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Repeating patterns of synovial joints are a highly conserved feature of articulated digits, with variations in joint number and location giving rise to a diverse range of digit morphologies and limb functions across the tetrapod clade. During development, joints form iteratively within the growing digit ray, as a population of distal progenitors alternately specifies joint and phalanx cell fates to segment the digit into distinct elements. Whilst numerous molecular pathways have been implicated in this fate choice, it remains unclear how they give rise to a repeating pattern. Here, using single cell RNA-sequencing and spatial gene expression profiling, we investigate the transcriptional dynamics of interphalangeal joint specificationin vivo. Combined with mathematical modelling, we predict that interactions within the BMP signaling pathway – between the ligand GDF5, the inhibitor NOG, and the intracellular effector pSMAD – result in a self-organizing Turing system that forms periodic joint patterns. Our model is able to recapitulate the spatiotemporal gene expression dynamics observedin vivo, as well as phenocopy digit malformations caused by BMP pathway perturbations. By contrastingin silicosimulations within vivomorphometrics of two morphologically distinct digits, we show how changes in signaling parameters and growth dynamics can result in variations in the size and number of phalanges. Together, our results reveal a self-organizing mechanism that underpins tetrapod digit patterning and its evolvability, and, more broadly, illustrate how Turing systems based on a single molecular pathway may generate complex repetitive patterns in a wide variety of organisms.

Motor neurons in the vertebrate spinal cord have long served as a paradigm to study the transcriptional logic of cell type specification and differentiation. At limb levels, pool-specific transcriptional signatures first restrict innervation to only one particular muscle in the periphery, and get refined, once muscle connection has been established. Accordingly, to study the transcriptional dynamics and specificity of the system, a method for establishing muscle target-specific motor neuron transcriptomes would be required.; To investigate target-specific transcriptional signatures of single motor neurons, here we combine ex-ovo retrograde axonal labeling in mid-gestation chicken embryos with manual isolation of individual fluorescent cells and Smart-seq2 single-cell RNA-sequencing. We validate our method by injecting the dorsal extensor metacarpi radialis and ventral flexor digiti quarti wing muscles and harvesting a total of 50 fluorescently labeled cells, in which we detect up to 12,000 transcribed genes. Additionally, we present visual cues and cDNA metrics predictive of sequencing success.; Our method provides a unique approach to study muscle target-specific motor neuron transcriptomes at a single-cell resolution. We anticipate that our method will provide key insights into the transcriptional logic underlying motor neuron pool specialization and proper neuromuscular circuit assembly and refinement.

SUMMARYThe early limb bud consists of mesenchymal progenitors (limb progenitors) derived from the lateral plate mesoderm (LPM) that produce most of the tissues of the mature limb bud. The LPM also gives rise to the mesodermal components of the trunk, flank and neck. However, the mesenchymal cells generated at these other axial levels cannot produce the variety of cell types found in the limb bud, nor can they be directed to form a patterned appendage-like structure, even when placed in the context of the signals responsible for organizing the limb bud. Here, by taking advantage of a direct reprogramming approach, we find a set of factors (Prdm16, Zbtb16, and Lin28) normally expressed in the early limb bud, that are capable of imparting limb progenitor-like properties to non-limb fibroblasts. Cells reprogrammed by these factors show similar gene expression profiles, and can differentiate into similar cell types, as endogenous limb progenitors. The further addition of Lin41 potentiates proliferation of the reprogrammed cells while suppressing differentiation. These results suggest that these same four key factors may play pivotal roles in the specification of endogenous limb progenitors.

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.

Transcriptomic signatures based on cellular mRNA expression profiles can be used to categorize cell types and states. Yet whether different functional groups of genes perform better or worse in this process remains largely unexplored. Here we test the core matrisome – that is, all genes coding for structural proteins of the extracellular matrix – for its ability to delineate distinct cell types in embryonic single-cell RNA-sequencing (scRNA-seq) data. We show that even though expressed core matrisome genes correspond to less than 2% of an entire cellular transcriptome, their RNA expression levels suffice to recapitulate essential aspects of cell type-specific clustering. Notably, using scRNA-seq data from the embryonic limb, we demonstrate that core matrisome gene expression outperforms random gene subsets of similar sizes and can match and exceed the predictive power of transcription factors. While transcription factor signatures generally perform better in predicting cell types at early stages of chicken and mouse limb development, i.e., when cells are less differentiated, the information content of the core matrisome signature increases in more differentiated cells. Moreover, using cross-species analyses, we show that these cell type-specific signatures are evolutionarily conserved. Our findings suggest that each cell type produces its own unique extracellular matrix, or matreotype, which becomes progressively more refined and cell type-specific as embryonic tissues mature., Matrix Biology Plus, 10, ISSN:2590-0285

