Differential regulation of the proteome and phosphosproteome along the dorso-ventral axis of the early Drosophila embryo

Manuscript abstract:

The initially homogeneous epithelium of the early Drosophila embryo differentiates into regional subpopulations with different behaviours and physical properties that are needed for morphogenesis. The factors at top of the genetic hierarchy that control these behaviours are known, but many of their targets are not. To understand how proteins work together to mediate differential cellular activities, we studied in an unbiased manner the proteomes and phosphoproteomes of the three main cell populations along the dorso-ventral axis during gastrulation using mutant embryos that represent the different populations. We detected 6111 protein groups and 6259 phosphosites of which 3399 and 3433 respectively, were differentially regulated. The changes in phosphosite abundance did not correlate with changes in host protein abundance, showing phosphorylation to be a regulatory step during gastrulation. Hierarchical clustering of protein groups and phosphosites identified clusters that contain known fate determinants such as Doc1, Sog, Snail and Twist. The recovery of the appropriate known marker proteins in each of the different mutants we used validated the approach, but also revealed that two mutations that both interfere with the dorsal fate pathway, Toll10B and serpin27aex do this in very different manners. Diffused network analyses within each cluster point to microtubule components as one of the main groups of regulated proteins. Functional studies on the role of microtubules provide the proof of principle that microtubules have different functions in different domains along the DV axis of the embryo.

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Supplementary tables 1-12 corresponding to the corresponding manuscript/article. Please refer to the doc file: GomezJMetal_guideline_Supplementary_Tables.docx for description of each table.

手稿摘要：
早期果蝇胚胎的初始均质上皮细胞会分化为具有不同行为特征与物理特性的区域亚群，这些特性是胚胎形态发生过程所必需的。调控这些细胞行为的遗传调控网络顶层因子已被探明，但其诸多下游靶标仍未被阐明。为解析蛋白质如何协同介导差异化细胞活动，本研究采用无偏研究策略，利用代表不同细胞群的突变体胚胎，对原肠胚形成（gastrulation）时期沿背腹轴（dorso-ventral axis）分布的三类主要细胞群的蛋白质组（proteome）与磷酸化蛋白质组（phosphoproteome）进行了分析。本研究共鉴定到6111个蛋白质组与6259个磷酸化位点，其中分别有3399个蛋白质组与3433个磷酸化位点呈现差异调控。磷酸化位点的丰度变化与宿主蛋白的丰度变化并无相关性，这表明磷酸化是原肠胚形成过程中的一类关键调控步骤。对蛋白质组与磷酸化位点进行层级聚类（hierarchical clustering）后，所得聚类簇中包含已知的细胞命运决定因子，例如Doc1、Sog、Snail与Twist。本研究在所用的各突变体样本中均成功检测到对应的已知标记蛋白，这验证了本实验方法的有效性；同时还发现，两个均干扰背侧命运通路的突变体——Toll10B与serpin27aex——的作用机制存在显著差异。对每个聚类簇进行弥散网络分析后发现，微管组分（microtubule components）是主要的受调控蛋白质类群之一。针对微管功能的功能性研究证实，微管在胚胎背腹轴的不同区域具有不同的功能。

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对应本手稿或论文的补充表格1至12。各表格的详细说明请参阅文档文件GomezJMetal_guideline_Supplementary_Tables.docx。