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Despite the great diversity of vertebrate limb proportion and our deep understanding of the genetic mechanisms that drive skeletal elongation, little is known about how individual bones reach different lengths in any species. Here, we directly compare the transcriptomes of homologous growth cartilages of the mouse (Mus musculus) and bipedal jerboa (Jaculus jaculus), the latter of which has "mouse-like" arms but extremely long metatarsals of the feet. Intersecting gene-expression differences in metatarsals and forearms of the two species revealed that about 10% of orthologous genes are associated with the disproportionately rapid elongation of neonatal jerboa feet. These include genes and enriched pathways not previously associated with endochondral elongation as well as those that might diversify skeletal proportion in addition to their known requirements for bone growth throughout the skeleton. We also identified transcription regulators that might act as "nodes" for sweeping differences in genome expression between species. Among these, Shox2, which is necessary for proximal limb elongation, has gained expression in jerboa metatarsals where it has not been detected in other vertebrates. We show that Shox2 is sufficient to increase mouse distal limb length, and a nearby putative cis-regulatory region is preferentially accessible in jerboa metatarsals. In addition to mechanisms that might directly promote growth, we found evidence that jerboa foot elongation may occur in part by de-repressing latent growth potential. The genes and pathways that we identified here provide a framework to understand the modular genetic control of skeletal growth and the remarkable malleability of vertebrate limb proportion.

SMAD4 regulates gene expression in response to BMP and TGFβ signal transduction, and is required for diverse morphogenetic processes, but its target genes have remained largely elusive. Here, we identify the SMAD4 target genes in mouse limb buds using an epitope-tagged Smad4 allele for ChIP-seq analysis in combination with transcription profiling. This analysis shows that SMAD4 predominantly mediates BMP signal transduction during early limb bud development. Unexpectedly, the expression of cholesterol biosynthesis enzymes is precociously downregulated and intracellular cholesterol levels are reduced in Smad4-deficient limb bud mesenchymal progenitors. Most importantly, our analysis reveals a predominant function of SMAD4 in upregulating target genes in the anterior limb bud mesenchyme. Analysis of differentially expressed genes shared between Smad4- and Shh-deficient limb buds corroborates this function of SMAD4 and also reveals the repressive effect of SMAD4 on posterior genes that are upregulated in response to SHH signaling. This analysis uncovers opposing trans-regulatory inputs from SHH- and SMAD4-mediated BMP signal transduction on anterior and posterior gene expression during the digit patterning and outgrowth in early limb buds.

In ribosomopathies, perturbed expression of ribosome components leads to tissue-specific phenotypes. What accounts for such tissue-selective manifestations as a result of mutations in the ribosome, a ubiquitous cellular machine, has remained a mystery. Combining mouse genetics and in vivo ribosome profiling, we observe limb-patterning phenotypes in ribosomal protein (RP) haploinsufficient embryos, and we uncover selective translational changes of transcripts that controlling limb development. Surprisingly, both loss of p53, which is activated by RP haploinsufficiency, and augmented protein synthesis rescue these phenotypes. These findings are explained by the finding that p53 functions as a master regulator of protein synthesis, at least in part, through transcriptional activation of 4E-BP1. 4E-BP1, a key translational regulator, in turn, facilitates selective changes in the translatome downstream of p53, and this thereby explains how RP haploinsufficiency may elicit specificity to gene expression. These results provide an integrative model to help understand how in vivo tissue-specific phenotypes emerge in ribosomopathies.

Establishing causal links between non-coding variants and human phenotypes is an increasing challenge. Here, we introduce a high-throughput mouse reporter assay for assessing the pathogenic potential of human enhancer variants in vivo and examine nearly a thousand variants in an enhancer repeatedly linked to polydactyly. We show that 71% of all rare non-coding variants previously proposed as causal lead to reporter gene expression in a pattern consistent with their pathogenic role. Variants observed to alter enhancer activity were further confirmed to cause polydactyly in knockin mice. We also used combinatorial and single-nucleotide mutagenesis to evaluate the in vivo impact of mutations affecting all positions of the enhancer and identified additional functional substitutions, including potentially pathogenic variants hitherto not observed in humans. Our results uncover the functional consequences of hundreds of mutations in a phenotype-associated enhancer and establish a widely applicable strategy for systematic in vivo evaluation of human enhancer variants.

