Publications by authors named "Denis Duboule"

124 Publications

Mesomelic dysplasias associated with the HOXD locus are caused by regulatory reallocations.

Nat Commun 2021 08 18;12(1):5013. Epub 2021 Aug 18.

School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland.

Human families with chromosomal rearrangements at 2q31, where the human HOXD locus maps, display mesomelic dysplasia, a severe shortening and bending of the limb. In mice, the dominant Ulnaless inversion of the HoxD cluster produces a similar phenotype suggesting the same origin for these malformations in humans and mice. Here we engineer 1 Mb inversion including the HoxD gene cluster, which positioned Hoxd13 close to proximal limb enhancers. Using this model, we show that these enhancers contact and activate Hoxd13 in proximal cells, inducing the formation of mesomelic dysplasia. We show that a secondary Hoxd13 null mutation in-cis with the inversion completely rescues the alterations, demonstrating that ectopic HOXD13 is directly responsible for this bone anomaly. Single-cell expression analysis and evaluation of HOXD13 binding sites suggests that the phenotype arises primarily by acting through genes normally controlled by HOXD13 in distal limb cells. Altogether, these results provide a conceptual and mechanistic framework to understand and unify the molecular origins of human mesomelic dysplasia associated with 2q31.
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http://dx.doi.org/10.1038/s41467-021-25330-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8373931PMC
August 2021

Induction of a chromatin boundary in vivo upon insertion of a TAD border.

PLoS Genet 2021 07 22;17(7):e1009691. Epub 2021 Jul 22.

Department of Genetics and Evolution, Faculty of Science, University of Geneva, Geneva, Switzerland.

Mammalian genomes are partitioned into sub-megabase to megabase-sized units of preferential interactions called topologically associating domains or TADs, which are likely important for the proper implementation of gene regulatory processes. These domains provide structural scaffolds for distant cis regulatory elements to interact with their target genes within the three-dimensional nuclear space and architectural proteins such as CTCF as well as the cohesin complex participate in the formation of the boundaries between them. However, the importance of the genomic context in providing a given DNA sequence the capacity to act as a boundary element remains to be fully investigated. To address this question, we randomly relocated a topological boundary functionally associated with the mouse HoxD gene cluster and show that it can indeed act similarly outside its initial genomic context. In particular, the relocated DNA segment recruited the required architectural proteins and induced a significant depletion of contacts between genomic regions located across the integration site. The host chromatin landscape was re-organized, with the splitting of the TAD wherein the boundary had integrated. These results provide evidence that topological boundaries can function independently of their site of origin, under physiological conditions during mouse development.
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http://dx.doi.org/10.1371/journal.pgen.1009691DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8330945PMC
July 2021

Time-sequenced transcriptomes of developing distal mouse limb buds: A comparative tissue layer analysis.

Dev Dyn 2021 Jul 12. Epub 2021 Jul 12.

Instituto de Biomedicina y Biotecnología de Cantabria, IBBTEC (CSIC-University of Cantabria-SODERCAN), Santander, Spain.

Background: The development of the amniote limb has been an important model system to study patterning mechanisms and morphogenesis. For proper growth and patterning, it requires the interaction between the distal sub-apical mesenchyme and the apical ectodermal ridge (AER) that involve the separate implementation of coordinated and tissue-specific genetic programs.

Results: Here, we produce and analyze the transcriptomes of both distal limb mesenchymal progenitors and the overlying ectodermal cells, following time-coursed dissections that cover from limb bud initiation to fully patterned limbs. The comparison of transcriptomes within each layer as well as between layers over time, allowed the identification of specific transcriptional signatures for each of the developmental stages. Special attention was given to the identification of genes whose transcription dynamics suggest a previously unnoticed role in the context of limb development and also to signaling pathways enriched between layers.

Conclusion: We interpret the transcriptomic data in light of the known development pattern and we conclude that a major transcriptional transition occurs in distal limb buds between E9.5 and E10.5, coincident with the switch from an early phase continuation of the signature of trunk progenitors, related to the initial proximo distal specification, to a late intrinsic phase of development.
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http://dx.doi.org/10.1002/dvdy.394DOI Listing
July 2021

Analysis of Polycerate Mutants Reveals the Evolutionary Co-option of HOXD1 for Horn Patterning in Bovidae.

Mol Biol Evol 2021 05;38(6):2260-2272

CNRS UMR 7044, ARCHIMEDE, MISHA, Strasbourg, France.

