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In the following paper, we present the result of an exhaustive review on how contemporary video games influence multiple social aspects such as health, psychology, family, entertainment, economics, and international conflicts. It also addresses the trends for the near future on the use and influence of video games in society, their consequences, and trends. Multiple scientific articles and websites specialized in the video game industry were reviewed, from which information has been obtained on how they contribute to society. The video game industry quickly incorporates solutions to many social problems directly and indirectly, and their incorporation into society will soon become a prevailing need.

Skates are cartilaginous fish whose body plan features enlarged wing-like pectoral fins, enabling them to thrive in benthic environments1,2. However, the molecular underpinnings of this unique trait remain unclear. Here we investigate the origin of this phenotypic innovation by developing the little skate Leucoraja erinacea as a genomically enabled model. Analysis of a high-quality chromosome-scale genome sequence for the little skate shows that it preserves many ancestral jawed vertebrate features compared with other sequenced genomes, including numerous ancient microchromosomes. Combining genome comparisons with extensive regulatory datasets in developing fins—including gene expression, chromatin occupancy and three-dimensional conformation—we find skate-specific genomic rearrangements that alter the three-dimensional regulatory landscape of genes that are involved in the planar cell polarity pathway. Functional inhibition of planar cell polarity signalling resulted in a reduction in anterior fin size, confirming that this pathway is a major contributor to batoid fin morphology. We also identified a fin-specific enhancer that interacts with several hoxa genes, consistent with the redeployment of hox gene expression in anterior pectoral fins, and confirmed its potential to activate transcription in the anterior fin using zebrafish reporter assays. Our findings underscore the central role of genome reorganization and regulatory variation in the evolution of phenotypes, shedding light on the molecular origin of an enigmatic trait.

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

14 pages, 8 figures, supplementary information https://doi.org/10.1038/s41588-022-01089-w.-- Data availability: Raw and aligned sequencing data are available at https://danio-code.zfin.org/dataExport/. The raw sequencing data produced for this study are available on the European Bioinformatics Institute (EBI) European Nucleotide Archive (ENA) under study numbers PRJNA824720, PRJNA821001, PRJNA821088, PRJNA821148 and PRJNA821034. Annotation tracks are available at http://genome.ucsc.edu/cgi-bin/hgTracks?db=danRer10&hubUrl=https://danio-code.zfin.org/trackhub/DANIO-CODE.hub.txt (danRer10) and http://genome.ucsc.edu/cgi-bin/hgTracks?db=danRer11&hubUrl=https://danio-code.zfin.org/trackhub/DANIO-CODE.hub.txt (danRer11). Code availability: The processing pipelines for the individual assays are available at https://gitlab.com/danio-code. The code used for the analysis is available at https://github.com/DANIO-CODE/DANIO-CODE_Data_analysis (https://doi.org/10.5281/zenodo.6424702). The script to generate the TrackHub is available at https://gitlab.com/danio-code/TrackHub, Zebrafish, a popular organism for studying embryonic development and for modeling human diseases, has so far lacked a systematic functional annotation program akin to those in other animal models. To address this, we formed the international DANIO-CODE consortium and created a central repository to store and process zebrafish developmental functional genomic data. Our data coordination center (https://danio-code.zfin.org) combines a total of 1,802 sets of unpublished and re-analyzed published genomic data, which we used to improve existing annotations and show its utility in experimental design. We identified over 140,000 cis-regulatory elements throughout development, including classes with distinct features dependent on their activity in time and space. We delineated the distinct distance topology and chromatin features between regulatory elements active during zygotic genome activation and those active during organogenesis. Finally, we matched regulatory elements and epigenomic landscapes between zebrafish and mouse and predicted functional relationships between them beyond sequence similarity, thus extending the utility of zebrafish developmental genomics to mammals, We thank our main funders, the Horizon 2020 MSCA-ITN project ZENCODE-ITN by the European Commission to F.M., B.L., C.O.D., J.M.V. and B.P. (GA no: 643062), BBSRC support (DanioPeaks, P61715) to B.L., F.M. and F.C.W., Wellcome Trust (Joint-Investigator award 106955/Z/15/Z) to F.M. and B.L. and AQUA-FAANG (Horizon 2020, GA 817923) to B.L., D.B. and F.M. and BBSRC (BB/R015457/1) to F.vE and Key Special Project for Introduced Talents Team to Z.C. (GML2019ZD0401) and PrecisionTox project by the European Commission (GA no: 965406), With the institutional support of the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S)

