Recombinant Danio rerio Homeobox protein Hox-A13b (hoxa13b)

What is the genomic organization of hoxa13b in Danio rerio?

Hoxa13b is one of several Hox13 paralogs in zebrafish (Danio rerio), located within the HoxA cluster. Genomic annotation studies have confirmed that zebrafish have a total of 49 hox genes distributed across multiple clusters due to a whole genome duplication event that occurred in teleost evolution. The hoxa13b gene contains one intron and is expressed from the endogenous hoxa13b locus in the tail region. Research methodologies for confirming genomic organization typically include genome database searches (e.g., TBLASTN searches in Ensembl), RT-PCR validation, and comparative genomic analyses to verify gene location and structure . For targeted genetic modifications, researchers have successfully inserted FLAG-2A-GFP sequences at the C terminus of the hoxa13b coding region to create knock-in lines that maintain the native expression pattern .

How is hoxa13b expression regulated during zebrafish development?

Hoxa13b expression is tightly regulated during zebrafish development with specific spatiotemporal patterns. Expression analysis using in situ hybridization demonstrates that hoxa13b is activated early during somitogenesis and is primarily expressed in the tailbud region of mid-somitogenesis embryos . Quantitative studies using qPCR have shown that hoxa13b, together with hoxd13a, are the most abundantly expressed Hox13 genes in the tailbud, although the absolute expression level remains relatively low compared to other developmental regulators .

The regulation involves both genetic and epigenetic mechanisms. CUT&RUN analyses have revealed that Hoxa13b can bind to its own regulatory regions, suggesting potential autoregulation. To study this regulation experimentally, researchers have developed transgenic lines such as the heat-shock inducible HS:hoxa13b-FLAG-GFP and the endogenous knock-in KI:hoxa13b-FLAG-GFP, allowing for controlled expression and visualization through the co-expressed GFP protein .

What is known about the evolutionary conservation of hoxa13b among teleosts?

Evolutionary analysis indicates that hoxa13b arose from a whole genome duplication event that occurred shortly before the most recent common ancestor of teleosts. This duplication resulted in the retention of many Hox cluster genes in teleosts, contributing to their morphological diversity. Comparative studies have shown that while some duplicated Hox genes demonstrate rapid divergence and neofunctionalization, others maintain conserved functions .

In zebrafish and related cypriniform fishes, molecular evolution studies of the duplicated HoxA13 paralogs (hoxa13a and hoxa13b) have revealed asymmetric rates of divergence. Research methodologies for studying evolutionary conservation typically include sequence alignment, phylogenetic analysis, and functional tests of orthologous genes across species. Data suggest that in early stages of zebrafish larval development, HoxA13b is the predominant paralog expressed, indicating potential subfunctionalization between the paralogs following duplication .

What are the most effective methods for creating hoxa13b mutants in zebrafish?

CRISPR/Cas9 genome editing has proven to be the most effective method for generating targeted hoxa13b mutations in zebrafish. This methodology has been successfully implemented in multiple studies:

• For knockout mutations, researchers have created lines with small insertions or deletions that disrupt the coding sequence. For example, a hoxa13b Δ16 mutation has been created that results in a complete loss of Hoxa13b function .

• For knock-in approaches, researchers have used the CRISPR/Cas9 system with specific gRNAs targeting the 5′ and 3′ UTRs of the endogenous hoxa13b locus, along with a donor plasmid containing the modified gene with an epitope tag .

The efficiency of creating mutations depends on several factors:

It's important to verify that mutant alleles do not trigger genetic compensation through nonsense-mediated decay, which has been observed for some genes. qPCR analysis of hoxa13b Δ16 embryos has shown that the mutant transcript is expressed at similar levels to wild-type, with no compensatory upregulation of other Hox13 genes .

How can binding sites of Hoxa13b be identified genome-wide in zebrafish embryos?

Identifying genome-wide binding sites for Hoxa13b has been challenging due to the paucity of high-quality antibodies and the limited number of cells in zebrafish tailbud tissue. Researchers have overcome these limitations through innovative approaches:

• Creation of transgenic lines expressing epitope-tagged Hoxa13b (FLAG-tagged) under either inducible (HS:hoxa13b-FLAG-GFP) or endogenous (KI:hoxa13b-FLAG-GFP) control .

• Adaptation of CUT&RUN (Cleavage Under Targets and Release Using Nuclease) technology for zebrafish embryos, which permits identification of in vivo binding sites using limited cell numbers .

The CUT&RUN methodology involves:

• Isolation of nuclei from transgenic embryos expressing FLAG-tagged Hoxa13b

• Binding of an anti-FLAG antibody to the epitope-tagged protein

• Addition of protein A-MNase to cleave DNA around binding sites

• Isolation and sequencing of the released DNA fragments

Using this approach, researchers identified 1871 Hoxa13b-occupied peaks from the heat-shock line, with 689 overlapping peaks also found in the knock-in line. Analysis revealed that only 12.6% of binding sites were located within 3 kb of transcription start sites, with over 60% in distal intergenic regions. Motif analysis showed that the Hox binding motif was the most common sequence in these regions .

What expression systems are optimal for producing recombinant Hoxa13b protein?

For functional studies of Hoxa13b protein, several expression systems have been effectively employed:

• In vivo zebrafish expression systems:

  • Heat-shock inducible transgenic lines allow for temporal control of expression

  • Endogenous knock-in lines maintain normal spatiotemporal regulation

  • Both approaches can incorporate epitope tags for protein detection and purification

• Bacterial expression systems:

  • E. coli BL21(DE3) with pET vector systems for expressing the DNA-binding homeodomain

  • Fusion tags (His6, GST, MBP) improve solubility and facilitate purification

  • Expression at lower temperatures (16-18°C) improves proper folding

• Mammalian and insect cell systems:

  • HEK293T cells for mammalian expression with post-translational modifications

  • Sf9 insect cells with baculovirus for higher yields of full-length protein

Key considerations for obtaining functional recombinant Hoxa13b include:

• Codon optimization for the expression system

• Inclusion of nuclear localization signals for proper subcellular localization

• Addition of epitope tags that don't interfere with DNA binding

• Protein purification under conditions that maintain DNA-binding activity

For in vitro binding studies, the properly folded homeodomain is essential for recognizing specific DNA sequences. Recombinant Hoxa13b has been shown to bind both "traditional" Hox motifs (with a T at a specific position) and posterior Hox-specific motifs (with a C at that position) .

