Multiscale view of protein change during Xenopus laevis embryogenesis

Abstract

Embryogenesis is the transformation of an egg into a juvenile animal, a process that requires proliferation, differentiation, cell motility and tissue level rearrangement. Changes in protein and post-translational modification amounts are directly responsible for these events. While much is known about levels of mRNA across the embryo as well as in specific tissues relevant to these events, protein and post-translational modification levels cannot currently be inferred from mRNA levels. Additionally, the implications of protein change are multi-level; events occurring in a specific tissue can impact events in other tissues and thus have a broader than expected impact on embryo development. Thus, understanding embryogenesis requires observing development at the protein and post-translational level at multiple different scales. In this thesis we use observation of both normal and perturbed whole embryos to connect proteins and their post-translational modifications to events during embryogenesis using the model vertebrate system Xenopus laevis. In Chapters 2 and 3 we employ quantitative multiplexed mass spectrometry to measure thousands of proteins and protein phosphorylations across ten or eleven timepoints and on two very different timescales: the twenty minutes following fertilization and the weeklong period from the oocyte to the juvenile tadpole. This data provides a wealth of information about specific entities and reveals properties of the proteome and phospho-proteome. In order to connect this genomic-era perspective to a long history of absolute protein and phosphorylation measurements, we used both existing and new data to ascribe absolute amounts to the different phenomena that we observe. To estimate the absolute amount of change in phosphorylation, we developed a novel method for estimating the fraction of a species of protein that have a certain post-translational modification (i.e. the “occupancy” of the modification). Chapters 4 and 5 are concerned with using perturbation of specific proteins to study embryogenesis. In Chapter 4 we address gaps in knowledge about the role of Wnt11 family signaling for anterior-posterior axis extension in Xenopus laevis. After using translation blocking morpholinos to provide evidence that both family members are involved in gastrulation, we further characterize a combined knock-down of both family members and find that, in addition to contributing to blastopore closure, Wnt11 family signaling is involved in the initial extension of the archenteron (the future gut). This later requirement of Wnt11 family signaling was revealed by a novel interphase localization of the cleavage furrow protein anillin in the epithelium lining of the archenteron. We are able to observe both tissue-autonomous and potentially non-tissue-autonomous effects of our perturbation with live imaging and fixed immunofluorescence of whole embryos. A limitation to this study is that we are not able to assess the effects of our translation-blocking morpholino-mediated knockdown on the levels of the Wnt11 family proteins in the embryo. In Chapter 5 we introduce and provide proof of principle data for the Tandem Protection Radio-Assay which addresses the challenge of confirming change in protein level in model systems with limited access to antibodies. This thesis demonstrates that new insights about embryogenesis can be revealed through observation of the unperturbed embryo-wide proteome and phospho-proteome and through observation of the consequences of specific protein perturbations on whole embryos.