Significance In this study, we show that the inactivation of the gli3 gene in medaka fish results in the formation of larger dorsal and paired fins. These mutant fins display multiple radial bones and fin rays which resemble polydactyly in Gli3-deficient mice. Our molecular and genetic analyses indicate that the size of fish fins is controlled by an ancient mechanism mediated by SHH-GLI signaling that appeared prior to the evolutionary appearance of paired fins. We also show that the key regulatory networks that mediate the expansion of digit progenitor cells in tetrapods were already in place in the fins of the last common ancestor between ray and lobe-finned fishes, suggesting an ancient similarity between distal fins and digits., One of the central problems of vertebrate evolution is understanding the relationship among the distal portions of fins and limbs. Lacking comparable morphological markers of these regions in fish and tetrapods, these relationships have remained uncertain for the past century and a half. Here we show that Gli3 functions in controlling the proliferative expansion of distal progenitors are shared among dorsal and paired fins as well as tetrapod limbs. Mutant knockout gli3 fins in medaka (Oryzias latipes) form multiple radials and rays, in a pattern reminiscent of the polydactyly observed in Gli3-null mutant mice. In limbs, Gli3 controls both anterior–posterior patterning and cell proliferation, two processes that can be genetically uncoupled. In situ hybridization, quantification of proliferation markers, and analysis of regulatory regions reveal that in paired and dorsal fins, gli3 plays a main role in controlling proliferation but not in patterning. Moreover, gli3 down-regulation in shh mutant fins rescues fin loss in a manner similar to how Gli3 deficiency restores digits in the limbs of Shh mutant mouse embryos. We hypothesize that the Gli3/Shh gene pathway preceded the origin of paired appendages and was originally involved in modulating cell proliferation. Accordingly, the distal regions of dorsal fins, paired fins, and limbs retain a deep regulatory and functional homology that predates the origin of paired appendages.

Zebrafish, a popular model for embryonic development and for modelling human diseases, has so far lacked a systematic functional annotation programme akin to those in other animal models. To address this, we formed the international DANIO-CODE consortium and created the first central repository to store and process zebrafish developmental functional genomic data. Our Data Coordination Center (https://danio-code.zfin.org) combines a total of 1,802 sets of unpublished and reanalysed published genomics data, which we used to improve existing annotations and show its utility in experimental design. We identified over 140,000 cis-regulatory elements in development, including novel classes with distinct features dependent on their activity in time and space. We delineated the distinction between regulatory elements active during zygotic genome activation and those active during organogenesis, identifying new aspects of how they relate to each other. Finally, we matched regulatory elements and epigenomic landscapes between zebrafish and mouse and predict functional relationships between them beyond sequence similarity, extending the utility of zebrafish developmental genomics to mammals.

Sight depends on the tight cooperation between photoreceptors and pigmented cells, which derive from common progenitors through the bifurcation of a single gene regulatory network into the neural retina (NR) and retinal-pigmented epithelium (RPE) programs. Although genetic studies have identified upstream nodes controlling these networks, their regulatory logic remains poorly investigated. Here, we characterize transcriptome dynamics and chromatin accessibility in segregating NR/RPE populations in zebrafish. We analyze cis-regulatory modules and enriched transcription factor motives to show extensive network redundancy and context-dependent activity. We identify downstream targets, highlighting an early recruitment of desmosomal genes in the flattening RPE and revealing Tead factors as upstream regulators. We investigate the RPE specification network dynamics to uncover an unexpected sequence of transcription factors recruitment, which is conserved in humans. This systematic interrogation of the NR/RPE bifurcation should improve both genetic counseling for eye disorders and hiPSCs-to-RPE differentiation protocols for cell-replacement therapies in degenerative diseases., This work is supported by the following grants: (I) To J.-R.M.-M.: From the Spanish Ministry of Science, Innovation, and Universities (MICINN): BFU2017-86339P with FEDER funds, MDM-2016-0687 and PY20_00006/Junta de Andalucía. (II) To O.B. Australian Research Council (ARC) Discovery Project (DP190103852). (III) To F.-J.D.-C.: Andalusian Ministry of Health, Equality and Social Policies (PI-0099-2018). (IV) To P.B.: BFU2016-75412-R with FEDER funds; PCIN-2015-176-C02-01/ERA-Net Neuron ImprovVision, and a CBMSO Institutional grant from the Fundación Ramón Areces. (V) To both J.-R.M.-M. and P.B.: BFU2016-81887-REDT, as well as Fundación Ramón Areces-2016 (Supporting L.B.).