In the course of evolution, pecorans (i.e., higher ruminants) developed a remarkable diversity of osseous cranial appendages, collectively referred to as "headgear," which likely share the same origin and genetic basis. However, the nature and function of the genetic determinants underlying their number and position remain elusive. Jacob and other rare populations of sheep and goats are characterized by polyceraty, the presence of more than two horns. Here, we characterize distinct POLYCERATE alleles in each species, both associated with defective HOXD1 function. We show that haploinsufficiency at this locus results in the splitting of horn bud primordia, likely following the abnormal extension of an initial morphogenetic field. These results highlight the key role played by this gene in headgear patterning and illustrate the evolutionary co-option of a gene involved in the early development of bilateria to properly fix the position and number of these distinctive organs of Bovidae.
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http://dx.doi.org/10.1093/molbev/msab021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8136503PMC
May 2021

Dbx2 regulation in limbs suggests interTAD sharing of enhancers.

Dev Dyn 2021 Sep 1;250(9):1280-1299. Epub 2021 Mar 1.

Department of Genetics and Evolution, University of Geneva, Geneva, Switzerland.

Background: During tetrapod limb development, the HOXA13 and HOXD13 transcription factors are critical for the emergence and organization of the autopod, the most distal aspect where digits will develop. Since previous work had suggested that the Dbx2 gene is a target of these factors, we set up to analyze in detail this potential regulatory interaction.

Results: We show that HOX13 proteins bind to mammalian-specific sequences at the vicinity of the Dbx2 locus that have enhancer activity in developing digits. However, the functional inactivation of the DBX2 protein did not elicit any particular phenotype related to Hox genes inactivation in digits, suggesting either redundant or compensatory mechanisms. We report that the neighboring Nell2 and Ano6 genes are also expressed in distal limb buds and are in part controlled by the same Dbx2 enhancers despite being localized into two different topologically associating domains (TADs) flanking the Dbx2 locus.

Conclusions: We conclude that Hoxa13 and Hoxd genes cooperatively activate Dbx2 expression in developing digits through binding to mammalian specific regulatory sequences in the Dbx2 neighborhood. Furthermore, these enhancers can overcome TAD boundaries in either direction to co-regulate a set of genes located in distinct chromatin domains.
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http://dx.doi.org/10.1002/dvdy.303DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8451760PMC
September 2021

Chromatin topology and the timing of enhancer function at the locus.

Proc Natl Acad Sci U S A 2020 12 23;117(49):31231-31241. Epub 2020 Nov 23.

Department of Genetics and Evolution, University of Geneva, 1211 Geneva, Switzerland;

The gene cluster is critical for proper limb formation in tetrapods. In the emerging limb buds, different subgroups of genes respond first to a proximal regulatory signal, then to a distal signal that organizes digits. These two regulations are exclusive from one another and emanate from two distinct topologically associating domains (TADs) flanking , both containing a range of appropriate enhancer sequences. The telomeric TAD (T-DOM) contains several enhancers active in presumptive forearm cells and is divided into two sub-TADs separated by a CTCF-rich boundary, which defines two regulatory submodules. To understand the importance of this particular regulatory topology to control gene transcription in time and space, we either deleted or inverted this sub-TAD boundary, eliminated the CTCF binding sites, or inverted the entire T-DOM to exchange the respective positions of the two sub-TADs. The effects of such perturbations on the transcriptional regulation of genes illustrate the requirement of this regulatory topology for the precise timing of gene activation. However, the spatial distribution of transcripts was eventually resumed, showing that the presence of enhancer sequences, rather than either their exact topology or a particular chromatin architecture, is the key factor. We also show that the affinity of enhancers to find their natural target genes can overcome the presence of both a strong TAD border and an unfavorable orientation of CTCF sites.
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http://dx.doi.org/10.1073/pnas.2015083117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7733857PMC
December 2020

Mammalian-specific ectodermal enhancers control the expression of genes in developing nails and hair follicles.

Proc Natl Acad Sci U S A 2020 12 16;117(48):30509-30519. Epub 2020 Nov 16.

Instituto de Biomedicina y Biotecnología de Cantabria, Consejo Superior de Investigaciones Científicas-Universidad de Cantabria-Sociedad para el Desarrollo de Cantabria, 39011 Santander, Spain;

Vertebrate genes are critical for 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 gene cluster was 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 cluster led to mice lacking nails (anonychia), a condition stronger than the previously reported loss of function of , which is the causative gene of the ectodermal dysplasia 9 (ECTD9) in human patients. We further identified two mammalian-specific ectodermal enhancers located upstream of the gene cluster, which together regulate 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 genes in the ectoderm, suggesting that these two enhancers may have evolved along with the mammalian taxon to provide the level of HOXC proteins necessary for the full development of hair and nail.
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http://dx.doi.org/10.1073/pnas.2011078117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7720164PMC
December 2020

A complex regulatory landscape involved in the development of mammalian external genitals.

