staff

Denis Duboule

Chairman in GenEv

  • T: +41 22 379 67 71
  • office 4004 (Sciences III)

Full Professor in Developmental Genomics

  • T: +41 22 379 67 71
  • office 4004 (Sciences III)
  • Reorganisation of Hoxd regulatory landscapes during the evolution of a snake-like body plan. Elife 2016 ;5():. 10.7554/eLife.16087. PMC4969037.

    Guerreiro I, Gitto S, Novoa A, Codourey J, Nguyen Huynh TH, Gonzalez F, Milinkovitch MC, Mallo M, Duboule D

    abstract

    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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  • A role for HOX13 proteins in the regulatory switch between TADs at the HoxD locus. Genes Dev. 2016 May;30(10):1172-86. gad.281055.116. 10.1101/gad.281055.116. PMC4888838.

    Beccari L, Yakushiji-Kaminatsui N, Woltering JM, Necsulea A, Lonfat N, Rodríguez-Carballo E, Mascrez B, Yamamoto S, Kuroiwa A, Duboule D

    abstract

    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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  • Topological Domains, Metagenes, and the Emergence of Pleiotropic Regulations at Hox Loci. Curr. Top. Dev. Biol. 2016 ;116():299-314. S0070-2153(15)00188-X. 10.1016/bs.ctdb.2015.11.022.

    Darbellay F, Duboule D

    abstract

    Hox gene clusters of jaw vertebrates display a tight genomic organization, which has no equivalent in any other bilateria genomes sequenced thus far. It was previously argued that such a topological consolidation toward a condensed, metagenic structure was due to the accumulation of long-range regulations flanking Hox loci, a phenomenon made possible by the successive genome duplications that occurred at the root of the vertebrate lineage, similar to gene neofunctionalization but applied to a coordinated multigenic system. Here, we propose that the emergence of such large vertebrate regulatory landscapes containing a range of global enhancers was greatly facilitated by the presence of topologically associating domains (TADs). These chromatin domains, mostly constitutive, may have been used as genomic niches where novel regulations could evolve due to both the preexistence of a structural backbone poised to integrate novel regulatory inputs, and a highly adaptive transcriptional readout. We propose a scenario for the coevolution of such TADs and the emergence of pleiotropy at ancestral vertebrate Hox loci.

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  • Visualizing the HoxD Gene Cluster at the Nanoscale Level. Cold Spring Harb. Symp. Quant. Biol. 2015 ;80():9-16. sqb.2015.80.027177. 10.1101/sqb.2015.80.027177.

    Fabre PJ, Benke A, Manley S, Duboule D

    abstract

    Transcription of HoxD cluster genes in limbs is coordinated by two topologically associating domains (TADs), neighboring the cluster and containing various enhancers. Here, we use a combination of microscopy approaches and chromosome conformation capture to assess the structural changes occurring in this global architecture in various functional states. We observed that despite their spatial juxtaposition, the TADs are consistently kept as distinct three-dimensional units. Hox genes located at their boundary can show significant spatial segregation over long distances, suggesting that physical elongation of the HoxD cluster occurs. The use of superresolution imaging (STORM [stochastic optical reconstruction microscopy]) revealed that the gene cluster can be in an either compact or elongated shape. The latter configuration is observed in transcriptionally active tissue and in embryonic stem cells, consistent with chromosome conformation capture results. Such morphological changes at HoxD in developing digits seem to be associated with its position at the boundary between two TADs and support the idea that chromatin dynamics is important in the establishment of transcriptional activity.

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  • Nanoscale spatial organization of the HoxD gene cluster in distinct transcriptional states. Proc. Natl. Acad. Sci. U.S.A. 2015 Nov;112(45):13964-9. 1517972112. 10.1073/pnas.1517972112. PMC4653165.

    Fabre PJ, Benke A, Joye E, Nguyen Huynh TH, Manley S, Duboule D

    abstract

    Chromatin condensation plays an important role in the regulation of gene expression. Recently, it was shown that the transcriptional activation of Hoxd genes during vertebrate digit development involves modifications in 3D interactions within and around the HoxD gene cluster. This reorganization follows a global transition from one set of regulatory contacts to another, between two topologically associating domains (TADs) located on either side of the HoxD locus. Here, we use 3D DNA FISH to assess the spatial organization of chromatin at and around the HoxD gene cluster and report that although the two TADs are tightly associated, they appear as spatially distinct units. We measured the relative position of genes within the cluster and found that they segregate over long distances, suggesting that a physical elongation of the HoxD cluster can occur. We analyzed this possibility by super-resolution imaging (STORM) and found that tissues with distinct transcriptional activity exhibit differing degrees of elongation. We also observed that the morphological change of the HoxD cluster in developing digits is associated with its position at the boundary between the two TADs. Such variations in the fine-scale architecture of the gene cluster suggest causal links among its spatial configuration, transcriptional activation, and the flanking chromatin context.

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  • Tetrapod axial evolution and developmental constraints; Empirical underpinning by a mouse model. Mech. Dev. 2015 Nov;138 Pt 2():64-72. S0925-4773(15)30007-1. 10.1016/j.mod.2015.07.006. PMC4678112.

    Woltering JM, Duboule D

    abstract

    The tetrapod vertebral column has become increasingly complex during evolution as an adaptation to a terrestrial life. At the same time, the evolution of the vertebral formula became subject to developmental constraints acting on the size of the cervical and thoraco-lumbar regions. In the course of our studies concerning the evolution of Hox gene regulation, we produced a transgenic mouse model expressing fish Hox genes, which displayed a reduced number of thoraco-lumbar vertebrae and concurrent sacral homeotic transformations. Here, we analyze this mutant stock and conclude that the ancestral, pre-tetrapodial Hox code already possessed the capacity to induce vertebrae with sacral characteristics. This suggests that alterations in the interpretation of the Hox code may have participated to the evolution of this region in tetrapods, along with potential modifications of the HOX proteins themselves. With its reduced vertebral number, this mouse stock violates a previously described developmental constraint, which applies to the thoraco-lumbar region. The resulting offset between motor neuron morphology, vertebral patterning and the relative positioning of hind limbs illustrates that the precise orchestration of the Hox-clock in parallel with other ontogenetic pathways places constraints on the evolvability of the body plan.

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  • Structure, function and evolution of topologically associating domains (TADs) at HOX loci. FEBS Lett. 2015 Oct;589(20 Pt A):2869-76. S0014-5793(15)00282-3. 10.1016/j.febslet.2015.04.024.

    Lonfat N, Duboule D

    abstract

    Hox genes encode transcription factors necessary for patterning the major developing anterior to posterior embryonic axis. In addition, during vertebrate evolution, various subsets of this gene family were co-opted along with the emergence of novel body structures, such as the limbs or the external genitalia. The morphogenesis of these axial structures thus relies in part upon the precisely controlled transcription of specific Hox genes, a mechanism involving multiple long-range enhancers. Recently, it was reported that such regulatory mechanisms were largely shared between different developing tissues, though with some specificities, suggesting the recruitment of ancestral regulatory modalities from one tissue to another. The analysis of chromatin architectures at HoxD and HoxA loci revealed the existence of two flanking topologically associating domains (TADs), precisely encompassing the adjacent regulatory landscapes. Here, we discuss the function of these TADs in the control of Hox gene regulation and we speculate about their capacity to serve as structural frameworks for the emergence of novel enhancers. In this view, TADs may have been used as genomic niches to evolve pleiotropic regulations found at many developmental loci.

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  • Clustering of mammalian Hox genes with other H3K27me3 targets within an active nuclear domain. Proc. Natl. Acad. Sci. U.S.A. 2015 Apr;112(15):4672-7. 1504783112. 10.1073/pnas.1504783112. PMC4403207.

    Vieux-Rochas M, Fabre PJ, Leleu M, Duboule D, Noordermeer D

    abstract

    Embryogenesis requires the precise activation and repression of many transcriptional regulators. The Polycomb group proteins and the associated H3K27me3 histone mark are essential to maintain the inactive state of many of these genes. Mammalian Hox genes are targets of Polycomb proteins and form local 3D clusters centered on the H3K27me3 mark. More distal contacts have also been described, yet their selectivity, dynamics, and relation to other layers of chromatin organization remained elusive. We report that repressed Hox genes form mutual intra- and interchromosomal interactions with other genes located in strong domains labeled by H3K27me3. These interactions occur in a central and active nuclear environment that consists of the HiC compartment A, away from peripheral lamina-associated domains. Interactions are independent of nearby H3K27me3-marked loci and determined by chromosomal distance and cell-type-specific scaling factors, thus inducing a moderate reorganization during embryogenesis. These results provide a simplified view of nuclear organization whereby Polycomb proteins may have evolved to repress genes located in gene-dense regions whose position is restricted to central, active, nuclear environments.

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  • Convergent evolution of complex regulatory landscapes and pleiotropy at Hox loci. Science 2014 Nov;346(6212):1004-6. 346/6212/1004. 10.1126/science.1257493.

    Lonfat N, Montavon T, Darbellay F, Gitto S, Duboule D

    abstract

    Hox genes are required during the morphogenesis of both vertebrate digits and external genitals. We investigated whether transcription in such distinct contexts involves a shared enhancer-containing landscape. We show that the same regulatory topology is used, yet with some tissue-specific enhancer-promoter interactions, suggesting the hijacking of a regulatory backbone from one context to the other. In addition, comparable organizations are observed at both HoxA and HoxD clusters, which separated through genome duplication in an ancestral invertebrate animal. We propose that this convergent regulatory evolution was triggered by the preexistence of some chromatin architecture, thus facilitating the subsequent recruitment of the appropriate transcription factors. Such regulatory topologies may have both favored and constrained the evolution of pleiotropic developmental loci in vertebrates.

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  • The genetics of murine Hox loci: TAMERE, STRING, and PANTHERE to engineer chromosome variants. Methods Mol. Biol. 2014 ;1196():89-102. 10.1007/978-1-4939-1242-1_6.

    Tschopp P, Duboule D

    abstract

    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.

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  • Considerations when investigating lncRNA function in vivo. Elife 2014 ;3():e03058. PMC4132285.

    Bassett AR, Akhtar A, Barlow DP, Bird AP, Brockdorff N, Duboule D, Ephrussi A, Ferguson-Smith AC, Gingeras TR, Haerty W, Higgs DR, Miska EA, Ponting CP

    abstract

    Although a small number of the vast array of animal long non-coding RNAs (lncRNAs) have known effects on cellular processes examined in vitro, the extent of their contributions to normal cell processes throughout development, differentiation and disease for the most part remains less clear. Phenotypes arising from deletion of an entire genomic locus cannot be unequivocally attributed either to the loss of the lncRNA per se or to the associated loss of other overlapping DNA regulatory elements. The distinction between cis- or trans-effects is also often problematic. We discuss the advantages and challenges associated with the current techniques for studying the in vivo function of lncRNAs in the light of different models of lncRNA molecular mechanism, and reflect on the design of experiments to mutate lncRNA loci. These considerations should assist in the further investigation of these transcriptional products of the genome.

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  • Attenuated sensing of SHH by Ptch1 underlies evolution of bovine limbs. Nature 2014 Jul;511(7507):46-51. nature13289. 10.1038/nature13289.

