Biology subjects

Darbellay, F.

Publications and source records attributed to Darbellay, F..

12 recordsLinked to original sources

bioRxiv2024

CTCF-DEPENDENT INSULATION OF Hoxb13 AND THE HETEROCHRONIC CONTROL OF TAIL LENGTH

In mammals, tail length is controlled by several genetic determinants, amongst which Hox13 genes located at the posterior extremities of Hox clusters, whose main function are to terminate the extension of the body axis. In this view, the precise timing in the transcriptional activation of these genes may impact upon body length. Unlike other Hox clusters, HoxB lacks all posterior genes between Hoxb9 and Hoxb13, two genes separated by a ca. 70 kb large DNA segment containing an unusually high number of CTCF sites, suggesting it isolates Hoxb13 from the rest of the cluster, thereby delaying its negative impact on trunk extension. We deleted the spacer DNA to induce a potential heterochronic gain of function of Hoxb13 at physiological concentration and observed a shortening of the tail as well as other abnormal phenotypes, which were all rescued by inactivating Hoxb13 in-cis with the deletion. A comparable gain of function was observed in mutant ES cells grown as pseudo-embryos in vitro, which allowed us to examine in details the importance of both the number and the orientation of CTCF sites in the insulating activity of the DNA spacer. A short cassette containing all the CTCF sites was sufficient to insulate Hoxb13 from the rest of HoxB and additional modifications of this CTCF cassette showed that two CTCF sites in convergent orientations are already capable of importantly delaying Hoxb13 activation in these conditions. We discuss the relative importance of genomic distance versus number and orientation of CTCF sites in preventing Hoxb13 to be activated too early during trunk extension and hence to modulate tail length.

developmental biology↗

bioRxiv2024

Temporal constraints on enhancer usage shape the regulation of limb gene transcription

Repertoires of transcriptional enhancers orchestrate gene expression during embryonic development, thereby shaping the forms and functions of organs. Within these repertoires individual enhancers display spatially distinct or overlapping activities that collectively build up the expression domain of cognate genes. However, the temporal specificity of these enhancers - how their activities change over developmental time to dynamically influence gene expression - remains uncharacterized. Here, we observed that temporally restricted enhancer repertoires are embedded at numerous loci associated with mouse limb development. To monitor how such enhancer repertoires govern gene transcription in vivo across extensive developmental periods, we introduce the regulatory trajectory framework. This paradigm conceptually involves transcriptional initiation, marking the beginning of gene expression, followed by its maintenance over time, and ultimately decommissioning, leading to gene repression. To track and sort cells undergoing these distinct phases, we devised a transgenic recorder approach at the Shox2 model locus. Through this method, we discovered that cells maintaining Shox2 transcription in early and late limb development relies on distinct, temporally restricted enhancer repertoires. We demonstrate that eliminating early-or late-acting enhancers only transiently affects Shox2 expression indicating that these enhancer repertoires function independently. Additionally, we found that changes in the 3D topology of the locus associate with enhancer activities and that a rapid loss of enhancer-promoter contacts occurs during decommissioning. Finally, we show that the decommissioning of the Shox2 locus can be actively driven by Hoxd13, a gene which expression is known to antagonize Shox2. Overall, our work uncovers the dependency of developmental genes on enhancers with temporally restricted activities to generate complex expression patterns over time and shed light on the dynamics of enhancer-promoter interactions.

developmental biology↗

bioRxiv2024

Liebenberg syndrome severity arises from variations in Pitx1 locus topology and ectopically transcribing cells

Enhancer hijacking, a common cause of gene misregulation linked to disease, occurs when non-matching enhancers and promoters interact ectopically. This interaction is made possible by genetic changes that alter the arrangement or insulation of gene regulatory landscapes. While the concept of enhancer hijacking is well understood, the specific reasons behind the variation in phenotypic severity or the point at which those phenotypes become evident remain unexplored. In this work, we expand on the ectopic activation of the hindlimb-specific transcription factor Pitx1 by one of its own enhancers, Pen, in forelimb tissues that causes the Liebenberg syndrome. We combine a previously developed in-embryo cell-tracing approach to a series of inversions and relocations to show that reduction in Pitx1-Pen relative genomic positioning leads to increased proportions of Pitx1 forelimb-expressing cells and more severe phenotypical outcomes. We demonstrate that the Pitx1 locus assumes an active topology when enhancer-promoter contacts are required for transcription and that its promoter generates consistent transcription levels across different alleles. Finally, we show that changes in 3D chromatin structure and enhancer-promoter contacts are not the result of Pitx1 transcriptional activity. In summary, our work shows that variation in enhancer-promoter interactions can lead to pathogenic locus activation in variable proportions of cells which, in turn, define phenotypic severity.

