Comparative genomics of the Hedgehog loci in chordates and the origins of Shh regulatory novelties

The origin and evolution of the complex regulatory landscapes of some vertebrate developmental genes, often spanning hundreds of Kbp and including neighboring genes, remain poorly understood. The Sonic Hedgehog (Shh) genomic regulatory block (GRB) is one of the best functionally characterized examples, with several discrete enhancers reported within its introns, vast upstream gene-free region and neighboring genes (Lmbr1 and Rnf32). To investigate the origin and evolution of this GRB, we sequenced and characterized the Hedgehog (Hh) loci from three invertebrate chordate amphioxus species, which share several early expression domains with Shh. Using phylogenetic footprinting within and between chordate lineages, and reporter assays in zebrafish probing >30 Kbp of amphioxus Hh, we report large sequence and functional divergence between both groups. In addition, we show that the linkage of Shh to Lmbr1 and Rnf32, necessary for the unique gnatostomate-specific Shh limb expression, is a vertebrate novelty occurred between the two whole-genome duplications

Metazoan evolution of glutamate receptors reveals unreported phylogenetic groups and divergent lineage-specific events

Glutamate receptors are divided in two unrelated families: ionotropic (iGluR), driving synaptic transmission, and metabotropic (mGluR), which modulate synaptic strength. The present classification of GluRs is based on vertebrate proteins and has remained unchanged for over two decades. Here we report an exhaustive phylogenetic study of GluRs in metazoans. Importantly, we demonstrate that GluRs have followed different evolutionary histories in separated animal lineages. Our analysis reveals that the present organization of iGluRs into six classes does not capture the full complexity of their evolution. Instead, we propose an organization into four subfamilies and ten classes, four of which have never been previously described. Furthermore, we report a sister class to mGluR classes I-III, class IV. We show that many unreported proteins are expressed in the nervous system, and that new Epsilon receptors form functional ligand-gated ion channels. We propose an updated classification of glutamate receptors that includes our findings

The round goby genome provides insights into mechanisms that may facilitate biological invasions

Background: The invasive benthic round goby (Neogobius melanostomus) is the most successful temperate invasive fish and has spread in aquatic ecosystems on both sides of the Atlantic. Invasive species constitute powerful in situ experimental systems to study fast adaptation and directional selection on short ecological timescales and present promising case studies to understand factors involved the impressive ability of some species to colonize novel environments. We seize the unique opportunity presented by the round goby invasion to study genomic substrates potentially involved in colonization success. Results We report a highly contiguous long-read-based genome and analyze gene families that we hypothesize to relate to the ability of these fish to deal with novel environments. The analyses provide novel insights from the large evolutionary scale to the small species-specific scale. We describe expansions in specific cytochrome P450 enzymes, a remarkably diverse innate immune system, an ancient duplication in red light vision accompanied by red skin fluorescence, evolutionary patterns of epigenetic regulators, and the presence of osmoregulatory genes that may have contributed to the round goby's capacity to invade cold and salty waters. A recurring theme across all analyzed gene families is gene expansions. Conclusions: The expanded innate immune system of round goby may potentially contribute to its ability to colonize novel areas. Since other gene families also feature copy number expansions in the round goby, and since other Gobiidae also feature fascinating environmental adaptations and are excellent colonizers, further long-read genome approaches across the goby family may reveal whether gene copy number expansions are more generally related to the ability to conquer new habitats in Gobiidae or in fish

Amphioxus functional genomics and the origins of vertebrate gene regulation.

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

Behavioural and functional evidence revealing the role of RBFOX1 variation in multiple psychiatric disorders and traits

Common variation in the gene encoding the neuron-specific RNA splicing factor RNA Binding Fox-1 Homolog 1 (RBFOX1) has been identified as a risk factor for several psychiatric conditions, and rare genetic variants have been found causal for autism spectrum disorder (ASD). Here, we explored the genetic landscape of RBFOX1 more deeply, integrating evidence from existing and new human studies as well as studies in Rbfox1 knockout mice. Mining existing data from large-scale studies of human common genetic variants, we confirmed gene-based and genome-wide association of RBFOX1 with risk tolerance, major depressive disorder and schizophrenia. Data on six mental disorders revealed copy number losses and gains to be more frequent in ASD cases than in controls. Consistently, RBFOX1 expression appeared decreased in post-mortem frontal and temporal cortices of individuals with ASD and prefrontal cortex of individuals with schizophrenia. Brain-functional MRI studies demonstrated that carriers of a common RBFOX1 variant, rs6500744, displayed increased neural reactivity to emotional stimuli, reduced prefrontal processing during cognitive control, and enhanced fear expression after fear conditioning, going along with increased avoidance behaviour. Investigating Rbfox1 neuron-specific knockout mice allowed us to further specify the role of this gene in behaviour. The model was characterised by pronounced hyperactivity, stereotyped behaviour, impairments in fear acquisition and extinction, reduced social interest, and lack of aggression; it provides excellent construct and face validity as an animal model of ASD. In conclusion, convergent translational evidence shows that common variants in RBFOX1 are associated with a broad spectrum of psychiatric traits and disorders, while rare genetic variation seems to expose to early-onset neurodevelopmental psychiatric disorders with and without developmental delay like ASD, in particular. Studying the pleiotropic nature of RBFOX1 can profoundly enhance our understanding of mental disorder vulnerability