One of the central problems of vertebrate evolution is understanding the relationship among the distal portions of fins and limbs. Lacking comparable morphological markers of these regions in fish and tetrapods, these relationships have remained uncertain for the past century and a half. Here we show that Gli3 functions in controlling the proliferative expansion of distal progenitors are shared among dorsal and paired fins as well as tetrapod limbs. Mutant knockout gli3 fins in medaka (Oryzias latipes) form multiple radials and rays, in a pattern reminiscent of the polydactyly observed in Gli3-null mutant mice. In limbs, Gli3 controls both anterior¿posterior patterning and cell proliferation, two processes that can be genetically uncoupled. In situ hybridization, quantification of proliferation markers, and analysis of regulatory regions reveal that in paired and dorsal fins, gli3 plays a main role in controlling proliferation but not in patterning. Moreover, gli3 down-regulation in shh mutant fins rescues fin loss in a manner similar to how Gli3 deficiency restores digits in the limbs of Shh mutant mouse embryos. We hypothesize that the Gli3/Shh gene pathway preceded the origin of paired appendages and was originally involved in modulating cell proliferation. Accordingly, the distal regions of dorsal fins, paired fins, and limbs retain a deep regulatory and functional homology that predates the origin of paired appendages., This work would have been impossible without the contribution of Jose Luis Gómez-Skarmeta, a beloved friend and mentor whose passion for science was only surpassed by his generosity and kindness. We thank members of the J.L.G.-S. laboratory for fruitful discussions. J.L.G.-S. received funding from the European Research Council under the European Union’s Horizon 2020 research and innovation program (Grant Agreement 740041) and the Spanish Ministerio de Economıa y Competitividad (Grant BFU2016-74961-P) and was also supported, together with J.L.-R. and J.R.M.- M., by institutional grant Unidad de Excelencia Marıa de Maeztu (MDM-2016- 0687, to the Department of Gene Regulation and Morphogenesis of the Centro Andaluz de Biologıa del Desarrollo). J.L.-R. and J.R.M.-M. are supported by the Spanish Ministerio de Ciencia e Innovacion (Grants BFU2017-82974-P PID2020-113497GB-I00 and BFU2017-86339-P/PID2020-112566GB-I00, respectively). N.S. is supported by grants from the Brinson Foundation. J.L. is financed by the Chilean FONDECYT agency (Grant 11180727).

Sight depends on the tight cooperation between photoreceptors and pigmented cells, an ancestral tandem in metazoans. In vertebrates, both cell types derive from common progenitors in which a single gene regulatory network (GRN) bifurcates into the neural retina (NR) and retinal-pigmented epithelium (RPE) specification programs. Classical genetic studies have identified the main upstream nodes controlling these networks; however, their detailed architecture and cis-regulatory logic remain poorly investigated. Here, we have characterized transcriptome dynamics (RNA-seq) and chromatin accessibility (ATAC-seq) in segregating NR/RPE populations in zebrafish. Analysis of differentially active cis-regulatory modules and enriched transcription factor (TF) motives suggest extensive network redundancy and context-dependent TF activity. Downstream targets identification highlights an early recruitment of desmosomal genes in the flattening RPE cells, revealing Tead factors as their upstream regulators. Dynamic investigation of GRNs uncovers an unexpected sequence of TF recruitment during RPE specification, which is conserved in humans. This systematic interrogation of the NR/RPE bifurcating networks should improve both counselling of congenital eye disorders and hiPSCs-to-RPE differentiation protocols for cell-replacement therapies applicable to retinal degenerative diseases.

Summary The notochord is an evolutionary novelty in vertebrates that functions as an important signaling center during development. Notochord ablation in chicken has demonstrated that it is crucial for pancreas development; however, the molecular mechanism has not been fully described. Here, we show that in zebrafish, the loss of function of nog2, a Bmp antagonist expressed in the notochord, impairs β cell differentiation, compatible with the antagonistic role of Bmp in β cell differentiation. In addition, we show that nog2 expression in the notochord is induced by at least one notochord enhancer and its loss of function reduces the number of pancreatic progenitors and impairs β cell differentiation. Tracing Nog2 diffusion, we show that Nog2 emanates from the notochord to the pancreas progenitor domain. Finally, we find a notochord enhancer in human and mice Nog genomic landscapes, suggesting that the acquisition of a Nog notochord enhancer occurred early in the vertebrate phylogeny and contributes to the development of complex organs like the pancreas., Graphical Abstract, Highlights • nog2 loss of function leads to a reduced number of pancreatic progenitor and β cells • A tissue-specific enhancer controls the expression of nog2 in the notochord • The nog2 notochord enhancer is required for establishing a correctly sized pancreas • Regulation of notochord expression of Nog might be similar in many vertebrate lineages, Amorim et al. find that Nog2 is expressed in the zebrafish notochord by the action of a tissue-specific enhancer, and it diffuses to the pancreatic domain and controls its size. The identification of Nog enhancers in other vertebrate lineages suggests a conserved mechanism for pancreas development in vertebrates.