developmental biology↗

bioRxiv2023

Cell Type- and Tissue-specific Enhancers in Craniofacial Development

The genetic basis of craniofacial birth defects and general variation in human facial shape remains poorly understood. Distant-acting transcriptional enhancers are a major category of non-coding genome function and have been shown to control the fine-tuned spatiotemporal expression of genes during critical stages of craniofacial development1-3. However, a lack of accurate maps of the genomic location and cell type-specific in vivo activities of all craniofacial enhancers prevents their systematic exploration in human genetics studies. Here, we combined histone modification and chromatin accessibility profiling from different stages of human craniofacial development with single-cell analyses of the developing mouse face to create a comprehensive catalogue of the regulatory landscape of facial development at tissue- and single cell-resolution. In total, we identified approximately 14,000 enhancers across seven developmental stages from weeks 4 through 8 of human embryonic face development. We used transgenic mouse reporter assays to determine the in vivo activity patterns of human face enhancers predicted from these data. Across 16 in vivo validated human enhancers, we observed a rich diversity of craniofacial subregions in which these enhancers are active in vivo. To annotate the cell type specificities of human-mouse conserved enhancers, we performed single-cell RNA-seq and single-nucleus ATAC-seq of mouse craniofacial tissues from embryonic days e11.5 to e15.5. By integrating these data across species, we find that the majority (56%) of human craniofacial enhancers are functionally conserved in mice, providing cell type- and embryonic stage-resolved predictions of their in vivo activity profiles. Using retrospective analysis of known craniofacial enhancers in combination with single cell-resolved transgenic reporter assays, we demonstrate the utility of these data for predicting the in vivo cell type specificity of enhancers. Taken together, our data provide an expansive resource for genetic and developmental studies of human craniofacial development. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=138 HEIGHT=200 SRC="FIGDIR/small/546603v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@7595a9org.highwire.dtl.DTLVardef@1b48b81org.highwire.dtl.DTLVardef@32fe86org.highwire.dtl.DTLVardef@1e41fed_HPS_FORMAT_FIGEXP M_FIG C_FIG

developmental biology↗

bioRxiv2023

Chondrogenic Enhancer Landscape of Limb and Axial Skeleton Development

The precisely orchestrated differentiation of chondrocytes during skeleton development is a critical determinant of human height and body shape and disruptions of this process can cause severe skeletal abnormalities. The ultimate size and shape of each of over 200 bones depends on the intricate spatiotemporal regulation of chondrogenic and chondrocyte differentiation genes, but the genomic architecture coordinating these events remains poorly defined. Here we provide a comprehensive map of transcriptional enhancers specifically active in chondrocytes and show that they provide a mechanistic framework through which noncoding genetic variants can influence human stature. We isolated limb and trunk fetal chondrocytes from mice with a Col2a1 fluorescent regulatory sensor and used RNA-seq to identify 780 genes that are specifically expressed during chondrogenesis. To create cell type-specific enhancer maps, we performed ATAC-seq to map open chromatin regions and ChIP-seq for H3K27ac, an enhancer-associated histone modification, and identified 2704 putative chondrogenic enhancer regions. Most (74%) of these enhancers showed pan-chondrogenic activity, with smaller populations being restricted to limb (18%) or trunk (8%) chondrocytes only. We found that chondrogenic enhancers are enriched for the binding of several chondrogenic transcription factors including SOX9. Finally, we find that genetic variation overlapping chondrogenic enhancers explains a higher fraction of the heritability of human adult height than the one overlapping non-chondrogenic enhancers. This data provides a comprehensive mapping of the chondrogenic enhancer repertoire, paving the way to interpreting the role of non-coding sequence polymorphisms in phenotypic variation and bone diseases.

genomics↗

bioRxiv2022

The little skate genome and the evolutionary emergence of wing-like fin appendages

