The move of vertebrates to a terrestrial lifestyle required major adaptations in their locomotory apparatus and reproductive organs. While the fin-to-limb transition has received considerable attention, little is known about the developmental and evolutionary origins of external genitalia. Similarities in gene expression have been interpreted as a potential evolutionary link between the limb and genitals; however, no underlying developmental mechanism has been identified. We re-examined this question using micro-computed tomography, lineage tracing in three amniote clades, and RNA-sequencing-based transcriptional profiling. Here we show that the developmental origin of external genitalia has shifted through evolution, and in some taxa limbs and genitals share a common primordium. In squamates, the genitalia develop directly from the budding hindlimbs, or the remnants thereof, whereas in mice the genital tubercle originates from the ventral and tail bud mesenchyme. The recruitment of different cell populations for genital outgrowth follows a change in the relative position of the cloaca, the genitalia organizing centre. Ectopic grafting of the cloaca demonstrates the conserved ability of different mesenchymal cells to respond to these genitalia-inducing signals. Our results support a limb-like developmental origin of external genitalia as the ancestral condition. Moreover, they suggest that a change in the relative position of the cloacal signalling centre during evolution has led to an altered developmental route for external genitalia in mammals, while preserving parts of the ancestral limb molecular circuitry owing to a common evolutionary origin.

Mammals have extremely limited regenerative capabilities; however, axolotls are profoundly regenerative and can replace entire limbs. The mechanisms underlying limb regeneration remain poorly understood, partly because the enormous and incompletely sequenced genomes of axolotls have hindered the study of genes facilitating regeneration. We assembled and annotated a de novo transcriptome using RNA-sequencing profiles for a broad spectrum of tissues that is estimated to have near-complete sequence information for 88% of axolotl genes. We devised expression analyses that identified the axolotl orthologs of cirbp and kazald1 as highly expressed and enriched in blastemas. Using morpholino anti-sense oligonucleotides, we find evidence that cirbp plays a cytoprotective role during limb regeneration whereas manipulation of kazald1 expression disrupts regeneration. Our transcriptome and annotation resources greatly complement previous transcriptomic studies and will be a valuable resource for future research in regenerative biology.

The Mexican tetra, Astyanax mexicanus, includes interfertile surface-dwelling and cave-dwelling morphs, enabling powerful studies aimed at uncovering genes involved in the evolution of cave-associated traits. Compared to surface fish, cavefish harbor several extreme traits within their skull, such as a protruding lower jaw, a wider gape, and an increase in tooth number. These features are highly variable between individual cavefish and even across different cavefish populations.

The early limb bud consists of mesenchymal limb progenitors derived from the lateral plate mesoderm (LPM). The LPM also gives rise to the mesodermal components of the flank and neck. However, the cells at these other levels cannot produce the variety of cell types found in the limb. Taking advantage of a direct reprogramming approach, we find a set of factors (Prdm16, Zbtb16, and Lin28a) normally expressed in the early limb bud and capable of imparting limb progenitor-like properties to mouse non-limb fibroblasts. The reprogrammed cells show similar gene expression profiles and can differentiate into similar cell types as endogenous limb progenitors. The further addition of Lin41 potentiates the proliferation of the reprogrammed cells. These results suggest that these same four factors may play pivotal roles in the specification of endogenous limb progenitors.

The molecular processes underlying regeneration remain largely unknown. Several potential factors have been elucidated by focusing on the regenerative function of genes originally identified in a developmental context. A complementary approach is to consider the roles of factors involved in wound healing. Here we focus on the Thrombospondins, a family of secreted extracellular matrix proteins that have been implicated in skin wound healing in mammals. We show that a subset of Thrombospondins are expressed at distinct times and in particular cell types during axolotl limb regeneration. Our studies have revealed the axolotl orthologs of thrombospondin-1 (tsp-1) and thrombospondin-4 (tsp-4) are highly upregulated during limb regeneration in patterns both distinct and similar to larval limb development. Our data suggest that thrombospondins may be key regulators of limb regeneration in axolotl, while their activation appears to be relegated solely to wound healing in vertebrates that have lost the ability to regenerate limbs.

