RESUMO
Sea urchins are emblematic models in developmental biology and display several characteristics that set them apart from other deuterostomes. To uncover the genomic cues that may underlie these specificities, we generated a chromosome-scale genome assembly for the sea urchin Paracentrotus lividus and an extensive gene expression and epigenetic profiles of its embryonic development. We found that, unlike vertebrates, sea urchins retained ancestral chromosomal linkages but underwent very fast intrachromosomal gene order mixing. We identified a burst of gene duplication in the echinoid lineage and showed that some of these expanded genes have been recruited in novel structures (water vascular system, Aristotle's lantern, and skeletogenic micromere lineage). Finally, we identified gene-regulatory modules conserved between sea urchins and chordates. Our results suggest that gene-regulatory networks controlling development can be conserved despite extensive gene order rearrangement.
RESUMO
With the completion of the genome sequencing projects, a new challenge for developmental biologists is to assign a function to the thousands of genes identified. Expression of exogenous mRNAs is a powerful, versatile and rapid technique that can be used to study gene function during development of the sea urchin. This chapter describes how this technique can be used to analyze gene function in echinoderm embryos, how it can be combined with cell transplantation to perform mosaic analysis and how it can be applied to identify downstream targets genes of transcription factors and signaling pathways. We describe specific examples of the use of overexpression of mRNA to analyze gene function, mention the benefits and current limitations of the technique and emphasize the importance of using different controls to assess the specificity of the effects observed. Finally, this chapter details the different steps, vectors and protocols for in vitro production of mRNA and phenotypic analysis.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento/genética , Genômica/métodos , RNA Mensageiro/genética , Ouriços-do-Mar/genética , Animais , Embrião não Mamífero , Redes Reguladoras de Genes/genética , Ouriços-do-Mar/crescimento & desenvolvimento , Transdução de Sinais/genética , Fatores de Transcrição/genéticaRESUMO
In sea urchins, the nuclear accumulation of ß-catenin in micromeres and macromeres at 4th and 5th cleavage activates the developmental gene regulatory circuits that specify all of the vegetal tissues (i.e. skeletogenic mesoderm, endoderm and non-skeletogenic mesoderm). Here, through the analysis of maternal Frizzled receptors as potential contributors to these processes, we found that, in Paracentrotus lividus, the receptor Frizzled1/2/7 is required by 5th cleavage for ß-catenin nuclearisation selectively in macromere daughter cells. Perturbation analyses established further that Frizzled1/2/7 signaling is required subsequently for the specification of the endomesoderm and then the endoderm but not for that of the non-skeletogenic mesoderm, even though this cell type also originates from the endomesoderm lineage. Complementary analyses on Wnt6 showed that this maternal ligand is similarly required at 5th cleavage for the nuclear accumulation of ß-catenin exclusively in the macromeres and for endoderm but not for non-skeletogenic mesoderm specification. In addition, Wnt6 misexpression reverses Frizzled1/2/7 downregulation-induced phenotypes. Thus, the results indicate that Wnt6 and Frizzled1/2/7 are likely to behave as the ligand-receptor pair responsible for initiating ß-catenin nuclearisation in macromeres at 5th cleavage and that event is necessary for endoderm specification. They show also that ß-catenin nuclearisation in micromeres and macromeres takes place through a different mechanism, and that non-skeletogenic mesoderm specification occurs independently of the nuclear accumulation of ß-catenin in macromeres at the 5th cleavage. Evolutionarily, this analysis outlines further the conserved involvement of the Frizzled1/2/7 subfamily, but not of specific Wnts, in the activation of canonical Wnt signaling during early animal development.
Assuntos
Desenvolvimento Embrionário/fisiologia , Endoderma/fisiologia , Receptores Frizzled/metabolismo , Paracentrotus/citologia , Paracentrotus/embriologia , Transdução de Sinais/fisiologia , beta Catenina/metabolismo , Animais , Embrião não Mamífero/citologia , Embrião não Mamífero/fisiologia , Endoderma/citologia , Receptores Frizzled/genética , Regulação da Expressão Gênica no Desenvolvimento , Paracentrotus/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Células-Tronco/citologia , Células-Tronco/fisiologia , beta Catenina/genéticaRESUMO
During development, cell migration plays an important role in morphogenetic processes. The construction of the skeleton of the sea urchin embryo by a small number of cells, the primary mesenchyme cells (PMCs), offers a remarkable model to study cell migration and its involvement in morphogenesis. During gastrulation, PMCs migrate and become positioned along the ectodermal wall following a stereotypical pattern that determines skeleton morphology. Previous studies have shown that interactions between ectoderm and PMCs regulate several aspects of skeletal morphogenesis, but little is known at the molecular level. Here we show that VEGF signaling between ectoderm and PMCs is crucial in this process. The VEGF receptor (VEGFR) is expressed exclusively in PMCs, whereas VEGF expression is restricted to two small areas of the ectoderm, in front of the positions where the ventrolateral PMC clusters that initiate skeletogenesis will form. Overexpression of VEGF leads to skeletal abnormalities, whereas inhibition of VEGF/VEGFR signaling results in incorrect positioning of the PMCs, downregulation of PMC-specific genes and loss of skeleton. We present evidence that localized VEGF acts as both a guidance cue and a differentiation signal, providing a crucial link between the positioning and differentiation of the migrating PMCs and leading to morphogenesis of the embryonic skeleton.