Skates are cartilaginous fish whose novel body plan features remarkably enlarged wing-like pectoral fins that allow them to thrive in benthic environments. The molecular underpinnings of this unique trait, however, remain elusive. Here we investigate the origin of this phenotypic innovation by developing the little skate Leucoraja erinacea as a genomically enabled model. Analysis of a high-quality chromosome-scale genome sequence for the little skate shows that it preserves many ancestral jawed vertebrate features compared with other sequenced genomes, including numerous ancient microchromosomes. Combining genome comparisons with extensive regulatory datasets in developing fins - gene expression, chromatin occupancy and three-dimensional (3D) conformation - we find skate-specific genomic rearrangements that alter the 3D regulatory landscape of genes involved in the planar cell polarity (PCP) pathway. Functional inhibition of PCP signaling resulted in marked reduction of anterior fin size, confirming this pathway as a major contributor of batoid fin morphology. We also identified a fin-specific enhancer that interacts with 3 HOX genes, consistent with the redeployment of Hox gene expression in anterior pectoral fins, and confirmed the potential of this element to activate transcription in the anterior fin using zebrafish reporter assays. Our findings underscore the central role of genome reorganizations and regulatory variation in the evolution of phenotypes, shedding light on the molecular origin of an enigmatic trait.

evolutionary biology↗

bioRxiv2021

Early mechanisms of whisker development: Prdm1 and its regulation in whisker development and evolutionary loss

Whiskers (vibrissae) are miniaturized organs that are designed for tactile sensing. Extremely conserved among mammals, they underwent a reduction in primates and disappeared in the human lineage. Furthermore, whiskers are highly innervated and their mechanoceptors signal to the primary somatosensory cortex, where a column of neurons called "barrel" represents each of them. This structure, known as barrel cortex, occupies a large portion of the somatosensory cortex of the rodent brain. Strikingly, Prdm1 conditional knockout mice are one of the rare transgenic strains that do not develop whisker hair follicles while still displaying a pelage (Robertson et al. 2007). Here we show that Prdm1 is expressed early on during whisker development, more precisely in clusters of mesenchymal cells before placode formation. Its conditional knockout leads to the loss of expression of Bmp2, Shh, Bmp4, Krt17, Edar, Gli1 though leaving the {beta}-catenin driven first dermal signal intact. Furthermore, we prove that Prdm1 expressing cells not only act as a signaling center but also as a multipotent progenitor population contributing to the formation of the dermal papilla, dermal sheath and pericytes of the vascular sinuses of vibrissae. We confirm by genetic ablation experiments that the absence of motile vibrissae (macro vibrissae) formation reverberates on the organization of nerve wiring in the mystacial pads and organization of the barrel cortex. We prove that Lef1 acts upstream of Prdm1 and identify a potential enhancer (named Leaf) that might be involved in the evolutionary process that led to the progressive reduction of snout size and vibrissae in primates.

developmental biology↗

bioRxiv2020

A gene desert required for regulatory control of pleiotropic Shox2 expression and embryonic survival

Gene deserts are defined as genomic regions devoid of protein coding genes and spanning more than 500 kilobases, collectively encompassing about 25% of the human genome. Approximately 30% of all gene deserts are enriched for conserved elements with cis-regulatory signatures. These are located predominantly near developmental transcription factors (TFs) but despite predicted critical functions, the transcriptional contributions and biological necessity of most gene deserts remain elusive. Here, we explore the cis-regulatory impact of a gene desert flanking the Shox2 gene, a TF indispensable for proximal limb, craniofacial and cardiac pacemaker development. Using a functional genomics approach in mouse embryos we identify the gene desert as a hub for numerous Shox2-overlapping enhancers arranged in a globular chromatin domain with tissue-specific features. In accordance, using endogenous CRISPR deletion, we demonstrate that the gene desert interval is essential for Shox2 transcriptional control in developing limbs, craniofacial compartments, and the heart. Phenotypically, gene desert ablation leads to pacemaker-related embryonic lethality due to Shox2 depletion in the cardiac sinus venosus. We show that this role is partially mediated through a distal gene desert enhancer, providing evidence for intra-gene desert regulatory robustness. Finally, we uncover a multi-layered functional role of the gene desert by revealing an additional requirement for stylopod morphogenesis, mediated through an array of proximal limb enhancers (PLEs). In summary, our study establishes the Shox2 gene desert as a fundamental genomic unit that controls pleiotropic gene expression through modular arrangement and coordinated dynamics of tissue-specific enhancers.

