Abstract
The coordinated movement of cells is indispensable for normal vertebrate gastrulation. Several important players and signaling pathways have been identified in convergence and extension (CE) cell movements during gastrulation, including non-canonical Wnt signaling. Fyn and Yes, members of the Src family of kinases, are key regulators of CE movements as well. Here we investigated signaling pathways in early development by comparison of the phosphoproteome of wild type zebrafish embryos with Fyn/Yes knockdown embryos that display specific CE cell movement defects. For quantitation we used differential stable isotope labeling by reductive amination of peptides. Equal amounts of labeled peptides from wild type and Fyn/Yes knockdown embryos were mixed and analyzed by on-line reversed phase TiO(2)-reversed phase LC-MS/MS. Phosphorylated and non-phosphorylated peptides were quantified, and significant changes in protein expression and/or phosphorylation were detected. We identified 348 phosphoproteins of which 69 showed a decrease in phosphorylation in Fyn/Yes knockdown embryos and 72 showed an increase in phosphorylation. Among these phosphoproteins were known regulators of cell movements, including Adducin and PDLIM5. Our results indicate that quantitative phosphoproteomics combined with morpholino-mediated knockdowns can be used to identify novel signaling pathways that act in zebrafish development in vivo.
Highlights
The coordinated movement of cells is indispensable for normal vertebrate gastrulation
Analysis of Differential Phosphorylation—To investigate signaling pathways that have a role in the regulation of convergence and extension (CE) cell movements, we set out to evaluate the changes in the phosphoproteome after morpholino-mediated knockdown of Fyn and Yes in 24-hpf zebrafish embryos
We used stable isotope labeling by reductive amination of peptides derived from the two experimental conditions and on-line automated RP-TiO2-RP-LC-MS/MS
Summary
The coordinated movement of cells is indispensable for normal vertebrate gastrulation. We investigated signaling pathways in early development by comparison of the phosphoproteome of wild type zebrafish embryos with Fyn/Yes knockdown embryos that display specific CE cell movement defects. Phosphoproteomics of Zebrafish Fyn/Yes Knockdown Embryos based on two-dimensional gel electrophoresis (DIGE), chemical approaches (ICAT and isobaric tags for relative and absolute quantitation (iTRAQ)) or stable isotope labeling by amino acids in cell culture (SILAC) [20]. SILAC has proven to be successful to study changes in the phosphoproteome upon induction of signal transduction pathways This labeling method has the advantage that after labeling cell states are mixed together and all other sample handlings are carried out simultaneously. Other methods use stable isotope metabolic labeling in which all amino acids and peptides are labeled by growing cells on media containing exclusively 14N/15N [21,22,23]. Because trypsin is usually used as the enzyme for digestion, most peptides will contain at least one label because each amino terminus and each lysine becomes labeled
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