An adaptive variant of the human Ectodysplasin receptor, EDARV370A, is one of the strongest candidates of recent positive selection from genome-wide scans. We have modeled EDAR370A in mice and characterized its phenotype and evolutionary origins in humans. Our computational analysis suggests the allele arose in central China approximately 30,000 years ago. Although EDAR370A has been associated with increased scalp hair thickness and changed tooth morphology in humans, its direct biological significance and potential adaptive role remain unclear. We generated a knockin mouse model and find that, as in humans, hair thickness is increased in EDAR370A mice. We identify new biological targets affected by the mutation, including mammary and eccrine glands. Building on these results, we find that EDAR370A is associated with an increased number of active eccrine glands in the Han Chinese. This interdisciplinary approach yields unique insight into the generation of adaptive variation among modern humans.

The evolution of degenerate characteristics remains a poorly understood phenomenon. Only recently has the identification of mutations underlying regressive phenotypes become accessible through the use of genetic analyses. Focusing on the Mexican cave tetra Astyanax mexicanus, we describe, here, an analysis of the brown mutation, which was first described in the literature nearly 40 years ago. This phenotype causes reduced melanin content, decreased melanophore number, and brownish eyes in convergent cave forms of A. mexicanus. Crosses demonstrate non-complementation of the brown phenotype in F(2) individuals derived from two independent cave populations: Pachón and the linked Yerbaniz and Japonés caves, indicating the same locus is responsible for reduced pigmentation in these fish. While the brown mutant phenotype arose prior to the fixation of albinism in Pachón cave individuals, it is unclear whether the brown mutation arose before or after the fixation of albinism in the linked Yerbaniz/Japonés caves. Using a QTL approach combined with sequence and functional analyses, we have discovered that two distinct genetic alterations in the coding sequence of the gene Mc1r cause reduced pigmentation associated with the brown mutant phenotype in these caves. Our analysis identifies a novel role for Mc1r in the evolution of degenerative phenotypes in blind Mexican cavefish. Further, the brown phenotype has arisen independently in geographically separate caves, mediated through different mutations of the same gene. This example of parallelism indicates that certain genes are frequent targets of mutation in the repeated evolution of regressive phenotypes in cave-adapted species.

Bone morphogenetic protein (BMP) family members, including BMP2, BMP4, and BMP7, are expressed throughout limb development. BMPs have been implicated in early limb patterning as well as in the process of skeletogenesis. However, due to complications associated with early embryonic lethality, particularly for Bmp2 and Bmp4, and with functional redundancy among BMP molecules, it has been difficult to decipher the specific roles of these BMP molecules during different stages of limb development. To circumvent these issues, we have constructed a series of mouse strains lacking one or more of these BMPs, using conditional alleles in the case of Bmp2 and Bmp4 to remove them specifically from the limb bud mesenchyme. Contrary to earlier suggestions, our results indicate that BMPs neither act as secondary signals downstream of Sonic Hedghog (SHH) in patterning the anteroposterior axis nor as signals from the interdigital mesenchyme in specifying digit identity. We do find that a threshold level of BMP signaling is required for the onset of chondrogenesis, and hence some chondrogenic condensations fail to form in limbs deficient in both BMP2 and BMP4. However, in the condensations that do form, subsequent chondrogenic differentiation proceeds normally even in the absence of BMP2 and BMP7 or BMP2 and BMP4. In contrast, we find that the loss of both BMP2 and BMP4 results in a severe impairment of osteogenesis.

