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Background Through precise implementation of distinct cell type specification programs, differentially regulated in both space and time, complex patterns emerge during organogenesis. Thanks to its easy experimental accessibility, the developing chicken limb has long served as a paradigm to study vertebrate pattern formation. Through decades’ worth of research, we now have a firm grasp on the molecular mechanisms driving limb formation at the tissue-level. However, to elucidate the dynamic interplay between transcriptional cell type specification programs and pattern formation at its relevant cellular scale, we lack appropriately resolved molecular data at the genome-wide level. Here, making use of droplet-based single-cell RNA-sequencing, we catalogue the developmental emergence of distinct tissue types and their transcriptome dynamics in the distal chicken limb, the so-called autopod, at cellular resolution. Results Using single-cell RNA-sequencing technology, we sequenced a total of 17,628 cells coming from three key developmental stages of chicken autopod patterning. Overall, we identified 23 cell populations with distinct transcriptional profiles. Amongst them were small, albeit essential populations like the apical ectodermal ridge, demonstrating the ability to detect even rare cell types. Moreover, we uncovered the existence of molecularly distinct sub-populations within previously defined compartments of the developing limb, some of which have important signaling functions during autopod pattern formation. Finally, we inferred gene co-expression modules that coincide with distinct tissue types across developmental time, and used them to track patterning-relevant cell populations of the forming digits. Conclusions We provide a comprehensive functional genomics resource to study the molecular effectors of chicken limb patterning at cellular resolution. Our single-cell transcriptomic atlas captures all major cell populations of the developing autopod, and highlights the transcriptional complexity in many of its components. Finally, integrating our data-set with other single-cell transcriptomics resources will enable researchers to assess molecular similarities in orthologous cell types across the major tetrapod clades, and provide an extensive candidate gene list to functionally test cell-type-specific drivers of limb morphological diversification.

The principle of homology is central to conceptualizing the comparative aspects of morphological evolution. The distinctions between homologous or non-homologous structures have become blurred, however, as modern evolutionary developmental biology (evo-devo) has shown that novel features often result from modification of pre-existing developmental modules, rather than arising completely de novo. With this realization in mind, the term ‘deep homology’ was coined, in recognition of the remarkably conserved gene expression during the development of certain animal structures that would not be considered homologous by previous strict definitions. At its core, it can help to formulate an understanding of deeper layers of ontogenetic conservation for anatomical features that lack any clear phylogenetic continuity. Here, we review deep homology and related concepts in the context of a gene expression-based homology discussion. We then focus on how these conceptual frameworks have profited from the recent rise of high-throughput next-generation sequencing. These techniques have greatly expanded the range of organisms amenable to such studies. Moreover, they helped to elevate the traditional gene-by-gene comparison to a transcriptome-wide level. We will end with an outlook on the next challenges in the field and how technological advances might provide exciting new strategies to tackle these questions. This article is part of the themed issue ‘Evo-devo in the genomics era, and the origins of morphological diversity’.

Cell type repertoires have expanded extensively in metazoan animals, with some clade-specific cells being crucial to evolutionary success. A prime example are the skeletogenic cells of vertebrates. Depending on anatomical location, these cells originate from three different precursor lineages, yet they converge developmentally towards similar cellular phenotypes. Furthermore, their ‘skeletogenic competency’ arose at distinct evolutionary timepoints, thus questioning to what extent different skeletal body parts rely on truly homologous cell types. Here, we investigate how lineage-specific molecular properties are integrated at the gene regulatory level, to allow for skeletogenic cell fate convergence. Using single-cell functional genomics, we find that distinct transcription factor profiles are inherited from the three precursor states and incorporated at lineage-specific enhancer elements. This lineage-specific regulatory logic suggests that these regionalized skeletogenic cells are distinct cell types, rendering them amenable to individualized selection, to define adaptive morphologies and biomaterial properties in different parts of the vertebrate skeleton. Vertebrate skeletogenic cells of different embryonic origins are distinct cell types, based on their lineage-specific gene regulatory logic that specifies them, and the potential for individualized evolutionary trajectories resulting therefrom.

It has been known for some time that limbs share at least some of their molecular patterning mechanism with external genitalia; here, this connection is examined in a variety of species, revealing that once-shared developmental trajectories could help to explain the observed patterning similarities. Embryonic origins of external genitalia It has been known for some time that limbs share at least some of their molecular patterning mechanisms with external genitalia. Clifford Tabin and colleagues examine the connection in a variety of amniotes (land vertebrates excluding amphibia) and show that the connection is more varied than one might imagine. In squamates (snakes and lizards) for example, the external genitalia are made from the tissues that the hindlimbs (or rudiments thereof) originate from, whereas in mammals they are made from tail-bud tissue. The determinant, it seems, is the relative position on the body axis of the cloaca — the primitively conjoint opening of urinary, digestive and reproductive tracts — which is an important 'organizing centre' in development. 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 1 , 2 , 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 3 , 4 , 5 , 6 ; 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.

Polycomb group (PcG) proteins are essential for the repression of key factors during early development. In Drosophila, the polycomb repressive complexes (PRC) associate with defined polycomb response DNA elements (PREs). In mammals, however, the mechanisms underlying polycomb recruitment at targeted loci are poorly understood. We have used an in vivo approach to identify DNA sequences of importance for the proper recruitment of polycomb proteins at the HoxD locus. We report that various genomic re-arrangements of the gene cluster do not strongly affect PRC2 recruitment and that relatively small polycomb interacting sequences appear necessary and sufficient to confer polycomb recognition and targeting to ectopic loci. In addition, a high GC content, while not sufficient to recruit PRC2, may help its local spreading. We discuss the importance of PRC2 recruitment over Hox gene clusters in embryonic stem cells, for their subsequent coordinated transcriptional activation during development.