How does hoxa13b contribute to axis elongation in zebrafish embryos?

Hoxa13b plays a critical role in posterior development and axis elongation in zebrafish embryos through genetic interactions with other key developmental regulators. Studies investigating the function of hoxa13b through loss-of-function approaches have revealed:

The mechanistic basis involves:

• Regulation of neuromesodermal progenitors (NMps) that contribute to both neural tube and paraxial mesoderm

• Maintenance of proper tbxta expression in the tailbud

• Control of the balance between mesodermal and neural fates during axis elongation

Temperature-sensitive experiments have demonstrated that hoxa13b mutants are hypersensitive to Tbxta reduction, with embryos placed at semi-permissive temperatures (18.5°C) showing a sharp reduction in tbxta expression in the NMps .

What is the role of hoxa13b in caudal fin development and evolution?

Hoxa13b contributes significantly to caudal fin development and evolution in zebrafish and related teleosts. Research has demonstrated:

• Hox13 genes, including hoxa13b, are expressed in distinct domains during caudal fin development, with expression patterns that correlate with specific morphological boundaries .

• While hoxa13b is more strongly expressed in the pectoral fin buds than in the tail, it still plays a role in caudal fin patterning through genetic interactions with other Hox13 paralogs .

• Loss-of-function studies of hoxa13b in combination with other Hox13 genes revealed phenotypes affecting caudal fin ray number, joint formation, and segment length .

Experimental approaches for studying hoxa13b's role in fin development include:

• In situ hybridization to map expression domains

• CRISPR/Cas9-mediated gene knockout

• Cell ablation experiments using the NTR/MTZ mechanism to partially remove hoxa13a-expressing cells

• Analysis of joint formation during both development and regeneration

Particularly noteworthy is the finding that partial ablation of hoxa13a-expressing cells results in shorter bone segments following regeneration, suggesting that Hox13 genes are involved in regulating segment length . Additionally, triple mutants with mutations in hoxa13a, hoxa13b, and hoxd13a show fin-specific defects in joint formation and patterning .

How does Hoxa13b protein function as a transcription factor?

Hoxa13b functions as a transcription factor by binding to specific DNA sequences and regulating the expression of target genes. Detailed molecular analyses have revealed:

• Hoxa13b binds to DNA through its homeodomain, recognizing both "traditional" Hox motifs and posterior Hox-specific motifs .

• CUT&RUN analysis identified 1871 Hoxa13b-occupied peaks in the zebrafish genome, with over 60% located in distal intergenic regions, suggesting that Hoxa13b primarily regulates gene expression through enhancer elements rather than proximal promoters .

• DNA footprinting analysis showed that Hoxa13b forms a 19-bp footprint on DNA, protecting this region from nuclease digestion .

• Hoxa13b can potentially regulate at least 576 candidate target genes in the zebrafish tailbud during mid-somitogenesis stages .

The transcriptional activity of Hoxa13b involves:

• Binding to enhancer elements often located far from the genes they regulate

• Potential cooperation with other transcription factors (co-factors)

• Recruitment of chromatin modifiers to activate or repress target genes

• Potential autoregulatory mechanisms, as Hoxa13b binding sites were found upstream of the hoxa13b locus itself

Experimental approaches for studying Hoxa13b transcription factor activity include:

• CUT&RUN for genome-wide binding site identification

• Epigenetic marker analysis to identify sites associated with transcriptional activation

• Reporter gene assays to validate enhancer function

• Gene expression analysis following hoxa13b manipulation to identify regulated genes

Why does single hoxa13b mutation show limited phenotypes despite its evolutionary conservation?

The limited phenotypes observed in single hoxa13b mutants despite the gene's evolutionary conservation represent a fascinating paradox in developmental biology. Several factors contribute to this phenomenon:

• Functional redundancy with other Hox13 paralogs: Zebrafish possess multiple Hox13 genes (hoxa13a, hoxa13b, hoxb13a, hoxc13a, hoxc13b, and hoxd13a) that show overlapping expression patterns. Loss of hoxa13b function can be compensated by these other paralogs, particularly hoxd13a .

• Genetic interaction with developmental pathways: The phenotypic consequences of hoxa13b loss become apparent only under certain genetic or environmental conditions. For example, hoxa13b mutants show severe defects when tbxta function is also reduced .

• Condition-dependent phenotypes: While hoxa13b mutants appear normal under standard laboratory conditions, they exhibit phenotypes when raised at lower temperatures (21°C), indicating a temperature-sensitive requirement for Hoxa13b function .

• Quantitative rather than qualitative effects: Hoxa13b may contribute to developmental robustness rather than being absolutely required for specific structures. This is evidenced by the synergistic effects seen when multiple Hox13 genes are mutated .

This apparent contradiction can be experimentally addressed through:

• Creation of multiple mutant combinations to uncover redundant functions

• Stress tests (temperature shifts, developmental timing alterations) to reveal cryptic phenotypes

• RNA-seq analysis to identify compensatory transcriptional changes

• Evolutionary comparisons across species with different Hox gene complements

These findings highlight the importance of testing genetic function under various conditions rather than relying solely on standard laboratory environments, which may mask subtle or condition-dependent phenotypes.

How do the functions of hoxa13b differ from or overlap with its paralog hoxa13a?

The functional relationship between hoxa13b and its paralog hoxa13a in zebrafish represents a classic case of post-duplication divergence and subfunctionalization. Research has revealed both overlapping and distinct functions:

Overlapping functions:

• Both genes are expressed in posterior tissues during development .

• Both contribute to fin development and patterning .

• Both belong to the same paralog group and recognize similar DNA binding motifs .

Distinct functions:

• Expression patterns show differences, with hoxa13b more strongly expressed in the tailbud during mid-somitogenesis, while hoxa13a shows stronger expression in fins .

• Functional studies indicate hoxa13a has a more prominent role in fin joint formation and regeneration, while hoxa13b has stronger genetic interactions with tbxta in posterior body development .

• Evolutionary analysis shows asymmetric rates of sequence divergence between the paralogs, with evidence suggesting hoxa13a has experienced more rapid sequence evolution .