    Lopez-Rios J, Duchesne A, Speziale D, Andrey G, Peterson KA, Germann P, Unal E, Liu J, Floriot S, Barbey S, Gallard Y, Müller-Gerbl M, Courtney AD, Klopp C, Rodriguez S, Ivanek R, Beisel C, Wicking C, Iber D, Robert B, McMahon AP, Duboule D, Zeller R

    abstract

    The large spectrum of limb morphologies reflects the wide evolutionary diversification of the basic pentadactyl pattern in tetrapods. In even-toed ungulates (artiodactyls, including cattle), limbs are adapted for running as a consequence of progressive reduction of their distal skeleton to symmetrical and elongated middle digits with hoofed phalanges. Here we analyse bovine embryos to establish that polarized gene expression is progressively lost during limb development in comparison to the mouse. Notably, the transcriptional upregulation of the Ptch1 gene, which encodes a Sonic hedgehog (SHH) receptor, is disrupted specifically in the bovine limb bud mesenchyme. This is due to evolutionary alteration of a Ptch1 cis-regulatory module, which no longer responds to graded SHH signalling during bovine handplate development. Our study provides a molecular explanation for the loss of digit asymmetry in bovine limb buds and suggests that modifications affecting the Ptch1 cis-regulatory landscape have contributed to evolutionary diversification of artiodactyl limbs.

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  • Temporal dynamics and developmental memory of 3D chromatin architecture at Hox gene loci. Elife 2014 ;3():e02557. PMC4017647.

    Noordermeer D, Leleu M, Schorderet P, Joye E, Chabaud F, Duboule D

    abstract

    Hox genes are essential regulators of embryonic development. Their step-wise transcriptional activation follows their genomic topology and the various states of activation are subsequently memorized into domains of progressively overlapping gene products. We have analyzed the 3D chromatin organization of Hox clusters during their early activation in vivo, using high-resolution circular chromosome conformation capture. Initially, Hox clusters are organized as single chromatin compartments containing all genes and bivalent chromatin marks. Transcriptional activation is associated with a dynamic bi-modal 3D organization, whereby the genes switch autonomously from an inactive to an active compartment. These local 3D dynamics occur within a framework of constitutive interactions within the surrounding Topological Associated Domains, indicating that this regulation process is mostly cluster intrinsic. The step-wise progression in time is fixed at various body levels and thus can account for the chromatin architectures previously described at a later stage for different anterior to posterior levels.DOI: http://dx.doi.org/10.7554/eLife.02557.001.

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  • Evolving Hox activity profiles govern diversity in locomotor systems. Dev. Cell 2014 Apr;29(2):171-87. S1534-5807(14)00160-9. 10.1016/j.devcel.2014.03.008. PMC4024207. NIHMS578792.

    Jung H, Mazzoni EO, Soshnikova N, Hanley O, Venkatesh B, Duboule D, Dasen JS

    abstract

    The emergence of limb-driven locomotor behaviors was a key event in the evolution of vertebrates and fostered the transition from aquatic to terrestrial life. We show that the generation of limb-projecting lateral motor column (LMC) neurons in mice relies on a transcriptional autoregulatory module initiated via transient activity of multiple genes within the HoxA and HoxC clusters. Repression of this module at thoracic levels restricts expression of LMC determinants, thus dictating LMC position relative to the limbs. This suppression is mediated by a key regulatory domain that is specifically found in the Hoxc9 proteins of appendage-bearing vertebrates. The profile of Hoxc9 expression inversely correlates with LMC position in land vertebrates and likely accounts for the absence of LMC neurons in limbless species such as snakes. Thus, modulation of both Hoxc9 protein function and Hoxc9 gene expression likely contributed to evolutionary transitions between undulatory and ambulatory motor circuit connectivity programs.

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  • SnapShot: Hox gene regulation. Cell 2014 Feb;156(4):856-856.e1. S0092-8674(14)00154-8. 10.1016/j.cell.2014.01.060.

    Andrey G, Duboule D

    abstract

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  • Conservation and divergence of regulatory strategies at Hox Loci and the origin of tetrapod digits. PLoS Biol. 2014 Jan;12(1):e1001773. 10.1371/journal.pbio.1001773. PBIOLOGY-D-13-02584. PMC3897358.

    Woltering JM, Noordermeer D, Leleu M, Duboule D

    abstract

    The evolution of tetrapod limbs from fish fins enabled the conquest of land by vertebrates and thus represents a key step in evolution. Despite the use of comparative gene expression analyses, critical aspects of this transformation remain controversial, in particular the origin of digits. Hoxa and Hoxd genes are essential for the specification of the different limb segments and their functional abrogation leads to large truncations of the appendages. Here we show that the selective transcription of mouse Hoxa genes in proximal and distal limbs is related to a bimodal higher order chromatin structure, similar to that reported for Hoxd genes, thus revealing a generic regulatory strategy implemented by both gene clusters during limb development. We found the same bimodal chromatin architecture in fish embryos, indicating that the regulatory mechanism used to pattern tetrapod limbs may predate the divergence between fish and tetrapods. However, when assessed in mice, both fish regulatory landscapes triggered transcription in proximal rather than distal limb territories, supporting an evolutionary scenario whereby digits arose as tetrapod novelties through genetic retrofitting of preexisting regulatory landscapes. We discuss the possibility to consider regulatory circuitries, rather than expression patterns, as essential parameters to define evolutionary synapomorphies.

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  • Snakes: hatching of a model system for Evo-Devo? Int. J. Dev. Biol. 2014 ;58(10-12):727-32. 150026dd. 10.1387/ijdb.150026dd.

    Guerreiro I, Duboule D

    abstract

    Evo-Devo studies rely on a collection of animal model systems belonging to different phylogenetic branches to try and understand how organisms carrying a similar set of genes and pathways can develop into such a variety of shapes and sizes. The squamate clade, however, has only recently started to receive the attention it deserves in particular due to extreme morphological and metabolic aspects and, consequently, the important insights that it could bring in different fields. The recent sequencing of several squamate genomes as well as the generation of high quality trancriptomes for different snake tissues now provide the necessary tools to complement biological studies. Here, we briefly report on recent work involving developing snake embryos to illustrate their interest to assess vertebrate developmental mechanisms. We also discuss the relevance to use snake species as Evo-Devo model systems and potential ways to cross the important limitations intrinsically associated with developmental and genetic studies of these fascinating animals.

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  • The king cobra genome reveals dynamic gene evolution and adaptation in the snake venom system. Proc. Natl. Acad. Sci. U.S.A. 2013 Dec;110(51):20651-6. 1314702110. 10.1073/pnas.1314702110. PMC3870661.

    Vonk FJ, Casewell NR, Henkel CV, Heimberg AM, Jansen HJ, McCleary RJ, Kerkkamp HM, Vos RA, Guerreiro I, Calvete JJ, Wüster W, Woods AE, Logan JM, Harrison RA, Castoe TA, de Koning AP, Pollock DD, Yandell M, Calderon D, Renjifo C, Currier RB, Salgado D, Pla D, Sanz L, Hyder AS, Ribeiro JM, Arntzen JW, van den Thillart GE, Boetzer M, Pirovano W, Dirks RP, Spaink HP, Duboule D, McGlinn E, Kini RM, Richardson MK

    abstract

    Snakes are limbless predators, and many species use venom to help overpower relatively large, agile prey. Snake venoms are complex protein mixtures encoded by several multilocus gene families that function synergistically to cause incapacitation. To examine venom evolution, we sequenced and interrogated the genome of a venomous snake, the king cobra (Ophiophagus hannah), and compared it, together with our unique transcriptome, microRNA, and proteome datasets from this species, with data from other vertebrates. In contrast to the platypus, the only other venomous vertebrate with a sequenced genome, we find that snake toxin genes evolve through several distinct co-option mechanisms and exhibit surprisingly variable levels of gene duplication and directional selection that correlate with their functional importance in prey capture. The enigmatic accessory venom gland shows a very different pattern of toxin gene expression from the main venom gland and seems to have recruited toxin-like lectin genes repeatedly for new nontoxic functions. In addition, tissue-specific microRNA analyses suggested the co-option of core genetic regulatory components of the venom secretory system from a pancreatic origin. Although the king cobra is limbless, we recovered coding sequences for all Hox genes involved in amniote limb development, with the exception of Hoxd12. Our results provide a unique view of the origin and evolution of snake venom and reveal multiple genome-level adaptive responses to natural selection in this complex biological weapon system. More generally, they provide insight into mechanisms of protein evolution under strong selection.

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  • A genetic approach to the recruitment of PRC2 at the HoxD locus. PLoS Genet. 2013 Nov;9(11):e1003951. 10.1371/journal.pgen.1003951. PGENETICS-D-13-01888. PMC3820793.

    Schorderet P, Lonfat N, Darbellay F, Tschopp P, Gitto S, Soshnikova N, Duboule D

    abstract

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

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  • Topology of mammalian developmental enhancers and their regulatory landscapes. Nature 2013 Oct;502(7472):499-506. nature12753. 10.1038/nature12753.

    de Laat W, Duboule D

    abstract

    How a complex animal can arise from a fertilized egg is one of the oldest and most fascinating questions of biology, the answer to which is encoded in the genome. Body shape and organ development, and their integration into a functional organism all depend on the precise expression of genes in space and time. The orchestration of transcription relies mostly on surrounding control sequences such as enhancers, millions of which form complex regulatory landscapes in the non-coding genome. Recent research shows that high-order chromosome structures make an important contribution to enhancer functionality by triggering their physical interactions with target genes.

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  • Multiple enhancers regulate Hoxd genes and the Hotdog LncRNA during cecum budding. Cell Rep 2013 Oct;5(1):137-50. S2211-1247(13)00500-7. 10.1016/j.celrep.2013.09.002.

    Delpretti S, Montavon T, Leleu M, Joye E, Tzika A, Milinkovitch M, Duboule D

    abstract

    Hox genes are required for the development of the intestinal cecum, a major organ of plant-eating species. We have analyzed the transcriptional regulation of Hoxd genes in cecal buds and show that they are controlled by a series of enhancers located in a gene desert flanking the HoxD cluster. The start site of two opposite long noncoding RNAs (lncRNAs), Hotdog and Twin of Hotdog, selectively contacts the expressed Hoxd genes in the framework of a topological domain, coinciding with robust transcription of these genes during cecum budding. Both lncRNAs are specifically transcribed in the cecum, albeit bearing no detectable function in trans. Hedgehogs have kept this regulatory potential despite the absence of the cecum, suggesting that these mechanisms are used in other developmental situations. In this context, we discuss the implementation of a common "budding toolkit" between the cecum and the limbs.

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  • Chromatin looping and organization at developmentally regulated gene loci. Wiley Interdiscip Rev Dev Biol ;2(5):615-30. 10.1002/wdev.103.

    Noordermeer D, Duboule D

    abstract

    Developmentally regulated genes are often controlled by distant enhancers, silencers and insulators, to implement their correct transcriptional programs. In recent years, the development of 3C and derived techniques (4C, 5C, HiC, ChIA-PET, etc.) has confirmed that chromatin looping is an important mechanism for the transfer of regulatory information in mammalian cells. At many developmentally regulated gene loci, transcriptional activation is indeed accompanied by the formation of chromatin loops between genes and distant enhancers. Similarly, dynamic looping between insulator elements and changes in local 3D organization may be observed upon variation in transcriptional activity. Chromatin looping also occurs at silent gene loci, where its function remains less understood. In lineage-committed cells, partial 3D configurations are detected at loci that are activated at later stages. However, these partial configurations usually lack promoter-enhancer loops that accompany transcriptional activation, suggesting they have structural functions. Definitive evidence for a repressive role of chromatin looping is still lacking. Chromatin loops have been reported at repressed loci but, alternatively, they may act as a distraction for active loops. Together, these mechanisms allow fine-tuning of regulatory programs, thus providing further diversity in the transcriptional control of developmentally regulated gene loci.

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  • Combined function of HoxA and HoxB clusters in neural crest cells. Dev. Biol. 2013 Oct;382(1):293-301. S0012-1606(13)00360-6. 10.1016/j.ydbio.2013.06.027.

