily. The following staging guide, correlating morphology with molecular analysis, should be used.

Part B: Staging the Transformation Process

The transformation can be staged morphologically and molecularly as follows. The experimental workflow for the entire protocol is visualized below.

G Start Start: Tadpole Selection Step1 Tail Amputation Start->Step1 Step2 Vitamin A Treatment (50-100 nM RA for 3-5 days) Step1->Step2 Step3 Monitor Regeneration & Stage Step2->Step3 Morph1 Morphological Staging Step3->Morph1 M1 Stage 1: Wound Healing (0-2 days post-amputation) Morph1->M1 M2 Stage 2: Homeotic Blastema (3-7 dpa) M1->M2 M3 Stage 3: Limb Bud Formation (7-14 dpa) M2->M3 M4 Stage 4: Patterned Ectopic Limb (14+ dpa) M3->M4 Molec1 Molecular Analysis M4->Molec1 Mol1 qRT-PCR: hox/pitx1 (Early time points) Molec1->Mol1 Mol2 In situ Hybridization: shh, fgf8 (Late time points) Mol1->Mol2 Histo Histological Analysis Mol2->Histo

  • Stage 1: Wound Healing (Days 0-2 Post-Amputation)

    • Morphology: Formation of a wound epithelium over the amputation plane. No visible blastema.
    • Molecular Analysis (Key Monitoring Point): By the end of this stage, qRT-PCR should reveal significant downregulation of posterior Hox genes in the treated group compared to the control. This is a critical early indicator of successful fate alteration [6].

  • Stage 2: Homeotic Blastema Formation (Days 3-7)

    • Morphology: Accumulation of mesenchymal cells forming a blastema. The blastema may appear distinct from a typical tail blastema.
    • Molecular Analysis: Upregulation of pitx1 is typically detected via qRT-PCR, following the Hox gene downregulation. This confirms the initiation of a hind limb developmental program [6].

  • Stage 3: Limb Bud Formation (Days 7-14)

    • Morphology: The blastema forms a distinct, condensed bud structure that protrudes laterally, resembling an early limb bud rather than a tapered tail regenerate.
    • Molecular Analysis: In situ hybridization is used at this stage to detect the spatial expression of key limb patterning genes like shh (in the ZPA) and fgf8 (in the AER) within the ectopic bud.

  • Stage 4: Patterned Ectopic Limb (Days 14+)

    • Morphology: The bud elongates and differentiates, showing clear patterning. In successful transformations, this results in structures with digits and joints, confirmed by cartilage staining (e.g., Alcian Blue).
    • Molecular & Histological Analysis: Continued expression of patterning genes. Histological sectioning reveals the formation of limb-like skeletal elements, muscles, and connective tissues, confirming a complete homeotic transformation.

The Scientist's Toolkit: Research Reagent Solutions

Table 3: Key reagents and tools for studying homeotic transformation.

Tool / Reagent Function in Research Application Note
all-trans Retinoic Acid Induces homeotic transformation by altering Hox gene expression. Critical to optimize concentration and exposure time for specific anuran species [6] [31].
Hox Gene Primers (qPCR) Quantifies changes in axial patterning genes. Focus on posterior Hox genes (e.g., Hoxc10, Hoxc11) as early molecular markers [6].
pitx1 Riboprobe (ISH) Labels cells committing to hind limb fate. A key downstream target; its expression confirms the limb identity switch [6].
RNA Biosensors (e.g., Mango, Broccoli) Live imaging of RNA dynamics in real-time. Emerging tool to monitor gene expression (e.g., pitx1 mRNA) without fixation, enabling live tracking of transformation [33].
Alcian Blue & Alizarin Red Stains cartilage and bone in differentiated structures. Used to validate the final outcome by revealing the patterned skeletal elements of the ectopic limb [31].
Antituberculosis agent-6Antituberculosis agent-6Antituberculosis agent-6 is a potent antimycobacterial compound for research. This product is For Research Use Only, not for human consumption.
Irak4-IN-26Irak4-IN-26, MF:C22H23N5O3, MW:405.4 g/molChemical Reagent

The protocol for monitoring and staging the vitamin A-induced homeotic transformation of tails into limbs provides a robust experimental framework for investigating the fundamental principles of cell fate and positional information. The cornerstone of this phenomenon is the retinoic acid-driven manipulation of Hox gene expression, which acts as a master switch to reprogram the developmental trajectory of the regenerating tail blastema [6] [32]. By integrating precise morphological staging with targeted molecular analyses of key genes like hox and pitx1, researchers can systematically dissect the mechanisms that enable such profound changes in tissue identity. This model system continues to offer invaluable insights with potential implications for regenerative medicine, evolutionary developmental biology, and the understanding of how signaling molecules like retinoids can orchestrate large-scale morphological changes.

The precise quantification of Hox gene expression is fundamental to developmental biology research, particularly in studies investigating the homeotic transformation of tails into limbs induced by vitamin A in anuran models [6]. In these studies, molecular tools such as qPCR and in situ hybridization are indispensable for detecting changes in gene expression that underlie dramatic morphological alterations [6]. This protocol details the application of these key molecular techniques within the context of vitamin A research, enabling researchers to reliably quantify expression dynamics of Hox genes and related limb development genes during ectopic limb formation.

Key Molecular Tools and Their Applications

The selection of appropriate molecular tools depends on the research question, whether it requires spatial resolution, precise quantification, or genome-wide expression profiling.

Table: Key Molecular Tools for Quantifying Hox Gene Expression

Method Key Application Resolution Throughput Key Output
qPCR Precise quantification of transcript levels for specific Hox genes [34] Bulk tissue or cell population Medium Expression fold changes
In Situ Hybridization Spatial localization of Hox mRNA within tissue sections [5] Tissue/cellular Low Spatial expression patterns
RNA Sequencing Unbiased profiling of all Hox transcripts and related genes [2] Single-cell to bulk tissue High Genome-wide expression data
In Situ Sequencing Spatial mapping of multiple Hox genes simultaneously [2] Single-cell High Spatial transcriptomics data

Detailed Experimental Protocols

Quantitative PCR (qPCR) for Hox Gene Expression

Principle

qPCR enables precise quantification of specific Hox gene transcript levels in tissue samples through reverse transcription of RNA to cDNA, followed by PCR amplification with fluorescent probes [34]. This method is ideal for tracking expression changes of specific Hox genes, such as the downregulation of posterior Hox genes prior to ectopic limb bud appearance in vitamin A-treated anurans [6].

Protocol

  • RNA Extraction

    • Homogenize tail or limb bud tissue (20-50 mg) in TRIzol reagent
    • Isolate total RNA following manufacturer's protocol
    • Determine RNA concentration and purity (A260/A280 ratio >1.8)
    • Treat with DNase I to remove genomic DNA contamination

  • cDNA Synthesis

    • Use 1 μg total RNA in 20 μL reaction volume
    • Employ reverse transcriptase and oligo(dT) primers
    • Incubate: 25°C for 10 min, 50°C for 60 min, 70°C for 15 min
    • Dilute cDNA 1:5 with nuclease-free water before qPCR

  • qPCR Reaction

    • Prepare reaction mix: 10 μL SYBR Green Master Mix, 1 μL cDNA, 0.5 μL each forward and reverse primer (10 μM), 8 μL nuclease-free water
    • Cycling conditions: 95°C for 10 min; 40 cycles of 95°C for 15 sec, 60°C for 1 min
    • Include melt curve analysis to verify amplification specificity
    • Perform technical triplicates for each biological sample

  • Data Analysis

    • Calculate ΔΔCt values normalized to housekeeping genes (GAPDH, β-actin)
    • Express results as fold change relative to control groups

G cluster_primers Primer Design Considerations Tissue Tissue RNA RNA Tissue->RNA Homogenize in TRIzol cDNA cDNA RNA->cDNA Reverse Transcription qPCR qPCR cDNA->qPCR Amplify with Hox primers Para1 Design primers spanning exon-exon junctions Para2 Account for high sequence similarity between Hox paralogs Data Data qPCR->Data Analyze ΔΔCt values

Figure: qPCR Workflow for Hox Gene Analysis

In Situ Hybridization for Spatial Hox Gene Expression

Principle

In situ hybridization enables precise spatial localization of Hox mRNA transcripts within tissue sections, preserving anatomical context [5]. This is particularly valuable for understanding regional Hox code patterns during vitamin A-induced transformations.

Protocol

  • Probe Synthesis

    • Clone 500-1000 bp Hox gene-specific fragments into transcription vector
    • Linearize plasmid DNA with appropriate restriction enzymes
    • Synthesize digoxigenin (DIG)-labeled RNA probes using T7/SP6 RNA polymerase
    • Hydrolyze probes to ~300 bp fragments for better tissue penetration

  • Tissue Preparation

    • Fix anuran tail/limb tissues in 4% paraformaldehyde for 24h at 4°C
    • Dehydrate through ethanol series, clear in xylene, embed in paraffin
    • Section tissues at 5-8 μm thickness using microtome
    • Mount sections on positively charged slides

  • Hybridization

    • Deparaffinize sections and rehydrate through graded ethanol series
    • Treat with proteinase K (10 μg/mL) for 15 min at 37°C
    • Prehybridize for 2h at 65°C in hybridization buffer
    • Hybridize with DIG-labeled probe (100-500 ng/mL) overnight at 65°C

  • Detection

    • Wash stringently: 2× SSC at 65°C, then 0.1× SSC at 65°C
    • Block with 10% normal sheep serum in TBST for 1h
    • Incubate with anti-DIG-alkaline phosphatase antibody (1:2000) overnight at 4°C
    • Develop with NBT/BCIP substrate until signal appears
    • Counterstain with nuclear fast red, mount with aqueous mounting medium

Application in Vitamin A Research on Anuran Models

Experimental Design for Vitamin A Studies

When investigating homeotic transformation of tails into limbs induced by vitamin A in anuran species such as Rana ornativentris, molecular quantification of Hox gene expression should be strategically timed [6]:

Table: Key Gene Targets in Vitamin A-Induced Homeotic Transformation

Gene Category Specific Targets Expected Expression Change Biological Significance
Posterior Hox Genes Hoxa11, Hoxa13, Hoxd10-Hoxd13 Downregulation prior to ectopic limb bud appearance [6] Loss of tail identity
Hind Limb Genes Pitx1, Tbx4 Upregulated following Hox changes [6] Establishment of hind limb program
TGF-β Signaling GDF11, Smad2, Smad4 Altered phosphorylation and activity [5] Regulation of posterior Hox genes

Signaling Pathways in Hox Gene Regulation

Vitamin A (retinoic acid) influences Hox gene expression through complex signaling interactions. Understanding these pathways is essential for interpreting experimental results.

G cluster_nuclear Nuclear Events VitA Vitamin A (Retinoic Acid) RAR RAR/RXR Receptors VitA->RAR GDF11 GDF11 VitA->GDF11 Modulates expression HoxChange Altered Hox Expression RAR->HoxChange Direct regulation PosteriorHox Posterior Hox Genes (Hox10-Hox13) HoxChange->PosteriorHox Alters axial identity pSmad2 pSmad2/Smad4 Complex GDF11->pSmad2 Binds and activates PRTG Protogenin (PRTG) PRTG->pSmad2 Enhances signaling pSmad2->PosteriorHox Transcriptional activation

Figure: Hox Gene Regulation by Vitamin A and Signaling Pathways

Research Reagent Solutions

Table: Essential Reagents for Hox Gene Expression Studies

Reagent Category Specific Products Application Notes
RNA Isolation TRIzol Reagent, RNeasy Mini Kit For high-quality RNA from anuran tissues rich in pigments and connective tissue
cDNA Synthesis High-Capacity cDNA Reverse Transcription Kit Use with random hexamers and oligo(dT) primers for comprehensive coverage
qPCR Reagents SYBR Green Master Mix, TaqMan Gene Expression Assays SYBR Green requires careful primer optimization; TaqMan offers higher specificity
In Situ Probes DIG RNA Labeling Kit, Fluorescent in situ hybridization kits DIG system provides excellent sensitivity for detecting low-abundance Hox transcripts
Antibodies Anti-DIG-AP, Phospho-Smad2 antibodies Essential for detecting in situ hybridization signals and signaling activity [5]
Vitamin A Reagents All-trans retinoic acid, Retinol Prepare fresh solutions and use appropriate vehicle controls in anuran studies

Troubleshooting and Quality Control

Method-Specific Considerations

  • qPCR: Account for high sequence similarity between Hox paralogs through careful primer design spanning unique regions. Verify amplification specificity with melt curve analysis and gel electrophoresis.
  • In Situ Hybridization: Include sense strand probes as negative controls. Optimize proteinase K concentration and digestion time to balance signal intensity with tissue morphology preservation.
  • RNA Sequencing: For studies investigating complete Hox expression patterns, utilize ribosomal RNA depletion rather than polyA selection to ensure coverage of non-polyadenylated transcripts [2].

Validation in Anuran Models

  • Species-Specific Reagents: Many commercial antibodies developed for mammalian systems may not recognize anuran epitopes; validate thoroughly before use.
  • Developmental Staging: Precise staging of anuran embryos is critical for reproducible results in vitamin A studies, as Hox expression patterns change rapidly during development.
  • Spatial Validation: Correlate qPCR findings with spatial techniques like in situ hybridization to confirm that expression changes occur in relevant tissue compartments.

Detecting the Onset of Limb-Specific Gene Programs (e.g., pitx1)

Application Notes

Understanding the initiation of limb-specific gene programs is fundamental to developmental biology and regenerative medicine. The transcription factor Pitx1 serves as a key determinant of hindlimb identity, and its ectopic expression is sufficient to induce a partial arm-to-leg transformation, as observed in Liebenberg syndrome [35]. Recent research demonstrates that the severity of such transformations is directly correlated with the proportion of cells ectopically expressing Pitx1, which is governed by the 3D topology of its locus and the activity of its enhancers [35]. Furthermore, studies in anuran models reveal that the manipulation of Hox gene expression using Vitamin A can precede and potentially induce the upregulation of Pitx1, leading to homeotic transformations where tails regenerate as limbs [6]. This provides a powerful experimental framework for investigating the genetic hierarchy governing limb identity. The following notes and protocols detail methods for quantifying and manipulating the onset of these critical gene programs.

Quantitative Data on Pitx1 Ectopic Expression and Phenotypic Severity

Table 1: Impact of Genetic Perturbations on Pitx1 Ectopic Expression in Mouse Forelimbs

Genetic Allele / Perturbation Description / Mechanism Pitx1-Enhancer Pen Distance Percentage of EGFP+ Cells in Forelimb (E12.5)
Pitx1EGFP; Inv1 Inversion placing Pen at RA4 location (~225 kb from Pitx1) [35] ~225 kb 6.4% [35]
Pitx1EGFP; Inv2 Larger inversion placing Pen at PDE location (~116 kb from Pitx1) [35] ~116 kb 27% [35]
Pitx1EGFP; ΔPen; Rel1 Relocation of Pen to RA4 site in ΔPen background [35] ~225 kb 2% [35]
Pitx1EGFP; ΔPen; Rel2 Relocation of Pen to PDE site in ΔPen background [35] ~116 kb 59% [35]
Pitx1EGFP; ΔPen; Rel3 Relocation of Pen 7.7 kb upstream of Pitx1 [35] ~10.5 kb Data not explicitly stated in provided results

Table 2: Key Gene Expression Changes in Anuran Tail-to-Limb Transformation

Experimental Model Intervention Key Genetic Findings Proposed Genetic Hierarchy
Rana ornativentris Tadpole Vitamin A administration post-tail amputation [6] Downregulation of posterior Hox genes precedes upregulation of Pitx1 in regenerating tail blastema [6] Vitamin A → Hox Gene Downregulation → Pitx1 Upregulation → Ectopic Limb Bud Formation [6]

Experimental Protocols

Protocol: Quantifying Ectopic Pitx1 Expression via Fluorescence-Activated Cell Sorting (FACS)

This protocol is adapted from methodologies used to analyze Pitx1 expression in engineered mouse models [35].

I. Purpose To isolate and quantify the proportion of cells ectopically expressing Pitx1 from developing limb tissues using a Pitx1EGFP reporter allele.

II. Materials

  • Pitx1EGFP Reporter Mice: Homozygous for the EGFP sensor allele [35].
  • Dissection Tools: Fine forceps and scissors for micro-dissection.
  • Digestion Buffer: e.g., Collagenase/Dispase in PBS.
  • FACS Buffer: PBS supplemented with 2% Fetal Bovine Serum (FBS).
  • Cell Strainer: 70-µm nylon mesh.
  • Fluorescence-Activated Cell Sorter.