developmental biology↗

bioRxiv2020

SPECIFIC ECTODERMAL ENHANCERS CONTROL THE EXPRESSION OF HoxC GENES IN DEVELOPING MAMMALIAN INTEGUMENTS

Vertebrate Hox genes are key players in the establishment of structures during the development of the main body axis. Subsequently, they play important roles either in organizing secondary axial structures such as the appendages, or during homeostasis in postnatal stages and adulthood. Here we set up to analyze their elusive function in the ectodermal compartment, using the mouse limb bud as a model. We report that the HoxC gene cluster was globally co-opted to be transcribed in the distal limb ectoderm, where it is activated following the rule of temporal colinearity. These ectodermal cells subsequently produce various keratinized organs such as nails or claws. Accordingly, deletion of the HoxC cluster led to mice lacking nails (anonychia) and also hairs (alopecia), a condition stronger than the previously reported loss of function of Hoxc13, which is the causative gene of the ectodermal dysplasia 9 (ECTD9) in human patients. We further identified two ectodermal, mammalian-specific enhancers located upstream of the HoxC gene cluster, which act synergistically to regulate Hoxc gene expression in the hair and nail ectodermal organs. Deletion of these regulatory elements alone or in combination revealed a strong quantitative component in the regulation of Hoxc genes in the ectoderm, suggesting that these two enhancers may have evolved along with mammals to provide the level of HOXC proteins necessary for the full development of hairs and nails. Significance StatementIn this study, we report a unique and necessary function for the HoxC gene cluster in the development of some ectodermal organs, as illustrated both by the hair and nail phenotype displayed by mice lacking the Hoxc13 function and by the congenital anonychia (absence of nails) in full HoxC cluster mutants. We show that Hoxc genes are activated in a colinear manner in the embryonic limb ectoderm and are subsequently transcribed in developing nails and hairs. We identify two mammalian-specific enhancers located upstream of the HoxC cluster with and exclusive ectodermal specificity. Individual or combined enhancer deletions suggest that they act in combination to raise the transcription level of several Hoxc genes during hairs and nails development.

developmental biology↗

bioRxiv2020

Analysis of zebrafish periderm enhancers facilitates identification of a regulatory variant near human KRT8/18

Genome wide association studies for non-syndromic orofacial cleft (OFC) have identified single nucleotide polymorphisms (SNPs) at loci where the presumed risk-relevant gene is expressed in oral periderm. The functional subsets of such SNPs are difficult to predict because the sequence underpinnings of periderm enhancers are unknown. We applied ATAC-seq to models of human palate periderm, including zebrafish periderm, mouse embryonic palate epithelia, and a human oral epithelium cell line, and to complementary mesenchymal cell types. We identified sets of enhancers specific to the epithelial cells and trained gapped-kmer support-vector-machine classifiers on these sets. We used the classifiers to predict the effect of 14 OFC-associated SNPs at 12q13 near KRT18. All the classifiers picked the same SNP as having the strongest effect, but the significance was highest with the classifier trained on zebrafish periderm. Reporter and deletion analyses support this SNP as lying within a periderm enhancer regulating KRT18/KRT8 expression.

developmental biology↗

bioRxiv2019

Comparative transcriptomics analyses across species, organs and developmental stages reveal functionally constrained lncRNAs

BackgroundTranscription of long non-coding RNAs (lncRNAs) is pervasive, but their functionality is disputed. As a class, lncRNAs show little selective constraint and negligible phenotypic effects upon perturbation. However, key biological roles were demonstrated for individual lncRNAs. Most validated lncRNAs were implicated in gene expression regulation, in pathways related to cellular pluripotency, differentiation and organ morphogenesis, suggesting that functional lncRNAs may be more abundant in embryonic development, rather than in adult organs.\n\nResultsHere, we perform a multi-dimensional comparative transcriptomics analysis, across five developmental time-points (two embryonic stages, newborn, adult and aged individuals), four organs (brain, kidney, liver and testes) and three species (mouse, rat and chicken). Overwhelmingly, lncRNAs are preferentially expressed in adult and aged testes, consistent with the presence of permissive transcription during spermatogenesis. LncRNAs are often differentially expressed among developmental stages and are less abundant in embryos and newborns compared to adult individuals, in agreement with a requirement for tighter expression control and less tolerance for noisy transcription early in development. However, lncRNAs expressed during embryonic development show increased levels of evolutionary conservation, both in terms of primary sequence and of expression patterns, and in particular at their promoter regions. We find that species-specific lncRNA transcription is frequent for enhancer-associated loci and occurs in parallel with expression pattern changes for neighboring protein-coding genes.\n\nConclusionsWe show that functionally constrained lncRNA loci are enriched in developing organ transcriptomes, and propose that many of these loci may function in an RNA-independent manner.

evolutionary biology↗

bioRxiv2019

Constrained Transcriptional Polarity In The Organization Of Mammalian Hox Gene Clusters

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 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 the HoxD cluster 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 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.

developmental biology↗