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The molecular mechanisms that maintain cellular identities and prevent dedifferentiation or transdifferentiation remain mysterious. However, both processes are transiently used during animal regeneration. Therefore, organisms that regenerate their organs, appendages, or even their whole body offer a fruitful paradigm to investigate the regulation of cell fate stability. Here, we used Hydra as a model system and show that Zic4, whose expression is controlled by Wnt3/β-catenin signaling and the Sp5 transcription factor, plays a key role in tentacle formation and tentacle maintenance. Reducing Zic4 expression suffices to induce transdifferentiation of tentacle epithelial cells into foot epithelial cells. This switch requires the reentry of tentacle battery cells into the cell cycle without cell division and is accompanied by degeneration of nematocytes embedded in these cells. These results indicate that maintenance of cell fate by a Wnt-controlled mechanism is a key process both during homeostasis and during regeneration.

The individual risk of developing drug addiction is highly determined by the epigenetic landscape1,2. Chromatin remodeling regulates drug-induced transcriptional and behavioral effects and the consequent development of addictive behaviors2,3. Several chromatin modifications in the ventral tegmental area and nucleus accumbens, including histone H3 methylation, H3 and H4 acetylation, have been implicated in drug addiction. Still, the contribution of other histones and their post-translational modifications (PTMs), such as monoubiquitination is unknown4–8. Here we found that H2A monoubiquitination in the paraventricular thalamus (PVT) plays a major role in cocaine-adaptive behaviors and local cocaine-evoked transcriptional repression. Mice undergoing chronic cocaine administration showed a specific increased monoubiquitination of H2A. Furthermore, we showed that this histone PTM is controlled, in the PVT, by an interaction between melanoma-associated antigen D1 (Maged1), a scaffold protein involved in drug addiction9, and USP7, a deubiquitinase10. Accordingly, Maged1 specific inactivation in thalamic vGluT2 neurons, or USP7 inhibition, blocked cocaine-evoked H2A monoubiquitination and abolished cocaine locomotor sensitization. Finally, we identified genetic variations ofMAGED1andUSP7associated with modified transition to cocaine addiction and cocaine-induced aggressive behavior in human subjects. These findings identified a new epigenetic modification in a non-canonical reward pathway of the brain and a potent marker of epigenetic risk factor for drug addiction in human.

Modifications in gene regulation are driving forces in the evolution of organisms. Part of these changes involve cis-regulatory elements (CREs), which contact their target genes through higher-order chromatin structures. However, how such architectures and variations in CREs contribute to transcriptional evolvability remains elusive. We use Hoxd genes as a paradigm for the emergence of regulatory innovations, as many relevant enhancers are located in a regulatory landscape highly conserved in amniotes. Here, we analysed their regulation in murine vibrissae and chicken feather primordia, two skin appendages expressing different Hoxd gene subsets, and compared the regulation of these genes in these appendages with that in the elongation of the posterior trunk. In the two former structures, distinct subsets of Hoxd genes are contacted by different lineage-specific enhancers, probably as a result of using an ancestral chromatin topology as an evolutionary playground, whereas the gene regulation that occurs in the mouse and chicken embryonic trunk partially relies on conserved CREs. A high proportion of these non-coding sequences active in the trunk have functionally diverged between species, suggesting that transcriptional robustness is maintained, despite considerable divergence in enhancer sequences.

The HoxD gene cluster is critical for proper limb formation in tetrapods. In the emerging limb buds, different sub-groups of Hoxd genes respond first to a proximal regulatory signal, then to a distal signal that organizes digits. These two regulations are exclusive from one another and emanate from two distinct TADs flanking HoxD, both containing a range of appropriate enhancer sequences. The telomeric TAD (T-DOM) contains several enhancers active in presumptive forearm cells and is divided into two sub-TADs separated by a CTCF-rich boundary, which defines two regulatory sub-modules. To understand the importance of this particular regulatory topology to control Hoxd gene transcription in time and space, we either deleted or inverted this sub-TAD boundary, eliminated the CTCF binding sites or inverted the entire T-DOM to exchange the respective positions of the two sub-TADs. The effects of such perturbations on the transcriptional regulation of Hoxd genes illustrate the requirement of this regulatory topology for the precise timing of gene activation. However, the spatial distribution of transcripts was eventually resumed, showing that the presence of enhancers sequences, rather than either their exact topology or a particular chromatin architecture, is the key factor. We also show that the affinity of enhancers to find their natural target genes can overcome the presence of both a strong TAD border and an unfavourable orientation of CTCF sites.SIGNIFICANCE STATEMENTMany genes important for vertebrate development are surrounded by series of remote enhancer sequences. Such regulatory landscapes and their target genes are usually located within the same chromatin domains, which appears to constrain the action of these regulatory sequences and hence to facilitate enhancer-promoter recognition and gene expression. We used the HoxD locus to assess the impact of modifying the regulatory topology upon gene activation in space and time. A series of chromosomal re-arrangements involving deletions and inversions reveals that the enhancer topology plays a role in the timing of gene activation. However, gene expression was often recovered, subsequently, illustrating the intrinsic capacity of some enhancers to find their target promoters despite an apparently adverse chromatin topology.