The Author(s) 2020., We investigated how the two rounds of whole-genome duplication that occurred at the base of the vertebrate lineage have impacted ancient microsyntenic associations involving developmental regulators (known as genomic regulatory blocks, GRBs). We showed that the majority of GRBs identified in the last common ancestor of chordates have been maintained as a single copy in humans. We found evidence that dismantling of the duplicated GRB copies occurred early in vertebrate evolution often through the differential retention of the regulatory gene but loss of the bystander gene’s exonic sequences. Despite the large evolutionary scale, the presence of duplicated highly conserved noncoding regions provided unambiguous proof for this scenario for multiple ancient GRBs. Remarkably, the dismantling of ancient GRB duplicates has contributed to the creation of large gene deserts associated with regulatory genes in vertebrates, providing a potentially widespread mechanism for the origin of these enigmatic genomic traits., The research has been funded by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (ERC-StG-LS2-637591 to M.I. and ERC-AdG-LS8-740041 to J.L.G.-S.), the Spanish Ministry of Science and Innovation (BFU2017-89201-P to M.I., RYC-2016-20089 and PGC2018-099392-A-I00 to I.M., BFU2016-74961-P to J.L.G.-S., and BFU2016-81887-REDT/AEI to J.L.G.-S. and M.I.), the People Programme (Marie Curie Actions) of the European Union’s Seventh Framework Programme (FP7/2007-2013) under REA grant agreement 608959, the “Centro de Excelencia Severo Ochoa 2013-2017” (SEV-2012-0208), and the “Unidad de Excelencia María de Maetzu 2017-2021” (MDM-2016-0687). We acknowledge the support of the CERCA Programme/Generalitat de Catalunya and of the Spanish Ministry of Economy, Industry and Competitiveness (MEIC) to the EMBL partnership.

[Background] : The organisation of vertebrate genomes into topologically associating domains (TADs) is believed to facilitate the regulation of the genes located within them. A remaining question is whether TAD organisation is achieved through the interactions of the regulatory elements within them or if these interactions are favoured by the pre-existence of TADs. If the latter is true, the fusion of two independent TADs should result in the rewiring of the transcriptional landscape and the generation of ectopic contacts., [Results]: We show that interactions within the PAX3 and FOXO1 domains are restricted to their respective TADs in normal conditions, while in a patient-derived alveolar rhabdomyosarcoma cell line, harbouring the diagnostic t(2;13)(q35;q14) translocation that brings together the PAX3 and FOXO1 genes, the PAX3 promoter interacts ectopically with FOXO1 sequences. Using a combination of 4C-seq datasets, we have modelled the three-dimensional organisation of the fused landscape in alveolar rhabdomyosarcoma., [Conclusions]: The chromosomal translocation that leads to alveolar rhabdomyosarcoma development generates a novel TAD that is likely to favour ectopic PAX3:FOXO1 oncogene activation in non-PAX3 territories. Rhabdomyosarcomas may therefore arise from cells which do not normally express PAX3. The borders of this novel TAD correspond to the original 5'- and 3'- borders of the PAX3 and FOXO1 TADs, respectively, suggesting that TAD organisation precedes the formation of regulatory long-range interactions. Our results demonstrate that, upon translocation, novel regulatory landscapes are formed allowing new intra-TAD interactions between the original loci involved., This work was supported by The Institute of Cancer Research and Cancer Research UK (Grant C1178/A4520). JJC was funded by grants from Spanish Ministerio de Ciencia e Innovación (BFU2011-22928) and the European Commission (PCIG10-GA-2011-303904). JLGS was funded by grants from Spanish Ministerio de Economía y Competitividad (BFU2013-41322-P) and the Andalusian Government (BIO-396). DPD and IIA were funded by grants from the Spanish Ministerio de Economia y Competitividad () and from the Junta de Andalucia (C2A). BV-B held a studentship from The Institute of Cancer Research., We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).

We investigated how the two rounds of whole genome duplication that occurred at the base of the vertebrate lineage have impacted ancient microsyntenic associations involving developmental regulators (known as genomic regulatory blocks, GRBs). We showed that the majority of GRBs present in the last common ancestor of chordates have been maintained as a single copy in humans. We found evidence that dismantling of the additional GRB copies occurred early in vertebrate evolution often through the differential retention of the regulatory gene but loss of the bystander gene’s exonic sequences. Despite the large evolutionary scale, the presence of duplicated highly conserved non-coding regions provided unambiguous proof for this scenario for dozens of ancient GRBs. Remarkably, the dismantling of ancient GRB duplicates has contributed to the creation of large gene deserts associated with regulatory genes in vertebrates, providing a widespread mechanism for the origin of these enigmatic genomic traits.