    Vieux-Rochas M, Mascrez B, Krumlauf R, Duboule D

    abstract

    The evolution of chordates was accompanied by critical anatomical innovations in craniofacial development, along with the emergence of neural crest cells. The potential of these cells to implement a craniofacial program in part depends upon the (non-)expression of Hox genes. For instance, the development of jaws requires the inhibition of Hox genes function in the first pharyngeal arch. In contrast, Hox gene products induce craniofacial structures in more caudal territories. To further investigate which Hox gene clusters are involved in this latter role, we generated HoxA;HoxB cluster double mutant animals in cranial neural crest cells. We observed the appearance of a supernumerary dentary-like bone with an endochondral ossification around a neo-Meckel's cartilage matrix and an attachment of neo-muscle demonstrating that HoxB genes enhance the phenotype induced by the deletion of the HoxA cluster alone. In addition, a cervical and hypertrophic thymus was associated with the supernumerary dentary-like bone, which may reflect its ancestral position near the filtrating system. Altogether these results show that the HoxA and HoxB clusters cooperated during evolution to lead to present craniofacial diversity.

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  • Transgene- and locus-dependent imprinting reveals allele-specific chromosome conformations. Proc. Natl. Acad. Sci. U.S.A. 2013 Jul;110(29):11946-51. 1310704110. 10.1073/pnas.1310704110. PMC3718164.

    Lonfat N, Montavon T, Jebb D, Tschopp P, Nguyen Huynh TH, Zakany J, Duboule D

    abstract

    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.

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  • A switch between topological domains underlies HoxD genes collinearity in mouse limbs. Science 2013 Jun;340(6137):1234167. 340/6137/1234167. 10.1126/science.1234167.

    Andrey G, Montavon T, Mascrez B, Gonzalez F, Noordermeer D, Leleu M, Trono D, Spitz F, Duboule D

    abstract

    Hox genes are major determinants of the animal body plan, where they organize structures along both the trunk and appendicular axes. During mouse limb development, Hoxd genes are transcribed in two waves: early on, when the arm and forearm are specified, and later, when digits form. The transition between early and late regulations involves a functional switch between two opposite topological domains. This switch is reflected by a subset of Hoxd genes mapping centrally into the cluster, which initially interact with the telomeric domain and subsequently swing toward the centromeric domain, where they establish new contacts. This transition between independent regulatory landscapes illustrates both the modularity of the limbs and the distinct evolutionary histories of its various pieces. It also allows the formation of an intermediate area of low HOX proteins content, which develops into the wrist, the transition between our arms and our hands. This regulatory strategy accounts for collinear Hox gene regulation in land vertebrate appendages.

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  • Role of a polymorphism in a Hox/Pax-responsive enhancer in the evolution of the vertebrate spine. Proc. Natl. Acad. Sci. U.S.A. 2013 Jun;110(26):10682-6. 1300592110. 10.1073/pnas.1300592110. PMC3696775.

    Guerreiro I, Nunes A, Woltering JM, Casaca A, Nóvoa A, Vinagre T, Hunter ME, Duboule D, Mallo M

    abstract

    Patterning of the vertebrate skeleton requires the coordinated activity of Hox genes. In particular, Hox10 proteins are essential to set the transition from thoracic to lumbar vertebrae because of their rib-repressing activity. In snakes, however, the thoracic region extends well into Hox10-expressing areas of the embryo, suggesting that these proteins are unable to block rib formation. Here, we show that this is not a result of the loss of rib-repressing properties by the snake proteins, but rather to a single base pair change in a Hox/Paired box (Pax)-responsive enhancer, which prevents the binding of Hox proteins. This polymorphism is also found in Paenungulata, such as elephants and manatees, which have extended rib cages. In vivo, this modified enhancer failed to respond to Hox10 activity, supporting its role in the extension of rib cages. In contrast, the enhancer could still interact with Hoxb6 and Pax3 to promote rib formation. These results suggest that a polymorphism in the Hox/Pax-responsive enhancer may have played a role in the evolution of the vertebrate spine by differently modulating its response to rib-suppressing and rib-promoting Hox proteins.

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  • Chromatin organization and global regulation of Hox gene clusters. Philos. Trans. R. Soc. Lond., B, Biol. Sci. 2013 ;368(1620):20120367. rstb.2012.0367. 10.1098/rstb.2012.0367. PMC3682730.

    Montavon T, Duboule D

    abstract

    During development, a properly coordinated expression of Hox genes, within their different genomic clusters is critical for patterning the body plans of many animals with a bilateral symmetry. The fascinating correspondence between the topological organization of Hox clusters and their transcriptional activation in space and time has served as a paradigm for understanding the relationships between genome structure and function. Here, we review some recent observations, which revealed highly dynamic changes in the structure of chromatin at Hox clusters, in parallel with their activation during embryonic development. We discuss the relevance of these findings for our understanding of large-scale gene regulation.

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  • Chromatin architectures and Hox gene collinearity. Curr. Top. Dev. Biol. 2013 ;104():113-48. B978-0-12-416027-9.00004-8. 10.1016/B978-0-12-416027-9.00004-8.

    Noordermeer D, Duboule D

    abstract

    Ever since the observation that collinearity, that is, the sequential activity of Hox genes based on their relative positions within their gene clusters, is conserved throughout most of the animal kingdom, the question has been raised as to what are the underlying molecular mechanisms. In recent years, technological advances have allowed to uncover changes in chromatin organization that accompany collinearity at Hox gene clusters. Here, we discuss insights in the dynamics of histone modifications and 3D organization in Drosophila and mammals and relate these findings to genomic organization of Hox gene clusters. Using these findings, we propose a framework for collinearity, based on five components: clustering, coating, compaction, compartmentalization, and contacts. We argue that these five components may be sufficient to provide a mechanistic ground for the readout of collinearity in Drosophila and vertebrates.

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  • Duplications of hox gene clusters and the emergence of vertebrates. Dev. Biol. 2013 Jun;378(2):194-9. S0012-1606(13)00133-4. 10.1016/j.ydbio.2013.03.004.

    Soshnikova N, Dewaele R, Janvier P, Krumlauf R, Duboule D

    abstract

    The vertebrate body plan is characterized by an increased complexity relative to that of all other chordates and large-scale gene amplifications have been associated with key morphological innovations leading to their remarkable evolutionary success. Here, we use compound full Hox clusters deletions to investigate how Hox genes duplications may have contributed to the emergence of vertebrate-specific innovations. We show that the combined deletion of HoxA and HoxB leads to an atavistic heart phenotype, suggesting that the ancestral HoxA/B cluster was co-opted to help in diversifying the complex organ in vertebrates. Other phenotypic effects observed seem to illustrate the resurgence of ancestral (plesiomorphic) features. This indicates that the duplications of Hox clusters were associated with the recruitment or formation of novel cis-regulatory controls, which were key to the evolution of many vertebrate features and hence to the evolutionary radiation of this group.

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  • Impact of copy number variations (CNVs) on long-range gene regulation at the HoxD locus. Proc. Natl. Acad. Sci. U.S.A. 2012 Dec;109(50):20204-11. 1217659109. 10.1073/pnas.1217659109. PMC3528568.

    Montavon T, Thevenet L, Duboule D

    abstract

    Copy number variations are genomic structural variants that are frequently associated with human diseases. Among these copy number variations, duplications of DNA segments are often assumed to lead to dosage effects by increasing the copy number of either genes or their regulatory elements. We produced a series of large targeted duplications within a conserved gene desert upstream of the murine HoxD locus. This DNA region, syntenic to human 2q31-32, contains a range of regulatory elements required for Hoxd gene transcription, and it is often disrupted and/or reorganized in human genetic conditions collectively known as the 2q31 syndrome. Unexpectedly, one such duplication led to a transcriptional down-regulation in developing digits by impairing physical interactions between the target genes and their upstream regulatory elements, thus phenocopying the effect obtained when these enhancer sequences are deleted. These results illustrate the detrimental consequences of interrupting highly conserved regulatory landscapes and reveal a mechanism where genomic duplications lead to partial loss of function of nearby located genes.

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  • A genetic basis for altered sexual behavior in mutant female mice. Curr. Biol. 2012 Sep;22(18):1676-80. S0960-9822(12)00743-9. 10.1016/j.cub.2012.06.067.

    Zakany J, Duboule D

    abstract

    Although neural substrates of mammalian female mating behavior have been described, the association between complex courtship activity and specific underlying mechanisms remains elusive. We have isolated a mouse line that unexpectedly shows altered female social behavior with increased investigation of males and increased genital biting. We investigated adult individuals by behavioral observation and genetic and molecular neuroanatomy methods. We report exacerbated inverse pursuits and incapacitating bites directed at the genitals of stud males. This extreme deviation from wild-type female courtship segregates with a deletion of the Hoxd1 to Hoxd9 genomic region. This dominant Atypical female courtship allele (HoxD(Afc)) induces ectopic Hoxd10 gene expression in several regions in newborn forebrain transitorily and stably in a sparse subpopulation of cells in the cornu ammonis fields of adult hippocampus, which may thus lead to an abnormal modulation in the sexual behavior of mutant females. The resulting compulsive sexual solicitation behavior displayed by the most affected individuals suggests new avenues to study the genetic and molecular bases of normal and pathological mammalian affect and raises the potential involvement of the hippocampus in the control of female courtship behavior. The potential relevance to human 2q.31.1 microdeletion syndrome is discussed.

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  • Landscapes and archipelagos: spatial organization of gene regulation in vertebrates. Trends Cell Biol. 2012 Jul;22(7):347-54. S0962-8924(12)00057-8. 10.1016/j.tcb.2012.04.003.

    Montavon T, Duboule D

    abstract

    Vertebrate genes controlling critical developmental processes are often regulated by complex sets of global enhancer sequences, located at a distance, within neighboring gene deserts. Recent technological advances have made it possible to investigate the spatial organization of these 'regulatory landscapes'. The integration of such datasets with information on chromatin status, transcriptional activity and nuclear localization of these loci, as well as the effects of genetic modifications thereof, may bring a more comprehensive understanding of tissue- and/or stage-specific gene regulation in both normal and pathological contexts. Here, we review the impact of recent technological advances on our understanding of large-scale gene regulation in vertebrates, by focusing on paradigmatic gene loci.

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  • A function for all posterior Hoxd genes during digit development? Dev. Dyn. 2012 Apr;241(4):792-802. 10.1002/dvdy.23756.

    Delpretti S, Zakany J, Duboule D

    abstract

    Four posterior Hoxd genes, from Hoxd13 to Hoxd10, are collectively regulated during the development of tetrapod digits. Besides the well-documented role of Hoxd13, the function of the neighboring genes has been difficult to evaluate due to the close genetic linkage and potential regulatory interferences. We used a combination of five small nested deletions in cis, involving from two to four consecutive genes of the Hoxd13 to Hoxd9 loci, in mice, to evaluate their combined functional importance.

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  • Bimodal control of Hoxd gene transcription in the spinal cord defines two regulatory subclusters. Development 2012 Mar;139(5):929-39. dev.076794. 10.1242/dev.076794.

    Tschopp P, Christen AJ, Duboule D

    abstract

    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.

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  • A regulatory archipelago controls Hox genes transcription in digits. Cell 2011 Nov;147(5):1132-45. S0092-8674(11)01273-6. 10.1016/j.cell.2011.10.023.

    Montavon T, Soshnikova N, Mascrez B, Joye E, Thevenet L, Splinter E, de Laat W, Spitz F, Duboule D

    abstract

    The evolution of digits was an essential step in the success of tetrapods. Among the key players, Hoxd genes are coordinately regulated in developing digits, where they help organize growth and patterns. We identified the distal regulatory sites associated with these genes by probing the three-dimensional architecture of this regulatory unit in developing limbs. This approach, combined with in vivo deletions of distinct regulatory regions, revealed that the active part of the gene cluster contacts several enhancer-like sequences. These elements are dispersed throughout the nearby gene desert, and each contributes either quantitatively or qualitatively to Hox gene transcription in presumptive digits. We propose that this genetic system, which we call a "regulatory archipelago," provides an inherent flexibility that may partly underlie the diversity in number and morphology of digits across tetrapods, as well as their resilience to drastic variations.

