During early development of Xenopus laevis, gastrulation is a key morphogenetic event which transforms the embryo into three of primary germ layers: ectoderm, mesoderm, and endoderm. In order for the physical separation of these layers to occur, cells have to acquire specific properties that distinguish one layer from another. These properties, which include cell adhesion and migration, should be reflected in the tissue-specific proteome. While genetic analysis has led to the determination of a number of proteins involved in germ layer formation, this method would not have identified those proteins regulated on a translational or post-translational level. In this study, we have developed a two-dimensional gel based comparative proteomic approach employing difference gel electrophoresis (DiGE) to identify proteins involved in germ layer morphogenesis during Xenopus gastrulation. Differences between the physical properties of the ectoderm and mesoderm are likely based on differences in the proteomes of the cell surface and/or cortex. We therefore analyzed plasma membrane enriched fractions, obtained using discontinuous sucrose density gradient centrifugation. The Decyder program was used to quantify expression changes with statistical confidence across multiple DiGE gels, provide independent confirmation of distinct expression patterns from the individual experiments, and demonstrate high reproducibility between replicate samples. The identity of 23 proteins, which were obtained from 33 analyzed spots, was determined using mass spectrometry. Our proteomic analysis of Xenopus ectoderm and mesoderm identified alterations in proteins involved in cytoskeletal organization, signal transduction, protein folding, vesicle trafficking, and in glycolysis. We have also demonstrated the feasibility of proteomics in Xenopus, and have therefore shown that proteomics may be a valuable tool for analysis of early development in this system.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.112317 |
| Date | January 2008 |
| Creators | Wang, Renee Wan-Jou, 1979- |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Master of Science (Department of Biology.) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 002712089, proquestno: AAIMR51354, Theses scanned by UMI/ProQuest. |
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