Elife 2020 04 17;9. Epub 2020 Apr 17.

School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland.

Developmental genes are often controlled by large regulatory landscapes matching topologically associating domains (TADs). In various contexts, the associated chromatin backbone is modified by specific enhancer-enhancer and enhancer-promoter interactions. We used a TAD flanking the mouse cluster to study how these regulatory architectures are formed and deconstructed once their function achieved. We describe this TAD as a functional unit, with several regulatory sequences acting together to elicit a transcriptional response. With one exception, deletion of these sequences didn't modify the transcriptional outcome, a result at odds with a conventional view of enhancer function. The deletion and inversion of a CTCF site located near these regulatory sequences did not affect transcription of the target gene. Slight modifications were nevertheless observed, in agreement with the loop extrusion model. We discuss these unexpected results considering both conventional and alternative explanations relying on the accumulation of poorly specific factors within the TAD backbone.
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http://dx.doi.org/10.7554/eLife.52962DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7185996PMC
April 2020

The regulatory landscapes of developmental genes.

Development 2020 02 3;147(3). Epub 2020 Feb 3.

Swiss Institute for Cancer Research (ISREC), School of Life Sciences, Federal Institute of Technology, Lausanne, 1015 Lausanne, Switzerland

Regulatory landscapes have been defined in vertebrates as large DNA segments containing diverse enhancer sequences that produce coherent gene transcription. These genomic platforms integrate multiple cellular signals and hence can trigger pleiotropic expression of developmental genes. Identifying and evaluating how these chromatin regions operate may be difficult as the underlying regulatory mechanisms can be as unique as the genes they control. In this brief article and accompanying poster, we discuss some of the ways in which regulatory landscapes operate, illustrating these mechanisms using genes important for vertebrate development as examples. We also highlight some of the techniques available to researchers for analysing regulatory landscapes.
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http://dx.doi.org/10.1242/dev.171736DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7033717PMC
February 2020

Fryns type mesomelic dysplasia of the upper limbs caused by inverted duplications of the HOXD gene cluster.

Eur J Hum Genet 2020 03 7;28(3):324-332. Epub 2019 Oct 7.

Service de Génétique, Hôpital Bretonneau, CHU, Tours, France.

The HoxD cluster is critical for vertebrate limb development. Enhancers located in both the telomeric and centromeric gene deserts flanking the cluster regulate the transcription of HoxD genes. In rare patients, duplications, balanced translocations or inversions misregulating HOXD genes are responsible for mesomelic dysplasia of the upper and lower limbs. By aCGH, whole-genome mate-pair sequencing, long-range PCR and fiber fluorescent in situ hybridization, we studied patients from two families displaying mesomelic dysplasia limited to the upper limbs. We identified microduplications including the HOXD cluster and showed that microduplications were in an inverted orientation and inserted between the HOXD cluster and the telomeric enhancers. Our results highlight the existence of an autosomal dominant condition consisting of isolated ulnar dysplasia caused by microduplications inserted between the HOXD cluster and the telomeric enhancers. The duplications likely disconnect the HOXD9 to HOXD11 genes from their regulatory sequences. This presumptive loss-of-function may have contributed to the phenotype. In both cases, however, these rearrangements brought HOXD13 closer to telomeric enhancers, suggesting that the alterations derive from the dominant-negative effect of this digit-specific protein when ectopically expressed during the early development of forearms, through the disruption of topologically associating domain structure at the HOXD locus.
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http://dx.doi.org/10.1038/s41431-019-0522-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7028936PMC
March 2020

Commentary on paper by Leroy C.

Authors:
Denis Duboule

Dev Biol 2019 10;454(1):1-14

School of Life Sciences, Federal Institute of Technology, Lausanne, 1015 Lausanne, Switzerland; Department of Genetics and Evolution, University of Geneva, 1211 Geneva 4, Switzerland; Collège de France, Paris, France.

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http://dx.doi.org/10.1016/j.ydbio.2019.07.016DOI Listing
October 2019

Impact of genome architecture on the functional activation and repression of Hox regulatory landscapes.

BMC Biol 2019 07 12;17(1):55. Epub 2019 Jul 12.

Laboratory of Developmental Genomics, Department of Genetics and Evolution, University of Geneva, 1211, Geneva 4, Switzerland.

Background: The spatial organization of the mammalian genome relies upon the formation of chromatin domains of various scales. At the level of gene regulation in cis, collections of enhancer sequences define large regulatory landscapes that usually match with the presence of topologically associating domains (TADs). These domains often contain ranges of enhancers displaying similar or related tissue specificity, suggesting that in some cases, such domains may act as coherent regulatory units, with a global on or off state. By using the HoxD gene cluster, which specifies the topology of the developing limbs via highly orchestrated regulation of gene expression, as a paradigm, we investigated how the arrangement of regulatory domains determines their activity and function.