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  • A genetic approach to the transcriptional regulation of Hox gene clusters. Annu. Rev. Genet. 2011 ;45():145-66. 10.1146/annurev-genet-102209-163429.

    Tschopp P, Duboule D

    abstract

    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.

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  • The dynamic architecture of Hox gene clusters. Science 2011 Oct;334(6053):222-5. 334/6053/222. 10.1126/science.1207194.

    Noordermeer D, Leleu M, Splinter E, Rougemont J, De Laat W, Duboule D

    abstract

    The spatial and temporal control of Hox gene transcription is essential for patterning the vertebrate body axis. Although this process involves changes in histone posttranslational modifications, the existence of particular three-dimensional (3D) architectures remained to be assessed in vivo. Using high-resolution chromatin conformation capture methodology, we examined the spatial configuration of Hox clusters in embryonic mouse tissues where different Hox genes are active. When the cluster is transcriptionally inactive, Hox genes associate into a single 3D structure delimited from flanking regions. Once transcription starts, Hox clusters switch to a bimodal 3D organization where newly activated genes progressively cluster into a transcriptionally active compartment. This transition in spatial configurations coincides with the dynamics of chromatin marks, which label the progression of the gene clusters from a negative to a positive transcription status. This spatial compartmentalization may be key to process the colinear activation of these compact gene clusters.

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  • Analysis of the dynamics of limb transcriptomes during mouse development. BMC Dev. Biol. 2011 ;11():47. 1471-213X-11-47. 10.1186/1471-213X-11-47. PMC3160909.

    Gyurján I, Sonderegger B, Naef F, Duboule D

    abstract

    The development of vertebrate limbs has been a traditional system to study fundamental processes at work during ontogenesis, such as the establishment of spatial cellular coordinates, the effect of diffusible morphogenetic molecules or the translation between gene activity and morphogenesis. In addition, limbs are amongst the first targets of malformations in human and they display a huge realm of evolutionary variations within tetrapods, which make them a paradigm to study the regulatory genome.

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  • Reshuffling genomic landscapes to study the regulatory evolution of Hox gene clusters. Proc. Natl. Acad. Sci. U.S.A. 2011 Jun;108(26):10632-7. 1102985108. 10.1073/pnas.1102985108. PMC3127922.

    Tschopp P, Fraudeau N, Béna F, Duboule D

    abstract

    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.

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  • Structural and functional differences in the long non-coding RNA hotair in mouse and human. PLoS Genet. 2011 May;7(5):e1002071. 10.1371/journal.pgen.1002071. PGENETICS-D-11-00310. PMC3102750.

    Schorderet P, Duboule D

    abstract

    Long non-coding RNAs regulate various biological processes such as dosage compensation, imprinting, and chromatin organization. HOTAIR, a paradigm of this new class of RNAs, is localized within the human HOXC gene cluster and was shown, in human cells, to regulate HOXD genes in trans via the recruitment of Polycomb Repressive Complex 2 (PRC2), followed by the trimethylation of lysine 27 of histone H3. We looked for the presence of Hotair in mice to assess whether this in trans mechanism was conserved, in particular at the developmental stages, when Hoxd genes must be tightly regulated. We show that the cognate mouse Hotair is poorly conserved in sequence; and its absence, along with the deletion of the HoxC cluster, has surprisingly little effect in vivo, neither on the expression pattern or transcription efficiency, nor on the amount of K27me3 coverage of different Hoxd target genes. We conclude that Hotair may have rapidly evolved within mammals and acquired a functional importance in humans that is not easily revealed in mice. Alternatively, redundant or compensatory mechanisms may mask its function when studied under physiological conditions.

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  • A systematic enhancer screen using lentivector transgenesis identifies conserved and non-conserved functional elements at the Olig1 and Olig2 locus. PLoS ONE 2010 ;5(12):e15741. 10.1371/journal.pone.0015741. PMC3012086.

    Friedli M, Barde I, Arcangeli M, Verp S, Quazzola A, Zakany J, Lin-Marq N, Robyr D, Attanasio C, Spitz F, Duboule D, Trono D, Antonarakis SE

    abstract

    Finding sequences that control expression of genes is central to understanding genome function. Previous studies have used evolutionary conservation as an indicator of regulatory potential. Here, we present a method for the unbiased in vivo screen of putative enhancers in large DNA regions, using the mouse as a model. We cloned a library of 142 overlapping fragments from a 200 kb-long murine BAC in a lentiviral vector expressing LacZ from a minimal promoter, and used the resulting vectors to infect fertilized murine oocytes. LacZ staining of E11 embryos obtained by first using the vectors in pools and then testing individual candidates led to the identification of 3 enhancers, only one of which shows significant evolutionary conservation. In situ hybridization and 3C/4C experiments suggest that this enhancer, which is active in the neural tube and posterior diencephalon, influences the expression of the Olig1 and/or Olig2 genes. This work provides a new approach for the large-scale in vivo screening of transcriptional regulatory sequences, and further demonstrates that evolutionary conservation alone seems too limiting a criterion for the identification of enhancers.

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  • A regulatory 'landscape effect' over the HoxD cluster. Dev. Biol. 2011 Mar;351(2):288-96. S0012-1606(10)01290-X. 10.1016/j.ydbio.2010.12.034.

    Tschopp P, Duboule D

    abstract

    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.

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  • Functional analysis of CTCF during mammalian limb development. Dev. Cell 2010 Dec;19(6):819-30. S1534-5807(10)00536-8. 10.1016/j.devcel.2010.11.009.

    Soshnikova N, Montavon T, Leleu M, Galjart N, Duboule D

    abstract

    CCCTC-binding factor (CTCF) is a nuclear zinc-finger protein that displays insulating activity in a variety of biological assays. For example, CTCF-binding sites have been suggested to isolate Hox gene clusters from neighboring transcriptional interference. We investigated this issue during limb development, where Hoxd genes must remain isolated from long-range effects to allow essential regulation within independent sub-groups. We used conditional Ctcf inactivation in incipient forelimbs and show that the overall pattern of Hoxd gene expression remains unchanged. Transcriptome analysis using tiling arrays covering chromosomes 2 and X confirmed the weak effect of CTCF depletion on global gene regulation. However, Ctcf deletion caused massive apoptosis, leading to a nearly complete loss of limb structure at a later stage. We conclude that, at least in this physiological context, rather than being an insulator, CTCF is required for cell survival via the direct transcriptional regulation of target genes critical for cellular homeostasis.

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  • The evo-devo comet. EMBO Rep. 2010 Jul;11(7):489. embor201094. 10.1038/embor.2010.94. PMC2897126.

    Duboule D

    abstract

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  • Homeobox genes d11-d13 and a13 control mouse autopod cortical bone and joint formation. J. Clin. Invest. 2010 Jun;120(6):1994-2004. 41554. 10.1172/JCI41554. PMC2877951.

    Villavicencio-Lorini P, Kuss P, Friedrich J, Haupt J, Farooq M, Türkmen S, Duboule D, Hecht J, Mundlos S

    abstract

    The molecular mechanisms that govern bone and joint formation are complex, involving an integrated network of signaling pathways and gene regulators. We investigated the role of Hox genes, which are known to specify individual segments of the skeleton, in the formation of autopod limb bones (i.e., the hands and feet) using the mouse mutant synpolydactyly homolog (spdh), which encodes a polyalanine expansion in Hoxd13. We found that no cortical bone was formed in the autopod in spdh/spdh mice; instead, these bones underwent trabecular ossification after birth. Spdh/spdh metacarpals acquired an ovoid shape and developed ectopic joints, indicating a loss of long bone characteristics and thus a transformation of metacarpals into carpal bones. The perichondrium of spdh/spdh mice showed abnormal morphology and decreased expression of Runt-related transcription factor 2 (Runx2), which was identified as a direct Hoxd13 transcriptional target. Hoxd11-/-Hoxd12-/-Hoxd13-/- triple-knockout mice and Hoxd13-/-Hoxa13+/- mice exhibited similar but less severe defects, suggesting that these Hox genes have similar and complementary functions and that the spdh allele acts as a dominant negative. This effect was shown to be due to sequestration of other polyalanine-containing transcription factors by the mutant Hoxd13 in the cytoplasm, leading to their degradation. These data indicate that Hox genes not only regulate patterning but also directly influence bone formation and the ossification pattern of bones, in part via Runx2.

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  • The origin of digits: expression patterns versus regulatory mechanisms. Dev. Cell 2010 Apr;18(4):526-32. S1534-5807(10)00153-X. 10.1016/j.devcel.2010.04.002.

    Woltering JM, Duboule D

    abstract

    In the emerging discipline of Evo-Devo, the analysis of gene expression patterns can be deceptive without a clear understanding of the underlying regulatory strategies. Here, we use the paradigm of hand and foot evolution to argue that the consideration of the regulatory mechanisms controlling developmental gene expression is essential to resolve comparative conundrums. In this context, we discuss the adaptive relevance of evolving stepwise, distinct developmental regulatory mechanisms to build an arm, i.e., a composite structure with functional coherence.

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  • Additive and global functions of HoxA cluster genes in mesoderm derivatives. Dev. Biol. 2010 May;341(2):488-98. S0012-1606(10)00154-5. 10.1016/j.ydbio.2010.03.006.

    Di-Poï N, Koch U, Radtke F, Duboule D

    abstract

    Hox genes encode transcription factors that play a central role in the specification of regional identities along the anterior to posterior body axis. In the developing mouse embryo, Hox genes from all four genomic clusters are involved in range of developmental processes, including the patterning of skeletal structures and the formation of several organs. However, the functional redundancy observed either between paralogous genes, or among neighboring genes from the same cluster, has hampered functional analyses, in particular when synergistic, cluster-specific functions are considered. Here, we report that mutant mice lacking the entire HoxA cluster in mesodermal lineages display the expected spectrum of postnatal respiratory, cardiac and urogenital defects, previously reported for single gene mutations. Likewise, mild phenotypes are observed in both appendicular and axial skeleton. However, a striking effect was uncovered in the hematopoietic system, much stronger than that seen for Hoxa9 inactivation alone, which involves stem cells (HSCs) as well as the erythroid lineage, indicating that several Hoxa genes are necessary for normal hematopoiesis to occur. Finally, the combined deletions of Hoxa and Hoxd genes reveal abnormalities in axial elongation as well as skin morphogenesis that are likely the results of defects in epithelial-mesenchymal interactions.

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  • Changes in Hox genes' structure and function during the evolution of the squamate body plan. Nature 2010 Mar;464(7285):99-103. nature08789. 10.1038/nature08789.

    Di-Poï N, Montoya-Burgos JI, Miller H, Pourquié O, Milinkovitch MC, Duboule D

    abstract

    Hox genes are central to the specification of structures along the anterior-posterior body axis, and modifications in their expression have paralleled the emergence of diversity in vertebrate body plans. Here we describe the genomic organization of Hox clusters in different reptiles and show that squamates have accumulated unusually large numbers of transposable elements at these loci, reflecting extensive genomic rearrangements of coding and non-coding regulatory regions. Comparative expression analyses between two species showing different axial skeletons, the corn snake and the whiptail lizard, revealed major alterations in Hox13 and Hox10 expression features during snake somitogenesis, in line with the expansion of both caudal and thoracic regions. Variations in both protein sequences and regulatory modalities of posterior Hox genes suggest how this genetic system has dealt with its intrinsic collinear constraint to accompany the substantial morphological radiation observed in this group.