Establishing causal links between non-coding variants and human phenotypes is an increasing challenge. Here, we introduce a high-throughput mouse reporter assay for assessing the pathogenic potential of human enhancer variants in vivo and examine nearly a thousand variants in an enhancer repeatedly linked to polydactyly. We show that 71% of all rare non-coding variants previously proposed as causal lead to reporter gene expression in a pattern consistent with their pathogenic role. Variants observed to alter enhancer activity were further confirmed to cause polydactyly in knockin mice. We also used combinatorial and single-nucleotide mutagenesis to evaluate the in vivo impact of mutations affecting all positions of the enhancer and identified additional functional substitutions, including potentially pathogenic variants hitherto not observed in humans. Our results uncover the functional consequences of hundreds of mutations in a phenotype-associated enhancer and establish a widely applicable strategy for systematic in vivo evaluation of human enhancer variants.

The fusion of digits or toes, syndactyly, can be part of complex syndromes, including van der Woude syndrome. A subset of van der Woude cases is caused by dominant-negative mutations in the epithelial transcription factor Grainyhead like-3 (GRHL3), and Grhl3-/-mice have soft-tissue syndactyly. Although impaired interdigital cell death of mesenchymal cells causes syndactyly in multiple genetic mutants, Grhl3-/- embryos had normal interdigital cell death, suggesting alternative mechanisms for syndactyly. We found that in digit separation, the overlying epidermis forms a migrating interdigital epithelial tongue (IET) when the epithelium invaginates to separate the digits. Normally, the non-adhesive surface periderm allows the IET to bifurcate as the digits separate. In contrast, in Grhl3-/- embryos, the IET moves normally between the digits but fails to bifurcate because of abnormal adhesion of the periderm. Our study identifies epidermal developmental processes required for digit separation.

The fusion of digits or toes, syndactyly, can be part of complex syndromes, including van der Woude syndrome. A subset of van der Woude cases is caused by dominant-negative mutations in the epithelial transcription factor Grainyhead like-3 (GRHL3), and Grhl3-/-mice have soft-tissue syndactyly. Although impaired interdigital cell death of mesenchymal cells causes syndactyly in multiple genetic mutants, Grhl3-/- embryos had normal interdigital cell death, suggesting alternative mechanisms for syndactyly. We found that in digit separation, the overlying epidermis forms a migrating interdigital epithelial tongue (IET) when the epithelium invaginates to separate the digits. Normally, the non-adhesive surface periderm allows the IET to bifurcate as the digits separate. In contrast, in Grhl3-/- embryos, the IET moves normally between the digits but fails to bifurcate because of abnormal adhesion of the periderm. Our study identifies epidermal developmental processes required for digit separation.

Establishing causal links between non-coding variants and human phenotypes is an increasing challenge. Here, we introduce a high-throughput mouse reporter assay for assessing the pathogenic potential of human enhancer variants in vivo and examine nearly a thousand variants in an enhancer repeatedly linked to polydactyly. We show that 71% of all rare non-coding variants previously proposed as causal lead to reporter gene expression in a pattern consistent with their pathogenic role. Variants observed to alter enhancer activity were further confirmed to cause polydactyly in knockin mice. We also used combinatorial and single-nucleotide mutagenesis to evaluate the in vivo impact of mutations affecting all positions of the enhancer and identified additional functional substitutions, including potentially pathogenic variants hitherto not observed in humans. Our results uncover the functional consequences of hundreds of mutations in a phenotype-associated enhancer and establish a widely applicable strategy for systematic in vivo evaluation of human enhancer variants.