Assuntos
Ectoderma/fisiologia , Mesoderma/fisiologia , Ouriços-do-Mar/embriologia , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Linhagem da Célula , Ectoderma/citologia , Ectoderma/metabolismo , Embrião não Mamífero , Gástrula , Hibridização In Situ , Mesoderma/citologia , Mesoderma/metabolismo , Microinjeções , Modelos Biológicos , Morfogênese , Oligonucleotídeos Antissenso/farmacologia , RNA Mensageiro/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Ouriços-do-Mar/citologiaRESUMO
The Receptor Tyrosine kinase (RTK) and TGF-beta signaling pathways play essential roles during development in many organisms and regulate a plethora of cellular responses. From the genome sequence of Strongylocentrotus purpuratus, we have made an inventory of the genes encoding receptor tyrosine kinases and their ligands, and of the genes encoding cytokines of the TGF-beta superfamily and their downstream components. The sea urchin genome contains at least 20 genes coding for canonical receptor tyrosine kinases. Seventeen of the nineteen vertebrate RTK families are represented in the sea urchin. Fourteen of these RTK among which ALK, CCK4/PTK7, DDR, EGFR, EPH, LMR, MET/RON, MUSK, RET, ROR, ROS, RYK, TIE and TRK are present as single copy genes while pairs of related genes are present for VEGFR, FGFR and INSR. Similarly, nearly all the subfamilies of TGF-beta ligands identified in vertebrates are present in the sea urchin genome including the BMP, ADMP, GDF, Activin, Myostatin, Nodal and Lefty, as well as the TGF-beta sensu stricto that had not been characterized in invertebrates so far. Expression analysis indicates that the early expression of nodal, BMP2/4 and lefty is restricted to the oral ectoderm reflecting their role in providing positional information along the oral-aboral axis of the embryo. The coincidence between the emergence of TGF-beta-related factors such as Nodal and Lefty and the emergence of the deuterostome lineage strongly suggests that the ancestral function of Nodal could have been related to the secondary opening of the mouth which characterizes this clade, a hypothesis supported by functional data in the extant species. The sea urchin genome contains 6 genes encoding TGF-beta receptors and 4 genes encoding prototypical Smad proteins. Furthermore, most of the transcriptional activators and repressors shown to interact with Smads in vertebrates have orthologues in echinoderms. Finally, the sea urchin genome contains an almost complete repertoire of genes encoding extracellular modulators of BMP signaling including Chordin, Noggin, Sclerotin, SFRP, Gremlin, DAN and Twisted gastrulation. Taken together, these findings indicate that the sea urchin complement of genes of the RTK and TGF-beta signaling pathways is qualitatively very similar to the repertoire present in vertebrates, and that these genes are part of the common genetool kit for intercellular signaling of deuterostomes.
Assuntos
Genoma , Proteínas Tirosina Quinases/genética , Ouriços-do-Mar/genética , Fator de Crescimento Transformador beta/genética , Sequência de Aminoácidos , Animais , Humanos , Filogenia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transdução de Sinais/genética , Vertebrados/genéticaRESUMO
The Wnt pathways are evolutionarily well-conserved signal transduction pathways that are known to play important roles in all Metazoans investigated to date. Here, we examine the Wnt pathway genes and target genes present in the genome of the echinoderm Strongylocentrotus purpuratus. Analysis of the Wnt genes revealed that eleven of the thirteen reported Wnt subfamilies are represented in sea urchin, with the intriguing identification of a Wnt-A ortholog thought to be absent in deuterostomes. A phylogenetic study of the Frizzled proteins, the Wnt receptors, performed throughout the animal kingdom showed that not all Frizzled subfamilies were present in the metazoan common ancestor, e.g. Fz3/6 emerged later during evolution. Using sequence analysis, orthologs of the vast majority of the cellular machinery involved in transducing the three types of Wnt pathways were found in the sea urchin genome. Furthermore, of about one hundred target genes identified in other organisms, more than half have clear echinoderm orthologs. Thus, these analyses produce new inputs in the evolutionary history of the Wnt genes in an animal occupying a position that offers great insights into the basal properties of deuterostomes.