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  • Epigenetic regulation of vertebrate Hox genes: a dynamic equilibrium. Epigenetics 2009 Nov;4(8):537-40. 10132.

    Soshnikova N, Duboule D

    abstract

    Temporal and spatial control of Hox gene expression is essential for correct patterning of many animals. In both Drosophila and vertebrates, Polycomb and Trithorax group complexes control the maintenance of Hox gene expression in appropriate domains. In vertebrates, dynamic changes in chromatin modifications are also observed during the sequential activation of Hox genes in the embryo, suggesting that progressive epigenetic modifications could regulate collinear gene activation.

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  • The Hox complex - an interview with Denis Duboule. Interviewed by Richardson, Michael K. Int. J. Dev. Biol. 2009 ;53(5-6):717-23. 072558mr. 10.1387/ijdb.072558mr.

    Duboule D

    abstract

    Denis Duboule is one of the most influential and highly-cited scientists in developmental biology. Born in Geneva in 1955, he holds dual Swiss and French nationality. His undergraduate studies in biology at the University of Geneva included research on mouse embryology. He later learned molecular techniques in the laboratory of Pierre Chambon, becoming a major player in characterising the newly-discovered vertebrate Hox genes. He helped discover their genomic clustering, realising that they had arisen by trans duplication. With Gaunt and Sharpe, he proposed that vertebrate Hox clusters might show spatial colinearity, and subsequently extended this concept to the timing of gene activation (temporal colinearity). Along with the Krumlauf laboratory, he reported the structural and functional conservation of the homeotic systems in flies and vertebrates. His lab was the first to describe nested patterns of Hox gene expression in the developing mouse limb, and later showed that digit-associated Hoxd gene expression was lacking in zebrafish paired fin development. His concept of phylotypic progression helps explain major evolutionary developmental phenomena in terms of Hox gene regulatory networks. His research helped reveal that the genital tubercle may, like the limb, be patterned by Hox genes. His lab developed targeted meiotic recombination (TAMERE), using it to make profound advances in our understanding of Hox gene regulation. Remote enhancers linked to digit patterning have been uncovered, together with a likely mechanism for colinearity. Denis lives in Geneva with his wife Brigitte Galliot, also a scientist, with their four children.

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  • Epigenetic temporal control of mouse Hox genes in vivo. Science 2009 Jun;324(5932):1320-3. 324/5932/1320. 10.1126/science.1171468.

    Soshnikova N, Duboule D

    abstract

    During vertebrate development, the temporal control of Hox gene transcriptional activation follows the genomic order of the genes within the Hox clusters. Although it is recognized that this "Hox clock" serves to coordinate body patterning, the underlying mechanism remains elusive. We have shown that successive Hox gene activation in the mouse embryo is closely associated with a directional transition in chromatin status, as judged by the dynamic progression of transcription-competent modifications: Increases in activation marks correspond to decreases in repressive marks. Furthermore, using a mouse in which a Hox cluster was split into two pieces, we document the necessity to maintain a clustered organization to properly implement this process. These results suggest that chromatin modifications are important parameters in the temporal regulation of this gene family.

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  • Uncoupling time and space in the collinear regulation of Hox genes. PLoS Genet. 2009 Mar;5(3):e1000398. 10.1371/journal.pgen.1000398. PMC2642670.

    Tschopp P, Tarchini B, Spitz F, Zakany J, Duboule D

    abstract

    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.

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  • Atypical relaxation of structural constraints in Hox gene clusters of the green anole lizard. Genome Res. 2009 Apr;19(4):602-10. gr.087932.108. 10.1101/gr.087932.108. PMC2665779.

    Di-Poï N, Montoya-Burgos JI, Duboule D

    abstract

    Hox genes control many aspects of embryonic development in metazoans. Previous analyses of this gene family revealed a surprising diversity in terms of gene number and organization between various animal species. In vertebrates, Hox genes are grouped into tightly organized clusters, claimed to be devoid of repetitive sequences. Here, we report the genomic organization of the four Hox loci present in the green anole lizard and show that they have massively accumulated retrotransposons, leading to gene clusters larger in size when compared to other vertebrates. In addition, similar repeats are present in many other development-related gene-containing regions, also thought to be refractory to such repetitive elements. Transposable elements are major sources of genetic variations, including alterations of gene expression, and hence this situation, so far unique among vertebrates, may have been associated with the evolution of the spectacular realm of morphological variations in the body plans of Squamata. Finally, sequence alignments highlight some divergent evolution in highly conserved DNA regions between vertebrate Hox clusters, which may coincide with the emergence of mammalian-specific features.

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  • Conserved elements within open reading frames of mammalian Hox genes. J. Biol. 2009 ;8(2):17. jbiol116. 10.1186/jbiol116. PMC2687771.

    Woltering JM, Duboule D

    abstract

    A recent study in BMC Evolutionary Biology shows that many of the open reading frames in mammalian Hox genes are more conserved than expected on the basis of their protein sequence. The presence of highly conserved DNA elements is thus not confined to the noncoding DNA in neighboring regions but clearly overlaps with coding sequences. These findings support an emerging view that gene regulatory and coding sequences are likely to be more intermingled than once believed.

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  • Rostral and caudal pharyngeal arches share a common neural crest ground pattern. Development 2009 Feb;136(4):637-45. 136/4/637. 10.1242/dev.028621. PMC4482666. CAMS4754.

    Minoux M, Antonarakis GS, Kmita M, Duboule D, Rijli FM

    abstract

    In vertebrates, face and throat structures, such as jaw, hyoid and thyroid cartilages develop from a rostrocaudal metameric series of pharyngeal arches, colonized by cranial neural crest cells (NCCs). Colinear Hox gene expression patterns underlie arch specific morphologies, with the exception of the first (mandibular) arch, which is devoid of any Hox gene activity. We have previously shown that the first and second (hyoid) arches share a common, Hox-free, patterning program. However, whether or not more posterior pharyngeal arch neural crest derivatives are also patterned on the top of the same ground-state remained an unanswered question. Here, we show that the simultaneous inactivation of all Hoxa cluster genes in NCCs leads to multiple jaw and first arch-like structures, partially replacing second, third and fourth arch derivatives, suggesting that rostral and caudal arches share the same mandibular arch-like ground patterning program. The additional inactivation of the Hoxd cluster did not significantly enhance such a homeotic phenotype, thus indicating a preponderant role of Hoxa genes in patterning skeletogenic NCCs. Moreover, we found that Hoxa2 and Hoxa3 act synergistically to pattern third and fourth arch derivatives. These results provide insights into how facial and throat structures are assembled during development, and have implications for the evolution of the pharyngeal region of the vertebrate head.

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  • Genotypic features of lentivirus transgenic mice. J. Virol. 2008 Jul;82(14):7111-9. JVI.00623-08. 10.1128/JVI.00623-08. PMC2446968.

    Sauvain MO, Dorr AP, Stevenson B, Quazzola A, Naef F, Wiznerowicz M, Schütz F, Jongeneel V, Duboule D, Spitz F, Trono D

    abstract

    Lentivector-mediated transgenesis is increasingly used, whether for basic studies as an alternative to pronuclear injection of naked DNA or to test candidate gene therapy vectors. In an effort to characterize the genetic features of this approach, we first measured the frequency of germ line transmission of individual proviruses established by infection of fertilized mouse oocytes. Seventy integrants from 11 founder (G0) mice were passed to 111 first generation (G1) pups, for a total of 255 events corresponding to an average rate of transmission of 44%. This implies that integration had most often occurred at the one- or two-cell stage and that the degree of genotypic mosaicism in G0 mice obtained through this approach is generally minimal. Transmission analysis of eight individual proviruses in 13 G2 mice obtained by a G0-G1 cross revealed only 8% of proviral homozygosity, significantly below the 25% expected from purely Mendelian transmission, suggesting counter-selection due to interference with the functions of targeted loci. Mapping of 239 proviral integration sites in 49 founder animals revealed that about 60% resided within annotated genes, with a marked tendency for clustering in the middle of the transcribed region, and that integration was not influenced by the transcriptional orientation. Transcript levels of a set of arbitrarily chosen target genes were significantly higher in two-cell embryos than in embryonic stem cells or adult somatic cells, suggesting that, as previously noted in other settings, lentiviral vectors integrate preferentially into regions of the genome that are transcriptionally active or poised for activation.

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  • Characterization of mouse Dactylaplasia mutations: a model for human ectrodactyly SHFM3. Mamm. Genome 2008 Apr;19(4):272-8. 10.1007/s00335-008-9106-0.

    Friedli M, Nikolaev S, Lyle R, Arcangeli M, Duboule D, Spitz F, Antonarakis SE

    abstract

    SHFM3 is a limb malformation characterized by the absence of central digits. It has been shown that this condition is associated with tandem duplications of about 500 kb at 10q24. The Dactylaplasia mice display equivalent limb defects and the two corresponding alleles (Dac1j and Dac2j) map in the region syntenic with the duplications in SHFM3. Dac1j was shown to be associated with an insertion of an unspecified ETn-like mouse endogenous transposon upstream of the Fbxw4 gene. Dac2j was also thought to be an insertion or a small inversion in intron 5 of Fbxw4, but the breakpoints and the exact molecular lesion have not yet been characterized. Here we report precise mapping and characterization of these alleles. We failed to identify any copy number differences within the SHFM3 orthologous genomic locus between Dac mutant and wild-type littermates, showing that the Dactylaplasia alleles are not associated with duplications of the region, in contrast with the described human SHFM3 cases. We further show that both Dac1j and Dac2j are caused by insertions of MusD retroelements that share 98% sequence identity. The differences between the nature of the human and mouse genomic abnormalities argue against models proposed so far that either envisioned SHFM3 as a local trisomy or Dac as a mutant allele of Fbxw4. Instead, both genetic conditions might lead to complex alterations of gene regulation mechanisms that would impair limb morphogenesis. Interestingly, the Dac2j mutation occurs within a highly conserved element that may represent a regulatory sequence for a neighboring gene.

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  • Epigenetic regulation of Hox gene activation: the waltz of methyls. Bioessays 2008 Mar;30(3):199-202. 10.1002/bies.20724.

    Soshnikova N, Duboule D

    abstract

    Genetic studies have revealed that the antagonistic interplay between PcG and TrxG/MLL complexes is essential for the proper maintenance of vertebrate Hox gene expression in time and space. Hox genes must be silenced in totipotent embryonic stem cells and, in contrast, rapidly activated during embryogenesis. Here we discuss some recently published articles that propose a novel mechanism for the induction of Hox gene transcription. These studies report a new family of histone demethylases that remove H3K27me3/me2 repressive marks at Hox promoters during differentiation of stem cells. Though the overall importance of these enzymes for proper embryogenesis was demonstrated, their precise role in Hox gene epigenetic regulation during development still remains to be firmly established.

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  • Global control regions and regulatory landscapes in vertebrate development and evolution. Adv. Genet. 2008 ;61():175-205. S0065-2660(07)00006-5. 10.1016/S0065-2660(07)00006-5.

    Spitz F, Duboule D

    abstract

    During the course of evolution, many genes that control the development of metazoan body plans were co-opted to exert novel functions, along with the emergence or modification of structures. Gene amplification and/or changes in the cis-regulatory modules responsible for the transcriptional activity of these genes have certainly contributed in a major way to evolution of gene functions. In some cases, these processes led to the formation of groups of adjacent genes that appear to be controlled by both global and shared mechanisms.

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  • Ectopic nuclear reorganisation driven by a Hoxb1 transgene transposed into Hoxd. J. Cell. Sci. 2008 Mar;121(Pt 5):571-7. jcs.023234. 10.1242/jcs.023234. PMC2258412. UKMS1525.