Experimental approaches to distinguish their functions include:

• Detailed expression pattern analysis using in situ hybridization

• Single and double mutant phenotypic analysis

• Paralog-specific rescue experiments

• CUT&RUN or ChIP-seq analysis to compare binding sites

The data indicates that while there is some functional redundancy, these paralogs have undergone subfunctionalization since their duplication, with each acquiring specialized roles in different developmental contexts. This highlights how gene duplication can contribute to evolutionary innovation through the partitioning of ancestral functions between duplicated genes .

What explains the contradictory findings on hoxa13b expression levels in different studies?

Several studies report varying levels of hoxa13b expression in zebrafish, leading to apparent contradictions in the literature. These discrepancies can be attributed to multiple factors:

• Technical differences in detection methods:

  • RNA-seq provides genome-wide quantification but may have lower sensitivity for low-abundance transcripts

  • qPCR offers higher sensitivity but is dependent on primer efficiency

  • In situ hybridization provides spatial information but is less quantitative

• Developmental timing differences:

  • Expression of hoxa13b is dynamically regulated during development

  • Peak expression occurs during mid-somitogenesis but precise timing varies

  • Some studies sample at specific stages while others use time windows

• Different reference points for comparison:

  • Some studies compare hoxa13b to other Hox13 paralogs

  • Others compare to housekeeping genes or global transcriptome

  • Relative vs. absolute quantification methods yield different perspectives

• Genetic background variations:

  • Different wild-type zebrafish strains (e.g., TU, AB) have slight genetic differences

  • Laboratory-specific genetic drift may affect expression levels

  • Transgenic reporter lines may not perfectly recapitulate endogenous expression

To reconcile these contradictions, researchers should:

• Clearly specify detection methods, developmental stages, and reference genes

• Use multiple complementary techniques (RNA-seq, qPCR, in situ hybridization)

• Perform direct comparisons under identical conditions

• Consider single-cell approaches to address cellular heterogeneity

One consistent finding across studies is that hoxa13b expression is relatively low compared to many other developmental regulators, yet it plays important roles through genetic interactions with other factors, highlighting the non-linear relationship between expression level and developmental significance .

How can we leverage hoxa13b to understand evolutionary changes in body plan?

Hoxa13b research provides a unique window into understanding evolutionary changes in vertebrate body plans, particularly in the context of the teleost-specific whole genome duplication. Several research approaches can leverage hoxa13b to gain insights into evolutionary developmental biology:

• Comparative genomics and phylogenetics:

  • Sequence analysis of hoxa13b across diverse fish lineages to identify selective pressures

  • Correlation of sequence changes with morphological innovations

  • Reconstruction of ancestral sequences to test evolutionary hypotheses

• Cis-regulatory element analysis:

  • Identification of conserved and divergent enhancer elements controlling hoxa13b expression

  • Cross-species enhancer swap experiments to test functional evolution

  • ATAC-seq and CUT&RUN studies to map regulatory landscape changes

• Functional testing across species:

  • CRISPR/Cas9 mutation of hoxa13b in different fish species

  • Cross-species rescue experiments to test functional equivalence

  • Generation of chimeric Hoxa13b proteins to map functionally important domains

Particularly promising is the investigation of hoxa13b's role in caudal fin evolution. Research has shown that hox13 genes regulate the number and identity of posterior vertebrae and the development of the homocercal (symmetrical) caudal fin, a key innovation in teleost evolution . Mutations in hoxb13a and hoxc13a result in extra vertebrae and altered fin ray patterning, suggesting that changes in Hox13 gene function contributed to the evolution of the distinctive teleost tail structure .

This provides an opportunity to understand how changes in Hox gene function and regulation contributed to the remarkable diversity of body plans observed in teleost fishes, which represent nearly half of all vertebrate species.

What are the latest methodological advances for studying hoxa13b protein-protein interactions?

Recent methodological advances have significantly enhanced our ability to study Hoxa13b protein-protein interactions, offering new insights into its molecular function:

• BioID and TurboID proximity labeling:

  • Fusion of Hoxa13b with a biotin ligase that biotinylates nearby proteins

  • Allows identification of proximal proteins in living cells

  • Captures transient interactions often missed by co-immunoprecipitation

• Single-molecule imaging:

  • Visualization of individual Hoxa13b molecules in living cells

  • Tracking of binding dynamics and residence times on chromatin

  • Reveals heterogeneity in binding behavior

• Protein complementation assays:

  • Split fluorescent proteins or luciferase fused to Hoxa13b and potential partners

  • Signal generated only when proteins interact

  • Amenable to high-throughput screening

• Mass spectrometry-based approaches:

  • Crosslinking mass spectrometry (XL-MS) to capture direct protein contacts

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map interaction surfaces

  • Thermal proteome profiling to detect stabilization upon binding

• Cryo-electron microscopy:

  • Structural determination of Hoxa13b complexes with transcriptional co-factors

  • Visualization of Hoxa13b as part of larger regulatory complexes

  • Resolution of conformational changes upon DNA binding

These methods have begun to reveal that Hoxa13b likely functions as part of multi-protein complexes that may include other Hox proteins, general transcription factors, and chromatin modifiers. Understanding these interactions is crucial for deciphering how Hoxa13b achieves target specificity and regulatory precision despite recognizing relatively simple DNA motifs .

The identification of Hoxa13b-interacting proteins also provides potential targets for manipulating Hoxa13b function in experimental contexts, offering new approaches to study its roles in development and disease.

What are the implications of hoxa13b research for understanding human HOX gene disorders?