Evolutionary recruitment and assembly of embryonic alternative splicing programs

[eng] In the present work, we studied the evolution of alternative splicing (AS) from different perspectives. First, we performed a preliminary analysis where we identified alternative exons with tissue-specific regulation in five species of deuterostomes: Homo sapiens, Danio rerio, Callorhinchus milii, Branchiostoma lanceolatum and Strongylocentrotus purpuratus. Seven different organs from each animal were employed, trying to use homologous tissues when possible (especially among vertebrates). Results revealed a higher relative proportion of organ-biased AS regulation in the nervous system of chordates, with respect to the rest of organs. From the other hand, sea urchin species showed more balanced amounts of organ-specific AS regulation. Moreover, human samples showed a tissue-specific trend towards increased levels of differential exon skipping in general. In this thesis, we also investigated the expression and function of RbFox and Nova genes in non-vertebrate deuterostome organisms to study the evolutionary scenario of both families. The two gene families code for RNA-binding proteins that regulate wide sets of alternative splicing events in vertebrate organisms. We found Nova function to be related to gastrulation movements during embryogenesis, but not endoderm specification. In all adult Bilateria organisms studied, RbFox genes are strongly expressed in the nervous system. But, strikingly, non-vertebrate deuterostomes present developmental expression in mesoderm tissues, especially in the myogenic lineage. Interestingly, RbFox activity has been co-opted in the central nervous system of bony vertebrates, as well as in the skeletogenic mesoderm of S. purpuratus. In this last species, RbFox is necessary for both the formation of circumesophageal muscles and the development of the larva skeleton. At the splicing level, Nova ortholog in sea urchin regulates splicing of a very vast set of exons, while the number of RbFox-dependent exons is much lower. Among the targets of the latter, we detected the Fgfr1 gene, that have been previously described as necessary in terms of muscle differentiation in this organism. Finally, we studied Esrp gene family, which codes also for a splicing factor. We investigated the expression and function of this gene family in several species of deuterostomes. In zebrafish, Esrp1 and Esrp2, are involved in the organogenesis of multiple structures, arguably by controlling epithelial-mesenchymal interactions. In Ciona intestinalis, Esrp ectopic expression is able to modulate the motility of mesenchymal migratory cells. In amphioxus, Esrp is expressed in the precursor of the epidermic sensory neurons at the time they ingress in the dorsal ectoderm. In S. purpuratus, Esrp is found in both aboral ectoderm and in the pigment cells during gastrulation. In fact, the gene is needed for a complete integration of those cells in the non-neural ectoderm. We detected multiple regulated exons that are shared among bony vertebrate organisms in terms of Esrp-regulated splicing, and we even found a case conserved also in amphioxus in Fgfr genes. However, no regulated exons have been detected as conserved between vertebrates and sea urchin, although several common targets are found at the gene level.[spa] En la presente tesis se ha estudiado la evolución del splicing alternativo (AS) desde diferentes perspectivas. Primeramente, se ha hecho un análisis preliminar en el que se han identificado exones alternativos con regulación específica de tejido, en cinco especies de deuteróstomos: Homo sapiens, Danio rerio, Callorhinchus milii, Branchiostoma lanceolatum y Strongylocentrotus purpuratus. Se emplearon siete órganos diferentes para cada animal, intentando utilizar muestras de órganos homólogos cuando era posible (especialmente entre vertebrados). El resultado reveló una proporción relativa mayor de splicing alternativo específico de tejido en el sistema nervioso de los cordados, respecto al resto de órganos. En erizo de mar, en cambio, se observaron valores más parecidos de AS entre los diferentes tejidos. La especie humana en particular mostró una tendencia mucho mayor a un exclusión diferencial de exones en general. Por otro lado, también se ha estudiado la expresión y/o función de los genes RbFox y Nova en organismos deuteróstomos no vertebrados para estudiar el escenario macroevolutivo de dichas familias. Ambos genes codifican para proteínas de unión a RNA que regulan conjuntos amplios de eventos de AS en organismos vertebrados. Encontramos que la función de Nova en erizo de mar está relacionada con los movimientos de gastrulación embrionaria. En el caso de RbFox, la expresión y función descrita en organismos adultos parece diferir de la que sucede durante el desarrollo embrionario. En todos los adultos Bilaterales estudiados, los genes RbFox se expresan en el sistema nervioso central mayoritariamente, mientras que los deuteróstomos no vertebrados, la expresión se observa en mesodermo embrionario mayoritariamente. Es interesante destacar que la expresión de RbFox en embriones ha sido co-optada tanto en el sistema nervioso de vertebrados durante el desarrollo, así como en el mesodermo esqueletogénico del erizo de mar. En esta última especie, RbFox es necesario tanto para la formación de los músculos circumesofágicos como para la formación del esqueleto larvario. A nivel de splicing, el ortólogo de Nova en S. purpuratus regula el splicing de un conjunto muy grande de exones, mientras que el regulado por RbFox es más pequeño. Entre los targets de este último, se encuentra el gen Fgfr1, que ha sido descrito como necesario para la diferenciación muscular en esta especie. Por último, se estudió la família génica Esrp, que también codifica para un factor de splicing. En el presente trabajo, se ha estudiado la expresión y función de dicha familia génica en diversas especies de deuteróstomos. En zebrafish, Esrp1 y Esrp2 están implicados en la organogénesis de múltiples estructuras. En Ciona intestinalis, Esrp modula la motilidad de las células mesenquimales migratorias. En anfioxo, Esrp se expresa en las precursoras de las neuronas epidérmicas sensoriales en el momento de introducirse en el ectodermo dorsal. En S. purpuratus, Esrp se encuentra tanto en el ectodermo aboral como en las células pigmentarias. De hecho, es necesario para una integración completa de estas últimas en el ectodermo no neural. A nivel de programa de splicing regulado, hemos detectado diversos exones regulados comunes entre vertebrados, e incluso uno con anfioxo en los genes Fgfr. Sin embargo, no se han detectado exones regulados conservados entre vertebrados y erizo de mar, aunque sí hay dianas comunes a nivel de gen