The molecular mechanisms that maintain cell identities and prevent transdifferentiation remain mysterious. Interestingly, both dedifferentiation and transdifferentiation are transiently reshuffled during regeneration. Therefore, organisms that regenerate readily offer a fruitful paradigm to investigate the regulation of cell fate stability. Here, we used Hydra as a model system and show that Zic4 silencing is sufficient to induce transdifferentiation of tentacle into foot cells. We identified a Wnt-controlled Gene Regulatory Network that controls a transcriptional switch of cell identity. Furthermore, we show that this switch also controls the re-entry into the cell cycle. Our data indicate that maintenance of cell fate by a Wnt-controlled GRN is a key mechanism during both homeostasis and regeneration.One-Sentence SummaryA Wnt-controlled GRN controls fate maintenance in Hydra.

Mammalian Hox gene clusters contain a range of CTCF binding sites. In addition to their importance in organizing a TAD border, which isolates the most posterior genes from the rest of the cluster, the positions and orientations of these sites suggest that CTCF may be instrumental in the selection of various subsets of contiguous genes, which are targets of distinct remote enhancers located in the flanking regulatory landscapes. We examined this possibility by producing an allelic series of cumulative in cis mutations in these sites, up to the abrogation of CTCF binding in the five sites located on one side of the TAD border. In the most impactful alleles, the global chromatin architecture of the locus was modified, yet not drastically, illustrating that CTCF sites located on one side of a strong TAD border are sufficient to organize at least part of this insulation. Spatial colinearity in the expression of these genes along the major body axis was nevertheless maintained, despite abnormal expression boundaries. In contrast, strong effects were scored in the selection of target genes responding to particular enhancers, leading to the misregulation of Hoxd genes in specific structures. Altogether, while most enhancer–promoter interactions can occur in the absence of this series of CTCF sites, the binding of CTCF in the Hox cluster is required to properly transform a rather unprecise process into a highly discriminative mechanism of interactions, which is translated into various patterns of transcription accompanied by the distinctive chromatin topology found at this locus. Our allelic series also allowed us to reveal the distinct functional contributions for CTCF sites within this Hox cluster, some acting as insulator elements, others being necessary to anchor or stabilize enhancer–promoter interactions, and some doing both, whereas they all together contribute to the formation of a TAD border. This variety of tasks may explain the amazing evolutionary conservation in the distribution of these sites among paralogous Hox clusters or between various vertebrates.

Mammalian Hox gene clusters contain a range of CTCF binding sites. In addition to their importance in organizing a TAD border, which isolates the most posterior genes from the rest of the cluster, the positions and orientations of these sites suggest that CTCF may be instrumental in the selection of various subsets of contiguous genes, which are targets of distinct remote enhancers located in the flanking regulatory landscapes. We examined this possibility by producing an allelic series of cumulative in-cis mutations in these sites, up to the abrogation of CTCF binding in the five sites located on one side of the TAD border. In the most impactful alleles, the global chromatin architecture of the locus was modified, yet not drastically, illustrating that CTCF sites located on one side of a strong TAD border are sufficient to organize at least part of this insulation. Spatial colinearity in the expression of these genes along the major body axis was nevertheless maintained, despite abnormal expression boundaries. In contrast, strong effects were scored in the selection of target genes responding to particular enhancers, leading to the mis-regulation of Hoxd genes in specific structures. Altogether, while most enhancer-promoter interactions can occur in the absence of this series of CTCF sites, it seems that the binding of CTCF in the Hox cluster is required to properly transform a rather unprecise process into a highly discriminative mechanism of interactions, which is translated into various patterns of transcription accompanied by the distinctive chromatin topology found at this locus. Our allelic series also allowed us to reveal the distinct functional contributions for CTCF sites within this Hox cluster, some acting as insulator elements, others being necessary to anchor or stabilize enhancer-promoter interactions and some doing both, whereas all together contribute to the formation of a TAD border. This variety of tasks may explain the amazing evolutionary conservation in the distribution of these sites amongst paralogous Hox clusters or between various vertebrates.