Research on zebrafish hoxa13b has significant implications for understanding human HOX gene disorders, particularly those involving HOXA13:

• Hand-foot-genital syndrome (HFGS):

  • Caused by mutations in human HOXA13

  • Characterized by limb and urogenital abnormalities

  • Zebrafish hoxa13b models can provide insights into the developmental mechanisms

• Guttmacher syndrome:

  • Related to HFGS but with additional features

  • Associated with HOXA13 mutations

  • Zebrafish studies help distinguish between primary defects and secondary consequences

• HOX genes in cancer:

  • HOXA13 acts as an oncogene in several cancers, including bladder cancer

  • High HOXA13 expression is associated with non-muscle invasive bladder cancer, lower tumor grade, higher lymph node metastases, and recurrence risk

  • Zebrafish hoxa13b research can illuminate the normal functions that become dysregulated in cancer

The translational value of zebrafish hoxa13b research stems from:

• Conservation of molecular mechanisms between zebrafish and humans

• Ability to perform rapid genetic manipulations in zebrafish

• Opportunity to study gene function in the context of a whole organism

• Feasibility of high-throughput drug screening

For example, understanding how Hoxa13b regulates target genes through distal enhancers may provide insights into how human HOXA13 mutations affect gene expression in HFGS. Similarly, the identification of Hoxa13b binding motifs and co-factors could help predict the impact of specific HOXA13 mutations found in human patients.

Furthermore, the finding that hoxa13b genetically interacts with tbxta suggests that HOX gene disorders may be influenced by genetic modifiers, potentially explaining the variable expressivity observed in human patients with identical HOXA13 mutations.

What are the key unanswered questions about hoxa13b function in zebrafish development?

Despite significant progress in understanding hoxa13b function, several key questions remain unanswered:

• Complete target gene network:

  • What is the comprehensive set of genes directly regulated by Hoxa13b?

  • How does this network change during different developmental stages?

  • Which targets are shared with other Hox13 paralogs and which are unique?

• Mechanism of genetic interaction with tbxta:

  • Does Hoxa13b physically interact with Tbxta protein?

  • Do they co-regulate common target genes?

  • How do they cooperatively control cell fate decisions in the tailbud?

• Epigenetic regulation:

  • How does Hoxa13b affect the chromatin landscape?

  • Does it recruit specific chromatin modifiers to target sites?

  • How are hoxa13b-bound enhancers looped to their target promoters?

• Evolutionary diversification:

  • What specific sequence changes underlie functional differences between hoxa13b and its paralogs?

  • How have cis-regulatory changes altered hoxa13b expression across fish species?

  • What role did hoxa13b play in the evolution of teleost-specific features?

• Regenerative contexts:

  • What is the role of hoxa13b during fin regeneration?

  • How does its function in regeneration compare to its developmental role?

  • Could manipulation of hoxa13b enhance regenerative capacity?

Addressing these questions will require integrating multiple approaches:

• Single-cell technologies to capture cellular heterogeneity

• Genome-wide approaches to map regulatory networks

• Live imaging to track dynamic processes

• Comparative studies across species

• Novel genome editing strategies for precise manipulation

These investigations will not only enhance our understanding of hoxa13b but will also provide broader insights into how HOX genes contribute to development, evolution, and disease across vertebrates.

How might combinatorial CRISPR approaches advance our understanding of Hox13 gene redundancy?

Combinatorial CRISPR approaches offer powerful new avenues for dissecting the functional redundancy among Hox13 paralogs in zebrafish:

• Multiplexed gene editing:

  • Simultaneous targeting of all six Hox13 paralogs in zebrafish

  • Creation of allelic series with different combinations of mutations

  • Generation of conditional knockouts for temporal control

• Base editing technology:

  • Introduction of specific amino acid changes without DNA breaks

  • Creation of separation-of-function mutations affecting specific domains

  • Precise modification of binding motifs in enhancers regulated by Hox13 proteins

• Prime editing approaches:

  • Introduction of specific insertions or deletions with minimal off-target effects

  • Replacement of entire coding regions between paralogs to test functional equivalence

  • Engineering of chimeric Hox13 proteins to map functional domains

• CRISPR activation/inhibition systems:

  • CRISPRa to upregulate specific Hox13 paralogs in defined spatiotemporal patterns

  • CRISPRi to selectively repress individual paralogs

  • Combinatorial activation/inhibition to test compensatory relationships

These approaches could address fundamental questions about Hox13 redundancy:

Current evidence suggests substantial but incomplete redundancy among Hox13 paralogs. For example, hoxa13b mutants show limited phenotypes alone but enhanced defects when combined with hoxd13a mutations or under stress conditions . Similarly, hoxb13a and hoxc13a single mutants both show extra vertebrae but affect different regions of the axial skeleton .

Combinatorial CRISPR approaches would allow systematic dissection of these relationships, potentially revealing cryptic functions and resolving the apparent paradox between evolutionary conservation and limited single-mutant phenotypes.

What novel insights might single-cell technologies provide about hoxa13b function?

Single-cell technologies offer unprecedented opportunities to gain novel insights into hoxa13b function by resolving cellular heterogeneity and dynamic processes that are obscured in bulk analyses:

• Single-cell RNA sequencing (scRNA-seq):

  • Identification of distinct cell populations expressing hoxa13b

  • Characterization of transcriptional states in individual hoxa13b-expressing cells

  • Tracking of cell fate trajectories in wild-type versus hoxa13b mutant embryos

  • Detection of compensatory responses that may be diluted in bulk analysis

• Single-cell ATAC-seq (scATAC-seq):

  • Mapping of chromatin accessibility in hoxa13b-expressing cells

  • Identification of cell type-specific Hoxa13b binding sites

  • Correlation of chromatin states with gene expression patterns

• Single-cell CUT&RUN or CUT&Tag:

  • Direct profiling of Hoxa13b binding in individual cells

  • Correlation of binding patterns with cell states and fate decisions

  • Identification of cell type-specific co-factors

• Spatial transcriptomics:

  • Preservation of spatial context while obtaining transcriptomic data

  • Mapping of hoxa13b expression domains with high resolution

  • Correlation of expression patterns with anatomical features

• Live imaging with single-molecule resolution:

  • Visualization of Hoxa13b protein dynamics in living cells

  • Tracking of target gene activation in real-time

  • Correlation of protein localization with cellular behaviors

These approaches could answer key questions about hoxa13b function:

• How heterogeneous are hoxa13b-expressing cells in terms of gene expression and chromatin state?

• Does Hoxa13b binding exhibit cell-to-cell variability even within seemingly uniform populations?

• How does the loss of hoxa13b affect cell fate decisions at the single-cell level?

• What are the earliest transcriptional changes following hoxa13b activation or loss?

Single-cell approaches are particularly valuable for studying hoxa13b given its expression in relatively small cell populations and its involvement in dynamic developmental processes like axis elongation and somitogenesis. These technologies could reveal previously undetected cellular subpopulations with distinct responses to Hoxa13b, potentially explaining some of the complex and context-dependent phenotypes observed in genetic studies .