The fusion of digits or toes, syndactyly, can be part of complex syndromes, including van der Woude syndrome. A subset of van der Woude cases is caused by dominant-negative mutations in the epithelial transcription factor Grainyhead like-3 (GRHL3), and Grhl3-/-mice have soft-tissue syndactyly. Although impaired interdigital cell death of mesenchymal cells causes syndactyly in multiple genetic mutants, Grhl3-/- embryos had normal interdigital cell death, suggesting alternative mechanisms for syndactyly. We found that in digit separation, the overlying epidermis forms a migrating interdigital epithelial tongue (IET) when the epithelium invaginates to separate the digits. Normally, the non-adhesive surface periderm allows the IET to bifurcate as the digits separate. In contrast, in Grhl3-/- embryos, the IET moves normally between the digits but fails to bifurcate because of abnormal adhesion of the periderm. Our study identifies epidermal developmental processes required for digit separation.

The chicken wingless-2 (wg-2) mutation is inherited in an autosomal recessive fashion, and the resulting phenotype in mutant (wg-2/wg-2) individuals is a developmental syndrome characterized by absent wings, truncated legs, craniofacial as well as skin and feather defects, and kidney malformations. Mapping and genotyping established that the mutation resides within 227 kilobases (kb) of chromosome 12 in a wg-2 congenic inbred line. A capture array was designed to target and sequence the candidate region along with flanking DNA in 24 birds from the line. Many point mutations and insertions or deletions were identified, and analysis of the linked variants indicated a point mutation predicted to cause a premature stop codon in the RAF1 gene. Expression studies were conducted inclusive of all genes in the candidate region. Interestingly, RAF1 transcription was elevated, yet the protein was absent in the mutants relative to normal individuals. RAF1 encodes a protein integral to the Ras/Raf/MAPK signaling pathway controlling cellular proliferation, and notably, human RASopathies are developmental syndromes caused by germline mutations in genes of this pathway. Our work indicates RAF1 as the priority candidate causative gene for wg-2 and provides a new animal model to study an important signaling pathway implicated in limb development, as well as RASopathies.

The chicken wingless-2 (wg-2) mutation is inherited in an autosomal recessive fashion, and the resulting phenotype in mutant (wg-2/wg-2) individuals is a developmental syndrome characterized by absent wings, truncated legs, craniofacial as well as skin and feather defects, and kidney malformations. Mapping and genotyping established that the mutation resides within 227 kilobases (kb) of chromosome 12 in a wg-2 congenic inbred line. A capture array was designed to target and sequence the candidate region along with flanking DNA in 24 birds from the line. Many point mutations and insertions or deletions were identified, and analysis of the linked variants indicated a point mutation predicted to cause a premature stop codon in the RAF1 gene. Expression studies were conducted inclusive of all genes in the candidate region. Interestingly, RAF1 transcription was elevated, yet the protein was absent in the mutants relative to normal individuals. RAF1 encodes a protein integral to the Ras/Raf/MAPK signaling pathway controlling cellular proliferation, and notably, human RASopathies are developmental syndromes caused by germline mutations in genes of this pathway. Our work indicates RAF1 as the priority candidate causative gene for wg-2 and provides a new animal model to study an important signaling pathway implicated in limb development, as well as RASopathies.

Wingless-2 (wg-2) is an autosomal recessive mutation in chicken that results in an embryonic lethal condition. Affected individuals exhibit a multisystem syndrome characterized by absent wings, truncated legs, and craniofacial, kidney, and feather malformations. Previously, work focused on phenotype description, establishing the autosomal recessive pattern of Mendelian inheritance and placing the mutation on an inbred genetic background to create the congenic line UCD Wingless-2.331. The research described in this paper employed the complementary tools of breeding, genetics, and genomics to map the chromosomal location of the mutation and successively narrow the size of the region for analysis of the causative element. Specifically, the wg-2 mutation was initially mapped to a 7 Mb region of chromosome 12 using an Illumina 3 K SNP array. Subsequent SNP genotyping and exon sequencing combined with analysis from improved genome assemblies narrowed the region of interest to a maximum size of 227 kb. Within this region, 3 validated and 3 predicted candidate genes are found, and these are described. The wg-2 mutation is a valuable resource to contribute to an improved understanding of the developmental pathways involved in chicken and avian limb development as well as serving as a model for human development, as the resulting syndrome shares features with human congenital disorders.