Results: Proximal and distal cells in the developing limb express different levels of Hoxd genes, regulated by flanking 3' and 5' TADs, respectively. We characterized the effect of large genomic rearrangements affecting these two TADs, including their fusion into a single chromatin domain. We show that, within a single hybrid TAD, the activation of both proximal and distal limb enhancers globally occurred as when both TADs are intact. However, the activity of the 3' TAD in distal cells is generally increased in the fused TAD, when compared to wild type where it is silenced. Also, target gene activity in distal cells depends on whether or not these genes had previously responded to proximal enhancers, which determines the presence or absence of H3K27me3 marks. We also show that the polycomb repressive complex 2 is mainly recruited at the Hox gene cluster and can extend its coverage to far-cis regulatory sequences as long as confined to the neighboring TAD structure.

Conclusions: We conclude that antagonistic limb proximal and distal enhancers can exert their specific effects when positioned into the same TAD and in the absence of their genuine target genes. We also conclude that removing these target genes reduced the coverage of a regulatory landscape by chromatin marks associated with silencing, which correlates with its prolonged activity in time.
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http://dx.doi.org/10.1186/s12915-019-0677-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6626364PMC
July 2019

The constrained architecture of mammalian gene clusters.

Proc Natl Acad Sci U S A 2019 07 17;116(27):13424-13433. Epub 2019 Jun 17.

School of Life Sciences, Federal Institute of Technology, Lausanne, 1015 Lausanne, Switzerland;

In many animal species with a bilateral symmetry, 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 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 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 clusters and their regulation makes inversions likely detrimental to the proper implementation of this complex genetic system. We propose that the consolidation of 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 .
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http://dx.doi.org/10.1073/pnas.1904602116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6613147PMC
July 2019

Characterization of paralogous transcription factor encoding genes in zebrafish.

Gene X 2019 Jun;2:100011

Biology and Evolution of Marine Organisms, Zoological Station Anton Dohrn, 80121 Naples, Italy.

The paired-type homeodomain transcription factor Uncx is involved in multiple processes of embryogenesis in vertebrates. Reasoning that zebrafish genes and are orthologs of mouse , we studied their genomic environment and developmental expression. Evolutionary analyses indicate the zebrafish genes as being paralogs deriving from teleost-specific whole-genome duplication. Whole-mount mRNA hybridization of transcripts in zebrafish embryos reveals novel expression domains, confirms those previously known, and suggests sub-functionalization of paralogs. Using genetic mutants and pharmacological inhibitors, we investigate the role of signaling pathways on the expression of zebrafish genes in developing somites. In identifying putative functional role(s) of zebrafish genes, we hypothesized that they encode transcription factors that coordinate growth and innervation of somitic muscles.
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http://dx.doi.org/10.1016/j.gene.2019.100011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6543554PMC
June 2019

Tail Bud Progenitor Activity Relies on a Network Comprising Gdf11, Lin28, and Hox13 Genes.

Dev Cell 2019 02 17;48(3):383-395.e8. Epub 2019 Jan 17.

Instituto Gulbenkian de Ciencia, Rua da Quinta Grande 6, 2780-156 Oeiras, Portugal. Electronic address:

During the trunk-to-tail transition, axial progenitors relocate from the epiblast to the tail bud. Here, we show that this process entails a major regulatory switch, bringing tail bud progenitors under Gdf11 signaling control. Gdf11 mutant embryos have an increased number of such progenitors that favor neural differentiation routes, resulting in a dramatic expansion of the neural tube. Moreover, inhibition of Gdf11 signaling recovers the proliferation ability of these progenitors when cultured in vitro. Tail bud progenitor growth is independent of Oct4, relying instead on Lin28 activity. Gdf11 signaling eventually activates Hox genes of paralog group 13, which halt expansion of these progenitors, at least in part, by down-regulating Lin28 genes. Our results uncover a genetic network involving Gdf11, Lin28, and Hox13 genes controlling axial progenitor activity in the tail bud.
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http://dx.doi.org/10.1016/j.devcel.2018.12.004DOI Listing
February 2019

Characterization of paralogous uncx transcription factor encoding genes in zebrafish.

Gene 2019 8;721S:100011. Epub 2019 Mar 8.

