title: Identification of regulators of germ layer morphogenesis using proteomics in zebrafish.
abstract: 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 comparative proteomic approach to target proteins involved in germ layer morphogenesis during zebrafish gastrulation. Proteomes of ectodermal and mesendodermal progenitor cells were compared and 35 significantly regulated proteins were identified by mass spectrometry, including several proteins with predicted functions in cytoskeletal organization. A comparison of our proteomic results with data obtained in an accompanying microarray-based gene expression analysis revealed no significant overlap, confirming the complementary nature of proteomics and transcriptomics. The regulation of ezrin2, which was identified based on a reduction in spot intensity in mesendodermal cells, was independently validated. Furthermore, we show that ezrin2 is activated by phosphorylation in mesendodermal cells and is required for proper germ layer morphogenesis. We demonstrate the feasibility of proteomics in zebrafish, concluding that proteomics is a valuable tool for analysis of early development.
authors.affiliation: Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstr.108, 01307 Dresden, Germany.
authors.forename: Vinzenz
authors.initials: V
authors.lastname: Link
authors.forename: Lara
authors.initials: L
authors.lastname: Carvalho
authors.forename: Irinka
authors.initials: I
authors.lastname: Castanon
authors.forename: Petra
authors.initials: P
authors.lastname: Stockinger
authors.forename: Andrej
authors.initials: A
authors.lastname: Shevchenko
authors.forename: Carl-Philipp
authors.initials: CP
authors.lastname: Heisenberg
mesh_terms: D000818:Animals; D003598:Cytoskeletal Proteins / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology; D020869:Gene Expression Profiling; D018507:Gene Expression Regulation, Developmental; D023281:Genomics / Q000379:methods; D005855:Germ Layers / Q000166:cytology / Q000378:metabolism / Q000502:physiology*; D013058:Mass Spectrometry / Q000379:methods; D046228:Microarray Analysis; D009024:Morphogenesis; D016376:Oligonucleotides, Antisense / Q000235:genetics; D040901:Proteomics / Q000379:methods*; D015027:Zebrafish / Q000196:embryology* / Q000235:genetics / Q000378:metabolism; D029961:Zebrafish Proteins / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D000099149:Ezrin
chemical_list: D003598:Cytoskeletal Proteins; D016376:Oligonucleotides, Antisense; D029961:Zebrafish Proteins; D000099149:Ezrin