Wingless-2 (wg-2) is an autosomal recessive mutation in chicken that results in an embryonic lethal condition. Affected individuals exhibit a multisystem syndrome characterized by absent wings, truncated legs, and craniofacial, kidney, and feather malformations. Previously, work focused on phenotype description, establishing the autosomal recessive pattern of Mendelian inheritance and placing the mutation on an inbred genetic background to create the congenic line UCD Wingless-2.331. The research described in this paper employed the complementary tools of breeding, genetics, and genomics to map the chromosomal location of the mutation and successively narrow the size of the region for analysis of the causative element. Specifically, the wg-2 mutation was initially mapped to a 7 Mb region of chromosome 12 using an Illumina 3 K SNP array. Subsequent SNP genotyping and exon sequencing combined with analysis from improved genome assemblies narrowed the region of interest to a maximum size of 227 kb. Within this region, 3 validated and 3 predicted candidate genes are found, and these are described. The wg-2 mutation is a valuable resource to contribute to an improved understanding of the developmental pathways involved in chicken and avian limb development as well as serving as a model for human development, as the resulting syndrome shares features with human congenital disorders.

The combinatorial expression of Hox genes along the body axes is a major determinant of cell fate and plays a pivotal role in generating the animal body plan. Loss of HOXA13 and HOXD13 transcription factors (HOX13) leads to digit agenesis in mice, but how HOX13 proteins regulate transcriptional outcomes and confer identity to the distal-most limb cells has remained elusive. Here, we report on the genome-wide profiling of HOXA13 and HOXD13 in vivo binding and changes of the transcriptome and chromatin state in the transition from the early to the late-distal limb developmental program, as well as in Hoxa13-/-; Hoxd13-/- limbs. Our results show that proper termination of the early limb transcriptional program and activation of the late-distal limb program are coordinated by the dual action of HOX13 on cis-regulatory modules.

The combinatorial expression of Hox genes along the body axes is a major determinant of cell fate and plays a pivotal role in generating the animal body plan. Loss of HOXA13 and HOXD13 transcription factors (HOX13) leads to digit agenesis in mice, but how HOX13 proteins regulate transcriptional outcomes and confer identity to the distal-most limb cells has remained elusive. Here, we report on the genome-wide profiling of HOXA13 and HOXD13 in vivo binding and changes of the transcriptome and chromatin state in the transition from the early to the late-distal limb developmental program, as well as in Hoxa13-/-; Hoxd13-/- limbs. Our results show that proper termination of the early limb transcriptional program and activation of the late-distal limb program are coordinated by the dual action of HOX13 on cis-regulatory modules.

The evolution of body shape is thought to be tightly coupled to changes in regulatory sequences, but specific molecular events associated with major morphological transitions in vertebrates have remained elusive. We identified snake-specific sequence changes within an otherwise highly conserved long-range limb enhancer of Sonic hedgehog (Shh). Transgenic mouse reporter assays revealed that the in vivo activity pattern of the enhancer is conserved across a wide range of vertebrates, including fish, but not in snakes. Genomic substitution of the mouse enhancer with its human or fish ortholog results in normal limb development. In contrast, replacement with snake orthologs caused severe limb reduction. Synthetic restoration of a single transcription factor binding site lost in the snake lineage reinstated full in vivo function to the snake enhancer. Our results demonstrate changes in a regulatory sequence associated with a major body plan transition and highlight the role of enhancers in morphological evolution. PAPERCLIP.

The evolution of body shape is thought to be tightly coupled to changes in regulatory sequences, but specific molecular events associated with major morphological transitions in vertebrates have remained elusive. We identified snake-specific sequence changes within an otherwise highly conserved long-range limb enhancer of Sonic hedgehog (Shh). Transgenic mouse reporter assays revealed that the in vivo activity pattern of the enhancer is conserved across a wide range of vertebrates, including fish, but not in snakes. Genomic substitution of the mouse enhancer with its human or fish ortholog results in normal limb development. In contrast, replacement with snake orthologs caused severe limb reduction. Synthetic restoration of a single transcription factor binding site lost in the snake lineage reinstated full in vivo function to the snake enhancer. Our results demonstrate changes in a regulatory sequence associated with a major body plan transition and highlight the role of enhancers in morphological evolution. PAPERCLIP.