Frequently Asked Questions: Recombinant Danio rerio Homeobox protein Hox-A13b (hoxa13b) in Research

What is the genomic organization of hoxa13b in Danio rerio?

Hoxa13b is one of several Hox13 paralogs in zebrafish (Danio rerio), located within the HoxA cluster. Genomic annotation studies have confirmed that zebrafish have a total of 49 hox genes distributed across multiple clusters due to a whole genome duplication event that occurred in teleost evolution. The hoxa13b gene contains one intron and is expressed from the endogenous hoxa13b locus in the tail region. Research methodologies for confirming genomic organization typically include genome database searches (e.g., TBLASTN searches in Ensembl), RT-PCR validation, and comparative genomic analyses to verify gene location and structure . For targeted genetic modifications, researchers have successfully inserted FLAG-2A-GFP sequences at the C terminus of the hoxa13b coding region to create knock-in lines that maintain the native expression pattern .

How is hoxa13b expression regulated during zebrafish development?

Hoxa13b expression is tightly regulated during zebrafish development with specific spatiotemporal patterns. Expression analysis using in situ hybridization demonstrates that hoxa13b is activated early during somitogenesis and is primarily expressed in the tailbud region of mid-somitogenesis embryos . Quantitative studies using qPCR have shown that hoxa13b, together with hoxd13a, are the most abundantly expressed Hox13 genes in the tailbud, although the absolute expression level remains relatively low compared to other developmental regulators .

The regulation involves both genetic and epigenetic mechanisms. CUT&RUN analyses have revealed that Hoxa13b can bind to its own regulatory regions, suggesting potential autoregulation. To study this regulation experimentally, researchers have developed transgenic lines such as the heat-shock inducible HS:hoxa13b-FLAG-GFP and the endogenous knock-in KI:hoxa13b-FLAG-GFP, allowing for controlled expression and visualization through the co-expressed GFP protein .

What is known about the evolutionary conservation of hoxa13b among teleosts?

Evolutionary analysis indicates that hoxa13b arose from a whole genome duplication event that occurred shortly before the most recent common ancestor of teleosts. This duplication resulted in the retention of many Hox cluster genes in teleosts, contributing to their morphological diversity. Comparative studies have shown that while some duplicated Hox genes demonstrate rapid divergence and neofunctionalization, others maintain conserved functions .

In zebrafish and related cypriniform fishes, molecular evolution studies of the duplicated HoxA13 paralogs (hoxa13a and hoxa13b) have revealed asymmetric rates of divergence. Research methodologies for studying evolutionary conservation typically include sequence alignment, phylogenetic analysis, and functional tests of orthologous genes across species. Data suggest that in early stages of zebrafish larval development, HoxA13b is the predominant paralog expressed, indicating potential subfunctionalization between the paralogs following duplication .

What are the most effective methods for creating hoxa13b mutants in zebrafish?

CRISPR/Cas9 genome editing has proven to be the most effective method for generating targeted hoxa13b mutations in zebrafish. This methodology has been successfully implemented in multiple studies:

• For knockout mutations, researchers have created lines with small insertions or deletions that disrupt the coding sequence. For example, a hoxa13b Δ16 mutation has been created that results in a complete loss of Hoxa13b function .

• For knock-in approaches, researchers have used the CRISPR/Cas9 system with specific gRNAs targeting the 5′ and 3′ UTRs of the endogenous hoxa13b locus, along with a donor plasmid containing the modified gene with an epitope tag .

The efficiency of creating mutations depends on several factors:

It's important to verify that mutant alleles do not trigger genetic compensation through nonsense-mediated decay, which has been observed for some genes. qPCR analysis of hoxa13b Δ16 embryos has shown that the mutant transcript is expressed at similar levels to wild-type, with no compensatory upregulation of other Hox13 genes .

How can binding sites of Hoxa13b be identified genome-wide in zebrafish embryos?

Identifying genome-wide binding sites for Hoxa13b has been challenging due to the paucity of high-quality antibodies and the limited number of cells in zebrafish tailbud tissue. Researchers have overcome these limitations through innovative approaches:

• Creation of transgenic lines expressing epitope-tagged Hoxa13b (FLAG-tagged) under either inducible (HS:hoxa13b-FLAG-GFP) or endogenous (KI:hoxa13b-FLAG-GFP) control .

• Adaptation of CUT&RUN (Cleavage Under Targets and Release Using Nuclease) technology for zebrafish embryos, which permits identification of in vivo binding sites using limited cell numbers .

The CUT&RUN methodology involves:

• Isolation of nuclei from transgenic embryos expressing FLAG-tagged Hoxa13b

• Binding of an anti-FLAG antibody to the epitope-tagged protein

• Addition of protein A-MNase to cleave DNA around binding sites

• Isolation and sequencing of the released DNA fragments

Using this approach, researchers identified 1871 Hoxa13b-occupied peaks from the heat-shock line, with 689 overlapping peaks also found in the knock-in line. Analysis revealed that only 12.6% of binding sites were located within 3 kb of transcription start sites, with over 60% in distal intergenic regions. Motif analysis showed that the Hox binding motif was the most common sequence in these regions .

What expression systems are optimal for producing recombinant Hoxa13b protein?

For functional studies of Hoxa13b protein, several expression systems have been effectively employed:

• In vivo zebrafish expression systems:

  • Heat-shock inducible transgenic lines allow for temporal control of expression

  • Endogenous knock-in lines maintain normal spatiotemporal regulation

  • Both approaches can incorporate epitope tags for protein detection and purification

• Bacterial expression systems:

  • E. coli BL21(DE3) with pET vector systems for expressing the DNA-binding homeodomain

  • Fusion tags (His6, GST, MBP) improve solubility and facilitate purification

  • Expression at lower temperatures (16-18°C) improves proper folding

• Mammalian and insect cell systems:

  • HEK293T cells for mammalian expression with post-translational modifications

  • Sf9 insect cells with baculovirus for higher yields of full-length protein

Key considerations for obtaining functional recombinant Hoxa13b include:

• Codon optimization for the expression system

• Inclusion of nuclear localization signals for proper subcellular localization

• Addition of epitope tags that don't interfere with DNA binding

• Protein purification under conditions that maintain DNA-binding activity

For in vitro binding studies, the properly folded homeodomain is essential for recognizing specific DNA sequences. Recombinant Hoxa13b has been shown to bind both "traditional" Hox motifs (with a T at a specific position) and posterior Hox-specific motifs (with a C at that position) .