    Morey C, Da Silva NR, Kmita M, Duboule D, Bickmore WA

    abstract

    The extent to which the nuclear organisation of a gene impacts on its ability to be expressed, or whether nuclear organisation merely reflects gene expression states, remains an important but unresolved issue. A model system that has been instrumental in investigating this question utilises the murine Hox gene clusters encoding homeobox-containing proteins. Nuclear reorganisation and chromatin decondensation, initiated towards the 3' end of the clusters, accompanies activation of Hox genes in both differentiation and development, and might be linked to mechanisms underlying colinearity. To investigate this, and to delineate the cis-acting elements involved, here we analyse the nuclear behaviour of a 3' Hoxb1 transgene transposed to the 5' end of the Hoxd cluster. We show that this transgene contains the cis-acting elements sufficient to initiate ectopic local nuclear reorganisation and chromatin decondensation and to break Hoxd colinearity in the primitive streak region of the early embryo. Significantly, in rhombomere 4, the transgene is able to induce attenuated nuclear reorganisation and decondensation of Hoxd even though there is no detectable expression of the transgene at this site. This shows that reorganisation of chromosome territories and chromatin decondensation can be uncoupled from transcription itself and suggests that they can therefore operate upstream of gene expression.

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  • Modeling Hox gene regulation in digits: reverse collinearity and the molecular origin of thumbness. Genes Dev. 2008 Feb;22(3):346-59. 22/3/346. 10.1101/gad.1631708. PMC2216694.

    Montavon T, Le Garrec JF, Kerszberg M, Duboule D

    abstract

    During the development of mammalian digits, clustered Hoxd genes are expressed following a collinear regulatory strategy, leading to both the growth of digits and their morphological identities. Because gene dosage is a key parameter in this system, we used a quantitative approach, associated with a collection of mutant stocks, to investigate the nature of the underlying regulatory mechanism(s). In parallel, we elaborated a mathematical model of quantitative collinearity, which was progressively challenged and validated by the experimental approach. This combined effort suggested a two-step mechanism, which involves initially the looping and recognition of the cluster by a complex including two enhancer sequences, followed by a second step of microscanning of genes located nearby. In this scenario, the respective rank of the genes, with respect to the 5' extremity of the cluster, is primordial, as well as different gene-specific affinities. This model accounts for the quantitative variations observed in our many mutant strains, and reveals the molecular constraint leading to thumbness; i.e., why a morphological difference must occur between the most anterior digit and the others. We also show that the same model applies to the collinear regulation of Hox genes during the emergence of external genitalia, though with some differences likely illustrating the distinct functionalities of these structures in adults.

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  • Distinct roles and regulations for HoxD genes in metanephric kidney development. PLoS Genet. 2007 Dec;3(12):e232. 07-PLGE-RA-0477. 10.1371/journal.pgen.0030232. PMC2151092.

    Di-Poï N, Zákány J, Duboule D

    abstract

    Hox genes encode homeodomain-containing proteins that control embryonic development in multiple contexts. Up to 30 Hox genes, distributed among all four clusters, are expressed during mammalian kidney morphogenesis, but functional redundancy between them has made a detailed functional account difficult to achieve. We have investigated the role of the HoxD cluster through comparative molecular embryological analysis of a set of mouse strains carrying targeted genomic rearrangements such as deletions, duplications, and inversions. This analysis allowed us to uncover and genetically dissect the complex role of the HoxD cluster. Regulation of metanephric mesenchyme-ureteric bud interactions and maintenance of structural integrity of tubular epithelia are differentially controlled by some Hoxd genes during renal development, consistent with their specific expression profiles. We also provide evidence for a kidney-specific form of colinearity that underlies the differential expression of two distinct sets of genes located on both sides and overlapping at the Hoxd9 locus. These insights further our knowledge of the genetic control of kidney morphogenesis and may contribute to understanding certain congenital kidney malformations, including polycystic kidney disease and renal hypoplasia.

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  • Differentiation and gene regulation. Curr. Opin. Genet. Dev. 2007 Oct;17(5):369-72. S0959-437X(07)00173-6. 10.1016/j.gde.2007.10.001.

    Grosveld FG, Duboule D

    abstract

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  • Hox gene function in vertebrate gut morphogenesis: the case of the caecum. Development 2007 Nov;134(22):3967-73. dev.010991. 10.1242/dev.010991.

    Zacchetti G, Duboule D, Zakany J

    abstract

    The digestive tract is made of different subdivisions with various functions. During embryonic development, the developing intestine expresses combinations of Hox genes along its anterior to posterior axis, suggesting a role for these genes in this regionalization process. In particular, the transition from small to large intestine is labelled by the transcription of all Hoxd genes except Hoxd12 and Hoxd13, the latter two genes being transcribed only near the anus. Here, we describe two lines of mice that express Hoxd12 ectopically within this morphological transition. As a consequence, budding of the caecum is impeded, leading to complete agenesis in homozygous individuals. This effect is concurrent with a dramatic reduction of both Fgf10 and Pitx1 expression. Furthermore, the interactions between ;anterior' Hox genes and ectopic Hoxd12 suggest a model whereby anterior and posterior Hox products compete in controlling Fgf10 signalling, which is required for the growth of this organ in mice. These results illuminate components of the genetic cascade necessary for the emergence of this gut segment, crucial for many vertebrates.

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  • Tinkering with constraints in the evolution of the vertebrate limb anterior-posterior polarity. Novartis Found. Symp. 2007 ;284():130-7; discussion 138-41, 158-63.

    Duboule D, Tarchini B, Zàkàny J, Kmita M

    abstract

    Genes belonging to both HoxA and HoxD clusters are required for proper vertebrate limb development. Mice lacking all, or parts of, Hoxa and Hoxd functions in forelimbs, as well as mice with a gain of function of these genes in the early limb bud, have helped us to understand functional and regulatory issues associated with these genes, such that, for example, the tight mechanistic interdependency that exists between the production of the limb and its anterior to posterior (AP) polarity. Our studies suggest that the evolutionary recruitment of Hox gene function into growing appendages was crucial to implement hedgehog signalling, subsequently leading to the distal extension of tetrapod appendages, with an already built-in AP polarity. We propose that this process results from the evolutionary co-option, in the developing limbs, of a particular regulatory mechanism (collinearity), which is necessary to pattern the developing trunk. This major regulatory constraint imposed a polarity to our limbs as the most parsimonious solution to grow appendages.

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  • The role of Hox genes during vertebrate limb development. Curr. Opin. Genet. Dev. 2007 Aug;17(4):359-66. S0959-437X(07)00116-5. 10.1016/j.gde.2007.05.011.

    Zakany J, Duboule D

    abstract

    The potential role of Hox genes during vertebrate limb development was brought into focus by gene expression analyses in mice (P Dolle, JC Izpisua-Belmonte, H Falkenstein, A Renucci, D Duboule, Nature 1989, 342:767-772), at a time when limb growth and patterning were thought to depend upon two distinct and rather independent systems of coordinates; one for the anterior-to-posterior axis and the other for the proximal-to-distal axis (see D Duboule, P Dolle, EMBO J 1989, 8:1497-1505). Over the past years, the function and regulation of these genes have been addressed using both gain-of-function and loss-of-function approaches in chick and mice. The use of multiple mutations either in cis-configuration in trans-configuration or in cis/trans configurations, has confirmed that Hox genes are essential for proper limb development, where they participate in both the growth and organization of the structures. Even though their molecular mechanisms of action remain somewhat elusive, the results of these extensive genetic analyses confirm that, during the development of the limbs, the various axes cannot be considered in isolation from each other and that a more holistic view of limb development should prevail over a simple cartesian, chess grid-like approach of these complex structures. With this in mind, the functional input of Hox genes during limb growth and development can now be re-assessed.

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  • The rise and fall of Hox gene clusters. Development 2007 Jul;134(14):2549-60. dev.001065. 10.1242/dev.001065.

    Duboule D

    abstract

    Although all bilaterian animals have a related set of Hox genes, the genomic organization of this gene complement comes in different flavors. In some unrelated species, Hox genes are clustered; in others, they are not. This indicates that the bilaterian ancestor had a clustered Hox gene family and that, subsequently, this genomic organization was either maintained or lost. Remarkably, the tightest organization is found in vertebrates, raising the embarrassingly finalistic possibility that vertebrates have maintained best this ancestral configuration. Alternatively, could they have co-evolved with an increased ;organization' of the Hox clusters, possibly linked to their genomic amplification, which would be at odds with our current perception of evolutionary mechanisms? When discussing the why's and how's of Hox gene clustering, we need to account for three points: the mechanisms of cluster evolution; the underlying biological constraints; and the developmental modes of the animals under consideration. By integrating these parameters, general conclusions emerge that can help solve the aforementioned dilemma.

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  • Interactions between HOXD and Gli3 genes control the limb apical ectodermal ridge via Fgf10. Dev. Biol. 2007 Jun;306(2):883-93. S0012-1606(07)00242-4. 10.1016/j.ydbio.2007.03.517.

    Zakany J, Zacchetti G, Duboule D

    abstract

    The development of the vertebrate limb is dependent upon two signaling centers, the apical ectodermal ridge (AER), which provides the underlying mesenchyme with essential growth factors, and the zone of polarizing activity (ZPA), the source of the Sonic hedgehog (SHH) product. Recent work involving gain and loss of function of Hox genes has emphasized their impact both on AER maintenance and Shh transcriptional activation. Here, we describe antagonistic interactions between posterior Hoxd genes and Gli3, suggesting that the latter product protects the AER from the deleterious effect of the formers, and we present evidence that Fgf10 is the mediator of HOX-dependent AER expansion. Furthermore, the striking similarity between some of the hereby observed Hox/Gli3-dependent morphogenetic defects and those displayed by fetuses with severely altered retinoic acid metabolism suggests a tight connection between these various pathways. The nature of these potential interactions is discussed in the context of proximal-distal growth and patterning.

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  • Transgenic analysis of Hoxd gene regulation during digit development. Dev. Biol. 2007 Jun;306(2):847-59. S0012-1606(07)00227-8. 10.1016/j.ydbio.2007.03.020.

    Gonzalez F, Duboule D, Spitz F

    abstract

    In tetrapods, posterior Hoxd genes (from groups 10 to 13) are necessary to properly pattern the developing autopods, including the number and identities of digits. Their coordinated expression is achieved by sharing a global control region (GCR), which was isolated and localized 200 kb 5' (centromeric) of the gene cluster. However, in transgenic assays, the GCR was unable to fully recapitulate all aspects of the endogenous Hoxd expression patterns during distal limb development. In this paper, we further analyze the regulatory potential of this locus and report the characterization of Prox, a second enhancer element that contributes to the transcriptional activity of posterior Hoxd genes in developing distal limb buds. We show that the GCR and Prox elements complement each other and work in combination to correctly establish the late phase of Hoxd genes expression. Based on DNA sequence conservation and transgenic assays, we discuss the functions of these regulatory regions as well as a potential evolutionary scheme accounting for their emergence along with the evolution of tetrapod limbs.

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  • Regulatory constraints in the evolution of the tetrapod limb anterior-posterior polarity. Nature 2006 Oct;443(7114):985-8. nature05247. 10.1038/nature05247.

    Tarchini B, Duboule D, Kmita M

    abstract

    The anterior to posterior (A-P) polarity of the tetrapod limb is determined by the confined expression of Sonic hedgehog (Shh) at the posterior margin of developing early limb buds, under the control of HOX proteins encoded by gene members of both the HoxA and HoxD clusters. Here, we use a set of partial deletions to show that only the last four Hox paralogy groups can elicit this response: that is, precisely those genes whose expression is excluded from most anterior limb bud cells owing to their collinear transcriptional activation. We propose that the limb A-P polarity is produced as a collateral effect of Hox gene collinearity, a process highly constrained by its crucial importance during trunk development. In this view, the co-option of the trunk collinear mechanism, along with the emergence of limbs, imposed an A-P polarity to these structures as the most parsimonious solution. This in turn further contributed to stabilize the architecture and operational mode of this genetic system.