III. Procedure

  • Harvest Tissue: At the desired embryonic stage (e.g., E12.5), sacrifice the dam according to ethical guidelines. Dissect embryos and micro-dissect forelimb buds into cold PBS [35].
  • Dissociate Tissue: Transfer forelimb buds to digestion buffer and incubate at 37°C for 15-20 minutes to create a single-cell suspension. Gently pipette to aid dissociation.
  • Prepare Single-Cell Suspension: Neutralize digestion with excess FACS buffer. Pass the cell suspension through a 70-µm cell strainer to remove clumps.
  • FACS Analysis: Resuspend cells in FACS buffer and analyze using a flow cytometer. Use untagged wild-type limb cells to establish baseline autofluorescence and set the EGFP-positive gate.
  • Data Collection: Record the percentage of cells falling within the EGFP-positive gate from at least three biological replicates.

IV. Analysis Compare the percentage of EGFP+ cells between control and experimental genotypes (e.g., those with structural variants or enhancer relocations) to assess the level of ectopic Pitx1 activation [35].

Protocol: Inducing and Analyzing Ectopic Limb Formation in Anuran Tadpoles

This protocol is based on research demonstrating homeotic transformation of tails into limbs using Vitamin A [6].

I. Purpose To induce ectopic limb formation in regenerating tadpole tails and analyze the subsequent gene expression changes, particularly in Hox genes and Pitx1.

II. Materials

  • Animals: Anuran tadpoles (e.g., Rana ornativentris) [6].
  • Vitamin A Solution: All-trans-retinoic acid (RA) dissolved in dimethyl sulfoxide (DMSO) and diluted in tank water.
  • Control Solution: DMSO diluted in tank water at the same concentration as the experimental solution.
  • Surgical Tools: Fine scalpel or scissors for tail amputation.
  • RNA Extraction Kit.
  • cDNA Synthesis Kit.
  • Quantitative PCR (qPCR) System and gene-specific primers (e.g., for posterior Hox genes, Pitx1).

III. Procedure

  • Tail Amputation: Anesthetize tadpoles. Using a sterile scalpel, amputate the tail posterior to the hindlimb buds.
  • Treatment Groups: Immediately after amputation, place tadpoles into one of two solutions:
    • Experimental Group: Tank water containing a defined concentration of Vitamin A/RA [6].
    • Control Group: Tank water containing an equivalent concentration of DMSO vehicle.
  • Sample Collection: At specific time points post-amputation (e.g., during blastema formation and early regeneration), collect the regenerating tail tissue.
  • Gene Expression Analysis:
    • Extract total RNA from the regenerating tissue.
    • Synthesize cDNA.
    • Perform qPCR using primers for genes of interest (e.g., posterior Hox genes, Pitx1). Normalize data to a housekeeping gene.

IV. Analysis Compare the expression levels of Hox genes and Pitx1 between Vitamin A-treated and control tadpoles. The expected result is a downregulation of posterior Hox genes followed by an upregulation of Pitx1 in the Vitamin A-treated group, preceding visible limb formation [6].

Signaling Pathways and Experimental Workflows

G Start Tail Amputation in Anuran Tadpole Intervention Vitamin A (RA) Treatment Start->Intervention Hox Downregulation of Posterior Hox Genes Intervention->Hox Pitx1 Upregulation of Pitx1 Expression Hox->Pitx1 Outcome Ectopic Limb Bud Formation (Homeotic Transformation) Pitx1->Outcome

Vitamin A Induces Tail-to-Limb Transformation

G SV Structural Variant (SV) (e.g., Inversion) Topology Altered 3D Chromatin Topology SV->Topology Hijacking Enhancer Hijacking (Pen-Pitx1 Ectopic Contact) Topology->Hijacking Expression Ectopic Pitx1 Expression in Forelimb Cells Hijacking->Expression Phenotype Liebenberg Syndrome (Arm-to-Leg Transformation) Expression->Phenotype

Enhancer Hijacking Causes Ectopic Pitx1 Expression

The Scientist's Toolkit: Research Reagent Solutions

Table 3: Essential Reagents for Studying Limb-Specific Gene Programs

Research Reagent Function and Application
Pitx1EGFP Reporter Allele A genetically engineered sensor that allows for the tracking, sorting, and quantification of Pitx1-expressing cells via EGFP fluorescence [35].
All-trans-Retinoic Acid (Vitamin A) A morphogen used in anuran and other models to manipulate Hox gene expression, inducing homeotic transformations and studying upstream regulators of Pitx1 [6].
siRNA / CRISPR-Cas9 Systems For targeted gene knockdown (e.g., Pitx1, Tbx4) or creation of structural variants (e.g., inversions, enhancer relocations) to study gene function and regulation [35] [36].
qPCR Primers for Hox & Pitx1 Genes Essential tools for quantifying temporal changes in gene expression during normal development or experimentally induced transformations [6].
Collagenase/Dispase Enzymes Used for the gentle dissociation of embryonic limb tissue into single-cell suspensions for downstream applications like FACS [35].
Hdac6-IN-14Hdac6-IN-14, MF:C24H30FN3O4, MW:443.5 g/mol
Boc-Gln-Gly-Arg-AMCBoc-Gln-Gly-Arg-AMC, MF:C28H40N8O8, MW:616.7 g/mol

Linking Hox Shifts to Tissue Remodeling and Cell Fate Reprogramming

This application note details the molecular mechanisms and experimental protocols for inducing homeotic transformations—specifically, the reprogramming of tadpole tail tissue into limbs—through vitamin A (retinoic acid) modulation of Hox gene expression. In anuran models, this process demonstrates the profound impact of Hox-regulated positional identity on cell fate and tissue remodeling. The core principle is that vitamin A, acting through retinoic acid signaling, can repattern the Hox code, a specific pattern of Hox gene expression that defines anatomical position along the anterior-posterior axis [37] [6]. This shift in the Hox code downstream leads to the activation of limb-specific genetic programs in a tissue normally fated to form a tail, resulting in a homeotic transformation [6]. The following sections provide a quantitative summary of the key molecular events, detailed methodologies for reproducing this phenomenon, and a visualization of the underlying signaling pathway.

Key Molecular Events & Quantitative Data

The following table summarizes the core quantitative findings and temporal sequence of molecular events during the vitamin A-induced homeotic transformation of tail to limb in Rana ornativentris.

Table 1: Key Molecular Events in Vitamin A-Induced Homeotic Transformation

Molecular Event Experimental Model Key Finding Timing/Measurement
Hox Gene Downregulation Rana ornativentris (Anuran) Downregulation of a posterior Hox gene precedes ectopic limb bud appearance [6]. Occurs prior to morphological changes and pitx1 upregulation.
Limb Gene Activation Rana ornativentris (Anuran) Subsequent upregulation of pitx1, a key hind limb identity gene [6]. Follows the initial shift in Hox gene expression.
Proliferation Regulation Drosophila CNS The Hox gene abdA slows neural stem cell (NSC) growth, lengthens G2 phase, and delays mitosis in abdominal segments [38]. Abdominal NSCs are smaller, divide slower; loss of abdA increases their size and division rate.
Prolonged Proliferation C. elegans vulval cells Constitutive expression of the Hox gene lin-39 prolongs the proliferative phase of somatic cells past their normal arrest point [39]. Vulval cell proliferation extends beyond the normal endpoint of the cell lineage.

Experimental Protocols

Protocol: Inducing Homeotic Transformation in Anuran Tadpoles

This protocol is adapted from Morioka et al., detailing the method for inducing ectopic limb formation in regenerating Rana ornativentris tadpole tails through vitamin A administration [6].

Objective: To investigate the molecular mechanisms of Hox-mediated tissue remodeling by inducing homeotic transformation.

Materials:

  • Animals: Anuran tadpoles (e.g., Rana ornativentris) at the appropriate developmental stage for tail regeneration.
  • Reagent: All-trans Retinoic Acid (RA) (Vitamin A).
  • Solvent: Dimethyl sulfoxide (DMSO) or ethanol for preparing RA stock solution.
  • Equipment: Stereomicroscope, microdissection tools, amputation apparatus, containers for tadpole housing, RNA extraction kit, quantitative PCR (qPCR) system.

Method:

  • Tadpole Preparation & Amputation:
    • Anesthetize tadpoles in a diluted solution of tricaine methanesulfonate (MS-222).
    • Under a stereomicroscope, amputate the tail using a sharp microdissection scalpel. Make a clean, transverse cut at the desired level along the tail.
    • Return tadpoles to fresh water to recover and initiate the regeneration process.

  • Vitamin A Administration:

    • Prepare a concentrated stock solution of RA in DMSO. Protect from light.
    • Dilute the RA stock in the tadpole housing water to the desired working concentration immediately before use. A typical concentration range is 1-10 µM, which must be determined empirically for the specific species and setup.
    • Expose the tadpoles to the RA-containing water following amputation. The timing and duration of exposure are critical and may vary (e.g., continuous exposure or a specific pulse).

  • Phenotypic Analysis:

    • Monitor the regenerating tail blastema daily for morphological changes.
    • A successful transformation will result in the formation of patterned limb structures (e.g., with digits) instead of a typical tail regenerate.
    • Fix samples for histological analysis or proceed to molecular analysis.

  • Molecular Analysis of Hox and Limb Genes:

    • At specific time points post-amputation (e.g., before, during, and after blastema formation), collect regenerating tissue samples.
    • Extract total RNA from the samples.
    • Perform cDNA synthesis and quantitative PCR (qPCR) to measure expression levels of:
      • Targets: Posterior Hox genes (e.g., Hoxc10), limb patterning genes (e.g., pitx1, tbx4).
      • Controls: Housekeeping genes (e.g., gapdh, ef1a).

Troubleshooting:

  • No Transformation: Optimize RA concentration, exposure window, and tadpole developmental stage.
  • Toxicity/Lethality: Reduce RA concentration or exposure time.
  • Weak/Partial Transformation: Ensure consistent RA delivery and health of tadpoles.
Protocol: Conditional Hox Gene Knockdown in Proliferating Cells

This protocol outlines a method for validating the necessity of Hox genes for sustaining cell proliferation, as demonstrated in C. elegans [39].

Objective: To test if Hox gene function is required to maintain proliferation in specified cell lineages.

Materials:

  • Strain: Transgenic animals (e.g., C. elegans) expressing a Hox gene (e.g., lin-39) fused to a degradation tag (e.g., ZF1) and a cell-specific GFP reporter.
  • Reagent: E. coli strain expressing double-stranded RNA (dsRNA) for the Hox gene of interest (e.g., lin-39(RNAi)).
  • Equipment: Standard microbiological culture supplies, microscope with fluorescence capability, microfluidic devices for live imaging (optional).

Method:

  • Synchronize a population of animals at the desired larval stage.
  • Induce Degradation: For strains with a heat-shock inducible degradation system (e.g., hsp-16p>zif-1), apply a heat shock to trigger the expression of ZIF-1, which targets the ZF1-tagged Hox protein for proteasomal degradation.
  • RNAi Enhancement: Place the heat-shocked animals on agar plates seeded with bacteria expressing the Hox-specific dsRNA to further reduce Hox levels via RNA interference.
  • Phenotypic Scoring: At a subsequent developmental time point, score the animals for phenotypic defects.
    • Quantitative Measure: Count the number of differentiated cells in the tissue of interest (e.g., vulval cells in C. elegans) and compare to control animals.
    • Control Groups: Include animals without the degradation transgene and animals grown on empty vector (EV) bacteria.

Signaling Pathway and Experimental Workflow Visualizations

Vitamin A-Induced Hox Shifts and Limb Reprogramming

G VitaminA Vitamin A (Retinoic Acid) HoxRepatterning Hox Code Repatterning VitaminA->HoxRepatterning PosteriorHox Downregulation of Posterior Hox Genes HoxRepatterning->PosteriorHox LimbProgram Activation of Limb Genetic Program (e.g., pitx1) PosteriorHox->LimbProgram TissueOutcome Homeotic Transformation (Tail Tissue → Limb) LimbProgram->TissueOutcome CellProliferation Altered Cell Proliferation & Tissue Remodeling LimbProgram->CellProliferation

Experimental Workflow for Hox Shift Analysis

G Start Tadpole Tail Amputation Treat Vitamin A Administration Start->Treat Monitor Monitor Regeneration (Phenotype Scoring) Treat->Monitor Sample Tissue Sampling at Key Time Points Monitor->Sample Analyze Molecular Analysis (qPCR for Hox/Limb Genes) Sample->Analyze Conclude Correlate Hox Shifts with Phenotype Analyze->Conclude

The Scientist's Toolkit: Research Reagent Solutions

Table 2: Essential Research Reagents for Hox Manipulation Studies

Reagent / Material Function / Application Example Use Case
All-trans Retinoic Acid (RA) A bioactive form of Vitamin A; acts as a morphogen to perturb the Hox code and induce homeotic transformations [6]. Inducing tail-to-limb transformation in anuran tadpoles.
Hox Reporter Strains Transgenic organisms with Hox genes tagged with fluorescent proteins (e.g., GFP) to visualize expression dynamics in live tissues [39]. Monitoring LIN-39::GFP expression in dividing C. elegans vulval cells.
Conditional Knockdown Systems Tools for spatially and temporally controlled gene inactivation (e.g., ZIF-1 degradation, RNAi, Cre-lox) [39]. Testing the necessity of a Hox gene for proliferation after cell fate specification.
Hox-Specific Antibodies Antibodies for immunohistochemistry (IHC) and Western Blot to detect Hox protein localization and levels. Validating Hox protein expression patterns in tissue sections.
Spatial Transcriptomics High-resolution mRNA assays (Visium, in-situ sequencing) to map the Hox code across cell types in a tissue [2]. Creating an atlas of HOX gene expression in the developing human spine.
Fgfr-IN-11FGFR-IN-11|Potent FGFR Inhibitor|Research CompoundFGFR-IN-11 is a potent, selective FGFR inhibitor for cancer research. It targets tyrosine kinase activity to suppress cell proliferation. For Research Use Only. Not for human or veterinary use.
HIV-1 integrase inhibitor 10HIV-1 integrase inhibitor 10, MF:C40H45N7O4, MW:687.8 g/molChemical Reagent

Overcoming Experimental Hurdles and Fine-Tuning Retinoic Acid Signaling

Retinoic acid (RA), the active metabolite of Vitamin A, serves as a critical signaling molecule in vertebrate embryonic development. Its function is fundamentally dualistic: precise spatial and temporal regulation is required for normal head and tail formation, whereas dysregulation leads to severe teratogenic effects. This duality is central to its role in patterning the anterior-posterior (A-P) axis, largely through the precise regulation of Hox gene expression [12]. In anuran models like Xenopus, RA signaling operates as a key modifier of developmental processes, making it a powerful experimental tool for investigating the principles of axial patterning. This document outlines the application of RA in manipulating Hox gene expression, providing detailed protocols and data frameworks for researchers in developmental biology and drug discovery.

Key Quantitative Data on RA-Induced Teratogenesis

The teratogenic effects of RA are concentration-dependent and associated with specific developmental windows. The following tables summarize quantitative data from zebrafish and model system studies, providing a reference for experimental design.

Table 1: Concentration-Dependent Teratogenic Effects of All-Trans Retinoic Acid (ATRA) in Zebrafish

Teratogenic Effect Most Sensitive Window (hpf) EC50 (µg/L) Key Observations
Tail Malformation (TM) 4 - 48 0.20 - 0.26 Most sensitive indicator of RA disruption [40]
Craniofacial Malformation (CFM) 4 - 48 ~1.00 - 1.21 Shares sensitivity window with posterior swim bladder [40]
Posterior Swim Bladder Non-inflation 4 - 48 ~1.00 - 1.21 Often co-occurs with craniofacial defects [40]

Table 2: Consequences of RA Signaling Disruption in Model Organisms

Condition Model System Major Phenotypic Outcomes
Reduced RA Signaling Xenopus Microcephaly, head truncation, failure of prechordal mesoderm migration [41]
Excess RA Signaling Mouse/Zebrafish Hindbrain expansion, tail malformations, homeotic transformations of vertebrae [41] [40] [8]
Embryonic Alcohol Exposure (Reduced RA) Xenopus FAS-like phenotypes, including microcephaly, due to competition for RA-biosynthetic enzymes [41]

Detailed Experimental Protocols

Protocol: RA Exposure inXenopusEmbryos for Hox Gene Manipulation

This protocol is designed to investigate the dual role of RA in head and tail development by altering Hox gene expression domains.

I. Materials and Reagents

  • Anuran Model: Healthy, staged Xenopus laevis or Xenopus tropicalis embryos.
  • RA Stock Solution: 10 mM all-trans Retinoic Acid (ATRA) in DMSO. Note: Protect from light and store at -20°C.
  • Control Solution: 0.1% DMSO in Danieau's solution.
  • Buffers: Danieau's solution (58 mM NaCl, 0.7 mM KCl, 0.4 mM MgSOâ‚„, 0.6 mM Ca(NO₃)â‚‚, 5.0 mM HEPES, pH 7.6) or Modified Barth's Saline (MBS).
  • Equipment: Sterile multi-well plates, precision pipettes, temperature-controlled incubator or environmental chamber set to 14-22°C.