Vertebrates have greatly elaborated the basic chordate body plan and evolved highly distinctive genomes that have been sculpted by two whole-genome duplications. Here we sequence the genome of the Mediterranean amphioxus (Branchiostoma lanceolatum) and characterize DNA methylation, chromatin accessibility, histone modifications and transcriptomes across multiple developmental stages and adult tissues to investigate the evolution of the regulation of the chordate genome. Comparisons with vertebrates identify an intermediate stage in the evolution of differentially methylated enhancers, and a high conservation of gene expression and its cis-regulatory logic between amphioxus and vertebrates that occurs maximally at an earlier mid-embryonic phylotypic period. We analyse regulatory evolution after whole-genome duplications, and find that—in vertebrates—over 80% of broadly expressed gene families with multiple paralogues derived from whole-genome duplications have members that restricted their ancestral expression, and underwent specialization rather than subfunctionalization. Counter-intuitively, paralogues that restricted their expression increased the complexity of their regulatory landscapes. These data pave the way for a better understanding of the regulatory principles that underlie key vertebrate innovations., This research was funded primarily by the European Research Council (ERC) under the European Union’s Horizon 2020 and Seventh Framework Program FP7 research and innovation programs (ERC-AdG-LS8-740041 to J.L.G.-S., ERC-StG-LS2-637591 to M.I., a Marie Sklodowska-Curie Grant (658521) to I.M. and a FP7/2007-2013-ERC-268513 to P.W.H.H.), the Spanish Ministerio de Economía y Competitividad (BFU2016-74961-P to J.L.G.-S., RYC-2016-20089 to I.M., BFU2014-55076-P and BFU2017-89201-P to M.I. and BFU2014-55738-REDT to J.L.G.-S, M.I. and J.R.M.-M), the ‘Centro de Excelencia Severo Ochoa 2013-2017’(SEV-2012-0208), the ‘Unidad de Excelencia María de Maetzu 2017-2021’(MDM-2016-0687), the People Program (Marie Curie Actions) of the European Union’s Seventh Framework Program FP7 under REA grant agreement number 607142 (DevCom) to J.L.G.-S., and the CNRS and the ANR (ANR16-CE12-0008-01) to H.E. O.B. was supported by an Australian Research Council Discovery Early Career Researcher Award (DECRA; DE140101962).

Despite their evolutionary, developmental and functional importance, the origin of vertebrate paired appendages remains uncertain. In mice, a single enhancer termed ZRS is solely responsible for Shh expression in limbs. Here, zebrafish and mouse transgenic assays trace the functional equivalence of ZRS across the gnathostome phylogeny. CRISPR/Cas9-mediated deletion of the medaka (Oryzias latipes) ZRS and enhancer assays identify the existence of ZRS shadow enhancers in both teleost and human genomes. Deletion of both ZRS and shadow ZRS abolishes shh expression and completely truncates pectoral fin formation. Strikingly, deletion of ZRS results in an almost complete ablation of the dorsal fin. This finding indicates that a ZRS-Shh regulatory module is shared by paired and median fins and that paired fins likely emerged by the co-option of developmental programs established in the median fins of stem gnathostomes. Shh function was later reinforced in pectoral fin development with the recruitment of shadow enhancers, conferring additional robustness., This project received funding from the European Research Council under the European Union’s Horizon 2020 research and innovation programme (grant agreement No. 740041), the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement #658521, the Spanish Ministerio de Economía y Competitividad (grants BFU2016-74961-P, BFU2014-53765-P and BFU2016-81887-REDT), the Andalusian Government (grant BIO-396), CNPq Universal Program Grant 403248/2016-7 and CAPES/Alexander von Humboldt Foundation fellowship (to I.S.). J.L. was supported by Becas Chile.

9 páginas, 4 figuras.-- This article is distributed under the terms of the Creative Commons Attribution Noncommercial License.-- et al., POU3F4 encodes a POU-domain transcription factor required for inner ear development. Defects in POU3F4 function are associated with X-linked deafness type 3 (DFN3). Multiple deletions affecting up to ~900-kb upstream of POU3F4 are found in DFN3 patients, suggesting the presence of essential POU3F4 enhancers in this region. Recently, an inner ear enhancer was reported that is absent in most DFN3 patients with upstream deletions. However, two indications suggest that additional enhancers in the POU3F4 upstream region are required for POU3F4 function during inner ear development. First, there is at least one DFN3 deletion that does not eliminate the reported enhancer. Second, the expression pattern driven by this enhancer does not fully recapitulate Pou3f4 expression in the inner ear. Here, we screened a 1-Mb region upstream of the POU3F4 gene for additional cis-regulatory elements and searched for novel DFN3 mutations in the identified POU3F4 enhancers. We found several novel enhancers for otic vesicle expression. Some of these also drive expression in kidney, pancreas and brain, tissues that are known to express Pou3f4. In addition, we report a new and smallest deletion identified so far in a DFN3 family which eliminates 3.9 kb, comprising almost exclusively the previous reported inner ear enhancer. We suggest that multiple enhancers control the expression of Pou3f4 in the inner ear and these may contribute to the phenotype observed in DFN3 patients. In addition, the novel deletion demonstrates that the previous reported enhancer, although not sufficient, is essential for POU3F4 function during inner ear development., This work was supported by grants from the Spanish Ministry of Education and Science (BFU2007-60042/BMC, Petri PET2007_0158, CSD2007-00008) and Junta de Andalucía (Proyecto de Excelencia CVI-3488) to JLG-S, and the Oticon Foundation (Denmark) to MBP. CABD is institutionally supported by CSIC, Universidad Pablo de Olavide and Junta de Andalucía.