Fifty years ago, Lewis Wolpert introduced the concept of "positional information" to explain how patterns form in a multicellular embryonic field. Using morphogen gradients, whose continuous distributions of positional values are discretized via thresholds into distinct cellular states, he provided, at the theoretical level, an elegant solution to the "French Flag problem." In the intervening years, many experimental studies have lent support to Wolpert's ideas. However, the embryonic patterning of highly repetitive morphological structures, as often occurring in nature, can reveal limitations in the strict implementation of his initial theory, given the number of distinct threshold values that would have to be specified. Here, we review how positional information is complemented to circumvent these inadequacies, to accommodate tissue growth and pattern periodicity. In particular, we focus on functional anatomical assemblies composed of such structures, like the vertebrate spine or tetrapod digits, where the resulting segmented architecture is intrinsically linked to periodic pattern formation and unidirectional growth. These systems integrate positional information and growth with additional patterning cues that, we suggest, increase robustness and evolvability. We discuss different experimental and theoretical models to study such patterning systems, and how the underlying processes are modulated over evolutionary timescales to enable morphological diversification.

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.

In many animal species with a bilateral symmetry,Hoxgenes 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 vertebrateHoxclusters 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 towards a tighter structure during the evolution of the vertebrate lineage. Here we investigate the importance of uniform transcriptional orientation by engineering several alleles within theHoxDcluster such as to invert one or several transcription unit(s), with or without a neighboring CTCF site. We observe that the association between the tight structure of mammalianHoxclusters and their regulation makes inversions likely detrimental to the proper implementation of this complex genetic system. We propose that the consolidation ofHoxclusters in vertebrates, including transcriptional polarity, evolved in conjunction with the emergence of global gene regulationviathe flanking regulatory landscapes, to optimize a coordinated response of selected subsets of target genes incis.

Androgen receptor (AR) signaling is a key driver of prostate cancer (PC). While androgen-deprivation therapy is transiently effective in advanced disease, tumors often progress to a lethal castration-resistant state (CRPC). We show that recurrent PC-driver mutations in speckle-type POZ protein (SPOP) stabilize the TRIM24 protein, which promotes proliferation under low androgen conditions. TRIM24 augments AR signaling, and AR and TRIM24 co-activated genes are significantly upregulated in CRPC. Expression of TRIM24 protein increases from primary PC to CRPC, and both TRIM24 protein levels and the AR/TRIM24 gene signature predict disease recurrence. Analyses in CRPC cells reveal that the TRIM24 bromodomain and the AR-interacting motif are essential to support proliferation. These data provide a rationale for therapeutic TRIM24 targeting in SPOP mutant and CRPC patients.

Sex chromosomes differentiated from different ancestral autosomes in various vertebrate lineages. Here, we trace the functional evolution of the XY Chromosomes of the green anole lizard (Anolis carolinensis), on the basis of extensive high-throughput genome, transcriptome and histone modification sequencing data and revisit dosage compensation evolution in representative mammals and birds with substantial new expression data. Our analyses show that Anolis sex chromosomes represent an ancient XY system that originated at least ≈160 million years ago in the ancestor of Iguania lizards, shortly after the separation from the snake lineage. The age of this system approximately coincides with the ages of the avian and two mammalian sex chromosomes systems. To compensate for the almost complete Y Chromosome degeneration, X-linked genes have become twofold up-regulated, restoring ancestral expression levels. The highly efficient dosage compensation mechanism of Anolis represents the only vertebrate case identified so far to fully support Ohno's original dosage compensation hypothesis. Further analyses reveal that X up-regulation occurs only in males and is mediated by a male-specific chromatin machinery that leads to global hyperacetylation of histone H4 at lysine 16 specifically on the X Chromosome. The green anole dosage compensation mechanism is highly reminiscent of that of the fruit fly, Drosophila melanogaster Altogether, our work unveils the convergent emergence of a Drosophila-like dosage compensation mechanism in an ancient reptilian sex chromosome system and highlights that the evolutionary pressures imposed by sex chromosome dosage reductions in different amniotes were resolved in fundamentally different ways. We also acknowledge computational support by the state of Baden-Württemberg through bwHPC and the German Research Foundation (DFG) through grant INST 35/1134-1 FUGG; in particular, our computational work on the bwForCluster was supported by M. Baumann and S. Richling from the Heidelberg University Computational Center (Universitätsrechenzentrum, URZ). P.T. acknowledges generous support from Prof. Cliff Tabin. The overall research project was supported by grants from the European Research Council (Grant: 615253, OntoTransEvol) and Swiss National Science Foundation (Grants: 146474) to H.K., as well as the CONACyT-SEP Basic Science grant (No. 254240) awarded to D.C.