II. Procedure

  • Embryo Preparation: Obtain and culture embryos using standard methods. Dejelly embryos chemically or manually at the appropriate developmental stage.
  • Treatment Solution Preparation: From the 10 mM ATRA stock, prepare working concentrations in Danieau's solution. A typical teratogenic range is 0.1 - 1.0 µM. Vortex thoroughly. Include a vehicle control (0.1% DMSO in Danieau's).
  • Experimental Exposure:
    • Distribute healthy, staged embryos (e.g., gastrula to early neurula stages, stages 10-15) into multi-well plates.
    • Gently replace the medium with the prepared RA treatment or control solutions.
    • Incubate embryos for the desired duration. For studies on head development, early exposure (gastrula) is critical. For tail and posterior development, slightly later exposures (neurula) can be informative.
  • Termination and Fixation: After exposure, wash embryos 3-5 times with fresh Danieau's solution. Fix embryos for subsequent analysis (e.g., in 4% paraformaldehyde for in situ hybridization or immunostaining).

III. Analysis and Validation

  • Phenotypic Scoring: Score fixed or live embryos for malformations (e.g., microcephaly, tail curvature, overall shortening) under a dissection microscope.
  • Molecular Analysis:
    • In situ Hybridization: To visualize shifts in Hox gene expression domains (e.g., anterior expansion of posterior Hox genes under high RA) [8] [12].
    • Gene Expression Knockdown: Use morpholino antisense oligonucleotides or CRISPR/Cas9 targeting RA-synthesizing enzymes (e.g., aldh1a2, aldh1a3) to model reduced RA signaling and confirm specificity [41].

Protocol: Quantitative Analysis of Endogenous RA from Embryonic Tissue

Accurately measuring endogenous RA levels is crucial for validating experimental manipulations.

I. Materials and Reagents

  • Tissue: Pooled Xenopus embryos or dissected embryonic regions (10-20 mg minimum).
  • Internal Standard: 4,4-dimethyl-RA or a stable-isotope labeled RA.
  • Extraction Solvents: HPLC-grade hexane, ethanol, acetonitrile, and formic acid.
  • Equipment: Ground glass homogenizers, LC-MS/MS system equipped with an APCI or ESI source.

II. Procedure

  • Homogenization: Homogenize tissue on ice in cold 0.9% saline to create a 10-25% homogenate.
  • Two-Step Acid-Base Extraction:
    • Add internal standard and 1-3 mL of 0.025 M KOH in ethanol to the homogenate.
    • Extract with 10 mL of hexane; remove and retain the organic phase (contains non-polar retinoids).
    • Acidify the aqueous phase with 4 M HCl and perform a second extraction with 10 mL of hexane to recover polar RA [42].
  • Sample Concentration: Evaporate the second hexane phase under a gentle stream of nitrogen. Reconstitute the dry extract in 60 µL of acetonitrile.
  • LC-MS/MS Analysis:
    • Column: Supelcosil ABZ+PLUS or equivalent C18 column.
    • Mobile Phase: (A) Water with 0.1% formic acid; (B) Acetonitrile with 0.1% formic acid.
    • Gradient: Use a linear gradient from 50% B to 98% B over 10-15 minutes.
    • MS Detection: Operate in positive APCI or negative ESI mode using Multiple Reaction Monitoring (MRM). For ATRA, monitor the transition m/z 301.2 → 205.1 [42] [43].

III. Advanced Method for Isomer Separation

  • To resolve RA isomers (all-trans, 9-cis, 13-cis), which have distinct biological activities, employ Differential Mobility Spectrometry (DMS/SelexION Technology) coupled to LC-MS/MS. This provides an orthogonal separation dimension, drastically reducing matrix interference and allowing for precise quantitation of each isomer [43].

Signaling Pathways and Workflows

RA Biosynthesis and Signaling Pathway

The following diagram illustrates the core pathway of RA biosynthesis and its mechanism of action in regulating target genes like Hox and Cdx1.

G Retinol Dietary Vitamin A (Retinol) Retinal Retinaldehyde Retinol->Retinal ADH/RDH atRA All-trans RA (atRA) Retinal->atRA RALDH1/2/3 RAR RAR/RXR Heterodimer atRA->RAR Binds CYP26 CYP26 Enzymes (RA Catabolism) atRA->CYP26 Degradation RARE RARE on DNA RAR->RARE Binds TargetGenes Target Gene Expression (Hox genes, Cdx1) RARE->TargetGenes CYP26->atRA Feedback Control

Experimental Workflow for RA Functional Analysis

This workflow outlines the key steps from hypothesis testing to data analysis in an RA manipulation experiment.

G A 1. Embryo Collection and Staging B 2. Experimental Perturbation A->B C 3. Phenotypic Analysis B->C D 4. Molecular Analysis B->D E 5. RA Level Quantification B->E F 6. Data Integration & Validation C->F D->F E->F

The Scientist's Toolkit: Essential Research Reagents

Table 3: Key Reagent Solutions for RA Signaling Research in Anuran Models

Reagent / Solution Function & Application in Research
all-trans Retinoic Acid (ATRA) The primary endogenous bioactive retinoid. Used to experimentally elevate RA signaling and induce teratogenic or patterning effects [40] [20].
DEAB (Diethylaminobenzaldehyde) A specific inhibitor of retinaldehyde dehydrogenases (RALDHs). Used to block endogenous RA synthesis and model reduced RA signaling [41].
RALDH2/RALDH3 Morpholinos or CRISPR/Cas9 Gene-specific knockdown/knockout tools to target RA biosynthesis in specific embryonic territories (e.g., organizer) and study loss-of-function phenotypes [41].
Retinoid Receptor Antagonists (e.g., BMS493) Pan-antagonist of retinoic acid receptors (RARs). Used to block RA signaling downstream, confirming the specificity of RA-mediated effects [8].
Hox Gene Probes Labeled RNA probes for in situ hybridization. Essential for visualizing the anterior-posterior shifts in Hox gene expression domains upon RA manipulation [8] [12].
LC-MS/MS with DMS The gold-standard methodology for the sensitive, specific, and quantitative measurement of endogenous RA and its isomers from limited biological samples like embryonic tissue [42] [43].

Optimizing Dosage and Timing to Achieve Specific Phenotypes

The ability to manipulate embryonic development to achieve specific phenotypic outcomes is a powerful tool in developmental biology. Research using anuran models (frogs and toads) has demonstrated that administration of vitamin A (retinoids) can induce dramatic homeotic transformations, such as the formation of ectopic limbs in regenerating tadpole tails instead of a normal tail regenerate [6]. This phenomenon is driven by the ability of retinoids to profoundly alter the expression of Hox genes, key transcriptional regulators of body patterning [6] [32]. This application note provides a detailed protocol for leveraging this effect, framing the methodology within the broader context of manipulating Hox gene expression to control developmental outcomes. It is designed to assist researchers and drug development professionals in reproducing and adapting this technique for specific experimental goals.

Molecular Mechanism: Retinoid Regulation of Hox Genes

Retinoic Acid (RA), the active derivative of Vitamin A, functions as a powerful signaling molecule during embryogenesis. It directly influences the expression of Hox genes by binding to Retinoic Acid Receptors (RARs) and Retinoid X Receptors (RXRs). This complex then binds to specific DNA sequences known as Retinoic Acid Response Elements (RAREs) located in the regulatory regions of Hox genes [32].

For instance, enhancers containing a DR5-type RARE have been identified at the 3' end of both the Hoxa and Hoxb gene clusters. These RAREs are responsible for driving the RA-responsive expression of genes like Hoxa-1 and Hoxb-1 [32]. In the context of anuran tadpole tail regeneration, Vitamin A administration leads to the downregulation of posterior Hox genes prior to the appearance of ectopic limb buds. This shift in Hox expression is a critical upstream event that precedes the activation of limb-specific genes such as pitx1, a key determinant of hind limb identity [6]. The proposed signaling pathway is summarized below.

G Vitamin_A Vitamin A (Retinoids) RAR_RXR RAR/RXR Receptor Complex Vitamin_A->RAR_RXR Metabolized to RA RARE Binding to RARE (in Hox Gene Enhancers) RAR_RXR->RARE Hox_Change Altered Hox Gene Expression RARE->Hox_Change Transcription Regulation Pitx1 Upregulation of pitx1 (Hind Limb Marker) Hox_Change->Pitx1 Precedes and induces Phenotype Homeotic Transformation (Ectopic Limb Formation) Pitx1->Phenotype

Quantitative Data for Phenotype Optimization

Achieving a specific phenotype is highly dependent on precise dosing and timing. The following table consolidates key quantitative data from foundational research to guide experimental design.

Table 1: Optimized Dosage and Timing Parameters for Phenotype Induction in Anuran Models

Phenotypic Outcome Recommended Compound Effective Concentration Critical Treatment Window Key Molecular Hallmarks
Ectopic Hind Limb Formation in regenerating tail tissue [6] Vitamin A (Retinoic Acid) Species-specific optimization required Immediately following tail amputation, prior to bud stage regeneration [6] 1. Downregulation of posterior Hox genes [6]2. Subsequent upregulation of pitx1 [6]

Detailed Experimental Protocol

The following protocol is adapted from established methodologies in anuran and Xenopus research, focusing on the key steps for inducing and analyzing homeotic transformations.

Stage 1: Animal Care and Embryo Handling
  • Induce Mating: Administer subcutaneous injections of Human Chorionic Gonadotropin (HCG)—600 U for females and 400 U for males—into the dorsal lymph sac of adult anurans (e.g., Rana or Xenopus) [44].
  • Collect Embryos: Place injected frogs in a holding tank overnight. Collect embryos 9-12 hours post-HCG injection.
  • Dejelly and Maintain: Gently wash embryos in 2% cysteine (pH 8.0) for 2-4 minutes to remove jelly coats. Rinse thoroughly 3x in 0.1x Marc's Modified Ringer's solution (0.1x MMR) and incubate in 0.1x MMR with 50 µg/mL gentamycin at 14-22°C [44].
  • Stage Embryos: Allow embryos to develop to the desired stage. For tadpole tail amputation, stage 40-50 (post-embryonic, feeding tadpoles) is typically used. Stage according to standard morphological tables (e.g., Nieuwkoop and Faber for Xenopus) [44].
Stage 2: Tail Amputation and Vitamin A Treatment
  • Amputate Tails: Under a dissecting microscope, use a sterile scalpel or razor blade to amputate the tail in the posterior half of the tail fin.
  • Prepare Vitamin A Stock: Dissolve all-trans Retinoic Acid (RA) in DMSO to create a concentrated stock solution (e.g., 10 mM). Protect from light.
  • Treat Tadpoles: Immediately following amputation, transfer tadpoles to a treatment solution. The working RA solution should be prepared by diluting the stock in the tadpole housing medium (e.g., 0.1x MMR). Note: Concentration must be empirically determined for the specific species and desired phenotype; start within a range of 0.1-10 µM. A DMSO-only control group is essential.
  • Exposure Duration: A 24-hour pulse treatment is often sufficient. After treatment, rinse tadpoles and return them to fresh medium without RA for the remainder of the regeneration period.
Stage 3: Molecular Analysis of Phenotype

To confirm the molecular mechanism, analyze gene expression changes in the regenerating tissue.

  • Tissue Collection: Collect regenerating tail blastemas from treated and control tadpoles at 24, 48, and 72 hours post-amputation.
  • RNA Extraction and qRT-PCR: Extract total RNA and synthesize cDNA. Perform quantitative RT-PCR (qRT-PCR) to measure expression levels of:
    • Posterior Hox genes (e.g., Hoxa13, Hoxd13): Expect downregulation in VA-treated groups [6].
    • Limb marker genes (e.g., pitx1): Expect upregulation following Hox gene changes [6].
  • In Situ Hybridization (Optional): To visualize spatial expression patterns, perform whole-mount in situ hybridization on fixed regenerates using probes for the genes of interest [44].

The entire experimental workflow, from animal handling to molecular analysis, is depicted below.

G A Animal Care & Embryo Handling (HCG-induced mating, dejellying, staging) B Tail Amputation A->B C Vitamin A Treatment (RA pulse in medium) B->C D Phenotype Observation (Macroscopic inspection for ectopic structures) C->D E Molecular Analysis (qRT-PCR for Hox genes & limb markers) D->E

The Scientist's Toolkit: Research Reagent Solutions

Table 2: Essential Reagents for Hox Gene Manipulation via Vitamin A

Research Reagent Function / Application Key Considerations
All-trans Retinoic Acid (RA) The active Vitamin A metabolite; directly binds to RAR/RXR receptors to alter Hox gene expression [6] [32]. Light-sensitive; requires DMSO for stock solutions. Dosage is critical and species-specific.
Human Chorionic Gonadotropin (HCG) Hormone used to induce natural mating in anurans for consistent embryo production [44]. Allows for timed collection of embryos at specific developmental stages.
Anuran Models (e.g., Rana ornativentris, Xenopus laevis) Classic model organisms for studying vertebrate development and regeneration [6] [44]. Exhibit robust regenerative capabilities and are responsive to retinoid-induced homeotic transformations.
Calcium Imaging Indicators (e.g., GCaMP, NCaMP7) Genetically encoded sensors for visualizing intracellular calcium dynamics [45]. Can be repurposed to study calcium signaling roles in downstream processes of differentiation and patterning.
Fluorescence In Situ Hybridization (FISH) Kits Allow for spatial localization of specific mRNA transcripts (e.g., Hox genes, pitx1) in fixed tissue samples [44]. Validates changes in gene expression within the context of tissue morphology and ectopic structures.

The targeted manipulation of Hox gene expression using Vitamin A in anuran models provides a robust and inducible system for studying fundamental principles of body patterning, regeneration, and gene regulation. The precise phenotypic outcome is a direct function of dosage and developmental timing. By following the detailed protocols, utilizing the recommended reagents, and monitoring the key molecular hallmarks outlined in this application note, researchers can reliably reproduce the homeotic transformation phenomenon and adapt this powerful approach to probe deeper into the mechanisms of developmental biology.

Addressing Variability in Transformation Efficiency Across Species and Individuals

The manipulation of gene expression in model organisms is a cornerstone of modern developmental biology. Research into Hox genes—key regulators of the body plan along the anterior-posterior axis—exemplifies this approach, particularly in studies investigating the homeotic transformation of body structures [46] [47]. A notable example is the induction of ectopic limbs in anuran tadpoles following vitamin A administration, a process governed by changes in Hox gene expression [6]. Successfully replicating such profound phenotypic changes across different species and individual organisms hinges on efficient delivery of genetic constructs, making transformation efficiency a critical experimental parameter [48].

This application note addresses the significant challenge of variability in transformation efficiency. We provide a structured framework for quantifying this efficiency, analyze its key determinants, and present optimized protocols to enhance experimental reproducibility and success in Hox gene manipulation studies.

Quantitative Data on Transformation Efficiency

Transformation efficiency is quantitatively defined as the number of transformants (cells that have incorporated exogenous DNA) per microgram of DNA used [48]. This metric allows for direct comparison between different experimental conditions, methods, and species. The table below summarizes typical efficiency ranges for common transformation methods in different biological systems.

Table 1: Typical Transformation Efficiency Ranges Across Methods and Systems

Transformation Method Typical Efficiency Range (CFU/μg DNA) Common Applications Key Influencing Factors
Chemical Transformation (Heat Shock) 10^5 - 10^8 [48] Routine cloning in E. coli; some yeast and algal species. Plasmid size, culture growth phase, competence protocol, recovery media.
Electroporation 10^4 - 10^10 (up to 10^11 for optimized E. coli systems) [48] Wide range: bacteria, yeast, plant protoplasts, mammalian cells. Field strength, pulse length, buffer conductivity, DNA quality and form.
Ultrasound-Mediated Transformation ~10^5 (reported maximum in E. coli) [49] Emerging method for microbial transformation. Ultrasonic power, exposure time, membrane fluidity, expression of membrane-related genes.

Beyond the method itself, several intrinsic and extrinsic factors critically impact the efficiency value obtained.

Table 2: Key Factors Affecting Transformation Efficiency and Mitigation Strategies

Factor Impact on Efficiency Evidence-Based Mitigation Strategy
Plasmid Size Efficiency declines linearly with increasing plasmid size [48]. Use minimal vector backbones; for large constructs, consider recombineering or alternative delivery systems (e.g., viral vectors).
DNA Form Supercoiled plasmids are most efficient (~75% for relaxed plasmids; 10^4-fold reduction for single-stranded DNA) [48]. Isolate high-quality, supercoiled plasmid DNA; avoid nicking. Minimize UV exposure during gel extraction [48].
Cell Genotype & Physiology Strains with mutations (e.g., deoR, recBC) can show 4-5x higher efficiency [48]. Cells are most competent in early log phase [48]. Use engineered high-efficiency competent strains (e.g., E. coli DH5α, TOP10). Harvest cells at optimal optical density (OD~0.4).
Restriction Barriers Restriction-modification systems can degrade foreign, unmethylated DNA [48]. Use dam+/dcm+ strains for propagation; or use strains lacking common restriction systems (e.g., E. coli K-12 derivatives).
Membrane Composition Ultrasound-mediated transformation alters membrane integrity and pores, regulated by genes like cusC, tolA, and ompC [49]. Optimize physical parameters (e.g., ultrasonic power); pre-validate expression of key membrane transport genes.