Modifications in gene regulation during development are considered to be a driving force in the evolution of organisms. Part of these changes involve rapidly evolving cis-regulatory elements (CREs), which interact with their target genes through higher-order 3D chromatin structures. However, how such 3D architectures and variations in CREs contribute to transcriptional evolvability remains elusive. During vertebrate evolution, Hox genes were redeployed in different organs in a class-specific manner, while maintaining the same basic function in organizing the primary body axis. Since a large part of the relevant enhancers are located in a conserved regulatory landscape, this gene cluster represents an interesting paradigm to study the emergence of regulatory innovations. Here, we analysed Hoxd gene regulation in both murine vibrissae and chicken feather primordia, two mammalian- and avian-specific skin appendages which express different subsets of Hoxd genes, and compared their regulatory modalities with the regulations at work during the elongation of the posterior trunk, a mechanism highly conserved in amniotes. We show that in the former two structures, distinct subsets of Hoxd genes are contacted by different lineage-specific enhancers, likely as a result of using an ancestral chromatin topology as an evolutionary playground, whereas the regulations implemented in the mouse and chicken embryonic trunk partially rely on conserved CREs. Nevertheless, a high proportion of these non-coding sequences active in the trunk appear to have functionally diverged between the two species, suggesting that transcriptional robustness is maintained despite a considerable divergence in CREs’ sequence, an observation supported by a genome-wide comparative approach.

Background During tetrapod limb development, the HOXA13 and HOXD13 transcription factors are critical for the emergence and organization of the autopod, the most distal aspect where digits will develop. Since previous work had suggested that the Dbx2 gene is a target of these factors, we set up to analyze in detail this potential regulatory interaction. Results We show that HOX13 proteins bind to mammalian‐specific sequences at the vicinity of the Dbx2 locus that have enhancer activity in developing digits. However, the functional inactivation of the DBX2 protein did not elicit any particular phenotype related to Hox genes inactivation in digits, suggesting either redundant or compensatory mechanisms. We report that the neighboring Nell2 and Ano6 genes are also expressed in distal limb buds and are in part controlled by the same Dbx2 enhancers despite being localized into two different topologically associating domains (TADs) flanking the Dbx2 locus. Conclusions We conclude that Hoxa13 and Hoxd genes cooperatively activate Dbx2 expression in developing digits through binding to mammalian specific regulatory sequences in the Dbx2 neighborhood. Furthermore, these enhancers can overcome TAD boundaries in either direction to co‐regulate a set of genes located in distinct chromatin domains., Key Findings Hoxa13 and Hoxd genes cooperatively regulate Dbx2 expression in developing digits via eutherian specific enhancers.Dbx2 is expressed in different digit joint precursors but its function there is not essential.Dbx2 enhancers also control the expression of the Nell2 and Ano6 genes, which are located in different TADs, thus overcoming the boundary effect.Dbx2 chromatin architecture and enhancers evolved in the mammalian lineage.

Significance Many genes important for vertebrate development are surrounded by multiple series of remote enhancer sequences. Such regulatory landscapes and their target genes are usually located within the same chromatin domains, which appears to constrain the action of these regulatory sequences and hence to facilitate enhancer–promoter recognition and gene expression. We used the HoxD locus to assess the impact of modifying the regulatory topology upon gene activation in space and time. A series of chromosomal rearrangements involving deletions and inversions reveals that the enhancer topology plays a role in the timing of gene activation. However, gene expression was often recovered subsequently, illustrating the intrinsic capacity of some enhancers to find their target promoters despite an apparently adverse chromatin topology., The HoxD gene cluster is critical for proper limb formation in tetrapods. In the emerging limb buds, different subgroups of Hoxd genes respond first to a proximal regulatory signal, then to a distal signal that organizes digits. These two regulations are exclusive from one another and emanate from two distinct topologically associating domains (TADs) flanking HoxD, both containing a range of appropriate enhancer sequences. The telomeric TAD (T-DOM) contains several enhancers active in presumptive forearm cells and is divided into two sub-TADs separated by a CTCF-rich boundary, which defines two regulatory submodules. To understand the importance of this particular regulatory topology to control Hoxd gene transcription in time and space, we either deleted or inverted this sub-TAD boundary, eliminated the CTCF binding sites, or inverted the entire T-DOM to exchange the respective positions of the two sub-TADs. The effects of such perturbations on the transcriptional regulation of Hoxd genes illustrate the requirement of this regulatory topology for the precise timing of gene activation. However, the spatial distribution of transcripts was eventually resumed, showing that the presence of enhancer sequences, rather than either their exact topology or a particular chromatin architecture, is the key factor. We also show that the affinity of enhancers to find their natural target genes can overcome the presence of both a strong TAD border and an unfavorable orientation of CTCF sites.