Assuntos
Genoma , Ouriços-do-Mar/genética , Proteínas Wnt/genética , Sequência de Aminoácidos , Animais , Sequência Conservada , Embrião não Mamífero/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ouriços-do-Mar/classificação , Ouriços-do-Mar/embriologia , Homologia de Sequência de AminoácidosRESUMO
Studies in Caenorhabditis elegans and vertebrates have established that the MAP kinase-related protein NLK counteracts Wnt signalling by downregulating the transcription factor TCF. Here, we present evidence that during early development of the sea urchin embryo, NLK is expressed in the mesodermal precursors in response to Notch signalling and directs their fate by downregulating TCF. The expression pattern of nlk is strikingly similar to that of Delta and the two genes regulate the expression of each other. nlk overexpression, like ectopic activation of Notch signalling, provoked massive formation of mesoderm and associated epithelial mesenchymal transition. NLK function was found to be redundant with that of the MAP kinase ERK during mesoderm formation and to require the activity of the activating kinase TAK1. In addition, the sea urchin NLK, like its vertebrate counterpart, antagonizes the activity of the transcription factor TCF. Finally, activating the expression of a TCF-VP16 construct at blastula stages strongly inhibits endoderm and mesoderm formation, indicating that while TCF activity is required early for launching the endomesoderm gene regulatory network, it has to be downregulated at blastula stage in the mesodermal lineage. Taken together, our results indicate that the evolutionarily conserved TAK/NLK regulatory pathway has been recruited downstream of the Notch/Delta pathway in the sea urchin to switch off TCF-beta-catenin signalling in the mesodermal territory, allowing precursors of this germ layer to segregate from the endomesoderm.
Assuntos
Proteínas de Membrana/metabolismo , Mesoderma/fisiologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Paracentrotus , Receptores Notch/metabolismo , Transdução de Sinais/fisiologia , Fatores de Transcrição TCF/metabolismo , Sequência de Aminoácidos , Animais , Indução Embrionária , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Hibridização In Situ , Peptídeos e Proteínas de Sinalização Intracelular , Lítio/metabolismo , MAP Quinase Quinase Quinases/genética , MAP Quinase Quinase Quinases/metabolismo , Proteínas de Membrana/genética , Proteínas Quinases Ativadas por Mitógeno/genética , Dados de Sequência Molecular , Paracentrotus/citologia , Paracentrotus/embriologia , Paracentrotus/enzimologia , Fenótipo , Receptores Notch/genética , Alinhamento de Sequência , Fatores de Transcrição TCF/genética , beta Catenina/metabolismoRESUMO
This paper reports a preliminary in silico analysis of the sea urchin kinome. The predicted protein kinases in the sea urchin genome were identified, annotated and classified, according to both function and kinase domain taxonomy. The results show that the sea urchin kinome, consisting of 353 protein kinases, is closer to the Drosophila kinome (239) than the human kinome (518) with respect to total kinase number. However, the diversity of sea urchin kinases is surprisingly similar to humans, since the urchin kinome is missing only 4 of 186 human subfamilies, while Drosophila lacks 24. Thus, the sea urchin kinome combines the simplicity of a non-duplicated genome with the diversity of function and signaling previously considered to be vertebrate-specific. More than half of the sea urchin kinases are involved with signal transduction, and approximately 88% of the signaling kinases are expressed in the developing embryo. These results support the strength of this nonchordate deuterostome as a pivotal developmental and evolutionary model organism.
Assuntos
Proteínas Quinases/genética , Ouriços-do-Mar/crescimento & desenvolvimento , Ouriços-do-Mar/genética , Animais , Embrião não Mamífero , Regulação da Expressão Gênica no Desenvolvimento , Fosforilação , Filogenia , Proteínas Quinases/classificação , Ouriços-do-Mar/classificação , Ouriços-do-Mar/embriologia , Transdução de SinaisRESUMO
Wnt signaling pathways play key roles in numerous developmental processes both in vertebrates and invertebrates. Their signals are transduced by Frizzled proteins, the cognate receptors of the Wnt ligands. This study focuses on the role of a member of the Frizzled family, Fz5/8, during sea urchin embryogenesis. During development, Fz5/8 displays restricted expression, beginning at the 60-cell stage in the animal domain and then from mesenchyme blastula stage, in both the animal domain and a subset of secondary mesenchyme cells (SMCs). Loss-of-function analyses in whole embryos and chimeras reveal that Fz5/8 is not involved in the specification of the main embryonic territories. Rather, it appears to be required in SMCs for primary invagination of the archenteron, maintenance of endodermal marker expression and apical localization of Notch receptors in endodermal cells. Furthermore, among the three known Wnt pathways, Fz5/8 appears to signal via the planar cell polarity pathway. Taken together, the results suggest that Fz5/8 plays a crucial role specifically in SMCs to control primary invagination during sea urchin gastrulation.