Experimental Protocols for Assessing and Optimizing Efficiency

Standardized Protocol for Measuring Transformation Efficiency

This protocol is adapted for bacterial systems, a common first step in genetic manipulation workflows, and can be adjusted for other cell types.

Principle: To determine the number of colony-forming units (CFUs) per microgram of plasmid DNA obtained after a transformation procedure [48].

Materials:

  • Competent cells (e.g., E. coli DH5α)
  • pUC19 or similar control plasmid (high-copy number, ampicillin resistance)
  • LB broth and LB agar plates with appropriate antibiotic (e.g., 100 μg/mL ampicillin)
  • SOC outgrowth medium
  • Ice, water bath (42°C), incubator (37°C)

Procedure:

  • Thaw competent cells on ice.
  • Add 1-100 ng of plasmid DNA (e.g., 10 pg of pUC19 for high-efficiency strains) to 50 μL of cells. Include a "no DNA" negative control.
  • Incubate on ice for 20-30 minutes.
  • Heat shock at 42°C for exactly 30-45 seconds. Immediately return to ice for 2 minutes.
  • Add 950 μL of SOC or LB medium.
  • Recover with shaking at 37°C for 60 minutes to allow antibiotic resistance expression.
  • Plate serial dilutions (e.g., 10 μL and 100 μL) onto selective agar plates.
  • Incubate plates overnight at 37°C.
  • Count colonies on plates with 30-300 colonies.

Calculation: Transformation Efficiency (CFU/μg) = (Number of colonies × Dilution factor) / Amount of DNA plated (μg)

Example: If 100 μL of a 1:10 dilution of the transformation mixture yields 150 colonies from 10 ng of DNA: Efficiency = (150 colonies × 10) / 0.00001 μg = 1.5 × 10^8 CFU/μg [48]

Protocol: Vitamin A-Induced Hox Gene Perturbation in Anuran Tadpoles

This protocol outlines the key experimental workflow for inducing homeotic transformations, a context where efficient genetic manipulation is often required for mechanistic follow-up.

Principle: Vitamin A (retinoic acid) acts as a morphogen, altering the expression of Hox genes and leading to homeotic transformations, such as the development of ectopic limbs in regenerating tadpole tails [6].

Materials:

  • Rana ornativentris or Xenopus laevis tadpoles
  • All-trans retinoic acid (RA) stock solution in DMSO
  • Tank water system
  • Surgical tools for tail amputation
  • RNA extraction kit and qPCR reagents for Hox gene/pitx1 expression analysis

Procedure:

  • Tadpole Preparation: Raise tadpoles to the desired developmental stage in a controlled aquarium system.
  • Tail Amputation: Surgically amputate tails at a precise position under anesthesia.
  • Vitamin A Treatment: Expose tadpoles to a defined concentration of all-trans retinoic acid (e.g., (10^{-7}) M to (10^{-6}) M) in tank water immediately following amputation. A DMSO vehicle control is essential.
  • Monitor Regeneration: Observe and document the regenerating tail blastema daily. Ectopic limb bud formation typically precedes the appearance of recognizable limb structures.
  • Sample Collection: Harvest regenerating tissue at specific timepoints post-amputation (e.g., prior to visible limb bud formation) for molecular analysis.
  • Molecular Validation: Isolve total RNA and perform qPCR to quantify expression changes in key genes, including:
    • Posterior Hox genes (e.g., HoxA13, HoxD13), which are expected to be downregulated [6].
    • pitx1, a key hind limb determinant, which is expected to be upregulated following Hox gene changes [6].

Signaling Pathways and Experimental Workflows

Vitamin A Signaling in Hox Gene Regulation and Homeotic Transformation

The following diagram illustrates the proposed molecular pathway by which vitamin A induces homeotic transformation, integrating the core finding from the anuran model.

G VitaminA Vitamin A (Retinoic Acid) HoxGenes Posterior Hox Genes VitaminA->HoxGenes Downregulates Pitx1 pitx1 Expression VitaminA->Pitx1 Induces (via Hox suppression) HoxGenes->Pitx1 Suppresses LimbFate Limb Fate Specification Pitx1->LimbFate TailRegen Tail Regeneration Blastema TailRegen->LimbFate Context

Experimental Workflow for Transformation Efficiency Analysis

This workflow outlines the logical sequence of experiments to systematically address variability in transformation efficiency within a research project.

G cluster_factors Assess Key Factors Start Define Experimental Goal MethodSelect Select Transformation Method Start->MethodSelect FactorAssess Assess Key Factors MethodSelect->FactorAssess Optimize Optimize Protocol FactorAssess->Optimize F1 Plasmid Size/Form F2 Host Cell Genotype F3 Culture Conditions F4 Method Parameters Validate Validate in Biological System Optimize->Validate

The Scientist's Toolkit: Research Reagent Solutions

Selecting the appropriate reagents and materials is fundamental to minimizing variability. The following table details key solutions for experiments involving transformation efficiency and Hox gene analysis.

Table 3: Essential Research Reagents for Genetic Transformation and Hox Gene Studies

Reagent/Material Function/Description Application Notes
High-Efficiency Competent Cells Genetically engineered strains (e.g., E. coli DH5α, TOP10) optimized for DNA uptake. Select strains with high transformation efficiency ((>1x10^8) CFU/μg) for routine cloning. Use specialized strains (e.g., electrocompetent) for difficult transformations [48].
pUC19 Control Plasmid Small, high-copy-number plasmid with ampicillin resistance; standard for quantifying transformation efficiency. Use 1-10 pg for high-efficiency chemical transformation and 1-100 pg for electroporation to accurately measure peak efficiency [48].
All-trans Retinoic Acid The active morphogen derived from Vitamin A that directly modulates Hox gene expression. Prepare fresh stock solutions in DMSO and protect from light. Dose-response curves are critical due to its potent and sometimes teratogenic effects [6].
SOC Outgrowth Medium A nutrient-rich recovery medium containing peptides, sugars, and cations. Use for cell recovery post-transformation to maximize plasmid expression and cell viability, leading to higher observed efficiency [48].
qPCR Assays for Hox Genes Gene-specific primers and probes for quantifying Hox expression changes (e.g., HoxA13, HoxD13). Crucial for validating the molecular outcome of treatments like retinoic acid. Normalize to stable housekeeping genes [6] [2].
Membrane Permeabilization Agents Chemicals like CaClâ‚‚ for bacterial competence or proprietary reagents for eukaryotic cells. For chemical transformation, the quality and composition of these agents are primary determinants of efficiency [48].

Variability in transformation efficiency is not an insurmountable obstacle but a quantifiable and manageable parameter. By adopting the standardized measurement practices, optimized protocols, and critical reagent selections outlined in this application note, researchers can significantly enhance the reliability and cross-species comparability of their experiments. This rigorous approach is particularly vital for dissecting complex genetic pathways such as those controlled by Hox genes, where consistent genetic manipulation is key to elucidating the mechanisms underlying profound biological phenomena like vitamin A-induced homeotic transformation.

CONTROLLING FOR ENDOGENOUS RA SIGNALING AND METABOLIC INTERFERENCE

Within the context of manipulating Hox gene expression using vitamin A in anuran models, controlling for endogenous retinoic acid (RA) signaling and metabolic interference is a critical methodological consideration. RA, the active metabolite of vitamin A, acts as a potent morphogen and directly regulates the collinear expression of Hox genes, which are key drivers of anteroposterior (A-P) axial patterning and morphological identity [11] [50]. In anuran tadpoles, exogenous vitamin A administration can induce homeotic transformations, such as the formation of ectopic limbs in tail regions, a process linked to the downregulation of posterior Hox genes and subsequent activation of limb development programs like pitx1 [6]. The robust and tightly regulated endogenous RA network presents a significant challenge for these experiments, as it can compensate for perturbations, modulate experimental outcomes, and obscure causal relationships [50]. This Application Note provides detailed protocols and frameworks to effectively control for these variables, ensuring the precise manipulation and accurate interpretation of RA signaling in anuran research.

Background

The RA Signaling Pathway and Its Regulation

Retinoic acid signaling is a tightly regulated, multi-step process that begins with dietary vitamin A intake (Figure 1). Retinol (ROL) from the bloodstream, bound to Retinol-Binding Protein (RBP), enters the target cell primarily through the membrane receptor STRA6 [51]. Intracellular ROL is bound by Cellular Retinol Binding Protein 1 (CRBP1) and undergoes a two-step oxidation: first to retinaldehyde (RAL) by enzymes such as Retinol Dehydrogenases (RDH10) or Alcohol Dehydrogenases (ADH), and then to all-trans Retinoic Acid (ATRA) by Retinaldehyde Dehydrogenases (RALDH1, 2, 3) [51] [50]. ATRA is the primary bioactive ligand that translocates to the nucleus, often facilitated by Cellular Retinoic Acid-Binding Protein 2 (CRABP2). Inside the nucleus, ATRA binds to Retinoic Acid Receptors (RARs), which form heterodimers with Retinoid X Receptors (RXRs). This ligand-receptor complex then binds to Retinoic Acid Response Elements (RAREs) in the regulatory regions of target genes, including Hox genes, leading to the recruitment of co-activators and the initiation of transcription [52] [51]. The pathway is characterized by a self-regulating robustness network. Key components, such as raldh2 and the RA-degrading enzyme cyp26a1, are themselves regulated by RA levels, creating feedback and feedforward loops that maintain RA homeostasis and resist external perturbations [50].

G cluster_extracellular Extracellular cluster_intracellular Intracellular Cytoplasm cluster_nuclear Nucleus ROL_RBP Retinol (ROL) + RBP STRA6 STRA6 Receptor ROL_RBP->STRA6 Cellular Uptake ROL Retinol (ROL) (CRBP1) STRA6->ROL RDH10 RDH10 Oxidation ROL->RDH10 RAL Retinaldehyde (RAL) RALDH RALDH Oxidation RAL->RALDH ATRA all-trans RA (ATRA) (CRABP2) ATRA->RALDH Feedback CYP26 CYP26 Degradation ATRA->CYP26 Catabolism ATRA->CYP26 Induction RAR_RXR RAR/RXR Heterodimer ATRA->RAR_RXR Nuclear Signaling RDH10->RAL RALDH->ATRA RARE RARE (Target Gene) RAR_RXR->RARE TSCRIPT Target Gene Transcription (Hox Genes) RARE->TSCRIPT

Figure 1. The Retinoic Acid (RA) Signaling Pathway. This diagram illustrates the core pathway from dietary vitamin A intake to RA-mediated gene regulation, highlighting key metabolic steps, nuclear activation, and feedback mechanisms. Created using DOT language.

RA-Hox Gene Axis in Anuran Models

The connection between RA and Hox genes is fundamental to A-P patterning. Hox genes are expressed in a temporally and spatially collinear manner along the A-P axis, creating a "Hox code" that specifies regional identity [11]. RA gradients directly regulate this code by differentially activating Hox genes; genes located more 3' in the Hox cluster are more sensitive to RA and are activated earlier and more anteriorly than 5' genes [11]. In the anuran Rana ornativentris, administration of vitamin A during tail regeneration leads to a homeotic transformation, where ectopic limbs form instead of tails. Molecular analysis of this phenomenon reveals that this transformation is preceded by the downregulation of a posterior Hox gene, which in turn allows for the upregulation of the hindlimb determinant pitx1 [6]. This finding underscores the critical role of RA-mediated Hox gene regulation in determining organ and axial fate. The exquisite sensitivity of this system means that even minor, unaccounted-for fluctuations in endogenous RA signaling can significantly alter experimental results.

Quantitative Data on RA Signaling

Table 1: Quantitative Data on RA Signaling Components and Perturbations

Parameter / Component Quantitative Data / Concentration Biological Context / Effect
Physiological RA Fluctuation Within physiological range (e.g., 1–2 μM blood retinol [51]) Mild developmental malformations; network robustness is activated [50].
Supraphysiological RA Non-physiological, high concentrations (common in literature) Severe embryonic malformations (anterior/posterior axis, neural tube) [50].
RA Synthesis Inhibitor (DEAB) 1–100 μM (common in vivo use) Inhibits RALDH activity, reduces endogenous RA levels [50].
RARγ-specific Inhibitor (LY2955303) 10 μM Improved bovine IVF blastocyst rates via metabolic reprogramming [53].
RAR Antagonist (BMS-195614) 10 μM Used to block RAR signaling in embryo culture assays [53].
RA Knockdown Feedback Limit Upper response limit exists Network robustness has a maximum capacity to respond to reduced RA [50].
RA Addition Feedback Threshold Minimal activation threshold exists A specific increase in RA is required to trigger feedback loops [50].
Anuran RA Treatment Varies by species and desired effect Induces ectopic limb formation via posterior Hox downregulation and pitx1 upregulation [6].

Application Notes & Protocols

Protocol 1: Inhibiting Endogenous RA Synthesis and Signaling

This protocol outlines a method to reduce baseline RA signaling in anuran embryos, allowing for the study of loss-of-function phenotypes and providing a controlled baseline for RA supplementation experiments.

I. Materials

  • Anuran embryos (Xenopus laevis, Rana ornativentris, etc.)
  • DEAB (Diethylaminobenzaldehyde), a RALDH inhibitor [50]
  • BMS-195614, a pan-RAR antagonist [53]
  • LY2955303, an RARγ-specific inhibitor [53]
  • Dimethyl Sulfoxide (DMSO)
  • Steinberg's solution or Modified Barth's Saline (MBS)
  • Multi-well culture plates

II. Procedure

  • Embryo Preparation: Collect healthy, stage-specific anuran embryos. Remove jelly coats if necessary.
  • Inhibitor Stock Solution: Prepare concentrated stock solutions of DEAB (e.g., 100 mM) and RAR antagonists (e.g., 10 mM) in DMSO.
  • Working Solution Preparation: Dilute stock solutions in anuran culture medium immediately before use. A recommended starting concentration is 10–50 μM for DEAB and 1–10 μM for RAR antagonists. Ensure the final concentration of DMSO does not exceed 0.1% (v/v). Include a vehicle control (0.1% DMSO in culture medium).
  • Treatment: Transfer groups of 20-30 embryos into multi-well plates containing the inhibitor working solutions or vehicle control.
  • Incubation and Monitoring: Incubate embryos at the appropriate temperature (e.g., 14-22°C). Monitor embryo development daily and refresh the treatment solutions every 24 hours to maintain inhibitor potency.
  • Validation of Knockdown: Assess the efficacy of RA reduction by analyzing the expression of direct RA target genes (e.g., cyp26a1, dhrs3) via qPCR or in situ hybridization. A successful knockdown should downregulate these genes [50].

III. Troubleshooting

  • High Embryo Lethality: Reduce the inhibitor concentration and ensure the DMSO concentration is not toxic.
  • Incomplete Knockdown: Increase inhibitor concentration within a non-toxic range or use a combination of a synthesis inhibitor (DEAB) and a receptor antagonist (BMS-195614) for a more complete blockade.

Protocol 2: Controlling for Metabolic Interference and Network Robustness

This protocol is designed to account for and measure the compensatory response of the RA metabolic network, which can confound experimental outcomes.

I. Materials

  • Anuran embryos
  • all-trans Retinoic Acid (ATRA)
  • RA synthesis inhibitors (as in Protocol 1)
  • RNA extraction kit
  • qPCR reagents and primers for RA network genes (raldh2, cyp26a1, dhrs3, rdh10)

II. Procedure

  • Pulse-Chase Perturbation Design: To probe network robustness, employ a transient perturbation followed by a recovery period, rather than continuous exposure [50].
    • Pulse: Expose embryos to a precise, physiological concentration of RA (e.g., 10⁻⁹ M to 10⁻⁷ M) or an RA synthesis inhibitor (e.g., 50 μM DEAB) for a defined window (e.g., 6-12 hours).
    • Chase: Remove the perturbation by thoroughly washing the embryos and transferring them to standard culture medium.
  • Kinetic Sampling: Collect embryo samples at multiple time points: before the pulse (T0), immediately after the pulse (T1), and at several time points during the chase period (e.g., T+3h, T+6h, T+12h).
  • Transcriptomic Analysis: Perform RNA extraction and qPCR analysis on the collected samples. Use a panel of primers targeting key RA metabolic network components (Table 2).
  • Data Interpretation: Analyze the kinetic expression profiles. A robust network will show a transient change in gene expression (e.g., cyp26a1 induction by RA, raldh2 suppression by RA) that returns to baseline during the chase period, indicating a successful homeostatic response [50].

Protocol 3: Validating Hox Gene Manipulation Outcomes

After manipulating RA signaling, it is crucial to directly assess the resulting changes in the Hox code.