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  • A mouse model for human short-stature syndromes identifies Shox2 as an upstream regulator of Runx2 during long-bone development. Proc. Natl. Acad. Sci. U.S.A. 2006 Mar;103(12):4511-5. 0510544103. 10.1073/pnas.0510544103. PMC1450202.

    Cobb J, Dierich A, Huss-Garcia Y, Duboule D

    abstract

    Deficiencies or mutations in the human pseudoautosomal SHOX gene are associated with a series of short-stature conditions, including Turner syndrome, Leri-Weill dyschondrosteosis, and Langer mesomelic dysplasia. Although this gene is absent from the mouse genome, the closely related paralogous gene Shox2 displays a similar expression pattern in developing limbs. Here, we report that the conditional inactivation of Shox2 in developing appendages leads to a strong phenotype, similar to the human conditions, although it affects a different proximodistal limb segment. Furthermore, using this mouse model, we establish the cellular etiology of these defects and show that Shox2 acts upstream the Runx2 gene, a key regulator of chondrogenesis.

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  • Control of Hoxd genes' collinearity during early limb development. Dev. Cell 2006 Jan;10(1):93-103. S1534-5807(05)00473-9. 10.1016/j.devcel.2005.11.014.

    Tarchini B, Duboule D

    abstract

    Hoxd genes are essential for limb growth and patterning. They are activated following a complex transcriptional regulation, leading to expression domains that are collinear in both space and time. To understand the mechanism(s) underlying collinearity, we produced and analyzed a set of mouse strains containing systematic deletions and duplications within the HoxD cluster. We show that two waves of transcriptional activation, controlled by different mechanisms, generate the observed developmental expression patterns. The first wave is time-dependent, involves the action of opposite regulatory modules, and is essential for the growth and polarity of the limb up to the forearm. The second phase involves a different regulation and is required for the morphogenesis of digits. We propose that these two phases reflect the different phylogenetic histories of proximal versus distal limb structures and discuss the biological relevance of these collinear patterns, particularly for the origin of the anterior-to-posterior limb polarity.

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  • [A STRING lifts the veil on the mechanisms controlling Hox genes expression]. Med Sci (Paris) 2006 Jan;22(1):14-6. 00/00/08/7C/. 10.1051/medsci/200622114.

    Spitz F, Herkenne C, Hinard C, Duboule D

    abstract

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  • Head-tail patterning of the vertebrate embryo: one, two or many unresolved problems? Int. J. Dev. Biol. 2006 ;50(1):3-15. 052095cs. 10.1387/ijdb.052095cs.

    Stern CD, Charité J, Deschamps J, Duboule D, Durston AJ, Kmita M, Nicolas JF, Palmeirim I, Smith JC, Wolpert L

    abstract

    When, where and how is the head-tail axis of the embryo set up during development? These are such fundamental and intensely studied questions that one might expect them to have been answered long ago. Not so; we still understand very little about the cellular or molecular mechanisms that lead to the orderly arrangement of body elements along the head-tail axis in vertebrates. In this paper, we outline some of the major outstanding problems and controversies and try to identify some reasons why it has been so difficult to resolve this important issue.

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  • HoxD cluster scanning deletions identify multiple defects leading to paralysis in the mouse mutant Ironside. Genes Dev. 2005 Dec;19(23):2862-76. 19/23/2862. 10.1101/gad.351105. PMC1315393.

    Tarchini B, Huynh TH, Cox GA, Duboule D

    abstract

    A spontaneous semidominant mutation (Ironside, Irn) was isolated in mice, leading to severe hindlimb paralysis following multiple deletions in cis at the HoxD locus. To understand its cellular and molecular etiology, we embarked on a comparative analysis using systematic HoxD cluster deletions, produced via targeted meiotic recombination (TAMERE). Different lines of mice were classified according to the severity of their paralyses, and subsequent analyses revealed that multiple causative factors were involved, alone or in combination, in the occurrence of this pathology. Among them are the loss of Hoxd10 function, the sum of remaining Hoxd gene activity, and the ectopic gain of function of the neighboring gene Evx2, all contributing to the mispositioning, the absence, or misidentification of specific lumbo-sacral pools of motoneurons, nerve root homeosis, and hindlimb innervation defects. These results highlight the importance of a systematic approach when studying such clustered gene families, and give insights into the function and regulation of Hox and Evx2 genes during early spinal cord development.

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  • Inversion-induced disruption of the Hoxd cluster leads to the partition of regulatory landscapes. Nat. Genet. 2005 Aug;37(8):889-93. ng1597. 10.1038/ng1597.

    Spitz F, Herkenne C, Morris MA, Duboule D

    abstract

    The developmental regulation of vertebrate Hox gene transcription relies on the interplay between local and long-range controls. To study this complex genomic organization, we designed a strategy combining meiotic and targeted recombinations to induce large chromosomal rearrangements in vivo without manipulating embryonic stem cells. With this simple approach (called STRING), we engineered a large 7-cM inversion, which split the Hoxd cluster into two independent pieces. Expression analyses showed a partition of global enhancers, allowing for their precise topographic allocation on either side of the cluster. Such a functional organization probably contributed to keeping these genes clustered in the course of vertebrate evolution. This approach can be used to study the relationship between genome architecture and gene expression, such as the effects of genome rearrangements in human diseases or during evolution.

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  • Early developmental arrest of mammalian limbs lacking HoxA/HoxD gene function. Nature 2005 Jun;435(7045):1113-6. nature03648. 10.1038/nature03648.

    Kmita M, Tarchini B, Zàkàny J, Logan M, Tabin CJ, Duboule D

    abstract

    Vertebrate HoxA and HoxD cluster genes are required for proper limb development. However, early lethality, compensation and redundancy have made a full assessment of their function difficult. Here we describe mice that are lacking all Hoxa and Hoxd functions in their forelimbs. We show that such limbs are arrested early in their developmental patterning and display severe truncations of distal elements, partly owing to the absence of Sonic hedgehog expression. These results indicate that the evolutionary recruitment of Hox gene function into growing appendages might have been crucial in implementing hedgehog signalling, subsequently leading to the distal extension of tetrapod appendages. Accordingly, these mutant limbs may be reminiscent of an ancestral trunk extension, related to that proposed for arthropods.

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  • Comparative analysis of genes downstream of the Hoxd cluster in developing digits and external genitalia. Development 2005 Jul;132(13):3055-67. 132/13/3055. 10.1242/dev.01885.

    Cobb J, Duboule D

    abstract

    Mammalian Hox genes encode transcription factors that are crucial for proper morphogenesis along the various body axes. Despite their extensive structural and functional characterization, the nature of their target genes remains elusive. We have addressed this question by using DNA microarrays to screen for genes whose expression in developing distal forelimbs and genital eminences was significantly modified in the absence of the full Hoxd gene complement. This comparative approach not only identified specific candidate genes, but also allowed the examination of whether a similar Hox expression pattern in distinct tissues leads to the modulation of the same or different downstream genes. We report here a set of potential target genes, most of which were not previously known to play a role in the early stages of either limb or genital bud development. Interestingly, we find that the majority of these candidate genes are differentially expressed in both structures, although often at different times. This supports the idea that both appendices involve similar genetic controls, both upstream and downstream of the Hox gene family. These results highlight the surprising mechanistic relationship between these rather different body parts and suggest a common developmental strategy to build up the most distal appendicular structures of the body, i.e. the digits and the penis/clitoris.

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  • Developmental biology: reproduction in clusters. Nature 2005 Apr;434(7034):715-6. 434715a. 10.1038/434715a.

    Spitz F, Duboule D

    abstract

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  • Tracing microRNA patterns in mice. Nat. Genet. 2004 Oct;36(10):1033-4. 10.1038/ng1004-1033. ng1004-1033.

    Cobb J, Duboule D

    abstract

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  • The European dimension for the mouse genome mutagenesis program. Nat. Genet. 2004 Sep;36(9):925-7. 10.1038/ng0904-925. ng0904-925. PMC2716028. UKMS27268.

    Auwerx J, Avner P, Baldock R, Ballabio A, Balling R, Barbacid M, Berns A, Bradley A, Brown S, Carmeliet P, Chambon P, Cox R, Davidson D, Davies K, Duboule D, Forejt J, Granucci F, Hastie N, de Angelis MH, Jackson I, Kioussis D, Kollias G, Lathrop M, Lendahl U, Malumbres M, von Melchner H, Müller W, Partanen J, Ricciardi-Castagnoli P, Rigby P, Rosen B, Rosenthal N, Skarnes B, Stewart AF, Thornton J, Tocchini-Valentini G, Wagner E, Wahli W, Wurst W

    abstract

    The European Mouse Mutagenesis Consortium is the European initiative contributing to the international effort on functional annotation of the mouse genome. Its objectives are to establish and integrate mutagenesis platforms, gene expression resources, phenotyping units, storage and distribution centers and bioinformatics resources. The combined efforts will accelerate our understanding of gene function and of human health and disease.

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  • Mouse limb deformity mutations disrupt a global control region within the large regulatory landscape required for Gremlin expression. Genes Dev. 2004 Jul;18(13):1553-64. 10.1101/gad.299904. 299904. PMC443518.

    Zuniga A, Michos O, Spitz F, Haramis AP, Panman L, Galli A, Vintersten K, Klasen C, Mansfield W, Kuc S, Duboule D, Dono R, Zeller R

    abstract

    The mouse limb deformity (ld) mutations cause limb malformations by disrupting epithelial-mesenchymal signaling between the polarizing region and the apical ectodermal ridge. Formin was proposed as the relevant gene because three of the five ld alleles disrupt its C-terminal domain. In contrast, our studies establish that the two other ld alleles directly disrupt the neighboring Gremlin gene, corroborating the requirement of this BMP antagonist for limb morphogenesis. Further doubts concerning an involvement of Formin in the ld limb phenotype are cast, as a targeted mutation removing the C-terminal Formin domain by frame shift does not affect embryogenesis. In contrast, the deletion of the corresponding genomic region reproduces the ld limb phenotype and is allelic to mutations in Gremlin. We resolve these conflicting results by identifying a cis-regulatory region within the deletion that is required for Gremlin activation in the limb bud mesenchyme. This distant cis-regulatory region within Formin is also altered by three of the ld mutations. Therefore, the ld limb bud patterning defects are not caused by disruption of Formin, but by alteration of a global control region (GCR) required for Gremlin transcription. Our studies reveal the large genomic landscape harboring this GCR, which is required for tissue-specific coexpression of two structurally and functionally unrelated genes.

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  • A dual role for Hox genes in limb anterior-posterior asymmetry. Science 2004 Jun;304(5677):1669-72. 10.1126/science.1096049. 304/5677/1669.

    Zákány J, Kmita M, Duboule D

    abstract

    Anterior-to-posterior patterning, the process whereby our digits are differently shaped, is a key aspect of limb development. It depends on the localized expression in posterior limb bud of Sonic hedgehog (Shh) and the morphogenetic potential of its diffusing product. By using an inversion of and a large deficiency in the mouse HoxD cluster, we found that a perturbation in the early collinear expression of Hoxd11, Hoxd12, and Hoxd13 in limb buds led to a loss of asymmetry. Ectopic Hox gene expression triggered abnormal Shh transcription, which in turn induced symmetrical expression of Hox genes in digits, thereby generating double posterior limbs. We conclude that early posterior restriction of Hox gene products sets up an anterior-posterior prepattern, which determines the localized activation of Shh. This signal is subsequently translated into digit morphological asymmetry by promoting the late expression of Hoxd genes, two collinear processes relying on opposite genomic topographies, upstream and downstream Shh signaling.