Biology and Evolution of Marine Organisms, Zoological Station Anton Dohrn, 80121 Naples, Italy. Electronic address:

The paired-type homeodomain transcription factor Uncx is involved in multiple processes of embryogenesis in vertebrates. Reasoning that zebrafish genes uncx4.1 and uncx are orthologs of mouse Uncx, we studied their genomic environment and developmental expression. Evolutionary analyses indicate the zebrafish uncx genes as being paralogs deriving from teleost-specific whole-genome duplication. Whole-mount in situ mRNA hybridization of uncx transcripts in zebrafish embryos reveals novel expression domains, confirms those previously known, and suggests sub-functionalization of paralogs. Using genetic mutants and pharmacological inhibitors, we investigate the role of signaling pathways on the expression of zebrafish uncx genes in developing somites. In identifying putative functional role(s) of zebrafish uncx genes, we hypothesized that they encode transcription factors that coordinate growth and innervation of somitic muscles.
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http://dx.doi.org/10.1016/j.gene.2019.100011DOI Listing
March 2019

Similarities and differences in the regulation of HoxD genes during chick and mouse limb development.

PLoS Biol 2018 11 26;16(11):e3000004. Epub 2018 Nov 26.

School of Life Sciences, Federal Institute of Technology, Lausanne, Lausanne, Switzerland.

In all tetrapods examined thus far, the development and patterning of limbs require the activation of gene members of the HoxD cluster. In mammals, they are regulated by a complex bimodal process that controls first the proximal patterning and then the distal structure. During the shift from the former to the latter regulation, this bimodal regulatory mechanism allows the production of a domain with low Hoxd gene expression, at which both telomeric (T-DOM) and centromeric regulatory domains (C-DOM) are silent. These cells generate the future wrist and ankle articulations. We analyzed the implementation of this regulatory mechanism in chicken, i.e., in an animal for which large morphological differences exist between fore- and hindlimbs. We report that although this bimodal regulation is globally conserved between the mouse and the chick, some important modifications evolved at least between these two model systems, in particular regarding the activity of specific enhancers, the width of the TAD boundary separating the two regulations, and the comparison between the forelimb versus hindlimb regulatory controls. At least one aspect of these regulations seems to be more conserved between chick and bats than with mouse, which may relate to the extent to which forelimbs and hindlimbs of these various animals differ in their morphologies.
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http://dx.doi.org/10.1371/journal.pbio.3000004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6283595PMC
November 2018

Multi-axial self-organization properties of mouse embryonic stem cells into gastruloids.

Nature 2018 10 3;562(7726):272-276. Epub 2018 Oct 3.

Department of Genetics, University of Cambridge, Cambridge, UK.

The emergence of multiple axes is an essential element in the establishment of the mammalian body plan. This process takes place shortly after implantation of the embryo within the uterus and relies on the activity of gene regulatory networks that coordinate transcription in space and time. Whereas genetic approaches have revealed important aspects of these processes, a mechanistic understanding is hampered by the poor experimental accessibility of early post-implantation stages. Here we show that small aggregates of mouse embryonic stem cells (ESCs), when stimulated to undergo gastrulation-like events and elongation in vitro, can organize a post-occipital pattern of neural, mesodermal and endodermal derivatives that mimic embryonic spatial and temporal gene expression. The establishment of the three major body axes in these 'gastruloids' suggests that the mechanisms involved are interdependent. Specifically, gastruloids display the hallmarks of axial gene regulatory systems as exemplified by the implementation of collinear Hox transcriptional patterns along an extending antero-posterior axis. These results reveal an unanticipated self-organizing capacity of aggregated ESCs and suggest that gastruloids could be used as a complementary system to study early developmental events in the mammalian embryo.
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http://dx.doi.org/10.1038/s41586-018-0578-0DOI Listing
October 2018

Rescue of an aggressive female sexual courtship in mice by CRISPR/Cas9 secondary mutation in vivo.

BMC Res Notes 2018 Mar 27;11(1):193. Epub 2018 Mar 27.

Department of Genetics and Evolution, University of Geneva, Geneva, Switzerland.

Objective: We had previously reported a mouse line carrying the Atypical female courtship (HoxD ) allele, where an ectopic accumulation of Hoxd10 transcripts was observed in a sparse population of cells in the adult isocortex, as a result of a partial deletion of the HoxD gene cluster. Female mice carrying this allele displayed an exacerbated paracopulatory behavior, culminating in a severe mutilation of the studs' external genitals. To unequivocally demonstrate that this intriguing phenotype was indeed caused by an illegitimate function of the HOXD10 protein, we use CRISPR/Cas9 technology to induce a microdeletion into the homeobox of the Hoxd10 gene in cis with the HoxD allele.