Evolutionary recruitment and assembly of embryonic alternative splicing programs: insights form the Deuterostomia lineage

In the present work, we studied the evolution of alternative splicing (AS) from different perspectives. First, we performed a preliminary analysis where we identified alternative exons with tissue-specific regulation in five species of deuterostomes: Homo sapiens, Danio rerio, Callorhinchus milii, Branchiostoma lanceolatum and Strongylocentrotus purpuratus. Seven different organs from each animal were employed, trying to use homologous tissues when possible (especially among vertebrates). Results revealed a higher relative proportion of organ-biased AS regulation in the nervous system of chordates, with respect to the rest of organs. From the other hand, sea urchin species showed more balanced amounts of organ-specific AS regulation. Moreover, human samples showed a tissue-specific trend towards increased levels of differential exon skipping in general. In this thesis, we also investigated the expression and function of RbFox and Nova genes in non-vertebrate deuterostome organisms to study the evolutionary scenario of both families. The two gene families code for RNA-binding proteins that regulate wide sets of alternative splicing events in vertebrate organisms. We found Nova function to be related to gastrulation movements during embryogenesis, but not endoderm specification. In all adult Bilateria organisms studied, RbFox genes are strongly expressed in the nervous system. But, strikingly, non-vertebrate deuterostomes present developmental expression in mesoderm tissues, especially in the myogenic lineage. Interestingly, RbFox activity has been co-opted in the central nervous system of bony vertebrates, as well as in the skeletogenic mesoderm of S. purpuratus. In this last species, RbFox is necessary for both the formation of circumesophageal muscles and the development of the larva skeleton. At the splicing level, Nova ortholog in sea urchin regulates splicing of a very vast set of exons, while the number of RbFox-dependent exons is much lower. Among the targets of the latter, we detected the Fgfr1 gene, that have been previously described as necessary in terms of muscle differentiation in this organism. Finally, we studied Esrp gene family, which codes also for a splicing factor. We investigated the expression and function of this gene family in several species of deuterostomes. In zebrafish, Esrp1 and Esrp2, are involved in the organogenesis of multiple structures, arguably by controlling epithelial-mesenchymal interactions. In Ciona intestinalis, Esrp ectopic expression is able to modulate the motility of mesenchymal migratory cells. In amphioxus, Esrp is expressed in the precursor of the epidermic sensory neurons at the time they ingress in the dorsal ectoderm. In S. purpuratus, Esrp is found in both aboral ectoderm and in the pigment cells during gastrulation. In fact, the gene is needed for a complete integration of those cells in the non-neural ectoderm. We detected multiple regulated exons that are shared among bony vertebrate organisms in terms of Esrp-regulated splicing, and we even found a case conserved also in amphioxus in Fgfr genes. However, no regulated exons have been detected as conserved between vertebrates and sea urchin, although several common targets are found at the gene level.En la presente tesis se ha