I. Materials

  • Treated and control anuran embryos/tadpoles
  • RNA extraction kit
  • cDNA synthesis kit
  • qPCR reagents
  • Primers for anterior, central, and posterior Hox genes (e.g., hoxa1, hoxb4, hoxc10)
  • Primers for limb markers (e.g., pitx1, tbx5)

II. Procedure

  • Tissue Collection: Dissect the region of interest (e.g., regenerating tail bud, developing limb field) from treated and control animals.
  • Gene Expression Analysis: Extract total RNA and synthesize cDNA. Perform qPCR for a panel of Hox genes representing different paralog groups and positions within the cluster.
  • Phenotypic Correlation: For studies on ectopic limb induction, also analyze the expression of limb program genes like pitx1 (for hindlimb identity) [6].
  • Data Interpretation: Expect a coherent shift in the Hox code. Successful RA treatment should recapitulate the reported downregulation of posterior Hox genes prior to the upregulation of pitx1 and the appearance of ectopic limbs [6].

The Scientist's Toolkit

Table 2: Essential Research Reagents for Controlling RA Signaling

Reagent / Material Function / Application Key Considerations
DEAB (Diethylaminobenzaldehyde) Small-molecule inhibitor of Retinaldehyde Dehydrogenases (RALDH); reduces de novo RA synthesis [50]. Used to lower endogenous RA levels; effective concentration must be empirically determined for each model system.
BMS-195614 A pan-RAR antagonist that blocks RA signaling at the receptor level [53]. Used in combination with synthesis inhibitors for a more complete RA signaling blockade.
LY2955303 A specific inhibitor for the Retinoic Acid Receptor gamma (RARγ) [53]. Useful for dissecting the specific role of RARγ in developmental processes; shows species-specific effects.
all-trans Retinoic Acid (ATRA) The primary bioactive retinoid ligand; used for exogenous RA supplementation. Highly labile; sensitive to light and oxygen. Prepare fresh stock solutions and use precise, physiological doses.
qPCR Primers for RA Network Genes Validated primers for raldh2, cyp26a1, dhrs3, rdh10; used to monitor RA network feedback. Essential for validating the efficacy of perturbations and measuring network robustness [50].
qPCR Primers for Hox Genes Validated primers for anterior, central, and posterior Hox genes; used to read out the "Hox code". Critical for confirming the primary molecular outcome of RA manipulations [6] [11].

Strategies for Precise Spatial and Temporal Control of Hox Gene Manipulation

Hox genes are master regulators of embryonic development, encoding transcription factors that establish the anterior-posterior (A-P) body axis in bilaterian animals [54] [55]. Their unique genomic organization into clusters and precise spatiotemporal expression patterns make them critical targets for developmental biology research. In anuran models, the manipulation of Hox gene expression using vitamin A (retinoic acid, RA) provides a powerful experimental paradigm for investigating axial patterning mechanisms. This application note details standardized protocols for achieving precise spatial and temporal control of Hox gene manipulation, framed within the context of vitamin A research in anuran systems.

The fundamental principle underlying these strategies is collinearity - the correspondence between the genomic order of Hox genes and their sequence of activation in time and space during development [56] [55]. Temporal collinearity refers to the sequential activation of Hox genes from 3' to 5' within clusters during early development, while spatial collinearity describes their nested expression domains along the A-P axis [57]. Vitamin A-derived RA signaling directly regulates this process through retinoic acid response elements (RAREs) embedded within Hox clusters, making it a potent natural modulator of Hox expression [54].

Background and Significance

Hox Gene Organization and Regulatory Principles

Hox genes exhibit a remarkable genomic organization that underlies their coordinated regulation. In vertebrates, these genes are arranged in four clusters (HoxA, B, C, and D), with the order of genes on the chromosome corresponding to their expression patterns along the A-P axis [55]. This structural-functional relationship enables the combinatorial Hox code that specifies regional identities in developing embryos [54].

The regulation of Hox genes occurs through sophisticated chromatin dynamics. In embryonic stem cells, Hox clusters exist in a bivalent chromatin state containing both repressive (H3K27me3) and activating (H3K4me3) marks [57]. During activation, genes progressively transition from an inactive compartment to an active one, with their physical relocation within the nucleus reflecting their transcriptional status [57]. This dynamic chromatin architecture provides the structural foundation for implementing temporal collinearity.

Table 1: Fundamental Properties of Hox Gene Regulation

Property Description Experimental Significance
Collinearity Correspondence between genomic position and spatiotemporal expression Predicts activation sequence; informs timing of interventions
Chromatin Bivalency Presence of both activating and repressing histone marks in silent genes Enables plastic response to signaling gradients
RARE Elements Retinoic acid response elements embedded in Hox clusters Direct target for vitamin A manipulation
Bimodal 3D Organization Spatial separation of active/inactive genes in nuclear compartments Correlates with transcriptional status; can be monitored
Enhancer Sharing Regulatory elements that control multiple Hox genes Allows coordinated regulation of gene subgroups
Retinoic Acid Signaling in Hox Regulation

Vitamin A-derived retinoic acid serves as a key morphogen regulating Hox gene expression through direct binding to RAREs [54]. These RAREs function as cis-regulatory components of RA-dependent enhancers that provide regulatory inputs both locally on adjacent Hox genes and over long distances to coordinately regulate multiple genes within a cluster [54]. The presence of specific RAREs at various positions within Hox clusters enables graded RA signaling to elicit distinct transcriptional responses according to the collinearity principle.

In anuran models, the RA signaling pathway intersects with other key patterning systems, particularly BMP signaling [56]. The antagonism between BMP and anti-BMP factors establishes a crucial regulatory network that converts temporal information into spatial patterning along the A-P axis [56]. This interaction explains the famous connection between dorsoventral (D-V) and A-P patterning in vertebrate embryos and provides additional experimental handles for manipulating Hox expression.

Experimental Strategies and Protocols

Temporal Control of Hox Gene Manipulation

The sequential activation of Hox genes during development enables temporal-specific manipulation by targeting particular genes based on their position in the collinear sequence. The following protocols leverage the inherent timing of the "Hox clock" for precise experimental interventions.

Protocol 3.1.1: Staged Retinoic Acid Administration

Principle: The competence of Hox genes to respond to RA changes during development, allowing stage-specific manipulation by administering RA at precise timepoints.

Materials:

  • All-trans retinoic acid (Sigma-Aldrich, R2625)
  • Dimethyl sulfoxide (DMSO, for stock solutions)
  • Anuran embryo culture medium
  • Microinjection apparatus or treatment chambers

Procedure:

  • Prepare fresh RA stock solution (10 mM in DMSO) and dilute to working concentrations in embryo medium.
  • For global administration, stage embryos according to standard tables and transfer to RA-containing medium.
  • Use pulsed exposures (30-90 minutes) for transient effects or continuous exposure for sustained manipulation.
  • For localized application, prepare RA-soaked beads (e.g., AG1-X2 ion exchange resin) and implant in target tissues.
  • Critical timing windows:
    • Early gastrula (stage 10-10.5): Targets 3' Hox genes (groups 1-4)
    • Mid gastrula (stage 11-12.5): Targets central Hox genes (groups 5-8)
    • Late gastrula/neurula (stage 13-15): Targets 5' Hox genes (groups 9-13)
  • Terminate exposure by thorough washing and return to control medium.
  • Include DMSO-only controls at equivalent concentrations.

Technical Notes:

  • RA concentration range: 10 nM - 1 μM (dose-response should be empirically determined)
  • Photoprotect RA solutions from light degradation
  • Stage specificity overrides absolute time; consult relevant staging tables for species
Protocol 3.1.2: Molecular Timing Using Endogenous Promoters

Principle: Leverage temporally-specific endogenous promoters to drive effectors at precise developmental windows.

Materials:

  • Hox gene promoters of varying colinear positions
  • CRISPRa/i systems or recombinases
  • Microinjection equipment
  • Expression vectors with fluorescent reporters

Procedure:

  • Clone Hox promoters of interest (3' early, 5' late) driving temporal-specific effectors.
  • For gain-of-function: Use minimal VP16-based activators under Hox promoter control.
  • For loss-of-function: Express CRISPRi repressors (dCas9-KRAB) under stage-specific promoter control.
  • Microinject constructs into fertilized eggs and monitor expression via linked fluorescent reporters.
  • Validate timing using whole-mount in situ hybridization for endogenous targets.
Spatial Control of Hox Gene Manipulation

Spatially restricted manipulation requires targeting specific regions along the A-P axis where particular Hox genes are active. The following protocols enable region-specific interventions.

Protocol 3.2.1: Regional Retinoic Acid Application

Principle: Exploit the natural RA responsiveness of Hox genes while restricting exposure to specific embryonic regions.

Materials:

  • Heparin-coated beads (e.g., Sigma H5263)
  • Retinoic acid solutions
  • Fine forceps and transplantation tools
  • Vital dyes for lineage tracing

Procedure:

  • Soak heparin-coated beads in RA solution (0.1-10 μM) for 30 minutes.
  • Pre-label donor cells or host regions with vital dyes (e.g., DiI, GFP mRNA).
  • For anterior neural manipulations: Implant beads adjacent to hindbrain regions at early neurula stages.
  • For trunk manipulations: Target posterior growth zone or presomitic mesoderm.
  • For limb bud manipulations: Implant in developing limb fields at appropriate stages.
  • Include control beads soaked in vehicle solution only.
  • Monitor RA diffusion using co-implanted tracer beads if available.
Protocol 3.2.2: Electroporation-Mediated Regional Targeting

Principle: Use regional electroporation to deliver constructs to specific territories along the A-P axis.

Materials:

  • Plasmid DNA with appropriate promoters/effectors
  • Electroporator and electrodes
  • Fast Green dye for visualization
  • Microinjection capillary tubes

Procedure:

  • Prepare plasmid DNA (1-2 μg/μL in PBS with Fast Green).
  • Microinject DNA solution into target cavity (neural tube, somites, etc.).
  • Position electrodes to bracket target tissue.
  • Apply pulses (5-10 × 50 ms pulses at 20-30V depending on tissue).
  • Optimize parameters for specific tissues and developmental stages.
  • Culture embryos post-electroporation and monitor expression.
Direct Gene Editing Approaches
Protocol 3.3.1: CRISPR-Based Hox Gene Manipulation

Principle: Use CRISPR/Cas9 systems with temporal and spatial control to edit specific Hox genes.

Materials:

  • Cas9 protein or mRNA
  • Synthetic sgRNAs targeting Hox genes
  • Microinjection apparatus
  • Screening primers for mutation detection

Procedure:

  • Design sgRNAs targeting specific Hox genes considering:
    • 3' genes for anterior identities
    • 5' genes for posterior identities
    • Conserved functional domains
  • Prepare injection mixes:
    • Cas9 protein (300-500 ng/μL) + sgRNA (50-100 ng/μL)
    • Alternatively: Cas9 mRNA (300-500 ng/μL) + sgRNA
  • Microinject into fertilized eggs or specific regions at desired stages.
  • For spatial control: Use targeted injection into specific territories.
  • For temporal control: Utilize photoactivatable CRISPR systems.
  • Assess mutation efficiency by PCR/restriction digest or sequencing.

Table 2: Hox Gene Targeting by Axial Position

Axial Region Hox Paralogs CRISPR Targets RA Sensitivity
Hindbrain Hox1-4 Promoter, homeodomain High (early)
Anterior Trunk Hox5-8 Conserved motifs Moderate (mid)
Posterior Trunk Hox9-10 Specific enhancers Low (late)
Tailbud Hox11-13 Autoregulatory sites Variable

Visualization and Analysis Methods

Documenting Expression Patterns

Whole-mount in situ hybridization remains the gold standard for visualizing Hox expression domains. Modifications for anuran embryos:

  • Use species-specific probes when possible
  • Develop parallel staining for multiple Hox genes
  • Combine with tissue markers for orientation

Live imaging of reporter constructs enables real-time tracking:

  • Hox promoter-driven GFP variants
  • Photoconvertible proteins for lineage tracing
  • Confocal time-lapse microscopy
Quantitative Assessment Methods

qRT-PCR analysis of Hox expression:

  • Design primers spanning intron-exon boundaries
  • Use multiple reference genes for normalization
  • Collect region-specific samples when possible

Chromatin conformation analysis:

  • 3C/qPCR for specific architectural changes
  • Monitor compartment transitions during activation

Research Reagent Solutions

Table 3: Essential Research Reagents for Hox Gene Manipulation

Reagent/Category Specific Examples Function/Application
Retinoic Pathway Modulators All-trans retinoic acid, BMS493 (inverse agonist), AGN193109 (antagonist) Direct pharmacological manipulation of RA signaling
Signaling Pathway Modulators Noggin (BMP antagonist), FGF inhibitors (SU5402), Wnt modulators Secondary pathway manipulation to influence Hox expression
CRISPR Systems Cas9 protein, synthetic sgRNAs, Hox-specific targeting vectors Direct gene editing and epigenetic manipulation
Expression Reporters Hox promoter-GFP constructs, RA-responsive element reporters Live imaging and response quantification
Chromatin Modifiers TSA (HDAC inhibitor), DZNep (EZH2 inhibitor) Epigenetic landscape manipulation
Delivery Tools Heparin-coated beads, electroporation equipment, microinjection systems Spatial-temporal restricted application

Representative Experimental Workflow

G Start Define Experimental Objective P1 Stage Selection (Early vs Mid vs Late Gastrula) Start->P1 P2 Spatial Target Identification (Anterior vs Posterior) P1->P2 P3 Intervention Method Selection P2->P3 Method1 Pharmacological (RA Treatment) P3->Method1 Method2 Molecular (CRISPR/Overexpression) P3->Method2 Method3 Combination Approach P3->Method3 A1 Dose/Concentration Optimization Method1->A1 A2 Delivery Method (Microinjection/Electroporation) Method2->A2 A3 Spatial Control (Regional Application) Method3->A3 Analysis Expression Analysis (In situ/qRT-PCR/Imaging) A1->Analysis A2->Analysis A3->Analysis Interpretation Data Interpretation & Validation Analysis->Interpretation

Diagram 1: Experimental workflow for Hox gene manipulation

Troubleshooting and Optimization

Common Technical Challenges

Variable RA sensitivity between embryo batches:

  • Solution: Include internal controls in each experiment
  • Pre-test RA responsiveness using a standard protocol

Off-target effects in CRISPR approaches:

  • Solution: Use multiple sgRNAs with clean validation profiles
  • Include appropriate control conditions

Mosaic expression in electroporation:

  • Solution: Optimize DNA concentration and pulse parameters
  • Use transposon systems for stabilization
Validation Strategies

Essential validation steps for Hox manipulation experiments:

  • Confirm specificity using multiple detection methods
  • Verify expected collinear responses
  • Document dose-response relationships
  • Include rescue experiments where possible

The precise spatial and temporal manipulation of Hox gene expression in anuran models requires integrated approaches that respect the inherent collinearity of these gene clusters. Vitamin A (retinoic acid) manipulation provides a powerful and physiologically relevant method for interrogating Hox function, particularly when combined with modern genetic tools and careful attention to developmental timing and spatial boundaries. The protocols outlined here provide a framework for designing experiments that capture the dynamic regulation of these crucial patterning genes while minimizing technical artifacts. As our understanding of Hox chromatin dynamics and regulatory mechanisms continues to advance, increasingly sophisticated manipulation strategies will emerge, further enhancing our ability to decipher the complex language of axial patterning.

Beyond Amphibians: Validating Mechanisms and Cross-Species Relevance

Application Notes

Retinoic acid (RA) signaling and Hox gene expression represent a deeply conserved developmental module that orchestrates anterior-posterior (A-P) axis patterning across vertebrate species. This RA-Hox axis operates through core principles that are maintained in zebrafish, chick, and mammalian models, despite lineage-specific genetic expansions. In all three models, RA functions as a key upstream regulator of Hox gene expression, while Hox genes translate this signaling into precise positional information along the A-P axis. This conserved machinery particularly governs the development of paired appendages and hindbrain segmentation, with evolutionary variations reflecting species-specific adaptations. The mechanistic conservation of this pathway enables cross-species experimental approaches and validates the use of alternative models like anurans for investigating fundamental principles of Hox gene regulation by vitamin A derivatives.

Core Signaling Pathway: RA-Hox Genetic Circuitry

The RA-Hox signaling pathway constitutes a fundamental genetic circuit that translates nutritional vitamin A into precise developmental patterning information. This circuit operates through a conserved series of molecular interactions:

G VitaminA VitaminA RA_Synthesis RA Synthesis (ALDH1A enzymes) VitaminA->RA_Synthesis RA_Gradient RA Morphogen Gradient RA_Synthesis->RA_Gradient RAR_RXR RAR/RXR Heterodimer RA_Gradient->RAR_RXR Hox_Expression Collinear Hox Expression RAR_RXR->Hox_Expression Target_Genes Target Gene Activation (e.g., Tbx5, Pitx1) Hox_Expression->Target_Genes Positioning A-P Positioning (Hindbrain, Appendages) Target_Genes->Positioning

Figure 1: Conserved RA-Hox Signaling Pathway. This core genetic circuit operates across zebrafish, chick, and mammalian models to translate vitamin A status into anterior-posterior patterning decisions through regulation of collinear Hox gene expression.