How does hoxa13b contribute to axis elongation in zebrafish embryos?

Hoxa13b plays a critical role in posterior development and axis elongation in zebrafish embryos through genetic interactions with other key developmental regulators. Studies investigating the function of hoxa13b through loss-of-function approaches have revealed:

The mechanistic basis involves:

• Regulation of neuromesodermal progenitors (NMps) that contribute to both neural tube and paraxial mesoderm

• Maintenance of proper tbxta expression in the tailbud

• Control of the balance between mesodermal and neural fates during axis elongation

Temperature-sensitive experiments have demonstrated that hoxa13b mutants are hypersensitive to Tbxta reduction, with embryos placed at semi-permissive temperatures (18.5°C) showing a sharp reduction in tbxta expression in the NMps .

What is the role of hoxa13b in caudal fin development and evolution?

Hoxa13b contributes significantly to caudal fin development and evolution in zebrafish and related teleosts. Research has demonstrated:

• Hox13 genes, including hoxa13b, are expressed in distinct domains during caudal fin development, with expression patterns that correlate with specific morphological boundaries .

• While hoxa13b is more strongly expressed in the pectoral fin buds than in the tail, it still plays a role in caudal fin patterning through genetic interactions with other Hox13 paralogs .

• Loss-of-function studies of hoxa13b in combination with other Hox13 genes revealed phenotypes affecting caudal fin ray number, joint formation, and segment length .

Experimental approaches for studying hoxa13b's role in fin development include:

• In situ hybridization to map expression domains

• CRISPR/Cas9-mediated gene knockout

• Cell ablation experiments using the NTR/MTZ mechanism to partially remove hoxa13a-expressing cells

• Analysis of joint formation during both development and regeneration

Particularly noteworthy is the finding that partial ablation of hoxa13a-expressing cells results in shorter bone segments following regeneration, suggesting that Hox13 genes are involved in regulating segment length . Additionally, triple mutants with mutations in hoxa13a, hoxa13b, and hoxd13a show fin-specific defects in joint formation and patterning .

How does Hoxa13b protein function as a transcription factor?

Hoxa13b functions as a transcription factor by binding to specific DNA sequences and regulating the expression of target genes. Detailed molecular analyses have revealed:

• Hoxa13b binds to DNA through its homeodomain, recognizing both "traditional" Hox motifs and posterior Hox-specific motifs .

• CUT&RUN analysis identified 1871 Hoxa13b-occupied peaks in the zebrafish genome, with over 60% located in distal intergenic regions, suggesting that Hoxa13b primarily regulates gene expression through enhancer elements rather than proximal promoters .

• DNA footprinting analysis showed that Hoxa13b forms a 19-bp footprint on DNA, protecting this region from nuclease digestion .

• Hoxa13b can potentially regulate at least 576 candidate target genes in the zebrafish tailbud during mid-somitogenesis stages .

The transcriptional activity of Hoxa13b involves:

• Binding to enhancer elements often located far from the genes they regulate

• Potential cooperation with other transcription factors (co-factors)

• Recruitment of chromatin modifiers to activate or repress target genes

• Potential autoregulatory mechanisms, as Hoxa13b binding sites were found upstream of the hoxa13b locus itself

Experimental approaches for studying Hoxa13b transcription factor activity include:

• CUT&RUN for genome-wide binding site identification

• Epigenetic marker analysis to identify sites associated with transcriptional activation

• Reporter gene assays to validate enhancer function

• Gene expression analysis following hoxa13b manipulation to identify regulated genes

Why does single hoxa13b mutation show limited phenotypes despite its evolutionary conservation?

The limited phenotypes observed in single hoxa13b mutants despite the gene's evolutionary conservation represent a fascinating paradox in developmental biology. Several factors contribute to this phenomenon:

• Functional redundancy with other Hox13 paralogs: Zebrafish possess multiple Hox13 genes (hoxa13a, hoxa13b, hoxb13a, hoxc13a, hoxc13b, and hoxd13a) that show overlapping expression patterns. Loss of hoxa13b function can be compensated by these other paralogs, particularly hoxd13a .

• Genetic interaction with developmental pathways: The phenotypic consequences of hoxa13b loss become apparent only under certain genetic or environmental conditions. For example, hoxa13b mutants show severe defects when tbxta function is also reduced .

• Condition-dependent phenotypes: While hoxa13b mutants appear normal under standard laboratory conditions, they exhibit phenotypes when raised at lower temperatures (21°C), indicating a temperature-sensitive requirement for Hoxa13b function .

• Quantitative rather than qualitative effects: Hoxa13b may contribute to developmental robustness rather than being absolutely required for specific structures. This is evidenced by the synergistic effects seen when multiple Hox13 genes are mutated .

This apparent contradiction can be experimentally addressed through:

• Creation of multiple mutant combinations to uncover redundant functions

• Stress tests (temperature shifts, developmental timing alterations) to reveal cryptic phenotypes

• RNA-seq analysis to identify compensatory transcriptional changes

• Evolutionary comparisons across species with different Hox gene complements

These findings highlight the importance of testing genetic function under various conditions rather than relying solely on standard laboratory environments, which may mask subtle or condition-dependent phenotypes.

How do the functions of hoxa13b differ from or overlap with its paralog hoxa13a?

The functional relationship between hoxa13b and its paralog hoxa13a in zebrafish represents a classic case of post-duplication divergence and subfunctionalization. Research has revealed both overlapping and distinct functions:

Overlapping functions:

• Both genes are expressed in posterior tissues during development .

• Both contribute to fin development and patterning .

• Both belong to the same paralog group and recognize similar DNA binding motifs .

Distinct functions:

• Expression patterns show differences, with hoxa13b more strongly expressed in the tailbud during mid-somitogenesis, while hoxa13a shows stronger expression in fins .

• Functional studies indicate hoxa13a has a more prominent role in fin joint formation and regeneration, while hoxa13b has stronger genetic interactions with tbxta in posterior body development .