BackgroundDuring tetrapod limb development, the HOXA13 and HOXD13 transcription factors are critical for the emergence and organization of the autopod, the most distal aspect where digits will develop. Since previous work had suggested that theDbx2gene is a target of these factors, we set up to analyze in detail this potential regulatory interaction.ResultsWe show that HOX13 proteins bind to eutherian-specific sequences at the vicinity of theDbx2locus that have enhancer activity in developing digits. However, the functional inactivation of the DBX2 protein did not elicit any particular phenotype related toHoxgenes inactivation in digits, suggesting either redundant or compensatory mechanisms. We report that the neighboringNell2andAno6genes are also expressed in distal limb buds and are, in part, controlled by the sameDbx2enhancers despite being localized into two different topologically associating domains (TADs) flanking theDbx2locus.ConclusionsWe conclude thatHoxa13andHoxdgenes cooperatively activateDbx2expression in developing digits through binding to eutherian specific regulators elements in theDbx2neighborhood. Furthermore, these enhancers can overcome TAD boundaries in either direction to co-regulate a set of genes located in distinct chromatin domains.Bullet pointsHoxa13andHoxdgenes cooperatively regulateDbx2expression in developing digits via eutherian specific enhancers.Dbx2is expressed in different digit joint precursors but its function there is not essential.Dbx2enhancers also control the expression of theNell2andAno6genes, which are located in different TADs, thus overcoming the boundary effect.Dbx2chromatin architecture and enhancers evolved in the mammalian lineage.Grant Sponsor and NumberSwiss National Research Foundation #310030B_138662.European Research Council grants RegulHox#588029

The emergence of multiple axes is an essential element in the establishment of the mammalian body plan. This process takes place shortly after implantation of the embryo within the uterus and relies on the activity of gene regulatory networks that coordinate transcription in space and time. Whereas genetic approaches have revealed important aspects of these processes(1), a mechanistic understanding is hampered by the poor experimental accessibility of early post-implantation stages. Here we show that small aggregates of mouse embryonic stem cells (ESCs), when stimulated to undergo gastrulation-like events and elongation in vitro, can organize a post-occipital pattern of neural, mesodermal and endodermal derivatives that mimic embryonic spatial and temporal gene expression. The establishment of the three major body axes in these 'gastruloids'(2,3) suggests that the mechanisms involved are interdependent. Specifically, gastruloids display the hallmarks of axial gene regulatory systems as exemplified by the implementation of collinear Hox transcriptional patterns along an extending anteroposterior axis. These results reveal an unanticipated self-organizing capacity of aggregated ESCs and suggest that gastruloids could be used as a complementary system to study early developmental events in the mammalian embryo.

In all tetrapods examined thus far, the development and patterning of limbs require the activation of gene members of theHoxDcluster. In mammals, they are controlled by a complex bimodal regulation, which controls first the proximal patterning, then the distal structure, allowing at the same time the formation of the wrist and ankle articulations. We analyzed the implementation of this regulatory mechanism in chicken, i.e. in an animal where large morphological differences exist between fore-and hindlimbs. We report that while this bimodal regulation is globally conserved between mammals and avian, some important modifications evolved at least between these two model systems, in particular regarding the activity of specific enhancers, the width of the TAD boundary separating the two regulations and the comparison between the forelimbversushindlimb regulatory controls. Some aspects of these regulations seem to be more conserved between chick and bats than with the mouse situation, which may relate to the extent to which forelimbs and hindlimbs of these various animals differ in their functions.AUTHOR SUMMARYThe morphologies of limbs largely vary either amongst tetrapod species, or even between the fore-and hindlimbs of the same animal species. In order to try and evaluate whether variations in the complex regulation ofHoxdgenes during limb development may contribute to these differences, we compared their transcriptional controls during both fore-and hindlimb buds development in either the mouse, or the chicken embryos. We combined transcriptome analyses with 3D genome conformation, histone modification profiles and mouse genetics and found that the regulatory mechanism underlyingHoxdgene expression was highly conserved in all contexts, though with some clear differences. For instance, we observed a variation in the TAD boundary interval between the mouse and the chick, as well as differences in the activity of a conserved enhancer element (CS93) situated within the T-DOM regulatory landscape. In contrast to the mouse, the chicken enhancer indeed displayed a stronger activity in fore-than in hindlimb buds, coinciding with the observed striking differences in the mRNA levels. Altogether, differences in both the timing and duration of TAD activities and in the width of their boundary may parallel the important decrease inHoxdgene transcription in chick hindlimbversusforelimb buds. These differences may also account for the slightly distinct regulatory strategies implemented by mammals and birds at this locus, potentially leading to substantial morphological variations.