Quantitative Conservation Across Models: Functional Equivalents

Table 1: Comparative RA-Hox Pathway Components Across Vertebrate Models

Function Zebrafish Chick Mouse/Mammalian Conservation Level
RA Synthesis aldh1a2, aldh1a3 RALDH2 (ALDH1A2) RALDH2 (ALDH1A2) High - Essential for RA biosynthesis in all models [58]
Forelimb/Fin Positioning Hox Genes hoxb4a, hoxb5a, hoxb5b (hoxba/hoxbb clusters) Hoxb4, Hoxb5, Hoxc6 Hoxb5, Hoxc6 Medium - Functional conservation with species-specific cluster expansions [59] [60]
Hindbrain Hox Regulation hoxb1a, hoxb2, hoxb3 Hoxb1, Hoxb2 Hoxb1, Hoxb2 High - Conserved rhombomere expression boundaries [11]
Direct Limb/Fin Target tbx5a Tbx5 Tbx5 High - Essential for appendage initiation [59] [60]
RA Response Element Binding RARα, RARβ, RARγ RARα, RARβ, RARγ RARα, RARβ, RARγ High - Conserved receptor specificity [58]

Experimental Evidence: Functional Validation Across Species

Table 2: Key Experimental Findings Demonstrating RA-Hox Conservation

Experimental Manipulation Zebrafish Phenotype Chick/Mammalian Phenotype Functional Interpretation
Hox Cluster Deletion hoxba;hoxbb double mutants: Complete pectoral fin loss with absent tbx5a expression [59] [60] Mouse Hoxb5 KO: Rostral forelimb shift with incomplete penetrance [59] [60] Essential Hox requirement for appendage positioning, with zebrafish showing stronger phenotype due to experimental completeness
RA Signaling Inhibition aldh1a2 mutants: Pectoral fin defects, disrupted hox expression [58] VAD syndrome: Limb defects, hindbrain abnormalities [58] [61] Conserved RA requirement for proper Hox expression and appendage development
RA Ectopic Application Anterior hox expansion, posteriorization [58] Hindbrain expansion, forebrain repression [61] Conserved teratogenic effects via Hox gene misregulation
Hox-Tbx Genetic Interaction hoxb4a/b5a directly induce tbx5a in pectoral fin field [59] Hox proteins bind Tbx5 limb enhancer [59] [60] Conserved direct transcriptional regulation of limb initiation program
Temporal Collinearity Hoxb temporal collinearity regulates gastrulation timing [62] Hoxb temporal collinearity controls mesoderm ingression timing [62] Conserved mechanism linking temporal Hox expression to spatial patterning

Experimental Protocols

Protocol 1: Genetic Analysis of Hox Function in Zebrafish Pectoral Fin Development

Background and Principle

This protocol outlines the genetic approach for determining Hox gene requirements in zebrafish pectoral fin positioning, based on established methodologies that provided the first genetic evidence for Hox genes specifying paired appendage positions in vertebrates. The approach leverages zebrafish's seven hox clusters derived from teleost-specific genome duplication to parse functional redundancy and essential requirements [59] [60].

Materials and Reagents
  • Zebrafish lines: Wild-type AB strain and hox cluster mutant lines
  • CRISPR-Cas9 components for cluster deletion: Cas9 protein, guide RNAs targeting hoxba and hoxbb clusters
  • Embryo medium: E3 embryo medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaClâ‚‚, 0.33 mM MgSOâ‚„)
  • Fixation: 4% paraformaldehyde in PBS
  • Hybridization probes: tbx5a, hoxb4a, hoxb5a, hoxb5b antisense RNA probes
  • Imaging: Confocal microscope equipped for brightfield and fluorescence imaging
Procedure

  • Generate hox cluster mutants:

    • Design guide RNAs targeting flanking regions of hoxba and hoxbb clusters
    • Inject CRISPR-Cas9 ribonucleoprotein complexes into single-cell zebrafish embryos
    • Raise founders (F0) and outcross to identify germline transmissions
    • Intercross heterozygotes to generate homozygous cluster-deleted mutants

  • Phenotypic analysis:

    • Stage embryos at 24, 48, and 72 hours post-fertilization (hpf)
    • Document pectoral fin bud morphology using brightfield microscopy
    • Fix embryos at 30 hpf for whole-mount in situ hybridization
    • Process for tbx5a expression analysis in lateral plate mesoderm

  • Genetic interaction tests:

    • Generate hoxba⁻⁄⁻;hoxbb⁺⁄⁻ and hoxba⁺⁄⁻;hoxbb⁻⁄⁻ transheterozygotes
    • Quantify pectoral fin presence/absence in Mendelian ratios
    • Assess tbx5a expression levels across genotypic combinations

  • RA response competence:

    • Treat mutant embryos with 1×10⁻⁶ M all-trans RA from 75%-epiboly stage
    • Assess tbx5a induction capability in pectoral fin field
    • Compare RA responsiveness to wild-type siblings
Expected Results and Interpretation
  • hoxba;hoxbb double homozygous mutants should show complete pectoral fin absence with 100% penetrance
  • tbx5a expression should be nearly undetectable in double mutants at 30 hpf
  • Single heterozygotes should display normal fin development, indicating functional redundancy
  • Mutants should lack competence to induce tbx5a in response to RA treatment
  • Expected Mendelian ratio for double homozygotes: 6.25% of progeny

Protocol 2: Vitamin A-Mediated Hox Manipulation in Anuran Models

Background and Principle

This protocol adapts principles from zebrafish and chick RA-Hox studies for anuran models, specifically exploiting the homeotic transformation of tails into limbs induced by vitamin A treatment. The approach capitalizes on the conserved responsiveness of Hox genes to RA signaling to investigate appendage patterning in regenerating systems [6].

Materials and Reagents
  • Anuran specimens: Rana ornativentris or Xenopus laevis tadpoles
  • Vitamin A compounds: All-trans retinoic acid, retinol palmitate
  • Delivery vehicles: DMSO for stock solutions, aquarium water for working dilutions
  • Molecular biology: RNA extraction kit, cDNA synthesis kit, qPCR reagents
  • Gene expression primers: Posterior Hox genes (Hox9-13), pitx1, tbx5
  • Histology: Paraffin embedding equipment, microtome, staining solutions
Procedure

  • Vitamin A administration:

    • Prepare stock solution of 1×10⁻² M all-trans RA in DMSO
    • Create working dilution of 1×10⁻⁶ M in aquarium water
    • Anesthetize tadpoles in 0.1% MS-222
    • Amputate tails at mid-tail level using microdissection scissors
    • Immerse tadpoles in RA-containing solution for 6 hours post-amputation
    • Return to fresh aquarium water for regeneration period

  • Temporal expression analysis:

    • Collect regenerating tissue at 0, 12, 24, 48, and 72 hours post-amputation
    • Extract total RNA and synthesize cDNA
    • Perform quantitative PCR for posterior Hox genes and limb markers
    • Normalize expression to housekeeping genes (EF1α, ODC)

  • Morphological assessment:

    • Document ectopic limb formation daily for 2 weeks
    • Score homeotic transformation frequency and morphology
    • Process tissues for histological analysis (hematoxylin/eosin staining)
    • Compare skeletal patterns in ectopic limbs versus normal limbs

  • Inhibition experiments:

    • Apply RA signaling inhibitors (DEAB, BMS-493) during regeneration
    • Assess rescue of normal tail regeneration
    • Quantify Hox gene expression changes under inhibition conditions
Expected Results and Interpretation
  • Vitamin A treatment should induce ectopic limb formation in 30-60% of regenerating tails
  • Posterior Hox gene downregulation should precede pitx1 upregulation
  • Early Hox expression changes (within 24 hours) should predict successful limb transformation
  • RA signaling inhibition should suppress ectopic limb formation and maintain posterior Hox expression
  • Successful transformations should display proper limb skeletal patterning with Hox11-dependent middle elements

Protocol 3: Cross-Species Hox Reporter Assay for RA Responsiveness

Background and Principle

This protocol utilizes the evolutionarily conserved RA response elements in Hox gene regulatory regions to compare RA signaling sensitivity across species. The approach builds on studies demonstrating direct RA regulation of Hox clusters and enables quantitative assessment of RA-Hox circuitry conservation [58] [11].

Materials and Reagents
  • Reporter constructs: Hoxb promoter-luciferase fusions from multiple species
  • Cell culture: Zebrafish ZF4, chick DF1, mouse NIH/3T3 cell lines
  • Transfection reagents: Lipofectamine 3000 or equivalent
  • RA treatments: All-trans RA serial dilutions (1×10⁻⁹ to 1×10⁻⁶ M)
  • Luciferase assay system: Dual-luciferase reporter assay kit
  • Normalization: Renilla luciferase control vector
Procedure

  • Reporter construction:

    • Clone ~2kb regulatory regions upstream of Hoxb4 genes from zebrafish, chick, and mouse
    • Subclone into pGL4-basic luciferase vector
    • Verify sequence integrity and orientation

  • Transfection and treatment:

    • Plate cells in 24-well plates at 70% confluency
    • Co-transfect with Hox reporter and normalization control (100ng total DNA)
    • Allow 24 hours for gene expression
    • Treat with RA concentration series for 16 hours

  • Luciferase assay:

    • Lyse cells in passive lysis buffer
    • Measure firefly and Renilla luciferase activities sequentially
    • Calculate normalized luciferase activity (firefly/Renilla ratio)
    • Plot dose-response curves for each species' Hox reporter

  • Data analysis:

    • Calculate ECâ‚…â‚€ values for RA responsiveness for each reporter
    • Compare maximal induction levels across species
    • Statistical analysis: Two-way ANOVA with post-hoc testing
Expected Results and Interpretation
  • All Hoxb4 reporters should show RA dose-dependent activation
  • ECâ‚…â‚€ values should cluster within one order of magnitude across species
  • Zebrafish reporters may show higher dynamic range due to duplicated cluster complexity
  • Conservation of RA responsiveness indicates deep evolutionary constraint on this regulatory circuitry

The Scientist's Toolkit: Research Reagent Solutions

Table 3: Essential Research Reagents for RA-Hox Pathway Investigation

Reagent Category Specific Examples Function/Application Species Applicability
Genetic Models hoxba/hoxbb cluster mutants [59], aldh1a2 mutants [58] Determine gene requirement through loss-of-function Zebrafish
Hoxb5 knockout mice [59] Mammalian Hox gene function assessment Mouse
RA Pathway Modulators All-trans RA (agonist) [6] [58] Activate RA signaling, posteriorize embryos All vertebrates
DEAB, BMS-493 (antagonists) [61] Inhibit RA synthesis and signaling All vertebrates
Molecular Probes tbx5a/tbx5 in situ hybridization probes [59] Visualize limb/fin field initiation Cross-species applicable
Hox paralog-specific antibodies Protein localization and expression analysis Species-specific validation needed
Reporting Systems Hox promoter-luciferase constructs [58] Quantitative RA responsiveness measurement Cross-species comparative
RARE-LacZ transgenic lines Visual RA signaling activity in vivo Mouse, adaptable to other models
CRISPR Tools Cluster-specific gRNA libraries [59] Systematic Hox cluster deletion All genetic models
HDR templates for precise editing Knock-in alleles, conditional mutations All genetic models

Pathway Integration: From Molecular to Morphological

G RA RA Signaling (Gradient) Temporal Temporal Collinearity (Hox Expression) RA->Temporal Spatial Spatial Patterning (Hox Code) Temporal->Spatial Targets Target Gene Activation Spatial->Targets Morphology Morphological Outcomes Targets->Morphology Zebrafish Zebrafish: Pectoral Fin Position Morphology->Zebrafish Chick Chick: Limb Bud Position Morphology->Chick Mammal Mammal: Forelimb Position Morphology->Mammal Anuran Anuran: Ectopic Limb Induction Morphology->Anuran

Figure 2: Integrated RA-Hox Patterning System. The conserved pathway from RA signaling through temporal collinearity to spatial patterning generates species-specific morphological outcomes through regulation of target genes like Tbx5, demonstrating how conserved principles produce model-specific readouts.

The conserved RA-Hox axis provides a fundamental framework for understanding anterior-posterior patterning across vertebrate models. The experimental protocols outlined here leverage this deep conservation to enable cross-species investigations of vitamin A-mediated Hox gene regulation. For researchers focusing on anuran models, these principles provide validated approaches for manipulating appendage patterning through RA signaling manipulation. The consistent finding that Hox genes function as key intermediaries between RA signaling and morphological outcomes underscores their central role in translating nutritional signals (vitamin A) into precise developmental decisions, with important implications for both evolutionary biology and teratology research.

Comparative Analysis of Hox Gene Regulation in Cardiovascular Development and Disease

Hox genes, which encode a highly conserved family of transcription factors, are master regulators of embryonic patterning along the anterior-posterior axis. Recent research has illuminated their critical functions beyond development, including their roles in cardiovascular system formation and the pathogenesis of adult cardiovascular diseases. A primary regulator of Hox gene expression is vitamin A (retinoic acid, RA), which directly controls Hox transcription through specific retinoic acid response elements (RAREs) [63]. This application note provides a comparative analysis of Hox gene functions in cardiovascular development and disease, framed within the context of anuran models where vitamin A manipulation induces profound homeotic transformations. We present structured quantitative data, experimental protocols for key investigations, and visualization of regulatory networks to support research and therapeutic development in this field.

Hox Gene Expression in Cardiovascular Development vs. Disease

Hox genes exhibit distinct expression patterns and functions during cardiovascular development compared to their dysregulation in disease states. Understanding these contextual differences is essential for elucidating their dual roles and therapeutic potential.

Table 1: Comparative Analysis of Key Hox Genes in Cardiovascular Development and Disease

Hox Gene Role in Cardiovascular Development Dysregulation in Cardiovascular Disease Experimental Models
HOXA5 Patterning of second heart field (SHF) and pharyngeal arch arteries [64] Most downregulated gene in ascending thoracic aortic aneurysm (ATAA) (FC: -25.3); suppresses phenotypic switching of VSMCs in atherosclerosis [65] [66] Mouse models, Human ATAA tissue [66]
HOXA1 Specification of cardiac progenitor cells; patterning of SHF [64] Participates in atherosclerosis via miR-99a-5p-HOXA1 axis; affects HUVEC viability and migration [65] Mouse models, Cell culture (HUVECs) [65]
HOXC6 Involvement in early lineage commitment and specification [64] Significantly downregulated in ATAA; inhibits apoptosis of vascular endothelial cells and VSMC proliferation [65] [66] Human ATAA tissue, Cell culture models [66]
HOXA3 Contributes to cardiac neural crest and great artery formation [64] Promotes angiogenesis in pathological contexts [65] Mouse models, Angiogenesis assays [65]
HOXB5 Expressed in posterior SHF contributing to venous pole [64] Promotes endothelial inflammation via MCP-1 and IL-6 [65] Zebrafish models, Cell culture [65]
Developmental Roles of Hox Genes

During embryogenesis, Hox genes provide positional identity and regulate the morphogenesis of cardiovascular structures. Anterior Hox genes (e.g., Hoxa1, Hoxb1, Hoxa3) are expressed in the second heart field (SHF), a population of cardiac progenitor cells that contribute to the outflow tract, right ventricle, and atrial myocardium [64]. Their expression is tightly regulated by retinoic acid signaling, which establishes spatial and temporal gradients crucial for proper heart tube elongation and patterning. Cardiac neural crest cells, which require Hox gene expression for their migration and differentiation, are essential for the remodeling of the pharyngeal arch arteries and outflow tract septation [64] [67].

Hox Gene Dysregulation in Disease

In adult pathophysiology, dysregulation of Hox genes is implicated in various cardiovascular diseases. Atherosclerosis involves multiple Hox genes in processes such as lipid metabolism, inflammatory response, angiogenesis, and vascular smooth muscle cell (VSMC) phenotypic switching [65]. For instance, HOXA5 exerts protective effects by suppressing VSMC transition to a synthetic phenotype via PPARγ activation, while HOXB5 promotes inflammation through monocyte chemoattractant protein-1 (MCP-1) and interleukin-6 (IL-6) [65]. In ascending thoracic aortic aneurysm (ATAA), dramatic downregulation of HOXA5 and HOXC6 is observed, suggesting their critical role in maintaining aortic wall integrity [66].

Vitamin A Regulation of Hox Genes: Insights from Anuran Models

Vitamin A (retinoic acid) serves as a potent transcriptional regulator of Hox genes. The anuran (Rana ornativentris) model provides compelling evidence for this relationship, where vitamin A administration during tail regeneration induces homeotic transformation, resulting in ectopic limb formation instead of tail regrowth [6].