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.

Critical steps in forming the vertebrate limb include the positioning of digits and the positioning of joints within each digit. Recent studies have proposed that the iterative series of digits is established by a Turing-like mechanism generating stripes of chondrogenic domains. However, re-examination of available data suggest that digits are actually patterned as evenly spaced spots, not stripes, which then elongate into rod-shaped digit rays by incorporating new cells at their tips. Moreover, extension of the digit rays and the patterning of the joints occur simultaneously at the distal tip, implying that an integrated model is required to fully understand these processes.

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.

The shape of the human face and skull is largely genetically determined, but the genetic drivers of craniofacial morphology remain poorly understood. Here we used a combination of epigenomic profiling, in vivo characterization of candidate enhancer sequences in transgenic mice, and targeted deletion experiments to examine the role of distant-acting enhancers in craniofacial development. We identified complex regulatory landscapes, consisting of enhancers that drive a remarkable spatial complexity of developmental expression patterns. Deletion of individual craniofacial enhancers from the mouse genome resulted in significant alterations of craniofacial shape, demonstrating their functional importance in defining face and skull morphology. These results demonstrate that enhancers play a pervasive role in mammalian craniofacial development and suggest that enhancer sequence variation contributes to human facial morphology.

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.

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.

Following their duplications at the base of the vertebrate clade, Hox gene clusters underwent remarkable sub- and neo-functionalization events. Many of these evolutionary innovations can be associated with changes in the transcriptional regulation of their genes, where an intricate relationship between the structure of the gene cluster and the architecture of the surrounding genomic landscape is at play. Here, we report on a portfolio of in vivo genome engineering strategies in mice, which have been used to probe and decipher the genetic and molecular underpinnings of the complex regulatory mechanisms implemented at these loci.

Following their duplications at the base of the vertebrate clade, Hox gene clusters underwent remarkable sub- and neo-functionalization events. Many of these evolutionary innovations can be associated with changes in the transcriptional regulation of their genes, where an intricate relationship between the structure of the gene cluster and the architecture of the surrounding genomic landscape is at play. Here, we report on a portfolio of in vivo genome engineering strategies in mice, which have been used to probe and decipher the genetic and molecular underpinnings of the complex regulatory mechanisms implemented at these loci.

Background: Linear DNA-based and Tol2-mediated transgenesis are powerful tools for the generation of transgenic zebrafish. However, the integration of multiple copies or transgenes at random genomic locations complicates comparative transgene analysis and makes long-term transgene stability unpredictable with variable expression. Targeted, site-directed transgene integration into pre-determined genomic loci can circumvent these issues. The phiC31 integrase catalyzes the unidirectional recombination reaction between heterotypic attP and attB sites and is an efficient platform for site-directed transgenesis. Results: We report the implementation of the phiC31 integrase-mediated attP/attB recombination for site-directed zebrafish transgenics of attB-containing transgene vectors into single genomic attP landing sites. We generated Tol2-based single-insertion attP transgenic lines and established their performance in phiC31 integrase-catalyzed integration of an attB-containing transgene vector. We found stable germline transmission into the next generation of an attB reporter transgene in 34% of all tested animals. We further characterized two functional attP landing site lines and determined their genomic location. Our experiments also demonstrate tissue-specific transgene applications as well as long-term stability of phiC31-mediated transgenes. Conclusions: Our results establish phiC31 integrase-controlled site-directed transgenesis into single, genomic attP sites as space-, time-, and labor-efficient zebrafish transgenesis technique. The described reagents are available for distribution to the zebrafish community. Developmental Dynamics 242:949–963, 2013. © 2013 Wiley Periodicals, Inc.

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.