Results: Females carrying this novel HoxD allele no longer mutilate males. We conclude that a brain malfunction leading to a severe pathological behavior can be caused by the mere binding to DNA of a transcription factor expressed ectopically. We also show that in HoxD mice, Hoxd10 was expressed in cells containing glutamate decarboxylase (Gad1) and Cholecystokinin (Cck) transcripts, corroborating our proposal that a small fraction of GABAergic neurons in adult hippocampus may participate to some aspects of female courtship.
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http://dx.doi.org/10.1186/s13104-018-3307-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5870235PMC
March 2018

Response to Peron et al.

Genet Med 2018 11;20(11):1481-1482

Department of Genome Sciences, University of Washington, Seattle, Washington, USA.

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http://dx.doi.org/10.1038/gim.2018.20DOI Listing
November 2018

The cluster is a dynamic and resilient TAD boundary controlling the segregation of antagonistic regulatory landscapes.

Genes Dev 2017 11 22;31(22):2264-2281. Epub 2017 Dec 22.

Department of Genetics and Evolution, University of Geneva, 1205 Geneva, Switzerland.

The mammalian cluster lies between two topologically associating domains (TADs) matching distinct enhancer-rich regulatory landscapes. During limb development, the telomeric TAD controls the early transcription of genes in forearm cells, whereas the centromeric TAD subsequently regulates more posterior genes in digit cells. Therefore, the TAD boundary prevents the terminal gene from responding to forearm enhancers, thereby allowing proper limb patterning. To assess the nature and function of this CTCF-rich DNA region in embryos we compared chromatin interaction profiles between proximal and distal limb bud cells isolated from mutant stocks where various parts of this boundary region were removed. The resulting progressive release in boundary effect triggered inter-TAD contacts, favored by the activity of the newly accessed enhancers. However, the boundary was highly resilient, and only a 400-kb deletion, including the whole-gene cluster, was eventually able to merge the neighboring TADs into a single structure. In this unified TAD, both proximal and distal limb enhancers nevertheless continued to work independently over a targeted transgenic reporter construct. We propose that the whole cluster is a dynamic TAD border and that the exact boundary position varies depending on both the transcriptional status and the developmental context.
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http://dx.doi.org/10.1101/gad.307769.117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5769770PMC
November 2017

Noncoding copy-number variations are associated with congenital limb malformation.

Genet Med 2018 06 12;20(6):599-607. Epub 2017 Oct 12.

Max Planck Institute for Molecular Genetics, Berlin, Germany.

PurposeCopy-number variants (CNVs) are generally interpreted by linking the effects of gene dosage with phenotypes. The clinical interpretation of noncoding CNVs remains challenging. We investigated the percentage of disease-associated CNVs in patients with congenital limb malformations that affect noncoding cis-regulatory sequences versus genes sensitive to gene dosage effects.MethodsWe applied high-resolution copy-number analysis to 340 unrelated individuals with isolated limb malformation. To investigate novel candidate CNVs, we re-engineered human CNVs in mice using clustered regularly interspaced short palindromic repeats (CRISPR)-based genome editing.ResultsOf the individuals studied, 10% harbored CNVs segregating with the phenotype in the affected families. We identified 31 CNVs previously associated with congenital limb malformations and four novel candidate CNVs. Most of the disease-associated CNVs (57%) affected the noncoding cis-regulatory genome, while only 43% included a known disease gene and were likely to result from gene dosage effects. In transgenic mice harboring four novel candidate CNVs, we observed altered gene expression in all cases, indicating that the CNVs had a regulatory effect either by changing the enhancer dosage or altering the topological associating domain architecture of the genome.ConclusionOur findings suggest that CNVs affecting noncoding regulatory elements are a major cause of congenital limb malformations.
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http://dx.doi.org/10.1038/gim.2017.154DOI Listing
June 2018

Control of growth and gut maturation by genes and the associated lncRNA .

Proc Natl Acad Sci U S A 2017 10 17;114(44):E9290-E9299. Epub 2017 Oct 17.

Department of Genetics and Evolution, University of Geneva, 1211 Geneva 4, Switzerland;

During embryonic development, genes participate in the building of a functional digestive system in metazoans, and genetic conditions involving these genes lead to important, sometimes lethal, growth retardation. Recently, this phenotype was obtained after deletion of , the antisense growth-associated long noncoding RNA (lncRNA) located between and In this study, we have analyzed the function of genes in delayed growth trajectories by looking at several nested targeted deficiencies of the mouse cluster. Mutant pups were severely stunted during the suckling period, but many recovered after weaning. After comparing seven distinct alleles, including CRISPR/Cas9 deletions involving , we identified as the critical component for the gut to maintain milk-digestive competence. This essential function could be abrogated by the dominant-negative effect of as shown by a genetic rescue approach, thus further illustrating the importance of posterior prevalence in gene function. A role for the lncRNA in the control of postnatal growth could not be corroborated.
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http://dx.doi.org/10.1073/pnas.1712511114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5676926PMC
October 2017

Embryonic timing, axial stem cells, chromatin dynamics, and the Hox clock.