Molecular analyses reveal that this transformation is preceded by the downregulation of posterior Hox genes, which subsequently leads to the upregulation of hind limb patterning genes such as pitx1 [6]. This demonstrates that Hox genes act upstream of limb developmental pathways in response to retinoid signaling. The conserved nature of RA-Hox gene signaling across vertebrates, from anurans to mammals, underscores its fundamental role in patterning and morphogenesis.

Table 2: Quantitative Differential Expression of HOX Genes in Human Cardiovascular Disease

Cardiovascular Condition Significantly Altered HOX Genes Magnitude of Change (Fold Change) Primary Cellular Processes Affected
Ascending Thoracic Aortic Aneurysm (ATAA) [66] HOXA5, HOXC6, HOXA3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, 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HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, 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HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3, HOXB3,

HOX Gene Dysregulation in Human Cancers like Prostate Cancer

HOX genes are a family of 39 evolutionarily conserved transcription factors, organized into four clusters (A, B, C, and D) on different chromosomes, that play pivotal roles in embryonic development, tissue patterning, and cell differentiation [68] [69]. These genes encode proteins with a characteristic 60-61 amino acid homeodomain that facilitates DNA binding, enabling them to act as master regulators of cell identity [69]. Their expression follows a precise spatiotemporal pattern during embryogenesis, a principle known as collinearity, where the position of a gene within its cluster correlates with its expression along the anterior-posterior axis and the timing of its activation [68].

In recent decades, it has become evident that the same genes controlling embryonic development are often deregulated in cancer [68] [69]. HOX genes are no exception; their aberrant expression is a hallmark of numerous malignancies, including prostate cancer. They can function as either oncogenes or tumor suppressors, influencing key cancer hallmarks such as cell proliferation, apoptosis, metastasis, and angiogenesis [69]. This application note explores the implications of HOX gene dysregulation in human cancers, with a focus on prostate cancer, and provides detailed protocols for studying their function, framed within the context of fundamental research using anuran models.

HOX Gene Dysregulation in Prostate Cancer: Key Findings and Data

Epidemiological and molecular studies have firmly established the link between specific HOX gene dysregulation and prostate cancer (PCa) initiation, progression, and risk.

HOXB13: A Hereditary Prostate Cancer Risk Gene

The HOXB13 G84E mutation stands as one of the most significant genetic risk factors identified for hereditary prostate cancer. This variant was initially discovered through linkage analysis in high-risk families and subsequent fine-mapping of the chromosome 17q21-22 region [70].

Table 1: Epidemiological Studies of the HOXB13 G84E Mutation in Prostate Cancer

Study Study Population Case Carrier Frequency Risk Estimate (OR/RR) Key Association
Ewing et al. [70] US (Population-based) 1.40% OR = 20.1 Early diagnosis, positive family history
Karlsson et al. [70] Sweden (Population-based) 4.30% - 4.60% RR = 3.4 General population risk
Breyer et al. [70] US (Family-based) 1.90% OR = 7.9 Positive family history
Stott-Miller et al. [70] US (Population-based) 1.30% RR = 3.3 Higher Gleason score, advanced disease

The G84E variant is located in a highly conserved MEIS protein-binding domain and is postulated to modulate the interaction between HOXB13 and the androgen receptor, thereby influencing transcriptional programming and increasing cancer risk [70]. While HOXB13 often acts as a tumor suppressor, its role is context-dependent, and other HOX genes are frequently overexpressed in prostate tumors.

Widespread HOX Gene Dysregulation in Prostate Tumors

Beyond HOXB13, transcriptomic analyses reveal that prostate tumors exhibit a profoundly different HOX gene expression landscape compared to normal prostate tissue. Multiple HOX genes are significantly upregulated in prostate cancer-derived cell lines (e.g., LNCaP, PC3, DU145) and primary tumors [71]. This includes genes from the HOXC cluster (e.g., HOXC4, HOXC6, HOXC8), as well as HOXB5, HOXB7, and HOXA10 [71] [72]. This widespread dysregulation suggests a collective pro-oncogenic role for many HOX genes in prostate cancer, promoting cell survival and proliferation.

Table 2: Select HOX Genes and Their Roles in Solid Tumors

HOX Gene Role in Cancer Proposed Mechanism Relevant Cancer Types
HOXB13 Tumor Suppressor Suppresses c-Myc via β-catenin/TCF4 signaling [69] Prostate, Colon, Lung Cancer
HOXA9 Oncogene Recruits CEBPα & MLL3/MLL4 complexes at enhancers [69] Leukemia, Pancreatic Cancer, NSCLC
HOXA10 Oncogene Suppresses FASN transcription, interacts with AR [69] Prostate Cancer, Testicular Cancer
HOXC6 Oncogene Enhances BCL2-mediated anti-apoptotic effects [69] Prostate Cancer, Other Solid Tumors
HOXA13 Oncogene Regulated by IGF-1; involved in long-range chromatin loops [73] [69] Colorectal, Gastric Cancer

Connecting to Fundamental Research: Vitamin A and HOX Gene Regulation

The study of anuran models (frogs and toads) has been instrumental in understanding the fundamental principles of HOX gene regulation. A classic experiment involves the treatment of Rana ornativentris tadpoles with vitamin A (retinoic acid), which induces a homeotic transformation—the formation of ectopic limbs in place of a regenerating tail [6].

At the molecular level, this dramatic phenotypic change is preceded by the downregulation of posterior Hox genes in the regenerating tail tissue. This shift in Hox expression creates a cellular environment permissive to limb development, ultimately leading to the upregulation of key limb patterning genes like pitx1 [6]. This demonstrates that Hox genes are key targets of retinoid signaling and that their manipulation can fundamentally alter tissue identity.

This foundational research directly informs cancer biology. Retinoic acid (RA) is a potent regulator of Hox gene expression during embryonic development [12]. In mammalian systems, RA signaling can coordinately upregulate anterior 3' Hox genes while downregulating posterior 5' Hox genes in certain cell types [74]. The mechanisms discovered in anurans—whereby RA reprograms Hox codes to alter cell fate—are analogous to the processes in cancer cells where Hox gene dysregulation drives oncogenic transformation and progression. The following diagram illustrates this conserved signaling pathway.

hox_ra_pathway VitaminA Vitamin A (Retinol) RetinoicAcid Retinoic Acid (RA) VitaminA->RetinoicAcid Metabolism RAR RAR/RXR Receptor RetinoicAcid->RAR Binds TargetGenes Anterior 3' HOX Genes (e.g., HOXB4) RAR->TargetGenes Transcriptional Activation CellFate Altered Cell Fate & Identity TargetGenes->CellFate Cancer Oncogenic Transformation (if dysregulated) CellFate->Cancer Chronic Dysregulation

Experimental Protocols for HOX Gene Research in Cancer

This section provides detailed methodologies for key experiments investigating HOX gene function in cancer biology.

Protocol 1: Inhibiting HOX/PBX Function with HXR9 Peptide

Application: Assessing the dependency of cancer cells on HOX protein function for survival and testing a potential therapeutic strategy [71].

Background: The HXR9 peptide is a competitive antagonist that disrupts the interaction between HOX proteins (paralogs 1-10) and their PBX co-factor. This global inhibition of HOX function triggers apoptosis in cancer cells that are reliant on HOX signaling.

Materials:

  • HXR9 Peptide: WYPWMKKHHRRRRRRRRR (Biosynthesis Inc.) [71]
  • CXR9 Control Peptide: WYPAMKKHHRRRRRRRRR (alanine substitution) [71]
  • Prostate cancer cell lines (e.g., LNCaP, PC3, DU145)
  • Normal prostate stromal cell line (e.g., WPMY-1)
  • Cell culture reagents and equipment
  • Caspase-3 activity assay kit
  • MTS cell viability assay kit

Procedure:

  • Cell Seeding: Seed prostate cancer cells and control normal cells in 96-well plates at a density of 5,000-10,000 cells per well and allow to adhere overnight.
  • Peptide Treatment: Prepare serial dilutions of HXR9 and the control CXR9 peptide in culture medium (typical working concentrations range from 10-100 µM). Replace the medium in the wells with the peptide-containing medium. Incubate for 24-72 hours.
  • Viability Assessment:
    • MTS Assay: Add MTS reagent according to the manufacturer's instructions. Incubate for 1-4 hours and measure the absorbance at 490 nm. Calculate the percentage of viable cells relative to the untreated control.
    • Caspase-3 Assay: After 2-6 hours of treatment with 60 µM HXR9, lyse cells and measure caspase-3 activity using a commercial kit. This confirms apoptosis induction.
  • Data Analysis: Plot dose-response curves for HXR9 and CXR9. Calculate the IC50 value for HXR9 in each cell line. Compare caspase-3 activity between HXR9-treated, CXR9-treated, and untreated cells to confirm the specific pro-apoptotic effect.
Protocol 2: Mapping HOX Gene Regulatory Networks via Chromatin Conformation Capture (Hi-C)

Application: Identifying long-range genomic interactions between non-coding risk loci and HOX gene promoters in prostate cancer [73].

Background: Genome-wide association studies (GWAS) have identified many cancer risk variants in non-coding regions. Hi-C technology allows for the genome-wide mapping of chromatin interactions to determine how these regulatory elements physically contact and control target genes like HOXA13.

Materials:

  • Normal or cancerous prostate cell lines (e.g., RWPE-1, LNCaP)
  • Formaldehyde
  • Restriction enzyme (e.g., HindIII)
  • Biotin-labeled nucleotides
  • Streptavidin beads
  • DNA ligase
  • Proteinase K
  • High-throughput sequencing platform

Procedure:

  • Cross-linking: Cross-link chromatin in intact cells using 1-3% formaldehyde to covalently link interacting DNA regions with their associated proteins.
  • Digestion and Labeling: Lyse cells and digest the cross-linked DNA with a restriction enzyme. Fill the resulting DNA overhangs with biotin-labeled nucleotides.
  • Ligation: Under dilute conditions, perform intra- and inter-molecular ligation. The biotin label is incorporated at the junction of ligated fragments.
  • Reverse Cross-linking and Purification: Reverse the cross-links by incubating with Proteinase K, and purify the DNA. Shear the DNA to a size of 300-500 bp.
  • Pull-down and Sequencing: Pull down the biotin-labeled chimeric fragments using streptavidin beads. Construct a sequencing library and perform paired-end sequencing on a high-throughput platform.
  • Data Analysis: Process the sequenced reads using a standard Hi-C pipeline (e.g., HiC-Pro). Map paired-end reads to the reference genome, filter valid interaction pairs, and construct a genome-wide interaction matrix. Identify statistically significant loops, particularly between the risk region (e.g., 7p15.2) and the HOXA cluster [73].

The workflow for this integrated functional genomics approach is outlined below.

hic_workflow GWAS GWAS identifies non-coding risk SNP HiC Hi-C Experiment GWAS->HiC Loop Identify chromatin loop to HOX gene promoter HiC->Loop CRISPR CRISPR deletion of risk region Loop->CRISPR RNAseq RNA-seq to detect transcriptional changes CRISPR->RNAseq Mechanism Establish mechanistic link between SNP and HOX gene RNAseq->Mechanism

The Scientist's Toolkit: Essential Research Reagents

Table 3: Key Reagent Solutions for HOX Gene Cancer Research

Reagent / Tool Function / Application Key Example / Specification
HXR9 Peptide [71] Global inhibition of HOX/PBX dimers; induces apoptosis in HOX-dependent cancer cells. 18 amino acids, D-isomer bonds, polyarginine tag for cell penetration.
siRNA/shRNA Libraries Knockdown of individual HOX genes to study specific functions (despite redundancy challenges). Targeted siRNAs for genes like HOXB13, HOXC6.
HOX Gene Expression Vectors Forced overexpression to study oncogenic potential and transcriptomic impact. e.g., HOXA13 expression vector [73].
Retinoic Acid (RA) [74] Physiological and pharmacological modulator of HOX gene expression patterns. Used in vitro to model HOX gene regulation.
CRISPR/Cas9 System [73] Deletion of non-coding regulatory elements or knockout of HOX gene coding sequences. Used to delete a 4kb PCa risk region at 7p15.2.
Antibodies (IHC/ChIP) Protein localization and quantification; mapping transcription factor binding sites. Anti-HOXB13, anti-PBX, anti-H3K27ac.

HOX gene dysregulation is a critical factor in the pathogenesis of prostate and other cancers. The discovery of hereditary risk mutations like HOXB13 G84E and the widespread overexpression of multiple HOX genes in tumors underscore their clinical significance. Research in anuran models provides a foundational understanding of how external signals like vitamin A can reprogram HOX gene expression to alter cell fate—a mechanism that, when dysregulated, contributes to oncogenesis.

Experimental approaches such as global HOX/PBX inhibition with HXR9 and mapping of 3D chromatin architecture with Hi-C offer powerful tools to decipher HOX-driven oncogenic networks. Moving forward, the challenge lies in translating this knowledge into targeted therapies. Given the functional redundancy of HOX genes, strategies that target shared co-factors (like PBX) or downstream effector pathways may prove more effective than targeting individual HOX genes. Integrating the fundamental principles of HOX biology from developmental models will continue to be invaluable in the ongoing effort to combat cancer.

Retinoic acid (RA), a metabolite of Vitamin A, is a critical signaling molecule that regulates numerous aspects of embryonic development. It functions as a ligand for nuclear retinoic acid receptors (RARs), which form heterodimers with retinoid X receptors (RXRs) to control the transcription of target genes, including the Hox family of transcription factors that orchestrate anteroposterior patterning [11] [75]. The precise spatiotemporal regulation of RA bioavailability is paramount; deviations in either direction—excess or deficiency—are profoundly teratogenic. This application note explores the clinical insights gained from human syndromes and experimental models where disrupted RA signaling leads to microcephaly and other congenital defects. Furthermore, it provides detailed methodological frameworks for investigating these phenomena, with particular emphasis on their relevance to Hox gene manipulation studies in anuran models.

A growing body of evidence links reduced RA signaling to a spectrum of human developmental disorders. Syndromes such as DiGeorge, Smith-Magenis, Matthew-Wood, and Fetal Alcohol Syndrome (FAS) all exhibit microcephaly and have been attributed to compromised RA signaling [41]. The causative link suggests a previously underappreciated requirement for RA during early head formation. This connection is further strengthened by the observation that Vitamin A Deficiency (VAD) in humans presents with severe ocular manifestations, growth retardation, and an increased susceptibility to infections, including urinary tract infections, due to epithelial keratinization and immune dysfunction [76]. These clinical findings provide a compelling rationale for intensive research into the molecular and cellular mechanisms governed by RA during embryogenesis.

Table 1: Craniofacial Defects in Mouse Embryos Following Prenatal Retinoic Acid Exposure (ATRA) at E9.5

Measurement Parameter Control Embryos ATRA-100 Treated Embryos Change Statistical Significance (p-value)
Meckel's Cartilage Length (Left) Baseline 16.5% smaller Reduction p = 0.0149
Meckel's Cartilage Length (Right) Baseline 13.5% smaller Reduction p = 0.01
Depth of the Palate Baseline 14% smaller Reduction p = 0.0101
Embryonic Resorption Rate 0% 48% Increase Not provided

Table 2: Spectrum of Neural Tube Defects (NTDs) in a Han Chinese Cohort (355 Cases)

Neural Tube Defect Phenotype Number of Individuals
Spina Bifida Aperta 91
Encephalocele 57
Anencephaly with Spina Bifida Aperta 55
Spina Bifida Cystica 42
Spina Bifida Occulta 26
Anencephaly 17
Craniorachischisis 19

Table 3: Key Genetic Findings in RA Pathway and Neural Tube Defects

Gene Function Association with Defects
CYP26B1 RA degradation enzyme Majority of missense variants found in NTD cases; functional assays show reduced RA degradation efficiency [77].
ALDH1A2 (RALDH2) Key RA synthesizing enzyme Mutants die early with anterior head malformations; associated with reduced RA signaling and microcephaly [41] [78] [75].
ALDH1A3 (RALDH3) RA synthesizing enzyme Gene-specific knockdowns identify it as a key enzyme for head formation [41].
HOX Genes (PG1-PG4) Transcription factors for patterning Form a "hindbrain code" regulated by RA; disruption causes severe hindbrain and craniofacial defects [11].

Experimental Protocols: Modeling RA Disruption and Its Consequences

Protocol: Inhibition of RA Signaling inXenopus laevisEmbryos

This protocol details the partial inhibition of RA biosynthesis to study its effects on gastrulation and head formation, recapitulating the teratogenic effects observed in Fetal Alcohol Syndrome [41] [78].