The tetrapod limb has long served as a paradigm to study vertebrate pattern formation and evolutionary diversification. The distal part of the limb, the so-called autopod, is of particular interest in this regard, given the numerous modifications in both its morphology and behavioral motor output. While the underlying alterations in skeletal form have received considerable attention, much less is known about the accompanying changes in the neuromuscular system. However, modifications in the skeleton need to be properly integrated with both muscle and nerve patterns, to result in a fully functional limb. This task is further complicated by the distinct embryonic origins of the three main tissue types involved—skeleton, muscles and nerves—and, accordingly, how they are patterned and connected with one another during development. To evaluate the degree of regulative crosstalk in this complex limb patterning process, here we analyze the developing limb neuromuscular system of Silkie breed chicken. These animals display a preaxial polydactyly, due to a polymorphism in the limb regulatory region of the Sonic Hedgehog gene. Using lightsheet microscopy and 3D-reconstructions, we investigate the neuromuscular patterns of extra digits in Silkie wings and legs, and compare our results to Retinoic Acid-induced polydactylies. Contrary to previous findings, Silkie autopod muscle patterns do not adjust to alterations in the underlying skeletal topology, while nerves show partial responsiveness. We discuss the implications of tissue-specific sensitivities to global limb patterning cues for our understanding of the evolution of novel forms and functions in the distal tetrapod limb.

During development of the vertebrate body axis, Hox genes are transcribed sequentially, in both time and space, following their relative positions within their genomic clusters. Analyses of animal genomes support the idea that Hox gene clustering is essential for coordinating the various times of gene activations. However, the eventual collinear ordering of the gene specific transcript domains in space does not always require genomic clustering. We analyzed these complex regulatory relationships by using mutant alleles at the mouse HoxD locus, including one that splits the cluster into two pieces. We show that both positive and negative regulatory influences, located on either side of the cluster, control an early phase of collinear expression in the trunk. Interestingly, this early phase does not systematically impact upon the subsequent expression patterns along the main body axis, indicating that the mechanism underlying temporal collinearity is distinct from those acting during the second phase. We discuss the potential functions and evolutionary origins of these mechanisms, as well as their relationship with similar processes at work during limb development.

Pax transcription factors are involved in a variety of developmental processes in bilaterians, including eye development, a role typically assigned to Pax-6. Although no true Pax-6 gene has been found in nonbilateral animals, some jellyfish have eyes with complex structures. In the cubozoan jellyfish Tripedalia, Pax-B, an ortholog of vertebrate Pax-2/5/8, had been proposed as a regulator of eye development. Here we have isolated three Pax genes (Pax-A, Pax-B, and Pax-E) from Cladonema radiatum, a hydrozoan jellyfish with elaborate eyes. Cladonema Pax-A is strongly expressed in the retina, whereas Pax-B and Pax-E are highly expressed in the manubrium, the feeding and reproductive organ. Misexpression of Cladonema Pax-A induces ectopic eyes in Drosophila imaginal discs, whereas Pax-B and Pax-E do not. Furthermore, Cladonema Pax-A paired domain protein directly binds to the 5' upstream region of eye-specific Cladonema opsin genes, whereas Pax-B does not. Our data suggest that Pax-A, but not Pax-B or Pax-E, is involved in eye development and/or maintenance in Cladonema. Phylogenetic analysis indicates that Pax-6, Pax-B, and Pax-A belong to different Pax subfamilies, which diverged at the latest before the Cnidaria–Bilateria separation. We argue that our data, showing the involvement of Pax genes in hydrozoan eye development as in bilaterians, supports the monophyletic evolutionary origin of all animal eyes. We then propose that during the early evolution of animals, distinct classes of Pax genes, which may have played redundant roles at that time, were flexibly deployed for eye development in different animal lineages.

The emergence of Vertebrata was accompanied by two rounds of whole-genome duplications. This enabled paralogous genes to acquire novel functions with high evolutionary potential, a process suggested to occur mostly by changes in gene regulation, rather than in protein sequences. In the case of Hox gene clusters, such duplications favored the appearance of distinct global regulations. To assess the impact of such \"regulatory evolution\" upon neo-functionalization, we developed PANTHERE (PAN-genomic Translocation for Heterologous Enhancer RE-shuffling) to bring the entire megabase-scale HoxD regulatory landscape in front of the HoxC gene cluster via a targeted translocation in vivo. At this chimeric locus, Hoxc genes could both interpret this foreign regulation and functionally substitute for their Hoxd counterparts. Our results emphasize the importance of evolving regulatory modules rather than their target genes in the process of neo-functionalization and offer a genetic tool to study the complexity of the vertebrate regulatory genome.

The principle of homology is central to conceptualizing the comparative aspects of morphological evolution. The distinctions between homologous or non-homologous structures have become blurred, however, as modern evolutionary developmental biology (evo-devo) has shown that novel features often result from modification of pre-existing developmental modules, rather than arising completely de novo. With this realization in mind, the term 'deep homology' was coined, in recognition of the remarkably conserved gene expression during the development of certain animal structures that would not be considered homologous by previous strict definitions. At its core, it can help to formulate an understanding of deeper layers of ontogenetic conservation for anatomical features that lack any clear phylogenetic continuity. Here, we review deep homology and related concepts in the context of a gene expression-based homology discussion. We then focus on how these conceptual frameworks have profited from the recent rise of high-throughput next-generation sequencing. These techniques have greatly expanded the range of organisms amenable to such studies. Moreover, they helped to elevate the traditional gene-by-gene comparison to a transcriptome-wide level. We will end with an outlook on the next challenges in the field and how technological advances might provide exciting new strategies to tackle these questions. This article is part of the themed issue 'Evo-devo in the genomics era, and the origins of morphological diversity'.