Proteomics of Asrij perturbed Drosophila lymph glands as a resource for identification of new regulators of hematopoiesis

Hematological disorders result in perturbed homeostasis of the blood system. However, a comprehensive understanding of how physiological and genetic mechanisms regulate blood cell precursor maintenance and differentiation is lacking. Owing to simplicity and ease of genetic analysis, the Drosophila melanogaster lymph gland (LG) is an excellent model to study hematopoiesis. The LG is a multi-lobed structure compartmentalized into precursor and differentiation zones whose geography and identity is regulated by multiple signalling pathways. While additional molecular and functional subtypes are expected there is a paucity of information on gene expression and regulation of hemocyte homeostasis. Hence, we quantitatively analyzed the LG proteome under conditions that maintain precursors or promote their differentiation in vivo, by perturbing expression of Asrij, a conserved endosomal regulator of hematopoiesis. Although technically demanding, we pooled samples obtained from 1500 larval dissections per genotype and using iTRAQ quantitative proteomics, determined the relative expression levels of polypeptides in Asrij knockout (KO) and overexpressing (OV) LGs in comparison to wild type (control). Mass spectrometry data analysis showed that at least 6.5% of the Drosophila proteome is expressed in wild type LGs.Of 2,133 proteins identified, 780 and 208 proteins were common to the previously reported cardiac tube and hemolymph proteomes, respectively, resulting in the identification of 1238 proteins exclusive to the LG. Perturbation of Asrij levels led to differential expression of 619 proteins, of which 23% have human homologs implicated in various diseases. Proteins regulating metabolism, immune system, signal transduction and vesicle-mediated transport were significantly enriched. Immunostaining of representative candidates from the enriched categories and previous reports confirmed 75% of our results and validated the LG proteome. Our study provides, for the first time, an in vivo proteomics resource for identifying novel regulators of hematopoiesis that will also be applicable to understanding vertebrate blood cell development.

血液系统疾病会导致血液系统内稳态失衡。然而，目前尚缺乏对生理与遗传机制如何调控血细胞前体维持与分化的全面认知。由于遗传分析简便易行，黑腹果蝇（Drosophila melanogaster）淋巴腺（lymph gland, LG）是研究造血作用的理想模型。该淋巴腺为多叶状结构，可分区为前体区与分化区，其空间分布与细胞特性受多条信号通路调控。尽管学界推测存在更多的分子与功能亚型，但目前关于血细胞内稳态的基因表达与调控机制的相关信息仍十分匮乏。因此，本研究通过干扰造血作用的保守内体调控因子Asrij的表达，在体内维持血细胞前体或促进其分化的条件下，对黑腹果蝇淋巴腺的蛋白质组进行了定量分析。尽管该实验技术难度较高，本研究收集了每个基因型对应的1500只幼虫解剖所得的样本，并通过iTRAQ定量蛋白质组学技术，测定了Asrij敲除（KO）与过表达（OV）淋巴腺中多肽相对于野生型（对照组）的相对表达水平。质谱数据分析显示，野生型淋巴腺中至少表达了黑腹果蝇蛋白质组的6.5%。在本次鉴定得到的2133种蛋白质中，分别有780种与208种蛋白质与此前报道的心管蛋白质组和血淋巴蛋白质组存在交集，最终得到1238种淋巴腺特有的蛋白质。干扰Asrij的表达水平可导致619种蛋白质发生差异表达，其中23%的蛋白质对应的人类同源基因与多种疾病存在关联。调控代谢、免疫系统、信号转导以及囊泡介导运输的蛋白质出现了显著富集。对富集类别及既往研究中的代表性候选蛋白进行免疫染色实验，证实了本研究75%的结果，验证了淋巴腺蛋白质组数据的可靠性。本研究首次提供了一个体内蛋白质组学资源，可用于鉴定造血作用的新型调控因子，同时也可为理解脊椎动物血细胞发育提供参考。