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In many animal species with a bilateral symmetry, Hox genes are clustered either at one or at several genomic loci. This organization has a functional relevance, as the transcriptional control applied to each gene depends upon its relative position within the gene cluster. It was previously noted that vertebrate Hox clusters display a much higher level of genomic organization than their invertebrate counterparts. The former are always more compact than the latter, they are generally devoid of repeats and of interspersed genes, and all genes are transcribed by the same DNA strand, suggesting that particular factors constrained these clusters toward a tighter structure during the evolution of the vertebrate lineage. Here, we investigate the importance of uniform transcriptional orientation by engineering several alleles within the HoxD cluster, such as to invert one or several transcription units, with or without a neighboring CTCF site. We observe that the association between the tight structure of mammalian Hox clusters and their regulation makes inversions likely detrimental to the proper implementation of this complex genetic system. We propose that the consolidation of Hox clusters in vertebrates, including transcriptional polarity, evolved in conjunction with the emergence of global gene regulation via the flanking regulatory landscapes, to optimize a coordinated response of selected subsets of target genes in cis .

BackgroundDuring development, complex organ patterns emerge through the precise temporal and spatial specification of different cell types. On an evolutionary timescale, these patterns can change, resulting in morphological diversification. It is generally believed that homologous anatomical structures are built – largely – by homologous cell types. However, whether a common evolutionary origin of such cell types is always reflected in the conservation of their intrinsic transcriptional specification programs is less clear.ResultsHere, using a paradigm of morphological diversification, the tetrapod limb, and singlecell RNA-sequencing data from two distantly related species, chicken and mouse, we assessed the transcriptional dynamics of homologous cell types during embryonic patterning. We developed a user-friendly bioinformatics workflow to detect gene co-expression modules and test for their conservation across developmental stages and species boundaries. Using mouse limb data as reference, we identified 19 gene co-expression modules with varying tissue or cell type-restricted activities. Testing for co-expression conservation revealed modules with high evolutionary turnover, while others seemed maintained – to different degrees, in module make-up, density or connectivity – over developmental and evolutionary timescales.ConclusionsWe present an approach to identify evolutionary and developmental dynamics in gene co-expression modules during patterning-relevant stages of homologous cell type specification.

The tetrapod limb has long served as a paradigm to study vertebrate pattern formation. During limb morphogenesis, a number of distinct tissue types are patterned and subsequently must be integrated to form coherent functional units. For example, the musculoskeletal apparatus of the limb requires the coordinated development of the skeletal elements, connective tissues, muscles and nerves. Here, using light-sheet microscopy and 3D-reconstructions, we concomitantly follow the developmental emergence of nerve and muscle patterns in chicken wings and legs, two appendages with highly specialized locomotor outputs. Despite a comparable flexor/extensor-arrangement of their embryonic muscles, wings and legs show a rotated innervation pattern for their three main motor nerve branches. To test the functional implications of these distinct neuromuscular topologies, we challenge their ability to adapt and connect to an experimentally altered skeletal pattern in the distal limb, the autopod. Our results show that, unlike autopod muscle groups, motor nerves are unable to fully adjust to a changed peripheral organisation, potentially constrained by their original projection routes. As the autopod has undergone substantial morphological diversifications over the course of tetrapod evolution, our results have implications for the coordinated modification of the distal limb musculoskeletal apparatus, as well as for our understanding of the varying degrees of motor functionality associated with human hand and foot malformations.

BackgroundThrough 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.ResultsUsing 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.ConclusionsWe 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.

When positioned into the integrin α-6 gene, an Hoxd9lacZ reporter transgene displayed parental imprinting in mouse embryos. While the expression from the paternal allele was comparable with patterns seen for the same transgene when present at the neighboring HoxD locus, almost no signal was scored at this integration site when the transgene was inherited from the mother, although the Itga6 locus itself is not imprinted. The transgene exhibited maternal allele-specific DNA hypermethylation acquired during oogenesis, and its expression silencing was reversible on passage through the male germ line. Histone modifications also corresponded to profiles described at known imprinted loci. Chromosome conformation analyses revealed distinct chromatin microarchitectures, with a more compact structure characterizing the maternally inherited repressed allele. Such genetic analyses of well-characterized transgene insertions associated with a de novo-induced parental imprint may help us understand the molecular determinants of imprinting.

Faithful expression of Hox genes in both time and space is essential for proper patterning of the primary body axis. Transgenic approaches in vertebrates have suggested that this collinear activation process is regulated in a largely gene cluster-autonomous manner. In contrast, more recently co-opted expression specificities, required in other embryonic structures, depend upon long-range enhancer sequences acting from outside the gene clusters. This regulatory dichotomy was recently questioned, since gene activation along the trunk seems to be partially regulated by signals located outside of the cluster. We investigated these alternative regulatory strategies by engineering a large inversion that precisely separates the murine HoxD complex from its centromeric neighborhood. Mutant animals displayed posterior transformations along with subtle deregulations of Hoxd genes, indicating an impact of the centromeric landscape on the fine-tuning of Hoxd gene expression. Proximal limbs were also affected, suggesting that this ‘landscape effect’ is generic and impacts upon regulatory mechanisms of various qualities and evolutionary origins.