Despite a longstanding interest in understanding how animals adapt to environments with limited nutrients, we have incomplete knowledge of the genetic basis of metabolic evolution. The Mexican tetra, Astyanax mexicanus, is a species of fish that consists of two morphotypes; eyeless cavefish that have adapted to a low-nutrient cave environment, and ancestral river-dwelling surface fish with abundant access to nutrients. Cavefish have evolved altered blood sugar regulation, starvation tolerance, increased fat accumulation, and superior body condition. To investigate the genetic basis of cavefish metabolic evolution we carried out a quantitative trait loci (QTL) analysis in surface/cave F2 hybrids. We genetically mapped seven metabolism-associated traits in hybrids that were challenged with a nutrient restricted diet.

Individual skeletal elements of the vertebrate limbs arise through a segmentation process introducing joints in specific locations. However, the molecular pathways controlling joint formation and subsequent joint maintenance are largely unknown. In this study, we focused on SOX11, and its contribution to the regulation of GDF5, a secreted signal necessary for proper joint formation and postnatal joint homeostasis.

Normal patterning of the developing limb requires a tight restriction of Sonic hedgehog (Shh) mRNA to the posterior margin of the limb bud. While several positive and negative regulatory factors have been identified which serve to position the Shh expression domain in the distal posterior limb, these factors cannot in themselves explain the tight restriction of Shh to the posterior margin, nor can they explain the similarly tight restriction of Shh to the anterior margin when the regulatory factors are disrupted or misexpressed. We suggest that the transcription factors Tbx2 and Tbx3 are excellent candidates for positively-acting factors responsible for limiting Shh expression to the margins of the limb bud. These closely related factors are indeed expressed at the anterior and posterior limb margins over a wide range of limb bud stages. Moreover, previous reports indicate that in addition, misexpression of Tbx2 beyond the limb margin is sufficient to anteriorly expand Shh, and conversely, antagonizing Tbx2 function leads to loss of Shh. In contrast to this idea, previous models have placed Tbx2 expression downstream of Shh and Bone Morphogenetic Protein (BMP) signaling. We find, however, that Tbx2 expression is neither affected by blocking Shh signaling with cyclopamine nor by genetic removal of several BMP activities in the limb bud. To understand the true source of the positional information responsible for limiting Tbx2, Tbx3 and Shh expression to the marginal mesenchyme of the limb bud, we undertook a series of grafting and extirpation experiments, which led to the identification of the dorsal-ventral (DV) border ectoderm exclusive of the apical ectodermal ridge (AER) as a new signaling center in the limb bud. We find that maintenance of Tbx2 expression in the limb mesoderm requires proximity to the non-AER D-V border. Using chick-quail graft chimeras, we find that a graft of the non-AER D-V border ectoderm to a location on the surface of the middle of the limb bud is sufficient to induce ectopic expression of Tbx2 in underlying mesoderm. These data demonstrate that the non-AER D-V border ectoderm is necessary and sufficient for Tbx2 expression at the anterior and posterior limb margins. Similarly, we find that a graft of the non-AER D-V border can expand the domain of Shh anteriorly when grafted just anterior to the ZPA. It is notable that Tbx2 expression does not extend distally to the mesoderm underlying the AER. Moreover, we find that grafts of the AER to more proximal locations result in downregulation of Tbx2 expression, suggesting that the AER produces a negatively-acting signal opposing the activity of the non-AER DV border ectoderm. Indeed, implantation of beads soaked in fibroblast growth factor 8 (Fgf8), expressed in the AER, downregulates Tbx2 expression. The data presented here identify the non-AER border of dorsal-ventral ectoderm as a new signaling center in limb development that localizes the ZPA to the limb margin. This finding explains the tight restriction of Shh expression to the posterior margin throughout limb outgrowth as well as the tight restriction of Shh expression to the anterior margin in many mutants exhibiting preaxial polydactyly.