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.

The self-organized morphogenesis of the vertebrate optic cup entails coupling the activation of the retinal gene regulatory network to the constriction-driven infolding of the retinal epithelium. Yet the genetic mechanisms underlying this coordination remain largely unexplored. Through phylogenetic footprinting and transgenesis in zebrafish, here we examine the cis-regulatory landscape of opo, an endocytosis regulator essential for eye morphogenesis. Among the different conserved enhancers identified, we isolate a single retina-specific element (H6-10137) and show that its activity depends on binding sites for the retinal determinant Vsx2. Gain- and loss-of-function experiments and ChIP analyses reveal that Vsx2 regulates opo expression through direct binding to this retinal enhancer. Furthermore, we show that vsx2 knockdown impairs the primary optic cup folding. These data support a model by which vsx2, operating through the effector gene opo, acts as a central transcriptional node that coordinates neural retina patterning and optic cup invagination in zebrafish., We thank the Portuguese Foundation for Science and Technology (FCT), the support given through the doctoral grant SFRH/BD/60489/2009 to I.G.-R. Work in J.L.G.-S.’s laboratory was supported by BFU2013-41322-P and Proyecto de Excelencia BIO-396. This work was supported by grants CSIC-010CR0002, BFU2008-04362, BFU2011-22916 and P11-CVI-7256 to J.R.M.-M.

PMCID: PMC4113484.-- et al., Genome-wide association studies (GWAS) have reproducibly associated variants within introns of FTO with increased risk for obesity and type 2 diabetes (T2D). Although the molecular mechanisms linking these noncoding variants with obesity are not immediately obvious, subsequent studies in mice demonstrated that FTO expression levels influence body mass and composition phenotypes. However, no direct connection between the obesity-associated variants and FTO expression or function has been made. Here we show that the obesity-associated noncoding sequences within FTO are functionally connected, at megabase distances, with the homeobox gene IRX3. The obesity-associated FTO region directly interacts with the promoters of IRX3 as well as FTO in the human, mouse and zebrafish genomes. Furthermore, long-range enhancers within this region recapitulate aspects of IRX3 expression, suggesting that the obesity-associated interval belongs to the regulatory landscape of IRX3. Consistent with this, obesity-associated single nucleotide polymorphisms are associated with expression of IRX3, but not FTO, in human brains. A direct link between IRX3 expression and regulation of body mass and composition is demonstrated by a reduction in body weight of 25 to 30% in Irx3-deficient mice, primarily through the loss of fat mass and increase in basal metabolic rate with browning of white adipose tissue. Finally, hypothalamic expression of a dominant-negative form of Irx3 reproduces the metabolic phenotypes of Irx3-deficient mice. Our data suggest that IRX3 is a functional long-range target of obesity-associated variants within FTO and represents a novel determinant of body mass and composition., This work was funded by grants from the National Institutes of Health (DK093972, HL119967, HL114010 and DK020595) to M.A.N. and (MH101820, MH090937 and DK20595) to N.J.C. J.L.G.-S. was funded by grants from the Spanish Ministerio de Economía y Competitividad (BFU2010-14839, CSD2007-00008) and the Andalusian Government (CVI-3488). C.-C.H. was supported by a grant from the Canadian Institute of Health Research. K.-H.K. is supported by a fellowship from the Heart and Stroke Foundation of Canada. S.S. is supported by an NIH postdoctoral training grant (T32HL007381)

et al., In multicellular organisms, cis-regulation controls gene expression in space and time. Despite the essential implication of cisregulation in the development and evolution of organisms and in human diseases, our knowledge about regulatory sequences largely derives from analyzing their activity individually and outside their genomic context. Indeed, the contribution of these sequences to the expression of their target genes in their genomic context is still largely unknown. Here we present a novel genetic screen designed to visualize and interrupt gene regulatory landscapes in vertebrates. In this screen, based on the random insertion of an engineered Tol2 transposon carrying a strong insulator separating two fluorescent reporter genes, we isolated hundreds of zebrafish lines containing insertions that disrupt the cis-regulation of tissue-specific expressed genes. We therefore provide a new easy-to-handle tool that will help to disrupt and chart the regulatory activity spread through the vast noncoding regions of the vertebrate genome., This study was supported by the Spanish and Andalusian Governments (JLGS grant numbers BFU2010-14839, CSD2007-00008, Proyecto de Excelencia CVI-3488, and JJC grant number BFU2011-22928), an EFSD/Lilly grant, and a Universidad Pablo de Olavide grant (JB grant number PPI0906). A.A. is an FPI fellow and J.B. is a Juan de la Cierva postdoctoral fellow (JCI-2009-04014) of the Consejo Superior de Investigaciones Cientificas. J.B. was also an FCT postdoctoral fellow (SFRH/BPD/38829/2007; POPH/FSE). M.L. is a Junta de Andalucia fellow.