Polyps of the cnidarian Hydra maintain their adult anatomy through two developmental organizers, the head organizer located apically and the foot organizer basally. The head organizer is made of two antagonistic cross-reacting components, an activator, driving apical differentiation and an inhibitor, preventing ectopic head formation. Here we characterize the head inhibitor by comparing planarian genes down-regulated when β-catenin is silenced to Hydra genes displaying a graded apical-to-basal expression and an up-regulation during head regeneration. We identify Sp5 as a transcription factor that fulfills the head inhibitor properties: leading to a robust multiheaded phenotype when knocked-down in Hydra, acting as a transcriptional repressor of Wnt3 and positively regulated by Wnt/β-catenin signaling. Hydra and zebrafish Sp5 repress Wnt3 promoter activity while Hydra Sp5 also activates its own expression, likely via β-catenin/TCF interaction. This work identifies Sp5 as a potent feedback loop inhibitor of Wnt/β-catenin signaling, a function conserved across eumetazoan evolution., Hydra regenerate various body parts on amputation by activation of the appropriate organiser, but how head formation is controlled is unclear. Here, the authors identify the transcription factor Sp5 as restricting head formation, by being activated by beta-catenin and then acting as a repressor of Wnt3.

TheHydrapolyp regenerates its head by transforming the gastric tissue below the wound into a head organizer made of two antagonistic cross-reacting components. The activator, previously characterized asWnt3, drives apical differentiation by acting locally and auto-catalytically. The uncharacterized inhibitor, produced under the control of the activator, prevents ectopic head formation. By crossing RNA-seq data obtained in aβ-catenin(RNAi) screen performed in planarians and a quantitative analysis of positional and temporal gene expression inHydra, we identified Sp5 as a transcription factor that fulfills the head inhibitor properties: a Wnt/β-catenin inducible expression, a graded apical-to-basal expression, a sustained up-regulation during head regeneration, a multi-headed phenotype when knocked-down, a repressing activity on Wnt3 expression. In mammalian cells,Hydraand zebrafish Sp5 repressWnt3promoter activity whileHydra Sp5also auto-activates its expression, possibly via β-catenin and/or Tcf/Lef1 interaction. This work identifies Sp5 as a novel potent feedback loop inhibitor of Wnt/β-catenin signaling across eumetazoans.

In all tetrapods examined thus far, the development and patterning of limbs require the activation of gene members of the HoxD cluster. In mammals, they are regulated by a complex bimodal process that controls first the proximal patterning and then the distal structure. During the shift from the former to the latter regulation, this bimodal regulatory mechanism allows the production of a domain with low Hoxd gene expression, at which both telomeric (T-DOM) and centromeric regulatory domains (C-DOM) are silent. These cells generate the future wrist and ankle articulations. We analyzed the implementation of this regulatory mechanism in chicken, i.e., in an animal for which large morphological differences exist between fore- and hindlimbs. We report that although this bimodal regulation is globally conserved between the mouse and the chick, some important modifications evolved at least between these two model systems, in particular regarding the activity of specific enhancers, the width of the TAD boundary separating the two regulations, and the comparison between the forelimb versus hindlimb regulatory controls. At least one aspect of these regulations seems to be more conserved between chick and bats than with mouse, which may relate to the extent to which forelimbs and hindlimbs of these various animals differ in their morphologies., A comparison of Hox gene regulation during the development of limbs in birds and mammals reveals that whereas the characteristic bimodal regulatory system, based on large chromatin domains, is largely conserved between these morphologically distinct structures, some differences are revealed in the way this is implemented in various vertebrates., Author summary The shapes of limbs vary greatly among tetrapod species, even between the forelimbs and hindlimbs of the same animal. Hox genes regulate the proper growth and patterning of tetrapod limbs. In order to evaluate whether variations in the complex regulation of a cluster of Hox genes—the Hoxd genes—during limb development contribute to the differences in limb shape, we compared their transcriptional control during limb bud development in the forelimbs and hindlimbs of mouse and chicken embryos. We found that the regulatory mechanism underlying Hoxd gene expression is highly conserved, but some clear differences exist. For instance, we observed a variation in the topologically associating domain (TAD; a self-interacting genomic region) boundary interval between the mouse and the chick, as well as differences in the activity of a conserved enhancer element situated within the telomeric regulatory domain. In contrast to the mouse, the chicken enhancer has a stronger activity in the forelimb buds than in the hindlimb buds, which is correlated with the striking differences in the mRNA levels of the genes. We conclude that differences in both the timing and duration of TAD activities and in the width of their boundary may parallel the important decrease in Hoxd gene transcription in chick hindlimb buds versus forelimb buds. These differences may also account for the slightly distinct regulatory strategies implemented by mammals and birds at this locus.