Assuntos
Padronização Corporal , Desenvolvimento Embrionário/fisiologia , Receptores Frizzled/metabolismo , Gástrula/fisiologia , Ouriços-do-Mar , Animais , Polaridade Celular , Receptores Frizzled/classificação , Receptores Frizzled/genética , Gástrula/citologia , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Mesoderma/citologia , Mesoderma/metabolismo , Filogenia , Receptores Notch/metabolismo , Ouriços-do-Mar/anatomia & histologia , Ouriços-do-Mar/embriologia , Ouriços-do-Mar/crescimento & desenvolvimento , Transdução de Sinais/fisiologia , Proteínas Wnt/genética , Proteínas Wnt/metabolismoRESUMO
This chapter summarizes four powerful assays for analyzing gene expression in cis-regulatory studies. The enzymatic assays (CAT, luciferase, lacZ) are currently limited by their application to embryo homogenates or fixed samples, but offer more robust analysis of gene activity than GFP. Assays based on CAT enzymatic activity or on CAT mRNA detection by WMISH are laborious but are well established for accurately quantifying gene expression and to determine spatial patterns at defined timepoints during development. LacZ assays are the current standard for spatially visualizing gene products in whole-mount fixed embryos. They are very sensitive but they provide limited temporal or quantitative information due to the perdurance of beta-galactosidase and the subtleties of the staining technique. Recently developed luciferase assays promise to be even more sensitive and accurate than the CAT and lacZ assays, and applicable to living cells and embryos. But, they have not yet been well established in invertebrate deuterostome research. GFP allows visualization of gene expression within living embryos. But because this is not an enzymatic assay, sensitivity can be a problem, particularly for weak promoters. Furthermore, imaging live embryos and quantifying gene expression in space and time (due to scattering of light by tissue, the perdurance of GFP, and other experimental details) is currently fraught with challenges. Ongoing improvements in imaging technology and the advent of multiple fluorescent proteins, as well as fluorescent and luminescent assays for vital imaging, will dramatically facilitate studies of gene expression in the coming decade.
Assuntos
Genes Reguladores/genética , Genes Reporter/genética , Biologia Molecular/métodos , Ouriços-do-Mar/genética , Animais , Cloranfenicol O-Acetiltransferase/genética , Proteínas de Fluorescência Verde/genética , Óperon Lac/genética , Luciferases/genética , Ouriços-do-Mar/metabolismoRESUMO
Expression of exogenous mRNAs has become part of the standard approach to studying gene function during development of the sea urchin. The method is simple and reliable, protocols for the preparation of synthetic mRNAs are well described, and the technique to transfer them into eggs is efficient. The protein encoded by these mRNAs can be designed to address a variety of biological questions and their DNA matrices are easily constructed by standard molecular biology techniques. The method aims to simulate gain or loss of gene function, and the phenotypes obtained are characterized using an increasing number of molecular markers. With the completion of the S. purpuratus genome project, the complete set of genes from the sea urchin will become available. Expression of mRNA will be an invaluable tool to study the function of newly identified genes and their protein products and to determine their positions within the networks of gene and protein interactions that control development.
Assuntos
Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Biologia Molecular/métodos , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Ouriços-do-Mar/genética , Animais , Embrião não Mamífero/embriologia , Marcação de Genes/métodos , Microinjeções/métodos , RNA Mensageiro/síntese química , Transdução de Sinais/genéticaRESUMO
Transcription factors of the T-domain family regulate many developmental processes. We have isolated from the sea urchin a new member of the Tbx2 subfamily: coquillette. Coquillette has a late zygotic expression whose localization is dynamic: at the blastula stage it is restricted to the aboral side of most of the presumptive ectoderm and endoderm territories and from gastrulation on, to the aboral-most primary mesenchyme cells. Perturbation of coquillette function delays gastrulation and strongly disorganizes the skeleton of the larva. Coquillette is sensitive to alteration of the oral-aboral (OA) axis and we identify goosecoid, which controls oral and aboral fates in the ectoderm, as a probable upstream regulator. Coquillette appears to be an integral part of the patterning system along the OA axis.