Cornelia de Lange Syndrome (CdLS) is characterized by a wide variety of structural and functional abnormalities in almost every organ system of the body. CdLS is now known to be caused by mutations that disrupt the function of the cohesin complex or its regulators, and studies of animal models and cell lines tell us that the effect of these mutations is to produce subtle yet pervasive dysregulation of gene expression. With many hundreds of mostly small gene expression changes occurring in every cell type and tissue, identifying the etiology of any particular birth defect is very challenging. Here we focus on limb abnormalities, which are commonly seen in CdLS. In the limb buds of the Nipbl-haploinsufficient mouse (Nipbl(+/-) mouse), a model for the most common form of CdLS, modest gene expression changes are observed in several candidate pathways whose disruption is known to cause limb abnormalities, yet the limbs of Nipbl(+/-) mice develop relatively normally. We hypothesized that further impairment of candidate pathways might produce limb defects similar to those seen in CdLS, and performed genetic experiments to test this. Focusing on Sonic hedgehog (Shh), Bone morphogenetic protein (Bmp), and Hox gene pathways, we show that decreasing Bmp or Hox function (but not Shh function) enhances polydactyly in Nipbl(+/-) mice, and in some cases produces novel skeletal phenotypes. However, frank limb reductions, as are seen in a subset of individuals with CdLS, do not occur, suggesting that additional signaling and/or gene regulatory pathways are involved in producing such dramatic changes. © 2016 Wiley Periodicals, Inc.

Cornelia de Lange Syndrome (CdLS) is characterized by a wide variety of structural and functional abnormalities in almost every organ system of the body. CdLS is now known to be caused by mutations that disrupt the function of the cohesin complex or its regulators, and studies of animal models and cell lines tell us that the effect of these mutations is to produce subtle yet pervasive dysregulation of gene expression. With many hundreds of mostly small gene expression changes occurring in every cell type and tissue, identifying the etiology of any particular birth defect is very challenging. Here we focus on limb abnormalities, which are commonly seen in CdLS. In the limb buds of the Nipbl-haploinsufficient mouse (Nipbl(+/-) mouse), a model for the most common form of CdLS, modest gene expression changes are observed in several candidate pathways whose disruption is known to cause limb abnormalities, yet the limbs of Nipbl(+/-) mice develop relatively normally. We hypothesized that further impairment of candidate pathways might produce limb defects similar to those seen in CdLS, and performed genetic experiments to test this. Focusing on Sonic hedgehog (Shh), Bone morphogenetic protein (Bmp), and Hox gene pathways, we show that decreasing Bmp or Hox function (but not Shh function) enhances polydactyly in Nipbl(+/-) mice, and in some cases produces novel skeletal phenotypes. However, frank limb reductions, as are seen in a subset of individuals with CdLS, do not occur, suggesting that additional signaling and/or gene regulatory pathways are involved in producing such dramatic changes. © 2016 Wiley Periodicals, Inc.

To date, only the five most posterior groups of Hox genes, Hox9-Hox13, have demonstrated loss-of-function roles in limb patterning. Individual paralog groups control proximodistal patterning of the limb skeletal elements. Hox9 genes also initiate the onset of Hand2 expression in the posterior forelimb compartment, and collectively, the posterior HoxA/D genes maintain posterior Sonic Hedgehog (Shh) expression. Here we show that an anterior Hox paralog group, Hox5, is required for forelimb anterior patterning. Deletion of all three Hox5 genes (Hoxa5, Hoxb5, and Hoxc5) leads to anterior forelimb defects resulting from derepression of Shh expression. The phenotype requires the loss of all three Hox5 genes, demonstrating the high level of redundancy in this Hox paralogous group. Further analyses reveal that Hox5 interacts with promyelocytic leukemia zinc finger biochemically and genetically to restrict Shh expression. These findings, along with previous reports showing that point mutations in the Shh limb enhancer lead to similar anterior limb defects, highlight the importance of Shh repression for proper patterning of the vertebrate limb.