BackgroundThe transcriptional activation of Hoxd genes during mammalian limb development involves dynamic interactions with the two Topologically Associating Domains (TADs) flanking the HoxD cluster. In particular, the activation of the most posterior Hoxd genes in developing digits is controlled by regulatory elements located in the centromeric TAD (C-DOM) through long-range contacts. To assess the structure-function relationships underlying such interactions, we measured compaction levels and TAD discreteness using a combination of chromosome conformation capture (4C-seq) and DNA FISH.ResultsWe challenged the robustness of the TAD architecture by using a series of genomic deletions and inversions that impact the integrity of this chromatin domain and that remodel the long-range contacts. We report multi-partite associations between Hoxd genes and up to three enhancers and show that breaking the native chromatin topology leads to the remodelling of TAD structure.ConclusionsOur results reveal that the re-composition of TADs architectures after severe genomic re-arrangements depends on a boundary-selection mechanism that uses CTCF-mediated gating of long-range contacts in combination with genomic distance and, to a certain extent, sequence specificity.

Background The transcriptional activation of HoxD genes during mammalian limb development involves dynamic interactions with two topologically associating domains (TADs) flanking the HoxD cluster. In particular, the activation of the most posterior HoxD genes in developing digits is controlled by regulatory elements located in the centromeric TAD (C-DOM) through long-range contacts. Results To assess the structure–function relationships underlying such interactions, we measured compaction levels and TAD discreteness using a combination of chromosome conformation capture (4C-seq) and DNA FISH. We assessed the robustness of the TAD architecture by using a series of genomic deletions and inversions that impact the integrity of this chromatin domain and that remodel long-range contacts. We report multi-partite associations between HoxD genes and up to three enhancers. We find that the loss of native chromatin topology leads to the remodeling of TAD structure following distinct parameters. Conclusions Our results reveal that the recomposition of TAD architectures after large genomic re-arrangements is dependent on a boundary-selection mechanism in which CTCF mediates the gating of long-range contacts in combination with genomic distance and sequence specificity. Accordingly, the building of a recomposed TAD at this locus depends on distinct functional and constitutive parameters. Electronic supplementary material The online version of this article (doi:10.1186/s13059-017-1278-z) contains supplementary material, which is available to authorized users.

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.

A well integrated and hierarchically organized gene regulatory network is responsible for the progressive specification of the forebrain. The transcription factor Six3 is one of the central components of this network. As such, Six3 regulates several components of the network, but its upstream regulators are still poorly characterized. Here we have systematically identified such regulators, taking advantage of the detailed functional characterization of the regulatory region of the medaka fish Six3.2 ortholog and of a time/cost-effective trans-regulatory screening, which complemented and overcame the limitations of in silico prediction approaches. The candidates resulting from this search were validated with dose-response luciferase assays and expression pattern criteria. Reconfirmed candidates with a matching expression pattern were also tested with chromatin immunoprecipitation and functional studies. Our results confirm the previously proposed direct regulation of Pax6 and further demonstrate that Msx2 and Pbx1 are bona fide direct regulators of early Six3.2 distribution in distinct domains of the medaka fish forebrain. They also point to other transcription factors, including Tcf3, as additional regulators of different spatial-temporal domains of Six3.2 expression. The activity of these regulators is discussed in the context of the gene regulatory network proposed for the specification of the forebrain., Spanish Ministerio de Economía y Competitividad (MINECO) Grants BFU2010-16031 and BFU2013-43213-P, cofounded by FEDER Funds; Comunidad Autónoma de Madrid (CAM) Grant CELL-DD S2010/BMD-2315; Fundaluce; Fundación ONCE; the Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER) del Instituto Carlos III (ISCIII); and an Institutional Grant from the Fundación Ramón Areces.

Increasing evidence in the last years indicates that the vast amount of regulatory information contained in mammalian genomes is organized in precise 3D chromatin structures. However, the impact of this spatial chromatin organization on gene expression and its degree of evolutionary conservation is still poorly understood. The Six homeobox genes are essential developmental regulators organized in gene clusters conserved during evolution. Here, we reveal that the Six clusters share a deeply evolutionarily conserved 3D chromatin organization that predates the Cambrian explosion. This chromatin architecture generates two largely independent regulatory landscapes (RLs) contained in two adjacent topological associating domains (TADs). By disrupting the conserved TAD border in one of the zebrafish Six clusters, we demonstrate that this border is critical for preventing competition between promoters and enhancers located in separated RLs, thereby generating different expression patterns in genes located in close genomic proximity. Moreover, evolutionary comparison of Six-associated TAD borders reveals the presence of CCCTC-binding factor (CTCF) sites with diverging orientations in all studied deuterostomes. Genome-wide examination of mammalian HiC data reveals that this conserved CTCF configuration is a general signature of TAD borders, underscoring that common organizational principles underlie TAD compartmentalization in deuterostome evolution., This study was supported by the Spanish and Andalusian Governments (BFU2013-41322-P and Proyecto de Excelencia BIO-396 to J.L.G.-S., BFU2013-43213-P to P.B., BFU2014-55738-REDT to J.L.G.-S. and P.B., and BFU2011-22928 to J.C. and M.L.-M.). EU-FP7-PEOPLE-2011-CIG Grant 303904 “EPAXIALMYF5KO” also supported J.C.