estudiado la evolución del splicing alternativo (AS) desde diferentes perspectivas. Primeramente, se ha hecho un análisis preliminar en el que se han identificado exones alternativos con regulación específica de tejido, en cinco especies de deuteróstomos: Homo sapiens, Danio rerio, Callorhinchus milii, Branchiostoma lanceolatum y Strongylocentrotus purpuratus. Se emplearon siete órganos diferentes para cada animal, intentando utilizar muestras de órganos homólogos cuando era posible (especialmente entre vertebrados). El resultado reveló una proporción relativa mayor de splicing alternativo específico de tejido en el sistema nervioso de los cordados, respecto al resto de órganos. En erizo de mar, en cambio, se observaron valores más parecidos de AS entre los diferentes tejidos. La especie humana en particular mostró una tendencia mucho mayor a un exclusión diferencial de exones en general. Por otro lado, también se ha estudiado la expresión y/o función de los genes RbFox y Nova en organismos deuteróstomos no vertebrados para estudiar el escenario macroevolutivo de dichas familias. Ambos genes codifican para proteínas de unión a RNA que regulan conjuntos amplios de eventos de AS en organismos vertebrados. Encontramos que la función de Nova en erizo de mar está relacionada con los movimientos de gastrulación embrionaria. En el caso de RbFox, la expresión y función descrita en organismos adultos parece diferir de la que sucede durante el desarrollo embrionario. En todos los adultos Bilaterales estudiados, los genes RbFox se expresan en el sistema nervioso central mayoritariamente, mientras que los deuteróstomos no vertebrados, la expresión se observa en mesodermo embrionario mayoritariamente. Es interesante destacar que la expresión de RbFox en embriones ha sido co-optada tanto en el sistema nervioso de vertebrados durante el desarrollo, así como en el mesodermo esqueletogénico del erizo de mar. En esta última especie, RbFox es necesario tanto para la formación de los músculos circumesofágicos como para la formación del esqueleto larvario. A nivel de splicing, el ortólogo de Nova en S. purpuratus regula el splicing de un conjunto muy grande de exones, mientras que el regulado por RbFox es más pequeño. Entre los targets de este último, se encuentra el gen Fgfr1, que ha sido descrito como necesario para la diferenciación muscular en esta especie. Por último, se estudió la família génica Esrp, que también codifica para un factor de splicing. En el presente trabajo, se ha estudiado la expresión y función de dicha familia génica en diversas especies de deuteróstomos. En zebrafish, Esrp1 y Esrp2 están implicados en la organogénesis de múltiples estructuras. En Ciona intestinalis, Esrp modula la motilidad de las células mesenquimales migratorias. En anfioxo, Esrp se expresa en las precursoras de las neuronas epidérmicas sensoriales en el momento de introducirse en el ectodermo dorsal. En S. purpuratus, Esrp se encuentra tanto en el ectodermo aboral como en las células pigmentarias. De hecho, es necesario para una integración completa de estas últimas en el ectodermo no neural. A nivel de programa de splicing regulado, hemos detectado diversos exones regulados comunes entre vertebrados, e incluso uno con anfioxo en los genes Fgfr. Sin embargo, no se han detectado exones regulados conservados entre vertebrados y erizo de mar, aunque sí hay dianas comunes a nivel de gen