• Evolutionary analysis shows asymmetric rates of sequence divergence between the paralogs, with evidence suggesting hoxa13a has experienced more rapid sequence evolution .

Experimental approaches to distinguish their functions include:

• Detailed expression pattern analysis using in situ hybridization

• Single and double mutant phenotypic analysis

• Paralog-specific rescue experiments

• CUT&RUN or ChIP-seq analysis to compare binding sites

The data indicates that while there is some functional redundancy, these paralogs have undergone subfunctionalization since their duplication, with each acquiring specialized roles in different developmental contexts. This highlights how gene duplication can contribute to evolutionary innovation through the partitioning of ancestral functions between duplicated genes .

What explains the contradictory findings on hoxa13b expression levels in different studies?

Several studies report varying levels of hoxa13b expression in zebrafish, leading to apparent contradictions in the literature. These discrepancies can be attributed to multiple factors:

• Technical differences in detection methods:

  • RNA-seq provides genome-wide quantification but may have lower sensitivity for low-abundance transcripts

  • qPCR offers higher sensitivity but is dependent on primer efficiency

  • In situ hybridization provides spatial information but is less quantitative

• Developmental timing differences:

  • Expression of hoxa13b is dynamically regulated during development

  • Peak expression occurs during mid-somitogenesis but precise timing varies

  • Some studies sample at specific stages while others use time windows

• Different reference points for comparison:

  • Some studies compare hoxa13b to other Hox13 paralogs

  • Others compare to housekeeping genes or global transcriptome

  • Relative vs. absolute quantification methods yield different perspectives

• Genetic background variations:

  • Different wild-type zebrafish strains (e.g., TU, AB) have slight genetic differences

  • Laboratory-specific genetic drift may affect expression levels

  • Transgenic reporter lines may not perfectly recapitulate endogenous expression

To reconcile these contradictions, researchers should:

• Clearly specify detection methods, developmental stages, and reference genes

• Use multiple complementary techniques (RNA-seq, qPCR, in situ hybridization)

• Perform direct comparisons under identical conditions

• Consider single-cell approaches to address cellular heterogeneity

One consistent finding across studies is that hoxa13b expression is relatively low compared to many other developmental regulators, yet it plays important roles through genetic interactions with other factors, highlighting the non-linear relationship between expression level and developmental significance .

How can we leverage hoxa13b to understand evolutionary changes in body plan?

Hoxa13b research provides a unique window into understanding evolutionary changes in vertebrate body plans, particularly in the context of the teleost-specific whole genome duplication. Several research approaches can leverage hoxa13b to gain insights into evolutionary developmental biology:

• Comparative genomics and phylogenetics:

  • Sequence analysis of hoxa13b across diverse fish lineages to identify selective pressures

  • Correlation of sequence changes with morphological innovations

  • Reconstruction of ancestral sequences to test evolutionary hypotheses

• Cis-regulatory element analysis:

  • Identification of conserved and divergent enhancer elements controlling hoxa13b expression

  • Cross-species enhancer swap experiments to test functional evolution

  • ATAC-seq and CUT&RUN studies to map regulatory landscape changes

• Functional testing across species:

  • CRISPR/Cas9 mutation of hoxa13b in different fish species

  • Cross-species rescue experiments to test functional equivalence

  • Generation of chimeric Hoxa13b proteins to map functionally important domains

Particularly promising is the investigation of hoxa13b's role in caudal fin evolution. Research has shown that hox13 genes regulate the number and identity of posterior vertebrae and the development of the homocercal (symmetrical) caudal fin, a key innovation in teleost evolution . Mutations in hoxb13a and hoxc13a result in extra vertebrae and altered fin ray patterning, suggesting that changes in Hox13 gene function contributed to the evolution of the distinctive teleost tail structure .

This provides an opportunity to understand how changes in Hox gene function and regulation contributed to the remarkable diversity of body plans observed in teleost fishes, which represent nearly half of all vertebrate species.

What are the latest methodological advances for studying hoxa13b protein-protein interactions?

Recent methodological advances have significantly enhanced our ability to study Hoxa13b protein-protein interactions, offering new insights into its molecular function:

• BioID and TurboID proximity labeling:

  • Fusion of Hoxa13b with a biotin ligase that biotinylates nearby proteins

  • Allows identification of proximal proteins in living cells

  • Captures transient interactions often missed by co-immunoprecipitation

• Single-molecule imaging:

  • Visualization of individual Hoxa13b molecules in living cells

  • Tracking of binding dynamics and residence times on chromatin

  • Reveals heterogeneity in binding behavior

• Protein complementation assays:

  • Split fluorescent proteins or luciferase fused to Hoxa13b and potential partners

  • Signal generated only when proteins interact

  • Amenable to high-throughput screening

• Mass spectrometry-based approaches:

  • Crosslinking mass spectrometry (XL-MS) to capture direct protein contacts

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map interaction surfaces

  • Thermal proteome profiling to detect stabilization upon binding

• Cryo-electron microscopy:

  • Structural determination of Hoxa13b complexes with transcriptional co-factors

  • Visualization of Hoxa13b as part of larger regulatory complexes

  • Resolution of conformational changes upon DNA binding

These methods have begun to reveal that Hoxa13b likely functions as part of multi-protein complexes that may include other Hox proteins, general transcription factors, and chromatin modifiers. Understanding these interactions is crucial for deciphering how Hoxa13b achieves target specificity and regulatory precision despite recognizing relatively simple DNA motifs .

The identification of Hoxa13b-interacting proteins also provides potential targets for manipulating Hoxa13b function in experimental contexts, offering new approaches to study its roles in development and disease.

What are the implications of hoxa13b research for understanding human HOX gene disorders?