Common genetic variation at human 14q22.2 tagged by rs4444235 is significantly associated with colorectal cancer (CRC) risk. Re-sequencing was used to comprehensively annotate the 17kb region of strong linkage disequilibrium encompassing rs4444235. Through bioinformatic analyses using H3K4Me1, H3K4Me3, and DNase-I hypersensitivity chromatin signatures and evolutionary conservation we identified seven candidate disease-causing single-nucleotide polymorphisms mapping to six regions within the 17-kb region predicted to have regulatory potential. Reporter gene studies of these regions demonstrated that the element to which rs4444235 maps acts as an allele-specific transcriptional enhancer. Allele-specific expression studies in CRC cell lines heterozygous for rs4444235 showed significantly increased expression of bone morphogenetic protein-4 (BMP4) associated with the risk allele (P, We acknowledge the National Health Service (NHS) funding to the National Institute for Health Research (NIHR) Biomedical Research Centre. Finally, we are grateful to all patients and individuals for participation. Cancer Research UK (C1298/A8362 supported by the Bobby Moore Fund) provided principal funding for the study. JLG-S acknowledges grants from the Spanish Ministry of Education and Science (BFU2010-14839 and CSD2007-00008) and Junta de Andalucía (CVI-3488). SJL is in receipt of a PhD studentship from Cancer Research UK. The research leading to these results has received funding from the European Union Seventh Framework Programme (FP7/207-2013) under grant no. 258236, FP7 collaborative project SYSCOL.

11 páginas, 7 figuras.-- This is an open-access article distributed under the terms of the Creative Commons Attribution License., POU3F4 is a member of the POU-homedomain transcription factor family with a prominent role in inner ear development. Mutations in the human POU3F4 coding unit leads to X-linked deafness type 3 (DFN3), characterized by conductive hearing loss and progressive sensorineural deafness. Microdeletions found 1 Mb 5′ upstream of the coding region also displayed the same phenotype, suggesting that cis-regulatory elements might be present in that region. Indeed, we and others have recently identified several enhancers at the 1 Mb 5′ upstream interval of the pou3f4 locus. Here we characterize the spatio-temporal patterns of these regulatory elements in zebrafish transgenic lines. We show that the most distal enhancer (HCNR 81675) is activated earlier and drives GFP reporter expression initially to a broad ear domain to progressively restrict to the sensory patches. The proximal enhancer (HCNR 82478) is switched later during development and promotes expression, among in other tissues, in sensory patches from its onset. The third enhancer (HCNR 81728) is also active at later stages in the otic mesenchyme and in the otic epithelium. We also characterize the signaling pathways regulating these enhancers. While HCNR 81675 is regulated by very early signals of retinoic acid, HCNR 82478 is regulated by Fgf activity at a later stage and the HCNR 81728 enhancer is under the control of Hh signaling. Finally, we show that Sox2 and Pax2 transcription factors are bound to HCNR 81675 genomic region during otic development and specific mutations to these transcription factor binding sites abrogates HCNR 81675 enhancer activity. Altogether, our results suggest that pou3f4 expression in inner ear might be under the control of distinct regulatory elements that fine-tune the spatio-temporal activity of this gene and provides novel data on the signaling mechanisms controlling pou3f4 function., This work has been supported by grants from MICINN to BA (BFU 2008-00714) and JLG-S (BFU2007-60042/BMC, Petri PET2007_0158, CSD2007-00008), a grant from the Junta de Andalucía to JLG-S (Proyecto de Excelencia CVI-3488) and a Juan de la Cierva postdoctoral fellowship to ARM.

The identification and characterization of the regulatory activity of genomic sequences is crucial for understanding how the information contained in genomes is translated into cellular function. The cisregulatory sequences control when, where, and how much genes are transcribed and can activate (enhancers) or repress (silencers) gene expression. Here, we describe a novel Tol2 transposon-based vector for assessing enhancer activity in the zebrafish (Danio rerio). This Zebrafish Enhancer Detector (ZED) vector harbors several key improvements, among them a sensitive and specific minimal promoter chosen for optimal enhancer activity detection, insulator sequences to shield the minimal promoter from position effects, and a positive control for transgenesis. Additionally, we demonstrate that highly conserved noncoding sequences homologous between humans and zebrafish largely with enhancer activity largely retain their tissue-specific enhancer activity during vertebrate evolution. More strikingly, insulator sequences from mouse and chicken, but not conserved in zebrafish, maintain their insulator capacity when tested in this model., This work was supported by grants from the Spanish Ministry of Education and Science (BFU2007-60042/BMC, Petri PET2007_0158, CSD2007-00008) and Junta de Andalucía (Proyecto de Excelencia CVI-3488) to J.L.G.-S. C.A.B.D. is institutionally supported by CSIC, Universidad Pablo de Olavide and Junta de Andalucía.