et al., Microphthalmos is a rare congenital anomaly characterized by reduced eye size and visual deficits of variable degree. Sporadic and hereditary microphthalmos have been associated with heterozygous mutations in genes fundamental for eye development. Yet,many cases are idiopathic or await the identification of molecular causes. Here we show that haploinsufficiency of Meis1, which encodes a transcription factor with evolutionarily conserved expression in the embryonic trunk, brain and sensory organs, including the eye, causes microphthalmic traits and visual impairment in adultmice. By combining analysis of Meis1 loss-offunction and conditional Meis1 functional rescue with ChIP-seq and RNA-seq approaches we show that, in contrast to its preferential association with Hox-Pbx BSs in the trunk, Meis1 binds to Hox/Pbxindependent sites during optic cup development. In the eye primordium, Meis1 coordinates, in a dose-dependent manner, retinal proliferation and differentiation by regulating genes responsible for human microphthalmia and components of the Notch signaling pathway. In addition, Meis1 is required for eye patterning by controlling a set of eye territory-specific transcription factors, so that in Meis1−/− embryos boundaries among the different eye territories are shifted or blurred.We propose that Meis1 is at the core of a genetic network implicated in eye patterning/microphthalmia, and represents an additional candidate for syndromic cases of these ocular malformations., This work was supported by grants from the Spanish Ministry of Economy and Competitiveness (MINECO) [BFU2010-16031; BFU2013-43213-P, supported by FEDER Funds], the CIBERER, Instituto de Salud Carlos III (ISCIII) to P.B. and an Institutional Grant from the Fundación Ramon Areces; by MINECO [BFU2012-31086] and ISCIII [RD12/0019/0005] to M.T.; by Comunidad Autonoma de Madrid (CAM) [S2010/BMD-2315] to P.B. and M.T.; by MINECO [BFU2013-41322-P] and Andalusian Government [BIO-396] to J.L.G.-S.; and by MINECO [BFU2014-55738-REDT] to P.B., J.L.G.-S. and F.C. M.G.-L. was supported by a grant from the CONACyT. O.B. is supported by an Australian Research Council Discovery Early Career Researcher Award-DECRA [DE140101962]. The CNIC is supported by MINECO and the Pro-CNIC Foundation.

The Sox2 transcription factor is active in stem/progenitor cells throughout the developing vertebrate central nervous system. However, its conditional deletion at E12.5 in mouse causes few brain developmental problems, with the exception of the postnatal loss of the hippocampal radial glia stem cells and the dentate gyrus. We deleted Sox2 at E9.5 in the telencephalon, using a Bf1-Cre transgene. We observed embryonic brain defects that were particularly severe in the ventral, as opposed to the dorsal, telencephalon. Important tissue loss, including the medial ganglionic eminence (MGE), was detected at E12.5, causing the subsequent impairment of MGE- derived neurons. The defect was preceded by loss of expression of the essential ventral determinants Nkx2.1 and Shh, and accompanied by ventral spread of dorsal markers. This phenotype is reminiscent of that of mice mutant for the transcription factor Nkx2.1 or for the Shh receptor Smo. Nkx2.1 is known to mediate the initial activation of ventral telencephalic Shh expression. A partial rescue of the normal phenotype at E14.5 was obtained by administration of a Shh agonist. Experiments in Medaka fish indicate that expression of Nkx2.1 is regulated by Sox2 in this species also. We propose that Sox2 contributes to Nkx2.1 expression in early mouse development, thus participating in the region-specific activation of Shh, thereby mediating ventral telencephalic patterning induction. © 2013. Published by The Company of Biologists Ltd., Cariplo Foundation (grant 20100673); ASTIL Regione Lombardia (SAL-19, N Prot FL 16874); Telethon (GGP12152); AssociazioneItaliana Ricerca sul Cancro (AIRC IG-5801); Spanish Ministerio de Economia y Competividad (BFU2010-16031); Comunidad Autonoma de Madrid (BDM-2315)

The vertebrate forebrain or prosencephalon is patterned at the beginning of neurulation into four major domains: the telencephalic, hypothalamic, retinal and diencephalic anlagen. These domains will then give rise to the majority of the brain structures involved in sensory integration and the control of higher intellectual and homeostatic functions. Understanding how forebrain pattering arises has thus attracted the interest of developmental neurobiologists for decades. As a result, most of its regulators have been identified and their hierarchical relationship is now the object of active investigation. Here, we summarize the main morphogenetic pathways and transcription factors involved in forebrain specification and propose the backbone of a possible gene regulatory network (GRN) governing its specification, taking advantage of the GRN principles elaborated by pioneer studies in simpler organisms. We will also discuss this GRN and its operational logic in the context of the remarkable morphological and functional diversification that the forebrain has undergone during evolution. © 2012 Elsevier Ireland Ltd., the Spanish MICINN (BFU2010-16031); Comunidad Autonoma de Madrid (CAM, CELL-DD S2010/BMD-2315); Fundaluce; Fundación ONCE; CIBERER.