Research on zebrafish hoxa13b has significant implications for understanding human HOX gene disorders, particularly those involving HOXA13:

• Hand-foot-genital syndrome (HFGS):

  • Caused by mutations in human HOXA13

  • Characterized by limb and urogenital abnormalities

  • Zebrafish hoxa13b models can provide insights into the developmental mechanisms

• Guttmacher syndrome:

  • Related to HFGS but with additional features

  • Associated with HOXA13 mutations

  • Zebrafish studies help distinguish between primary defects and secondary consequences

• HOX genes in cancer:

  • HOXA13 acts as an oncogene in several cancers, including bladder cancer

  • High HOXA13 expression is associated with non-muscle invasive bladder cancer, lower tumor grade, higher lymph node metastases, and recurrence risk

  • Zebrafish hoxa13b research can illuminate the normal functions that become dysregulated in cancer

The translational value of zebrafish hoxa13b research stems from:

• Conservation of molecular mechanisms between zebrafish and humans

• Ability to perform rapid genetic manipulations in zebrafish

• Opportunity to study gene function in the context of a whole organism

• Feasibility of high-throughput drug screening

For example, understanding how Hoxa13b regulates target genes through distal enhancers may provide insights into how human HOXA13 mutations affect gene expression in HFGS. Similarly, the identification of Hoxa13b binding motifs and co-factors could help predict the impact of specific HOXA13 mutations found in human patients.

Furthermore, the finding that hoxa13b genetically interacts with tbxta suggests that HOX gene disorders may be influenced by genetic modifiers, potentially explaining the variable expressivity observed in human patients with identical HOXA13 mutations.

What are the key unanswered questions about hoxa13b function in zebrafish development?

Despite significant progress in understanding hoxa13b function, several key questions remain unanswered:

• Complete target gene network:

  • What is the comprehensive set of genes directly regulated by Hoxa13b?

  • How does this network change during different developmental stages?

  • Which targets are shared with other Hox13 paralogs and which are unique?

• Mechanism of genetic interaction with tbxta:

  • Does Hoxa13b physically interact with Tbxta protein?

  • Do they co-regulate common target genes?

  • How do they cooperatively control cell fate decisions in the tailbud?

• Epigenetic regulation:

  • How does Hoxa13b affect the chromatin landscape?

  • Does it recruit specific chromatin modifiers to target sites?

  • How are hoxa13b-bound enhancers looped to their target promoters?

• Evolutionary diversification:

  • What specific sequence changes underlie functional differences between hoxa13b and its paralogs?

  • How have cis-regulatory changes altered hoxa13b expression across fish species?

  • What role did hoxa13b play in the evolution of teleost-specific features?

• Regenerative contexts:

  • What is the role of hoxa13b during fin regeneration?

  • How does its function in regeneration compare to its developmental role?

  • Could manipulation of hoxa13b enhance regenerative capacity?

Addressing these questions will require integrating multiple approaches:

• Single-cell technologies to capture cellular heterogeneity

• Genome-wide approaches to map regulatory networks

• Live imaging to track dynamic processes

• Comparative studies across species

• Novel genome editing strategies for precise manipulation

These investigations will not only enhance our understanding of hoxa13b but will also provide broader insights into how HOX genes contribute to development, evolution, and disease across vertebrates.

How might combinatorial CRISPR approaches advance our understanding of Hox13 gene redundancy?

Combinatorial CRISPR approaches offer powerful new avenues for dissecting the functional redundancy among Hox13 paralogs in zebrafish:

• Multiplexed gene editing:

  • Simultaneous targeting of all six Hox13 paralogs in zebrafish

  • Creation of allelic series with different combinations of mutations

  • Generation of conditional knockouts for temporal control

• Base editing technology:

  • Introduction of specific amino acid changes without DNA breaks

  • Creation of separation-of-function mutations affecting specific domains

  • Precise modification of binding motifs in enhancers regulated by Hox13 proteins

• Prime editing approaches:

  • Introduction of specific insertions or deletions with minimal off-target effects

  • Replacement of entire coding regions between paralogs to test functional equivalence

  • Engineering of chimeric Hox13 proteins to map functional domains

• CRISPR activation/inhibition systems:

  • CRISPRa to upregulate specific Hox13 paralogs in defined spatiotemporal patterns

  • CRISPRi to selectively repress individual paralogs

  • Combinatorial activation/inhibition to test compensatory relationships

These approaches could address fundamental questions about Hox13 redundancy:

Current evidence suggests substantial but incomplete redundancy among Hox13 paralogs. For example, hoxa13b mutants show limited phenotypes alone but enhanced defects when combined with hoxd13a mutations or under stress conditions . Similarly, hoxb13a and hoxc13a single mutants both show extra vertebrae but affect different regions of the axial skeleton .

Combinatorial CRISPR approaches would allow systematic dissection of these relationships, potentially revealing cryptic functions and resolving the apparent paradox between evolutionary conservation and limited single-mutant phenotypes.

What novel insights might single-cell technologies provide about hoxa13b function?

Single-cell technologies offer unprecedented opportunities to gain novel insights into hoxa13b function by resolving cellular heterogeneity and dynamic processes that are obscured in bulk analyses:

• Single-cell RNA sequencing (scRNA-seq):

  • Identification of distinct cell populations expressing hoxa13b

  • Characterization of transcriptional states in individual hoxa13b-expressing cells

  • Tracking of cell fate trajectories in wild-type versus hoxa13b mutant embryos

  • Detection of compensatory responses that may be diluted in bulk analysis

• Single-cell ATAC-seq (scATAC-seq):

  • Mapping of chromatin accessibility in hoxa13b-expressing cells

  • Identification of cell type-specific Hoxa13b binding sites

  • Correlation of chromatin states with gene expression patterns

• Single-cell CUT&RUN or CUT&Tag:

  • Direct profiling of Hoxa13b binding in individual cells

  • Correlation of binding patterns with cell states and fate decisions

  • Identification of cell type-specific co-factors

• Spatial transcriptomics:

  • Preservation of spatial context while obtaining transcriptomic data

  • Mapping of hoxa13b expression domains with high resolution

  • Correlation of expression patterns with anatomical features

• Live imaging with single-molecule resolution:

  • Visualization of Hoxa13b protein dynamics in living cells

  • Tracking of target gene activation in real-time

  • Correlation of protein localization with cellular behaviors

These approaches could answer key questions about hoxa13b function:

• How heterogeneous are hoxa13b-expressing cells in terms of gene expression and chromatin state?

• Does Hoxa13b binding exhibit cell-to-cell variability even within seemingly uniform populations?

• How does the loss of hoxa13b affect cell fate decisions at the single-cell level?

• What are the earliest transcriptional changes following hoxa13b activation or loss?