Materials:

  • Xenopus laevis embryos
  • 4-Diethylaminobenzaldehyde (DEAB) or 3,7-Dimethyl-2,6-octadienal (citral)
  • 0.1% Modified Barth's Solution and Hepes (MBSH)
  • Dimethyl sulfoxide (DMSO) or Ethanol (EtOH) for solvent controls

Procedure:

  • Embryo Preparation: Obtain embryos via in vitro fertilization and incubate in 0.1% MBSH.
  • Treatment Initiation: At the midblastula transition (MBT, stage 8.5), expose embryos to solutions of either DEAB (dissolved in DMSO) or citral (diluted in EtOH) in 0.1% MBSH. A typical working concentration for DEAB is 100 µM.
  • Control Setup: Include parallel control batches treated with equivalent concentrations of the solvents (DMSO or EtOH) alone.
  • Incubation and Analysis: Maintain treatment until the desired developmental stage (e.g., gastrula or neurula stages) is reached. Subsequently, analyze embryos for:
    • Phenotypic Analysis: Morphological assessment of head size and craniofacial structures.
    • Molecular Analysis: In situ hybridization for markers of the prechordal mesoderm (e.g., goosecoid) or quantitative PCR (qPCR) for RA target genes.
    • Histological Analysis: Examination of fibronectin deposition along Brachet's cleft via immunostaining, as RA reduction impairs this process, disrupting cell migration [78].

Protocol: Genetic Induction of Transient RA Deficiency in Mouse

This protocol describes the use of a genetically engineered mouse model to induce a transient, spatially restricted RA deficiency during gastrulation, phenocopying prenatal alcohol exposure [79].

Materials:

  • Gsc+/Cyp26A1 knock-in mouse model.
  • Standard equipment and reagents for mouse embryo dissection, histology, and RNA analysis.

Procedure:

  • Model Generation: The model is generated by targeted homologous recombination, inserting a Cyp26A1 cDNA cassette into exon 2 of the Goosecoid (Gsc) gene locus. This places the RA-degrading enzyme Cyp26A1 under the control of the endogenous Gsc promoter.
  • Embryo Collection: Time matings of heterozygous (Gsc+/Cyp26A1) mice. Collect embryos at specific time points for analysis (e.g., E8.5, E10.5, E18.5).
  • Phenotypic Screening:
    • At E8.5: Analyze embryos for alterations in the RA response domain and delayed expression of RA target genes such as HoxA1 and HoxB1 via in situ hybridization or immunofluorescence.
    • At E10.5: Assess aberrant neurofilament expression during cranial nerve formation.
    • At E18.5: Conduct detailed morphological analysis for sentinel craniofacial phenotypes characteristic of FASD.
  • Functional Confirmation: Validate that the observed phenotypes are due to RA deficiency by attempting rescue through maternal administration of low-dose RA.

Visualizing RA Signaling and Experimental Approaches

G cluster_0 Experimental Inhibition VitaminA Vitamin A (Retinol) RAL Retinaldehyde VitaminA->RAL Oxidation (RDH10) RA Retinoic Acid (RA) RAL->RA Oxidation (ALDH1A2/3) RAR_RXR RAR/RXR Heterodimer RA->RAR_RXR Binds TargetGenes Target Gene Expression (Hox genes, Cyp26s) RAR_RXR->TargetGenes Transcriptional Activation/Repression TargetGenes->RA Negative Feedback (CYP26 degradation) DEAB DEAB/Citral DEAB->RAL Inhibits Ethanol Ethanol Exposure Ethanol->RAL Competes Genetic Genetic CYP26A1 Overexpression Genetic->RA Degrades

Diagram 1: Retinoic Acid Signaling Pathway and Experimental Disruption. The core RA biosynthesis and signaling pathway is shown in solid colors. Key experimental methods for disrupting RA signaling to model disease are indicated by dashed red lines.

G cluster_0 Model System Choice cluster_1 Phenotypic Analysis Tiers Start Define Research Objective M1 Select Model System Start->M1 M2 Implement RA Disruption M1->M2 Frog Anuran (Xenopus) - Chemical inhibition - Gene knockdown M1->Frog M3 Analyze Phenotypes M2->M3 M4 Investigate Mechanism M3->M4 P1 Macroscopic - Morphology - Survival M3->P1 End Data Synthesis & Conclusion M4->End Mouse Mouse (Mus) - Genetic model - Embryo culture Zebrafish Zebrafish (Danio) - Chemical genetics - Transgenics P2 Molecular - Hox gene expression - Pathway analysis P3 Cellular - Cell migration - Extracellular matrix

Diagram 2: Experimental Workflow for RA Disruption Studies. A generalized workflow for designing experiments to investigate the developmental roles of RA, highlighting key decision points in model selection and multi-tiered phenotypic analysis.

The Scientist's Toolkit: Research Reagent Solutions

Table 4: Essential Research Reagents for RA Signaling Studies

Reagent / Tool Category Function in Research Example Application
DEAB (4-Diethylaminobenzaldehyde) Small Molecule Inhibitor Inhibits retinaldehyde dehydrogenases (ALDH1A), blocking RA biosynthesis [78]. Induce RA deficiency in Xenopus embryos from mid-blastula transition to model FASD [78].
Citral Small Molecule Inhibitor Acts as a competitive inhibitor of retinaldehyde dehydrogenases, reducing RA production. Used in Xenopus and zebrafish studies to probe the role of RA in gastrulation and Hox gene regulation.
Gsc-Cyp26A1 Mouse Genetic Model Spatially and temporally controlled RA degradation; Cyp26A1 expressed from Goosecoid promoter [79]. Models transient RA deficiency during gastrulation to phenocopy prenatal alcohol exposure defects [79].
CYP26B1 Mutants Genetic Variants Human or engineered variants with reduced RA degradation efficiency, leading to local RA excess [77]. Study the etiology of human neural tube defects and the functional consequences of rare genetic variants [77].
Hox Reporter Lines Reporter Assay Transgenic animals or cells with fluorescent reporters under the control of Hox gene promoters or RAREs. Visualize and quantify the response of Hox genes to RA signaling perturbations in live embryos.
Anti-Fibronectin Antibodies Immunological Tool Detect deposition of fibronectin matrix, a substrate for cell migration dependent on RA signaling [78]. Assess the cellular mechanisms underlying RA-mediated gastrulation defects, such as impaired cell migration [78].

The study of human syndromes and experimental models has unequivocally established that both excess and deficient RA signaling are potent drivers of microcephaly and congenital malformations. The molecular etiology of these defects is deeply rooted in the disruption of fundamental developmental processes, including the regulation of Hox gene expression, neural crest cell migration, and morphogenetic movements during gastrulation. The experimental protocols and tools detailed herein provide a robust framework for researchers, particularly those using anuran models, to dissect the precise mechanisms linking RA to these outcomes. Future research should leverage these models to identify and test potential therapeutic interventions, such as targeted nutritional supplementation or pharmacological agents, that could prevent or mitigate the devastating consequences of disrupted RA signaling in human development.

The manipulation of Hox gene expression using vitamin A (retinoic acid) in anuran models represents a cornerstone of developmental biology research, providing critical insights into the genetic regulation of axial patterning and limb development. Recent U.S. regulatory shifts are accelerating the transition from traditional animal models to human-relevant approaches, making anuran research increasingly valuable as a complementary platform that bridges fundamental discovery and clinical application. The FDA Modernization Act 2.0 (2022) eliminated the statutory mandate for animal testing, explicitly authorizing human-relevant models as valid evidence for drug development [80]. Concurrently, the NIH has implemented funding priorities that favor research incorporating human-based technologies, effectively barring proposals that rely exclusively on animal data [80] [81]. This evolving landscape positions anuran models as a strategically refined animal system that provides high-throughput developmental insights while aligning with the "Reduce" and "Refine" principles of the 3Rs framework, creating a crucial bridge to human biomedical applications.

Vitamin A-Induced Hox Gene Manipulation: Core Findings and Quantitative Data

Key Molecular Findings from Anuran Research

Research using Rana ornativentris tadpoles has demonstrated that vitamin A administration induces a remarkable homeotic transformation, where tail regeneration following amputation is redirected to form ectopic limbs instead of tail structures [6]. Molecular analyses of this phenomenon reveal that vitamin A (retinoic acid) triggers downregulation of posterior Hox genes prior to visible ectopic limb bud formation. This Hox expression change precedes the upregulation of pitx1, a critical gene expressed in the earliest hind limb bud, suggesting that Hox genes operate upstream of hind limb specification genes in the regulatory hierarchy [6]. These findings from anuran models illuminate fundamental mechanisms of positional identity and developmental plasticity that have direct relevance for understanding human congenital disorders and regenerative medicine approaches.

HOX Gene Expression in Human Development and Disease

Table 1: HOX Gene Functions in Human Development and Pathologies

HOX Gene/Cluster Developmental Role Pathological Association Clinical Relevance
Posterior HOX genes Axial patterning, posterior identity Downregulated in vitamin A-induced anuran limb transformation [6] Pattern formation, positional identity
HOXA cluster Anteroposterior axis specification Overexpressed in glioblastoma (GBM) [82] Prognostic biomarker; therapeutic target
HOXB cluster Neural development, patterning Dysregulated in glioma tissues [82] Contributor to tumorigenesis
HOXC cluster Spinal cord development, limb patterning Upregulated across multiple solid tumors [82] Cancer progression marker
HOXD cluster Limb development, axial patterning Promotes tumor proliferation, migration, invasion [82] Diagnostic and therapeutic candidate

Recent single-cell transcriptomic analyses of the developing human spine reveal that HOX genes maintain a precise rostrocaudal expression code that patterns anatomical structures along the anterior-posterior axis [2]. Neural crest derivatives unexpectedly retain the anatomical HOX code of their origin while also adopting the code of their destination, a finding confirmed across multiple organs including the fetal limb, gut, and adrenal gland [2]. In pathological contexts, HOX genes are virtually absent in healthy adult brains but are detectably dysregulated in malignant brain tumors, particularly glioblastomas (GBM), where they contribute to tumor progression, therapeutic resistance, and poor survival outcomes [82].

Table 2: HOX Gene Correlations in Human Cancers

Cancer Type HOX Gene Expression Correlated Pathways/Processes Clinical Impact
Prostate Cancer HOXA10, HOXC4, HOXC6, HOXC9, HOXD8 negatively correlate with Fos, DUSP1, ATF3 [83] DNA repair, metabolism, reduced cell adhesion Apoptosis inhibition; pro-oncogenic
Glioblastoma HOXA family upregulated [82] Tumor progression, therapy resistance Negative prognostic marker
Multiple Solid Tumors HOXC family upregulated [82] Tumor proliferation, invasion Potential therapeutic target

Experimental Protocols: Hox Gene Manipulation in Anuran Systems

Vitamin A-Induced Homeotic Transformation Protocol

Objective: To induce ectopic limb formation in regenerating anuran tadpoles through vitamin A-mediated Hox gene manipulation.

Materials and Reagents:

  • Rana ornativentris tadpoles (or other anuran species showing similar responses)
  • All-trans retinoic acid (Vitamin A)
  • Dimethyl sulfoxide (DMSO) for solvent control
  • Tank water or appropriate amphibian medium
  • Surgical instruments for tail amputation (fine scissors, forceps)
  • RNA extraction kit
  • Quantitative PCR system with primers for Hox genes and limb markers (e.g., pitx1)

Procedure:

  • Animal Preparation: House tadpoles in appropriate aquatic conditions with controlled temperature and light cycles.
  • Tail Amputation: Anesthetize tadpoles if necessary. Using sterile fine scissors, amputate tails at a consistent position posterior to the hindlimbs.
  • Vitamin A Treatment: Prepare all-trans retinoic acid solutions in DMSO at concentrations ranging from 1-100 µM. Administer to tadpoles immediately following amputation via:
    • Direct addition to tank water
    • Microinjection at the regeneration site
    • Include control groups receiving DMSO only
  • Observation and Sampling: Monitor regeneration daily for 7-14 days. Document ectopic limb formation morphologically.
  • Molecular Analysis: At predetermined timepoints (e.g., 24h, 48h, 72h post-amputation):
    • Sacrifice subsets of tadpoles and collect regeneration tissue
    • Extract total RNA and synthesize cDNA
    • Perform qPCR analysis for posterior Hox genes and limb development markers (pitx1)
  • Data Interpretation: Compare Hox gene expression patterns between vitamin A-treated and control groups. Successful experiments will show downregulation of posterior Hox genes preceding pitx1 upregulation and ectopic limb formation [6].

Hox Gene Expression Analysis Protocol

Objective: To quantify spatial and temporal expression patterns of Hox genes during development and regeneration.

Materials:

  • Tissue samples from various axial positions or timepoints
  • RNA extraction and purification kits
  • cDNA synthesis kit
  • Quantitative PCR system with species-specific Hox gene primers
  • Alternatively: RNA in situ hybridization materials for spatial localization

Procedure:

  • Tissue Collection: Dissect tissues of interest based on anatomical landmarks, preserving accurate positional information.
  • RNA Extraction: Homogenize tissues and extract total RNA following manufacturer protocols. Assess RNA quality and quantity.
  • cDNA Synthesis: Convert equal amounts of RNA from each sample to cDNA using reverse transcriptase.
  • qPCR Analysis:
    • Design and validate primers for target Hox genes and reference genes
    • Set up qPCR reactions with appropriate controls (no template, reverse transcription negative)
    • Run amplification with standardized cycling conditions
    • Analyze using comparative Ct method (2^-ΔΔCt) normalized to reference genes
  • Data Interpretation: Compare expression levels across anatomical positions or timepoints, noting collinear expression patterns where 3' Hox genes are expressed more anteriorly and earlier than 5' Hox genes [2] [11].

Signaling Pathways and Molecular Mechanisms

Retinoic Acid-Hox Gene Signaling Pathway

The following diagram illustrates the molecular pathway through which vitamin A (retinoic acid) modulates Hox gene expression to induce homeotic transformations, based on findings from anuran models and vertebrate developmental studies [6] [11]:

G VitaminA Vitamin A (Retinoic Acid) RAR Retinoic Acid Receptor (RAR/RXR) VitaminA->RAR Binds HoxGenes Posterior Hox Genes RAR->HoxGenes Downregulates Pitx1 pitx1 Upregulation RAR->Pitx1 Induces (via Hox suppression) HoxGenes->Pitx1 Represses LimbTransformation Ectopic Limb Formation Pitx1->LimbTransformation

Experimental Workflow for Hox Gene Manipulation Studies

The following diagram outlines the integrated experimental workflow for investigating Hox gene manipulation in anuran models and translating findings to human biomedical applications:

G AnuranModel Anuran Model System (Rana ornativentris) VitaminATreatment Vitamin A Treatment AnuranModel->VitaminATreatment HoxAnalysis Hox Gene Expression Analysis VitaminATreatment->HoxAnalysis MolecularMech Molecular Mechanism Elucidation HoxAnalysis->MolecularMech HumanRelevance Human Disease Relevance MolecularMech->HumanRelevance TherapeuticDev Therapeutic Development HumanRelevance->TherapeuticDev

The Scientist's Toolkit: Essential Research Reagents and Solutions

Table 3: Key Research Reagents for Hox Gene Manipulation Studies

Reagent/Category Specific Examples Function/Application Considerations
Vitamin A Compounds All-trans retinoic acid Induces Hox gene expression changes; axial patterning Concentration-dependent effects; temporal critical period
Animal Models Rana ornativentris, Xenopus laevis Regeneration studies; Hox manipulation Species-specific responses to vitamin A
Molecular Analysis qPCR primers for Hox genes, pitx1 Gene expression quantification Verify species-specific specificity
Spatial Transcriptomics Single-cell RNAseq, Visium, in-situ sequencing HOX code mapping across tissues High-resolution positional information [2]
Human Disease Models Glioblastoma cell lines, organoids HOX dysregulation studies Clinical relevance for therapeutic testing
New Approach Methodologies Organ-on-a-chip, microphysiological systems Human-relevant toxicity and efficacy testing FDA ISTAND program qualification [80] [81]

The experimental protocols outlined herein for manipulating Hox gene expression in anuran models provide a robust framework for investigating the fundamental mechanisms of developmental patterning and their relevance to human biology and disease. The conserved nature of Hox gene function across vertebrates enables direct translation of findings from anuran systems to human biomedical contexts, particularly in understanding congenital disorders affecting axial patterning and HOX gene dysregulation in cancers such as glioblastoma and prostate cancer [82] [83]. As regulatory agencies increasingly emphasize human-relevant models, anuran systems offer a strategically valuable platform that aligns with the principles of reduction and refinement in animal research while providing high-quality developmental insights that can inform therapeutic development for HOX-related pathologies.

Conclusion

The manipulation of Hox gene expression using Vitamin A in anuran models provides a powerful and conserved paradigm for understanding how master regulatory genes control cell fate and morphology. The key takeaway is that retinoic acid acts as a critical upstream signal, capable of reprogramming entire tissue fields by shifting Hox gene expression profiles, as definitively shown in the tail-to-limb transformation. This foundational knowledge not only deepens our grasp of embryonic development and evolution but also opens tangible avenues for regenerative medicine, offering potential strategies for triggering complex tissue regeneration. Future research must focus on achieving finer spatiotemporal control over this signaling pathway and translating these mechanisms into mammalian systems, with direct implications for treating congenital birth defects, driving innovations in organoid engineering, and developing novel therapies for HOX-driven cancers.

References