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  • Colinearity loops out. Dev. Cell 2004 Jun;6(6):738-40. 10.1016/j.devcel.2004.05.016. S1534580704001789.

    Duboule D, Deschamps J

    abstract

    Modulation of chromatin structure has long been proposed to underlie the colinear regulation of Hox genes during animal development. In a recent paper, Chambeyron and Bickmore explore this possibility in retinoic acid-induced ES cells. They show that, while chromatin remodeling confers transcriptional competence to the gene cluster, subsequent sequential extrusion of genes from their chromosome territory may determine their coordinated expression in time.

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  • The loss of circadian PAR bZip transcription factors results in epilepsy. Genes Dev. 2004 Jun;18(12):1397-412. 10.1101/gad.301404. 301404. PMC423191.

    Gachon F, Fonjallaz P, Damiola F, Gos P, Kodama T, Zakany J, Duboule D, Petit B, Tafti M, Schibler U

    abstract

    DBP (albumin D-site-binding protein), HLF (hepatic leukemia factor), and TEF (thyrotroph embryonic factor) are the three members of the PAR bZip (proline and acidic amino acid-rich basic leucine zipper) transcription factor family. All three of these transcriptional regulatory proteins accumulate with robust circadian rhythms in tissues with high amplitudes of clock gene expression, such as the suprachiasmatic nucleus (SCN) and the liver. However, they are expressed at nearly invariable levels in most brain regions, in which clock gene expression only cycles with low amplitude. Here we show that mice deficient for all three PAR bZip proteins are highly susceptible to generalized spontaneous and audiogenic epilepsies that frequently are lethal. Transcriptome profiling revealed pyridoxal kinase (Pdxk) as a target gene of PAR bZip proteins in both liver and brain. Pyridoxal kinase converts vitamin B6 derivatives into pyridoxal phosphate (PLP), the coenzyme of many enzymes involved in amino acid and neurotransmitter metabolism. PAR bZip-deficient mice show decreased brain levels of PLP, serotonin, and dopamine, and such changes have previously been reported to cause epilepsies in other systems. Hence, the expression of some clock-controlled genes, such as Pdxk, may have to remain within narrow limits in the brain. This could explain why the circadian oscillator has evolved to generate only low-amplitude cycles in most brain regions.

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  • Organizing axes in time and space; 25 years of colinear tinkering. Science 2003 Jul;301(5631):331-3. 10.1126/science.1085753. 301/5631/331.

    Kmita M, Duboule D

    abstract

    During vertebrate development, clustered genes from the Hox family of transcription factors are activated in a precise temporal and spatial sequence that follows their chromosomal order (the "Hox clock"). Recent advances in the knowledge of the underlying mechanisms reveal that the embryo uses a variety of strategies to implement this colinear process, depending on both the type and the evolutionary history of axial structures. The search for a universal mechanism has likely hampered our understanding of this enigmatic phenomenon, which may be caused by various and unrelated regulatory processes, as long as the final distribution of proteins (the HOX code) is preserved.

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  • Time for chronomics? Science 2003 Jul;301(5631):277. 10.1126/science.301.5631.277. 301/5631/277.

    Duboule D

    abstract

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  • A global control region defines a chromosomal regulatory landscape containing the HoxD cluster. Cell 2003 May;113(3):405-17. S0092867403003106.

    Spitz F, Gonzalez F, Duboule D

    abstract

    During limb development, coordinated expression of several Hoxd genes is required in presumptive digits. We searched for the underlying control sequences upstream from the cluster and found Lunapark (Lnp), a gene which shares limb and CNS expression specificities with both Hoxd genes and Evx2, another gene located nearby. We used a targeted enhancer-trap approach to identify a DNA segment capable of directing reporter gene expression in both digits and CNS, following Lnp, Evx2, and Hoxd-specific patterns. This DNA region showed an unusual interspecies conservation, including with its pufferfish counterpart. It contains a cluster of global enhancers capable of controlling transcription of several genes unrelated in structure or function, thus defining large regulatory domains. These domains were interrupted in the Ulnaless mutation, a balanced inversion that modified the topography of the locus. We discuss the heuristic value of these results in term of locus specific versus gene-specific regulation.

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  • An enhancer-titration effect induces digit-specific regulatory alleles of the HoxD cluster. Dev. Biol. 2003 Apr;256(2):212-20. S0012160602001367.

    Monge I, Kondo T, Duboule D

    abstract

    Mice carrying transgenes targeted upstream the HoxD cluster display abnormal digits, with alterations resembling those obtained with loss of functions of Hoxd genes. Because the HoxD cluster remained entirely untouched by the insertional events, we investigated whether these phenotypes were induced by regulatory modifications at a distance. We report here that these targeted relocations behaved as hypomorphic alleles of the distantly located gene Hoxd13 and showed that posterior Hoxd genes located in cis with the integration site were down-regulated. Genetic analyses suggested that this down-regulation resulted from the titration of the activity of a remote located enhancer sequence. These results indicate that the transcriptional efficiency of Hoxd genes in digits could be modulated by the presence of other, unrelated, promoters, within the regulatory landscape of this enhancer. Modifications in these latter transcription units may thus impact upon digit morphology, through misregulation of Hoxd genes, thus illustrating the "buffering effect" that such a global regulatory element can exert upon a short genomic interval.

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  • Serial deletions and duplications suggest a mechanism for the collinearity of Hoxd genes in limbs. Nature 2002 Nov;420(6912):145-50. 10.1038/nature01189. nature01189.

    Kmita M, Fraudeau N, Hérault Y, Duboule D

    abstract

    Hox genes, located at one end of the HoxD cluster, are essential for the development of the extremities of our limbs; that is, the digits. This 'collinear' correspondence is accompanied by a gradual decrease in the transcriptional efficiency of the genes. To decipher the underlying regulatory mechanisms, and thus to understand better how digits develop, we engineered a series of deletions and duplications in vivo. We find that HoxD genes compete for a remote enhancer that recognizes the locus in a polar fashion, with a preference for the 5' extremity. Modifications in either the number or topography of Hoxd loci induced regulatory reallocations affecting both the number and morphology of digits. These results demonstrate why genes located at the extremity of the cluster are expressed at the distal end of the limbs, following a gradual reduction in transcriptional efficiency, and thus highlight the mechanistic nature of collinearity in limbs.

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  • Evolutionary conserved sequences are required for the insulation of the vertebrate Hoxd complex in neural cells. Development 2002 Dec;129(23):5521-8.

    Kmita M, Tarchini B, Duboule D, Hérault Y

    abstract

    Transcriptional regulation of vertebrate Hox genes involves enhancer sequences located either inside or outside the gene clusters. In the mouse Hoxd complex, for example, series of contiguous genes are coordinately controlled by regulatory sequences located at remote distances. However, in different cellular contexts, Hox genes may have to be insulated from undesirable external regulatory influences to prevent ectopic gene activation, a situation that would likely be detrimental to the developing embryo. We show the presence of an insulator activity, at one extremity of the Hoxd complex, that is composed of at least two distinct DNA elements, one of which is conserved throughout vertebrate species. However, deletion of this element on its own did not detectably affect Hoxd gene expression, unless another DNA fragment located nearby was removed in cis. These results suggest that insulation of this important gene cluster relies, at least in part, upon a sequence-specific mechanism that displays some redundancy.

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  • Making progress with limb models. Nature 2002 Aug;418(6897):492-3. 10.1038/418492a. 418492a.

    Duboule D

    abstract

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  • The orphan nuclear receptor REV-ERBalpha controls circadian transcription within the positive limb of the mammalian circadian oscillator. Cell 2002 Jul;110(2):251-60. S0092867402008255.

    Preitner N, Damiola F, Lopez-Molina L, Zakany J, Duboule D, Albrecht U, Schibler U

    abstract

    Mammalian circadian rhythms are generated by a feedback loop in which BMAL1 and CLOCK, players of the positive limb, activate transcription of the cryptochrome and period genes, components of the negative limb. Bmal1 and Per transcription cycles display nearly opposite phases and are thus governed by different mechanisms. Here, we identify the orphan nuclear receptor REV-ERBalpha as the major regulator of cyclic Bmal1 transcription. Circadian Rev-erbalpha expression is controlled by components of the general feedback loop. Thus, REV-ERBalpha constitutes a molecular link through which components of the negative limb drive antiphasic expression of components of the positive limb. While REV-ERBalpha influences the period length and affects the phase-shifting properties of the clock, it is not required for circadian rhythm generation.

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  • A t(2;8) balanced translocation with breakpoints near the human HOXD complex causes mesomelic dysplasia and vertebral defects. Genomics 2002 Apr;79(4):493-8. 10.1006/geno.2002.6735. S0888754302967352.

    Spitz F, Montavon T, Monso-Hinard C, Morris M, Ventruto ML, Antonarakis S, Ventruto V, Duboule D

    abstract

    Mesomelic dysplasia is a severe shortening of forearms and forelegs, and is found in several distinct human syndromes. Here, we report the cloning of the breakpoints of a human t(2;8)(q31;p21) balanced translocation associated with mesomelic dysplasia of the upper limbs, as well as with vertebral defects. We show that this translocation does not disrupt any gene, hence it most likely exerts its deleterious effect by modifying gene regulation. The HOXD complex lies approximately 60 kb from the translocation breakpoint on chromosome 2. This cluster of genes has an important role in the development of both the vertebral column and the limbs. Only a few cases of mutations of these homeotic genes have been described so far in humans. However, gain- and loss-of-function of Hoxd genes in mice can induce mesomelic dysplasia-like phenotypes, suggesting that misexpression of HOXD genes may indeed be at the origin of this hereditary phenotype.

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  • Developmental biology in Geneva: a three century-long tradition. Int. J. Dev. Biol. 2002 Jan;46(1):5-13.

    Buscaglia M, Duboule D

    abstract

    It was in the first half of the 18th century when life sciences started to flourish in the independent republic of Geneva. However, it is difficult to identify a genuine school of developmental biologists during that era. Nevertheless, several prominent scientists over the past two and a half centuries have established and maintained a strong tradition of studies in embryological development and reproduction. In this short historical account, we briefly pay tribute to these famous forerunners, by emphasizing both the originality and quality of their work, as well as the many accompanying conceptual and methodological advances. We start with Abraham Trembley (1710-1784) and the discovery of Hydra and of regeneration, and with Charles Bonnet (1720-1793) who, amongst other contributions, first observed parthenogenetic development. In the 19th century, Carl Vogt (1817-1895) and Edouard Claparède (1832-1871) were well-known scientists in this field of research, whereas Hermann Fol (1845-1892) can be considered as one of the pioneers, if not the founder, of causal embryology, through his experiments on lateral asymmetry in manipulated chicken. More recently, Emile Guyénot (1885-1963) and Kitty Ponse (1897-1982) perpetuated this tradition, which is well alive nowadays in the city of Calvin.

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  • A nested deletion approach to generate Cre deleter mice with progressive Hox profiles. Int. J. Dev. Biol. 2002 Jan;46(1):185-91.

    Hérault Y, Kmita M, Sawaya CC, Duboule D

    abstract

    In mice, the loxP/Cre recombinase-dependent system of recombination offers powerful possibilities for engineering genetic configurations of interest. This system can also be advantageously used for conditional mutagenesis in vivo, whenever such an approach is required due to deleterious effects of either one mutation, or a combination thereof. Here, we report on the production of an allelic series of insertions of a Hoxd11/Cre fusion transgene at different positions within the HoxD complex, in order to produce the CRE recombinase with a 'Hox profile' progressively more extended. We used the R26R (R26R) reporter mouse line to functionally assess the distribution and efficiency of the CRE enzyme and discuss the usefulness of these various lines as deleter strains.

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