to determine the relationship between the abdominal obesity and cardiovascular risk factors in apparently healthy subjects from Mexico City.a total of 186 apparently healthy men and nonpregnant women from Mexico City, were enrolled in a cross-sectional study. A detailed medical history and physical examination were performed. Abdominal obesity was defined by waist circumferenceor = 80 cm for women andor = 90 cm for men.a total of 125 women (67.2 %) and 61 men (32.8 %) were enrolled. Of them, 151 (81.2 %) had insulin resistance and 130 (69.9 %) abdominal obesity. Among obese subjects 96 (46.2 %) showed metabolic syndrome. There were a high prevalence of hypertriglyceridemia (31 %) and low serum levels of HDL-cholesterol (58 %).the used cut point for abdominal obesity, despite identifying a high proportion of subjects with cardiovascular risk, did not recognize a high proportion of subjects with disorders in their lipid profile.

7 páginas, 4 figuras, 1 tabla.-- et al., Recent genome-wide scans for colorectal cancer (CRC) have revealed the SMAD7 (mothers against decapentaplegic homolog 7) gene as a locus associated with a modest, but highly significant increase in CRC risk. To identify the causal basis of the association between 18q21 variation and CRC, we resequenced the 17-kb region of linkage disequilibrium and evaluated all variants in 2532 CRC cases and 2607 controls. A novel C to G single nucleotide polymorphism (SNP) at 44,703,563 bp was maximally associated with CRC risk (P = 5.98 × 10−7; ≥1.5-fold more likely to be causal than other variants). Using transgenic assays in Xenopus laevis as a functional model, we demonstrate that the G risk allele leads to reduced reporter gene expression in the colorectum (P = 5.4 × 10−3). Electrophoretic mobility shift assays provided evidence for the role of Novel 1 in transcription factor binding. We propose that the novel SNP we have identified is the functional change leading to CRC predisposition through differential SMAD7 expression and, hence, aberrant TGF-beta signaling., Cancer Research UK (C1298/A8362 supported by the Bobby Moore Fund) provided principal funding for the study. Additional funding was provided by the European Union (CPRB LSHC-CT-2004-503465). J.L.G.-S. and F.C. acknowledge grants from the Spanish Ministry of Education and Science (BFU2007-60042/BMC, BFU2006-00349/BMC, CSD2007-00008) and Junta de Andalucía (CVI 00260 and CVI 2658). H.M. and T.v.W. acknowledge support by Dutch Cancer Society grant UL2003-2807.

9 páginas, 5 figuras.-- This is an open-access article distributed under the terms of the Creative Commons Attribution License.-- et al., Common genetic variation at human 8q23.3 is significantly associated with colorectal cancer (CRC) risk. To elucidate the basis of this association we compared the frequency of common variants at 8q23.3 in 1,964 CRC cases and 2,081 healthy controls. Reporter gene studies showed that the single nucleotide polymorphism rs16888589 acts as an allele-specific transcriptional repressor. Chromosome conformation capture (3C) analysis demonstrated that the genomic region harboring rs16888589 interacts with the promoter of gene for eukaryotic translation initiation factor 3, subunit H (EIF3H). We show that increased expression of EIF3H gene increases CRC growth and invasiveness thereby providing a biological mechanism for the 8q23.3 association. These data provide evidence for a functional basis for the non-coding risk variant rs16888589 at 8q23.3 and provides novel insight into the etiological basis of CRC., Cancer Research UK (C1298/A8362 supported by the Bobby Moore Fund), provided principal funding for the study. Additional funding was provided by the European Union (CPRB LSHC-CT-2004-503465). JLG-S acknowledges grants from the Spanish Ministry of Education and Science (BFU2007-60042/BMC, Petri PET2007-0158 and CSD2007-00008) and Junta de Andalucí­a (CVI-3488). MM acknowledges support from the ProCNIC Foundation. HM and TVW acknowledge support by Dutch Cancer Society grant UL2003-2807. MM acknowledges support from the Spanish Government (BFU2008-00838, CSD2007-00008) and the ProCNIC Foundation. Work in Finland was supported by grants from the Academy of Finland (Finnish Center of Excellence Program 2006-2011), the Finnish Cancer Society, the Sigrid Juselius Foundation, the European Commission (LSHG-CT-2004-512142), Ida Montin Foundation (ST), and Biomedicum Helsinki Foundation (ST).