To date, only the five most posterior groups of Hox genes, Hox9-Hox13, have demonstrated loss-of-function roles in limb patterning. Individual paralog groups control proximodistal patterning of the limb skeletal elements. Hox9 genes also initiate the onset of Hand2 expression in the posterior forelimb compartment, and collectively, the posterior HoxA/D genes maintain posterior Sonic Hedgehog (Shh) expression. Here we show that an anterior Hox paralog group, Hox5, is required for forelimb anterior patterning. Deletion of all three Hox5 genes (Hoxa5, Hoxb5, and Hoxc5) leads to anterior forelimb defects resulting from derepression of Shh expression. The phenotype requires the loss of all three Hox5 genes, demonstrating the high level of redundancy in this Hox paralogous group. Further analyses reveal that Hox5 interacts with promyelocytic leukemia zinc finger biochemically and genetically to restrict Shh expression. These findings, along with previous reports showing that point mutations in the Shh limb enhancer lead to similar anterior limb defects, highlight the importance of Shh repression for proper patterning of the vertebrate limb.

To date, only the five most posterior groups of Hox genes, Hox9-Hox13, have demonstrated loss-of-function roles in limb patterning. Individual paralog groups control proximodistal patterning of the limb skeletal elements. Hox9 genes also initiate the onset of Hand2 expression in the posterior forelimb compartment, and collectively, the posterior HoxA/D genes maintain posterior Sonic Hedgehog (Shh) expression. Here we show that an anterior Hox paralog group, Hox5, is required for forelimb anterior patterning. Deletion of all three Hox5 genes (Hoxa5, Hoxb5, and Hoxc5) leads to anterior forelimb defects resulting from derepression of Shh expression. The phenotype requires the loss of all three Hox5 genes, demonstrating the high level of redundancy in this Hox paralogous group. Further analyses reveal that Hox5 interacts with promyelocytic leukemia zinc finger biochemically and genetically to restrict Shh expression. These findings, along with previous reports showing that point mutations in the Shh limb enhancer lead to similar anterior limb defects, highlight the importance of Shh repression for proper patterning of the vertebrate limb.

Hox genes play a critical role in the development of the vertebrate axis and limbs, and previous studies have implicated them in the specification of positional identity, the control of growth, and the timing of differentiation. Axolotl limbs offer an opportunity to distinguish these alternatives because the sequence of skeletal differentiation is reversed along the anterior-posterior axis relative to that of other tetrapods. We report that during early limb development, expression patterns of HoxD genes in axolotls resemble those in amniotes and anuran amphibians. At later stages, the anterior boundary of Hoxd-11 expression is conserved with respect to morphological landmarks, but there is no anterior-distal expansion of the posterior domain of Hoxd-11 expression similar to that observed in mice and chicks. Since axolotls do not form an expanded paddle-like handplate prior to digit differentiation, we suggest that anterior expansion of expression in higher vertebrates is linked to the formation of the handplate, but is clearly not necessary for digit differentiation. We also show that the 5' HoxD genes are reexpressed during limb regeneration. The change in the expression pattern of Hoxd-11 during the course of regeneration is consistent with the hypothesis that the distal tip of the regenerate is specified first, followed by intercalation of intermediate levels of the pattern. Both Hoxd-8 and Hoxd-10 are expressed in non-regenerating wounds, but Hoxd-11 is specific for regeneration. It is also expressed in the posterior half of nerve-induced supernumerary outgrowths.