Developing cartilaginous and ossified skeletal anlagen is encapsulated within a membranous sheath of flattened, elongated cells called, respectively, the perichondrium and the periosteum. These periskeletal tissues are organized in distinct morphological layers that have been proposed to support distinct functions. Classical experiments, particularly those using an in vitro organ culture system, demonstrated that these tissues play important roles in regulating the differentiation of the subjacent skeletal elements. However, there has been a lack of molecular markers that would allow analysis of these interactions. To understand the molecular bases for the roles played by the periskeletal tissues, we generated microarrays from perichondrium and periosteum cDNA libraries and used them to compare the gene expression profiles of these two tissues. In situ hybridization analysis of genes identified on the microarrays revealed many unique markers for these tissues and demonstrated that the histologically distinct layers of the perichondrium and periosteum are associated with distinct molecular expression domains. Moreover our marker analysis identified new domains that had not been previously recognized as distinct within these tissues as well as a previously uncharacterized molecular domain along the lateral edges of the adjacent developing cartilage that experimental analysis showed to be dependent upon the perichondrium.

In the avian embryo, endothelial cells originate from several sources, including the lateral plate and somite mesoderm. In this study, we show that Gata transcription factors are expressed in the lateral plate and in vasculogenic regions of the avian somite and are able to promote a vascular endothelial fate when ectopically expressed in somite precursors. A fusion of GATA4 to the transcriptional activator VP16 promoted endothelium formation, indicating that GATA transcription factors promote vasculogenesis via activation of downstream targets, while a fusion of GATA4 to the transcriptional repressor engrailed repressed expression of Vascular Endothelial Growth Factor Receptor 2, a marker of endothelial precursors. These findings indicate a role for GATA transcription factors in the differentiation of the endothelium.

The gastrointestinal tract is enveloped by concentric and orthogonally aligned layers of smooth muscle; however, an understanding of the mechanisms by which these muscles become patterned and aligned in the embryo has been lacking. We find that Hedgehog acts through Bmp to delineate the position of the circumferentially oriented inner muscle layer, whereas localized Bmp inhibition is critical for allowing formation of the later-forming, longitudinally oriented outer layer. Because the layers form at different developmental stages, the muscle cells are exposed to unique mechanical stimuli that direct their alignments. Differential growth within the early gut tube generates residual strains that orient the first layer circumferentially, and when formed, the spontaneous contractions of this layer align the second layer longitudinally. Our data link morphogen-based patterning to mechanically controlled smooth muscle cell alignment and provide a mechanistic context for potentially understanding smooth muscle organization in a wide variety of tubular organs.

Identifying the genetic factors that underlie complex traits is central to understanding the mechanistic underpinnings of evolution. Cave-dwelling Astyanax mexicanus populations are well adapted to subterranean life and many populations appear to have evolved troglomorphic traits independently, while the surface-dwelling populations can be used as a proxy for the ancestral form. Here we present a high-resolution, chromosome-level surface fish genome, enabling the first genome-wide comparison between surface fish and cavefish populations. Using this resource, we performed quantitative trait locus (QTL) mapping analyses and found new candidate genes for eye loss such as dusp26. We used CRISPR gene editing in A. mexicanus to confirm the essential role of a gene within an eye size QTL, rx3, in eye formation. We also generated the first genome-wide evaluation of deletion variability across cavefish populations to gain insight into this potential source of cave adaptation. The surface fish genome reference now provides a more complete resource for comparative, functional and genetic studies of drastic trait differences within a species.

We have cloned a chick homologue of Drosophila dachshund (dac), termed Dach1. Dach1 is the orthologue of mouse and human Dac/Dach (hereafter referred to as Dach1). We show that chick Dach1 is expressed in a variety of sites during embryonic development, including the eye and ear. Previous work has demonstrated the existence of a functional network and genetic regulatory hierarchy in Drosophila in which eyeless (ey, the Pax6 orthologue), eyes absent (eya), and dac operate together to regulate Drosophila eye development, and that ey regulates the expression of eya and dac. We find that in the developing eye of both chick and mouse, expression domains of Dach1 overlap with those of Pax6, a gene required for normal eye development. Similarly, in the developing ear of both mouse and chick, Dach1 expression overlaps with the expression of another Pax gene, Pax2. In the mouse, Dach1 expression in the developing ear also overlaps with the expression of Eya1 (an eya homologue). Both Pax2 and Eya1 are required for normal ear development. Our expression studies suggest that the Drosophila Pax-eya-dac regulatory network may be evolutionarily conserved such that Pax genes, Eya1, and Dach1 may function together in vertebrates to regulate neural development. To address the further possibility that a regulatory hierarchy exists between Pax, Eya, and Dach genes, we have examined the expression of mouse Dach1 in Pax6, Pax2 and Eya1 mutant backgrounds. Our results indicate that Pax6, Pax2, and Eya1 do not regulate Dach1 expression through a simple linear hierarchy.