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.

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. 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 attention1,2, 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 genitals3,4,5,6; 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., Author Summary Hox genes are essential for the proper organization of structures along the developing vertebrate body axis. These genes must be activated at a precise time and their premature transcription is deleterious to the organism. Early on, Hox gene clusters are covered by Polycomb Repressive protein Complexes (PRCs), which help keep these genes silent. However, the mechanism(s) that selectively recruit PRCs to these particular genomic loci remains elusive. We have used a collection of mutant mice carrying a set of deletions inside and outside the HoxD cluster to try and detect the presence of any DNA sequence of particular importance in this mechanism. We conclude that a range of low affinity sequences synergize to recruit PRCs over the gene cluster, which makes this process very robust and resistant to genetic perturbations.

The study of chromatin and its regulators is key to understanding and manipulating transcription. We previously exploited the Krüppel-associated box (KRAB) transcriptional repressor domain, present in hundreds of vertebrate-specific zinc finger proteins, to assess the effect of its binding to gene bodies. These experiments revealed that the ectopic and doxycycline (dox)-controlled tet repressor KRAB fusion protein (tTRKRAB) can induce reversible and long-range silencing of cellular promoters. Here, we extend this system to in vivo applications and use tTRKRAB to achieve externally controllable repression of an endogenous mouse locus. We employed lentiviral-mediated transgenesis with promoterless TetO-containing gene traps to engineer a mouse line where the endogenous kinesin family member 2A (Kif2A) promoter drives a YFP reporter gene. When these mice were crossed to animals expressing the TetO-binding tTRKRAB repressor, this regulator was recruited to the Kif2A locus, and YFP expression was reduced. This effect was reversed when dox was given to embryos or adult mice, demonstrating that the cellular Kif2A promoter was only silenced upon repressor binding. Molecular analyses confirmed that tTRKRAB induced transcriptional repression through the spread of H3K9me3-containing heterochromatin, without DNA methylation of the trapped Kif2A promoter. Therefore, we demonstrate that targeting of tTRKRAB to a gene body in vivo results in reversible transcriptional repression through the spreading of facultative heterochromatin. This finding not only sheds light on KRAB-mediated transcriptional processes, but also suggests approaches for the externally controllable and reversible modulation of chromatin and transcription in vivo.

The importance of Hox genes in the specification of neuronal fates in the spinal cord has long been recognized. However, the transcriptional controls underlying their collinear expression domains remain largely unknown. Here we show in mice that the correspondence between the physical order of Hoxd genes and their rostral expression boundaries, although respecting spatial collinearity, does not display a fully progressive distribution. Instead, two major anteroposterior boundaries are detected, coinciding with the functional subdivision of the spinal cord. Tiling array analyses reveal two distinct blocks of transcription, regulated independently from one another, that define the observed expression boundaries. Targeted deletions in vivo that remove the genomic fragments separating the two blocks induce ectopic expression of posterior genes. We further evaluate the independent regulatory potential and transcription profile of each gene locus by a tiling array approach using a contiguous series of transgenes combined with locus-specific deletions. Our work uncovers a bimodal type of HoxD spatial collinearity in the developing spinal cord that relies on two separate ‘enhancer mini-hubs’ to ensure correct Hoxd gene expression levels while maintaining their appropriate anteroposterior boundaries.

The evolution of vertebrate genomes was accompanied by an astounding increase in the complexity of their regulatory modalities. Genetic redundancy resulting from large-scale genome duplications at the base of the chordate tree was repeatedly exploited by the functional redeployment of paralogous genes via innovations in their regulatory circuits. As a paradigm of such regulatory evolution, we have extensively studied those control mechanisms at work in-cis over vertebrate Hox gene clusters. Here, we review the portfolio of genetic strategies that have been developed to tackle the intricate relationship between genomic topography and the transcriptional activities in this gene family, and we describe some of the mechanistic insights we gained by using the HoxD cluster as an example. We discuss the high heuristic value of this system in our general understanding of how changes in transcriptional regulation can diversify gene function and thereby fuel morphological evolution.

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., Author Summary Hox genes encode proteins that control embryonic development along the head-to-tail axis. These genes are clustered in one site on the chromosome and their respective positions within the cluster determine their time and place of activation. Here, by using a large set of targeted mutations disturbing the integrity of the gene cluster, we show that the spatial organization of expression domains does not directly depend upon the timing of activation as was previously suggested. This uncoupling between space and time in the regulation of these Hox genes coincides with the existence of two major phases of regulation. The first is time-dependent and involves global regulatory influences, located outside the gene cluster, whereas the second relies upon more local regulatory elements, likely interspersed between the genes, inside the cluster. These results provide the bases for future analyses of collinear mechanisms and indicate that different types of collinearities are not necessarily related, neither in function, nor in their evolutionary histories.