Hox genes play a critical role in the development of the vertebrate axis and limbs, and previous studies have implicated them in the specification of positional identity, the control of growth, and the timing of differentiation. Axolotl limbs offer an opportunity to distinguish these alternatives because the sequence of skeletal differentiation is reversed along the anterior-posterior axis relative to that of other tetrapods. We report that during early limb development, expression patterns of HoxD genes in axolotls resemble those in amniotes and anuran amphibians. At later stages, the anterior boundary of Hoxd-11 expression is conserved with respect to morphological landmarks, but there is no anterior-distal expansion of the posterior domain of Hoxd-11 expression similar to that observed in mice and chicks. Since axolotls do not form an expanded paddle-like handplate prior to digit differentiation, we suggest that anterior expansion of expression in higher vertebrates is linked to the formation of the handplate, but is clearly not necessary for digit differentiation. We also show that the 5' HoxD genes are reexpressed during limb regeneration. The change in the expression pattern of Hoxd-11 during the course of regeneration is consistent with the hypothesis that the distal tip of the regenerate is specified first, followed by intercalation of intermediate levels of the pattern. Both Hoxd-8 and Hoxd-10 are expressed in non-regenerating wounds, but Hoxd-11 is specific for regeneration. It is also expressed in the posterior half of nerve-induced supernumerary outgrowths.

Homeobox genes are important in the regulation of outgrowth and pattern formation during limb development. It is likely that homeobox genes play an equally important role during limb regeneration. We have isolated and identified 17 different homeobox-containing genes expressed by cells of regenerating axolotl limbs. Of these, nearly half of the clones represent genes belonging to the HoxA complex, which are thought to be involved in pattern formation along the proximal-distal limb axis. In this paper we report on the expression patterns of two 5' members of this complex, HoxA13 and HoxA9. These genes are expressed in cells of developing limb buds and regenerating blastemas. The pattern of expression in developing axolotl limb buds is comparable to that in mouse and chick limb buds; the expression domain of HoxA13 is more distally restricted than that of HoxA9. As in developing mouse and chick limbs, HoxA13 likely functions in the specification of distal limb structures, and HoxA9 in the specification of more proximal structures. In contrast, during regeneration, HoxA13 and HoxA9 do not follow the rule of spatial colinearity observed in developing limbs. Instead, both genes are initially expressed in the same population of stump cells, giving them a distal Hox code regardless of the level of amputation. In addition, both are reexpressed within 24 hours after amputation, suggesting that reexpression may be synchronous rather than temporally colinear. Treatment with retinoic acid alters this Hox code to that of a more proximal region by the rapid and differential downregulation of HoxA13, at the same time that expression of HoxA9 is unaffected. HoxA reexpression occurs prior to blastema formation, 24-48 hours after amputation, and is an early molecular marker for dedifferentiation.

Homeobox genes are important in the regulation of outgrowth and pattern formation during limb development. It is likely that homeobox genes play an equally important role during limb regeneration. We have isolated and identified 17 different homeobox-containing genes expressed by cells of regenerating axolotl limbs. Of these, nearly half of the clones represent genes belonging to the HoxA complex, which are thought to be involved in pattern formation along the proximal-distal limb axis. In this paper we report on the expression patterns of two 5' members of this complex, HoxA13 and HoxA9. These genes are expressed in cells of developing limb buds and regenerating blastemas. The pattern of expression in developing axolotl limb buds is comparable to that in mouse and chick limb buds; the expression domain of HoxA13 is more distally restricted than that of HoxA9. As in developing mouse and chick limbs, HoxA13 likely functions in the specification of distal limb structures, and HoxA9 in the specification of more proximal structures. In contrast, during regeneration, HoxA13 and HoxA9 do not follow the rule of spatial colinearity observed in developing limbs. Instead, both genes are initially expressed in the same population of stump cells, giving them a distal Hox code regardless of the level of amputation. In addition, both are reexpressed within 24 hours after amputation, suggesting that reexpression may be synchronous rather than temporally colinear. Treatment with retinoic acid alters this Hox code to that of a more proximal region by the rapid and differential downregulation of HoxA13, at the same time that expression of HoxA9 is unaffected. HoxA reexpression occurs prior to blastema formation, 24-48 hours after amputation, and is an early molecular marker for dedifferentiation.