CERKL Knockdown Causes Retinal Degeneration in Zebrafish

The human CERKL gene is responsible for common and severe forms of retinal dystrophies. Despite intense in vitro studies at the molecular and cellular level and in vivo analyses of the retina of murine knockout models, CERKL function remains unknown. In this study, we aimed to approach the developmental and functional features of cerkl in Danio rerio within an Evo-Devo framework. We show that gene expression increases from early developmental stages until the formation of the retina in the optic cup. Unlike the high mRNA-CERKL isoform multiplicity shown in mammals, the moderate transcriptional complexity in fish facilitates phenotypic studies derived from gene silencing. Moreover, of relevance to pathogenicity, teleost CERKL shares the two main human protein isoforms. Morpholino injection has been used to generate a cerkl knockdown zebrafish model. The morphant phenotype results in abnormal eye development with lamination defects, failure to develop photoreceptor outer segments, increased apoptosis of retinal cells and small eyes. Our data support that zebrafish Cerkl does not interfere with proliferation and neural differentiation during early developmental stages but is relevant for survival and protection of the retinal tissue. Overall, we propose that this zebrafish model is a powerful tool to unveil CERKL contribution to human retinal degeneratio

The birth of a new gene cluster in mammalian e volution: exciting origin, intriguing shared regulation, (un)known functions, and implications in human disease

Resumen del póster presentado al Joint Congress of the Spanish Societies of Genetics, Cell Biology and Developmental Biology, celebrado en Gijón del 24 al 27 de octubre de 2017.-- et al.In recent years it is becoming clear that genome organisation and architecture made an insightful contribution to gene regulation during embryonic development, being in some cases responsible for the maintenance of gene clusters, through genes being linked by sharing of regulatory sequences, or by using global cluster regulatory landscapes. These genome architectures and its sudden changes may well be linked to morphological evolution, by changing networks of developmental regulatory genes. Among those phenomena, the sudden birth of new gene clusters has been scarcely reported. We wish here to introduce our latest research in these fields; the BGW gene cluster (name to be changed). The cluster suddenly appeared at the origin of Eutherian Mammals, and the initial genes we analysed, ArmcX 1-6, play a key role in mitochondrial dynamics in neurons. Surprisingly, the cluster encompasses not six, but nearly 20 genes poorly analysed. We will show here the true origin of this cluster, Also, we will analyse its maintenance in most eutherian mammals, explore conserved regulatory motifs shared by these genes, and show that they are expressed during mouse embryonic development, namely but not solely in the central nervous system. Finally, as some of the cluster genes are involved in the control of proliferation, apoptosis and neuronal differentiation, we will hypothesise that the origin of the cluster was correlated to the increase in complexity of the central nervous system of Eutherian mammals. CRISP-R transgenic mice of one of the genes excitingly suggest it may be implicated in human autism.Peer Reviewe

Origins and regulation of an eutherian novelty: the BGW cluster

Resumen del póster presentado al 11th Meeting of the Spanish Society for Developmental Biology, celebrado en Girona (España) del 19 al 21 de octubre de 2016.-- et al.Two related gene subfamilies known as BEX and TCEAL (also known as WEX) map to a genomic region specific to Eutheria (placental mammals), located on the X chromosome. These families are part of a gene cluster, named BGW cluster, together with the ARMCX family and HNRNPH2. Some of the BEX/TCEAL genes have been related to control the balance between proliferation and differentiation, while others promote apoptosis in a p75-dependent manner, but most of them remain poorly studied. The ARMCX family and HNRNPH2 are derived from retrocopies of the ARMC10 and HNRNPH1 genes respectively conserved across bilateria, and located in autosomal chromosomes?, whereas no orthologs have been found for the BEX/TCEAL family outside of Eutheria. However, all these genes share an intriguing feature: a sequence motif in their proximal promoter region that appears to be crucial for their expression, the BGW motif. To further understand the evolution of this gene cluster, we investigated the origin of the BEX/TCEAL genes and traced it to an atypical formation in the ancestor of eutherians. Furthermore, novel features associated with BEX/TCEAL suggest a more complete scenario for the origin of the cluster: the BGW motif was already present at the HNRNPH2 locus in the ancestor of therian mammals, being subsequently duplicated and coopted in the eutherian lineage by the BEX/TCEAL ancestor and, posteriorly, by the ARMCX ancestral gene. Finally, we also studied the expression of the BEX/TCEAL genes during mouse development using in situ hybridization. We found that they are highly expressed in the brain and placenta, which are structures that require a well-tuned control of cell cycle during their development in eutherian mammals. Here we propose a scenario for the origin of the BEX/TCEAL family and for the formation of the BGW cluster where they belong. Their uncommon origin, their pattern of expression, and their putative biological function during development makes these genes an interesting subject of study to understand how lineage-specific genes could contribute to mammalian evolution.Peer reviewe