Anatomical proportions are robustly maintained in individuals that vary enormously in size, both within a species and between members of related taxa. However, the mechanisms underlying scaling are still poorly understood. We have examined this phenomenon in the context of the patterning of the ventral neural tube in response to a gradient of the morphogen Sonic hedgehog (SHH) in the chick and zebra finch, two species that differ in size during the time of neural tube patterning. We find that scaling is achieved, at least in part, by altering the sensitivity of the target cells to SHH and appears to be achieved by modulating the ratio of the repressive and activating transcriptional regulators, GLI2 and GLI3. This mechanism contrasts with previous experimental and theoretical analyses of morphogenic scaling that have focused on compensatory changes in the morphogen gradient itself.

During somite development, a fibroblast growth factor (FGF) signal secreted from the myotome induces formation of a scleraxis (Scx)-expressing tendon progenitor population in the sclerotome, at the juncture between the future lineages of muscle and cartilage. While overexpression studies show that the entire sclerotome is competent to express Scx in response to FGF signaling, the normal Scx expression domain includes only the anterior and posterior dorsal sclerotome. To understand the molecular basis for this restriction, we examined the expression of a set of genes involved in FGF signaling and found that several members of the Fgf8 synexpression group are co-expressed with Scx in the dorsal sclerotome. Of particular interest were the Ets transcription factors Pea3 and Erm, which function as transcriptional effectors of FGF signaling. We show here that transcriptional activation by Pea3 and Erm in response to FGF signaling is both necessary and sufficient for Scx expression in the somite, and propose that the domain of the somitic tendon progenitors is regulated both by the restricted expression of Pea3 and Erm, and by the precise spatial relationship between these Ets transcription factors and the FGF signal originating in the myotome.

We have investigated the structural basis by which the counterclockwise direction of the amniote gut is established. The chirality of midgut looping is determined by left-right asymmetries in the cellular architecture of the dorsal mesentery, the structure that connects the primitive gut tube to the body wall. The mesenchymal cells of the dorsal mesentery are more condensed on the left side than on the right and, additionally, the overlying epithelium on the left side exhibits a columnar morphology, in contrast to a cuboidal morphology on the right. These properties are instructed by a set of transcription factors: Pitx2 and Isl1 specifically expressed on the left side, and Tbx18 expressed on the right, regulated downstream of the secreted protein Nodal which is present exclusively on the left side. The resultant differences in cellular organization cause the mesentery to assume a trapezoidal shape, tilting the primitive gut tube leftward.

Axolotls are poised to become the premiere model system for studying vertebrate appendage regeneration. However, very few molecular tools exist for studying crucial cell lineage relationships over regeneration or for robust and sustained misexpression of genetic elements to test their function. Furthermore, targeting specific cell types will be necessary to understand how regeneration of the diverse tissues within the limb is accomplished. We report that pseudotyped, replication-incompetent retroviruses can be used in axolotls to permanently express markers or genetic elements for functional study. These viruses, when modified by changing their coat protein, can infect axolotl cells only when they have been experimentally manipulated to express the receptor for that coat protein, thus allowing for the possibility of targeting specific cell types. Using viral vectors, we have found that progenitor populations for many different cell types within the blastema are present at all stages of limb regeneration, although their relative proportions change with time.