Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Transcription profiling of zebrafish germ layer morphogenesis

ABSTRACT: In gastrulation, distinct progenitor cell populations are induced and sorted into the three germ layers ectoderm, mesoderm and endoderm. In order to identify genes involved in germ layer specification and morphogenesis, we identified genes differentially expressed between ectodermal and mesendodermal progenitor cells. To do so, we first generated highly enriched pools of ectodermal and mesendodermal progenitor cells. Mesendodermal cells were generated by over-expressing the Nodal signal Cyclops in wild type embryos and ectodermal cells were taken from mz-one-eyed-pinhead (oep) mutant embryos. We then compared the transcriptome of ectodermal versus mesendodermal cells taken from embryos at 7 hours post fertilization (hpf). In wild type embryos at this stage (70% epiboly), the first ectodermal and mesendodermal progenitor cells have already been sorted into their respective germ layers and ingression of mesendodermal progenitors is still ongoing.

ORGANISM(S): Danio rerio

SUBMITTER: Carl-Philipp Heisenberg

PROVIDER: E-MEXP-171 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Identification of regulators of germ layer morphogenesis using proteomics in zebrafish.

Link Vinzenz V Carvalho Lara L Castanon Irinka I Stockinger Petra P Shevchenko Andrej A Heisenberg Carl-Philipp CP

Journal of cell science 20060425 Pt 10

During vertebrate gastrulation, a well-orchestrated series of morphogenetic changes leads to the formation of the three germ layers: the ectoderm, mesoderm and endoderm. The analysis of gene expression patterns during gastrulation has been central to the identification of genes involved in germ layer formation. However, many proteins are regulated on a translational or post-translational level and are thus undetectable by gene expression analysis. Therefore, we developed a 2D-gel-based comparati ...[more]

PMID: 16638810

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Project description:A balance between cell survival and apoptosis is essential for animal development. Although proper development involves multiple interactions between germ layers, little is known about the intercellular and intertissue signaling pathways that promote cell survival in neighboring or distant germ layers . We show that serum- and glucocorticoid-inducible kinase 1 (SGK1) promoted ectodermal cell survival during early Xenopus embryogenesis through a non-cell-autonomous mechanism. Dorsal depletion of SGK1 in Xenopus embryos resulted in shortened axes and reduced head structures with defective eyes, and ventral depletion led to defective tail morphologies. Although the gene encoding SGK1 was mainly expressed in the endoderm and dorsal mesoderm, knockdown of SGK1 caused excessive apoptosis in the ectoderm. SGK1-depleted ectodermal explants showed little or no apoptosis, suggesting non-cell-autonomous effects of SGK1 on ectodermal cells. Microarray analysis revealed that SGK1 knockdown increased the expression of genes encoding FADD and caspase-10, components of the death-inducing signaling complex (DISC). Inhibition of DISC function suppressed excessive apoptosis in SGK1-knockdown embryos. SGK1 acted through the transcription factor nuclear factor kappaB to stimulate production of bone morphogenetic protein 7 (BMP7), and overexpression of BMP7 in SGK1-knockdown embryos reduced the abundance of DISC components. We show that phosphoinositide 3-kinase (PI3K) functioned upstream of SGK1, thus revealing an endodermal and mesodermal pathway from PI3K to SGK1 to NF-kappaB that produces BMP7, which provides a survival signal to the ectoderm by decreasing DISC function. To identify genes whose expression levels are regulated by SGK1 in Xenopus development, we performed genome-wide analysis by using Affymetrix GeneChip oligonucleotide microarrays. We performed two independent experiments. For each microarray experiment, we radially injected control-morpholino or xSGK1-morpholinos into 4-cell embryos, cultured the embryos until stage 12, extracted total RNAs using Trizol (Invitrogen) and purified them on RNeasy columns (Qiagen). Synthesis of cDNA, in vitro transcription and biotin labeling of cRNA, and hybridization to the Xenopus laevis genome array (Affymetrix) were performed according to the Affymetrix protocol (Two-Cycle Target Labeling Assays). Hybridized arrays were scanned using an Affymetrix GeneChip Scanner. Scanned chip images were analyzed with GeneChip Operating Software (GCOS) v. 1.4.

Project description:The germ layer concept has been one of the foremost organizing principles in developmental biology, classification, systematics and evolution for 150 years. Of the three germ layers, the mesoderm is found in bilaterian animals but is absent in species in the phyla Cnidaria and Ctenophora, which has been taken as evidence that the mesoderm was the final germ layer to evolve. The origin of the ectoderm and endoderm layers, however, remains unclear with models supporting the antecedence of each as well as a simultaneous origin. We hypothesized that global analysis of gene expression in each layer throughout development may resolve the early events of animal evolution. Here, we determine the temporal and spatial components of gene expression spanning embryonic development for all C. elegans genes and use it to determine the evolutionary age of the germ layers. The gene expression programs of the mesoderm is generally induced after the ectoderm and endoderm germ layers, thus making it the last germ layer to both evolve and develop. Strikingly, the C. elegans endoderm and ectoderm expression programs do not co-induce; rather the endoderm activates earlier. We also observed early expression of endoderm orthologs during the embryology of Xenopus tropicalis, Nematostella vectensis, and the sponge Amphimedon queenslandica. Querying for the phylogenetic ages of specifically expressed genes revealed that the endoderm is comprised of older genes, supporting its antecedence among the germ layers. Taken together, we propose that the endoderm program has retained the feeding functions of the last common ancestor with the choanoflagellates, thus allowing for the specialization of an ectoderm germ layer. Our work reveals that the evolutionary appearance of the germ layers continues to constrain regulatory networks in metazoans. Two temporal assays of C. elegans embryonic development, starting at the zygote: (a) Embryos collected at fixed (~10 minute) time intervals. (b) Embryo segregates, up to five lines of blastomeres, isolated in reference to mitotic events. There were 184 samples in total, representing 100 distnict data points.

Dataset Information

Transcription profiling of zebrafish germ layer morphogenesis

Publications

Identification of regulators of germ layer morphogenesis using proteomics in zebrafish.

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