Genes Dev 2017 07;31(14):1406-1416

School of Life Sciences, Ecole Polytechnique Fédérale, Lausanne, 1015 Lausanne, Switzerland.

Collinear regulation of genes in space and time has been an outstanding question ever since the initial work of Ed Lewis in 1978. Here we discuss recent advances in our understanding of this phenomenon in relation to novel concepts associated with large-scale regulation and chromatin structure during the development of both axial and limb patterns. We further discuss how this sequential transcriptional activation marks embryonic stem cell-like axial progenitors in mammals and, consequently, how a temporal genetic system is further translated into spatial coordinates via the fate of these progenitors. In this context, we argue the benefit and necessity of implementing this unique mechanism as well as the difficulty in evolving an alternative strategy to deliver this critical positional information.
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http://dx.doi.org/10.1101/gad.303123.117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5588924PMC
July 2017

Large scale genomic reorganization of topological domains at the HoxD locus.

Genome Biol 2017 08 7;18(1):149. Epub 2017 Aug 7.

School of Life Sciences, Ecole Polytechnique Fédérale, 1015, Lausanne, Switzerland.

Background: The transcriptional activation of HoxD genes during mammalian limb development involves dynamic interactions with two topologically associating domains (TADs) flanking the HoxD cluster. In particular, the activation of the most posterior HoxD genes in developing digits is controlled by regulatory elements located in the centromeric TAD (C-DOM) through long-range contacts.

Results: To assess the structure-function relationships underlying such interactions, we measured compaction levels and TAD discreteness using a combination of chromosome conformation capture (4C-seq) and DNA FISH. We assessed the robustness of the TAD architecture by using a series of genomic deletions and inversions that impact the integrity of this chromatin domain and that remodel long-range contacts. We report multi-partite associations between HoxD genes and up to three enhancers. We find that the loss of native chromatin topology leads to the remodeling of TAD structure following distinct parameters.

Conclusions: Our results reveal that the recomposition of TAD architectures after large genomic re-arrangements is dependent on a boundary-selection mechanism in which CTCF mediates the gating of long-range contacts in combination with genomic distance and sequence specificity. Accordingly, the building of a recomposed TAD at this locus depends on distinct functional and constitutive parameters.
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http://dx.doi.org/10.1186/s13059-017-1278-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5547506PMC
August 2017

Integration of Shh and Fgf signaling in controlling gene expression in cultured limb cells.

Proc Natl Acad Sci U S A 2017 03 7;114(12):3139-3144. Epub 2017 Mar 7.

Department of Genetics, Harvard Medical School, Boston, MA 02115;

During embryonic development, fields of progenitor cells form complex structures through dynamic interactions with external signaling molecules. How complex signaling inputs are integrated to yield appropriate gene expression responses is poorly understood. In the early limb bud, for instance, Sonic hedgehog () is expressed in the distal posterior mesenchyme, where it acts as a mediator of anterior to posterior (AP) patterning, whereas fibroblast growth factor 8 () is produced by the apical ectodermal ridge (AER) at the distal tip of the limb bud to direct outgrowth along the proximal to distal (PD) axis. Here we use cultured limb mesenchyme cells to assess the response of the target genes to these two factors. We find that they act synergistically and that both factors are required to activate in limb mesenchymal cells. However, the analysis of the enhancer landscapes flanking the cluster reveals that the bimodal regulatory switch observed in vivo is only partially achieved under these in vitro conditions, suggesting an additional requirement for other factors.
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http://dx.doi.org/10.1073/pnas.1620767114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5373353PMC
March 2017

Hotair Is Dispensible for Mouse Development.

PLoS Genet 2016 Dec 15;12(12):e1006232. Epub 2016 Dec 15.

School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland.

Despite the crucial importance of Hox genes functions during animal development, the mechanisms that control their transcription in time and space are not yet fully understood. In this context, it was proposed that Hotair, a lncRNA transcribed from within the HoxC cluster regulates Hoxd gene expression in trans, through the targeting of Polycomb and consecutive transcriptional repression. This activity was recently supported by the skeletal phenotype of mice lacking Hotair function. However, other loss of function alleles at this locus did not elicit the same effects. Here, we re-analyze the molecular and phenotypic consequences of deleting the Hotair locus in vivo. In contrast with previous findings, we show that deleting Hotair has no detectable effect on Hoxd genes expression in vivo. In addition, we were unable to observe any significant morphological alteration in mice lacking the Hotair transcript. However, we find a subtle impact of deleting the Hotair locus upon the expression of the neighboring Hoxc11 and Hoxc12 genes in cis. Our results do not support any substantial role for Hotair during mammalian development in vivo. Instead, they argue in favor of a DNA-dependent effect of the Hotair deletion upon the transcriptional landscape in cis.
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http://dx.doi.org/10.1371/journal.pgen.1006232DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5157951PMC
December 2016

Control of Hoxd gene transcription in the mammary bud by hijacking a preexisting regulatory landscape.