The vertebrate forebrain is patterned during gastrulation into telencephalic, retinal, hypothalamic and diencephalic primordia. Specification of each of these domains requires the concerted activity of combinations of transcription factors (TFs). Paradoxically, some of these factors are widely expressed in the forebrain, which raises the question of how they can mediate regional differences. To address this issue, we focused on the homeobox TF Six3.2. With genomic and functional approaches we demonstrate that, in medaka fish, Six3.2 regulates, in a concentration-dependent manner, telencephalic and retinal specification under the direct control of Sox2. Six3.2 and Sox2 have antagonistic functions in hypothalamic development. These activities are, in part, executed by Foxg1 and Rx3, which seem to be differentially and directly regulated by Six3.2 and Sox2. Together, these data delineate the mechanisms by which Six3.2 diversifies its activity in the forebrain and highlight a novel function for Sox2 as one of the main regulators of anterior forebrain development. They also demonstrate that graded levels of the same TF, probably operating in partially independent transcriptional networks, pattern the vertebrate forebrain along the anterior-posterior axis. © 2012. Published by The Company of Biologists Ltd., Spanish Ministry of Science and Innovation (BFU2007-61774, BFU2010-16031); Comunidad Autonoma de Madrid (P-SAL-0190-2006); Centre for Biomedical Network Research on Rare Diseases

Timely generation of distinct neural cell types in appropriate numbers is fundamental for the generation of a functional retina. In vertebrates, the transcription factor Six6 is initially expressed in multipotent retina progenitors and then becomes restricted to differentiated retinal ganglion and amacrine cells. How Six6 expression in the retina is controlled and what are its precise functions are still unclear. To address this issue, we used bioinformatic searches and transgenic approaches in medaka fish (Oryzias latipes) to characterise highly conserved regulatory enhancers responsible for Six6 expression. One of the enhancers drove gene expression in the differentiating and adult retina. A search for transcription factor binding sites, together with luciferase, ChIP assays and gain-of-function studies, indicated that NeuroD, a bHLH transcription factor, directly binds an ‘E-box’ sequence present in this enhancer and specifically regulates Six6 expression in the retina. NeuroD-induced Six6 overexpression in medaka embryos promoted unorganized retinal progenitor proliferation and, most notably, impaired photoreceptor differentiation, with no apparent changes in other retinal cell types. Conversely, Six6 gain- and loss-of-function changed NeuroD expression levels and altered the expression of the photoreceptor differentiation marker Rhodopsin. In addition, knockdown of Six6 interfered with amacrine cell generation. Together, these results indicate that Six6 and NeuroD control the expression of each other and their functions coordinate amacrine cell generation and photoreceptor terminal differentiation.

13 páginas, 8 figuras.-- PMCID: PMC2894581, Dendrite branching and spine formation determines the function of morphologically distinct and specialized neuronal subclasses. However, little is known about the programs instructing specific branching patterns in vertebrate neurons and whether such programs influence dendritic spines and synapses. Using knockout and knockdown studies combined with morphological, molecular, and electrophysiological analysis, we show that the homeobox Cux1 and Cux2 are intrinsic and complementary regulators of dendrite branching, spine development, and synapse formation in layer II-III neurons of the cerebral cortex. Cux genes control the number and maturation of dendritic spines partly through direct regulation of the expression of Xlr3b and Xlr4b, chromatin remodeling genes previously implicated in cognitive defects. Accordingly, abnormal dendrites and synapses in Cux2−/− mice correlate with reduced synaptic function and defects in working memory. These demonstrate critical roles of Cux in dendritogenesis and highlight subclass-specific mechanisms of synapse regulation that contribute to the establishment of cognitive circuits., This work was supported by MICINN grants (SAF2005-0094, SAF2008-00211, PIE-200820I166, and BFU2007-61774), a grant from Mutua Madrilen˜ a Automovilı´stica (0328-2005), and a grant from the Spanish Comunidad de Madrid CCG08-CSIC/SAL-3464. B. Cubelos holds a fellowship from the CSIC (JAEDoc2008-020), and A. Sebastian-Serrano, from the MICINN (BES-2006-13901). C.A.W. was supported by 2RO1 NS032457 from the NINDS, and J.M. Redondo, by grant SAF2006-08348. C.A.W. is an Investigator of the Howard Hughes Medical Institute. The Centro Nacional de Investigaciones Cardiovasculares is supported by the MICINN and the Pro-CNIC Foundation.

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