Proc Natl Acad Sci U S A 2016 11 15;113(48):E7720-E7729. Epub 2016 Nov 15.

Department of Genetics and Evolution, University of Geneva,1211 Geneva, Switzerland.

Vertebrate Hox genes encode transcription factors operating during the development of multiple organs and structures. However, the evolutionary mechanism underlying this remarkable pleiotropy remains to be fully understood. Here, we show that Hoxd8 and Hoxd9, two genes of the HoxD complex, are transcribed during mammary bud (MB) development. However, unlike in other developmental contexts, their coexpression does not rely on the same regulatory mechanism. Hoxd8 is regulated by the combined activity of closely located sequences and the most distant telomeric gene desert. On the other hand, Hoxd9 is controlled by an enhancer-rich region that is also located within the telomeric gene desert but has no impact on Hoxd8 transcription, thus constituting an exception to the global regulatory logic systematically observed at this locus. The latter DNA region is also involved in Hoxd gene regulation in other contexts and strongly interacts with Hoxd9 in all tissues analyzed thus far, indicating that its regulatory activity was already operational before the appearance of mammary glands. Within this DNA region and neighboring a strong limb enhancer, we identified a short sequence conserved in therian mammals and capable of enhancer activity in the MBs. We propose that Hoxd gene regulation in embryonic MBs evolved by hijacking a preexisting regulatory landscape that was already at work before the emergence of mammals in structures such as the limbs or the intestinal tract.
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http://dx.doi.org/10.1073/pnas.1617141113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5137715PMC
November 2016

Reorganisation of Hoxd regulatory landscapes during the evolution of a snake-like body plan.

Elife 2016 08 1;5. Epub 2016 Aug 1.

Department of Genetics and Evolution, University of Geneva, Geneva, Switzerland.

Within land vertebrate species, snakes display extreme variations in their body plan, characterized by the absence of limbs and an elongated morphology. Such a particular interpretation of the basic vertebrate body architecture has often been associated with changes in the function or regulation of Hox genes. Here, we use an interspecies comparative approach to investigate different regulatory aspects at the snake HoxD locus. We report that, unlike in other vertebrates, snake mesoderm-specific enhancers are mostly located within the HoxD cluster itself rather than outside. In addition, despite both the absence of limbs and an altered Hoxd gene regulation in external genitalia, the limb-associated bimodal HoxD chromatin structure is maintained at the snake locus. Finally, we show that snake and mouse orthologous enhancer sequences can display distinct expression specificities. These results show that vertebrate morphological evolution likely involved extensive reorganisation at Hox loci, yet within a generally conserved regulatory framework.
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http://dx.doi.org/10.7554/eLife.16087DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4969037PMC
August 2016

A role for HOX13 proteins in the regulatory switch between TADs at the HoxD locus.

Genes Dev 2016 05 19;30(10):1172-86. Epub 2016 May 19.

Department of Genetics and Evolution, University of Geneva, 1211 Geneva 4, Switzerland; School of Life Sciences, Federal Institute of Technology, Lausanne, 1015 Lausanne, Switzerland;

During vertebrate limb development, Hoxd genes are regulated following a bimodal strategy involving two topologically associating domains (TADs) located on either side of the gene cluster. These regulatory landscapes alternatively control different subsets of Hoxd targets, first into the arm and subsequently into the digits. We studied the transition between these two global regulations, a switch that correlates with the positioning of the wrist, which articulates these two main limb segments. We show that the HOX13 proteins themselves help switch off the telomeric TAD, likely through a global repressive mechanism. At the same time, they directly interact with distal enhancers to sustain the activity of the centromeric TAD, thus explaining both the sequential and exclusive operating processes of these two regulatory domains. We propose a model in which the activation of Hox13 gene expression in distal limb cells both interrupts the proximal Hox gene regulation and re-enforces the distal regulation. In the absence of HOX13 proteins, a proximal limb structure grows without any sign of wrist articulation, likely related to an ancestral fish-like condition.
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http://dx.doi.org/10.1101/gad.281055.116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4888838PMC
May 2016
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