Molecular genetic study of reproductive system development in C. elegans and related nematode species

线虫及相关线虫物种生殖系统发育的分子遗传学研究

• 批准号: RGPIN-2020-06912
• 负责人: Gupta, Bhagwati
• 金额: $ 3.64万
• 依托单位: McMaster University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2021
• 资助国家: 加拿大
• 起止时间: 2021-01-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=741489
• 关键词: Molecular; genetic; study; reproductive; system

My NSERC-funded research program focuses on understanding genetic networks that control organ formation in eukaryotes by dissecting molecular mechanisms of similarities and differences in gene function in nematodes. To this end, we are conducting a comparative analysis of reproductive system development, specifically the hermaphrodite vulva, in the nematode (worm) Caenorhabditis elegans and its sister species C. briggsae. A large collection of   mutants were isolated earlier in our lab through forward genetic screens. The mutants belong to three distinct phenotypic classes: vulvaless (Vul), multivulva (Muv), and protruding vulva (Pvl). To date, 17 Vul, Muv and Pvl genes have been studied in some detail. Ten of these are cloned and function investigated in some detail (Seetharaman et al., Dev Biol 2010; Sharanya et al., G3 2012; Sharanya et al., Evol Chamberlin et al., manuscript under review). These genes function at different steps of the vulval induction process and participate in Wnt-beta-Catenin and EGFR-Ras signalling pathways. Interestingly, we have also uncovered evolutionary changes in some of the genes, leading us to propose that developmental mechanisms have diverged between the two nematode species. NSERC funding will enable us to further investigate the mechanism of evolutionary changes in gene function and pathways. Towards this, we will be focusing on the Muv genes that show differences in cell induction pattern compared to the C. elegans orthologs. In collaboration with Dr. Chamberlin's lab (Ohio State), we have molecularly characterized four of the Muv mutants that define a new phenotypic class termed 'inappropriate vulva proliferation (ivp).' The genes are Cbr-htz-1 (H2A.z), Cbr-spr-4 (REST/NRSF), Cbr-gon-14 (hAT transposon family), and Cbr-ivp-3 (Ribonuclease H-like superfamily). Although all four genes are conserved in C. elegans, there are major functional differences. While mutations in any of the C. elegans orthologs, except gon-14, cause normal vulva phenotype, C. briggsae mutants are nearly fully penetrant Muv. To understand the genetic pathway of ivp genes, RNA-seq experiment was carried out. The results revealed that C. briggsae Muv mutants exhibit misregulation of germline genes, leading us to propose that the Muv genes may act to promote differentiation of somatic cells. Future experiments will focus on understanding the mechanism of action of ivp genes in C. briggsae and their functional differences from C. elegans. To this end, we plan to characterize the interacting genes and downstream targets through a combination of forward and reverse genetic (such as CRISPR and RNAi) approaches. These experiments should reveal whether C. briggsae Muv genes define new pathways by altered regulation of known C. elegans orthologs or whether existing developmental pathways are used differently. Ultimately, the findings will uncover the conservation and divergence in developmental mechanisms in metazoans.

我的 NSERC 资助的研究项目重点是通过剖析线虫基因功能相似和差异的分子机制来了解控制真核生物器官形成的遗传网络。为此，我们正在对线虫秀丽隐杆线虫及其姐妹物种 C. briggsae 的生殖系统发育，特别是雌雄同体的外阴进行比较分析。 我们实验室早些时候通过正向遗传筛选分离出了大量突变体。突变体属于三个不同的表型类别：无外阴（Vul）、多外阴（Muv）和突出外阴（Pvl）。迄今为止，已经对 17 个 Vul、Muv 和 Pvl 基因进行了较为详细的研究。其中十个被克隆并进行了一些详细的功能研究（Seetharaman 等人，Dev Biol 2010；Sharanya 等人，G3 2012；Sharanya 等人，Evol Chamberlin 等人，手稿正在审查中）。这些基因在外阴诱导过程的不同步骤发挥作用，并参与 Wnt-β-Catenin 和 EGFR-Ras 信号通路。有趣的是，我们还发现了一些基因的进化变化，这使我们提出这两种线虫物种之间的发育机制存在差异。 NSERC 的资助将使我们能够进一步研究基因功能和途径的进化变化机制。为此，我们将重点关注与秀丽隐杆线虫直向同源物相比在细胞诱导模式上表现出差异的 Muv 基因。 我们与 Chamberlin 博士的实验室（俄亥俄州立大学）合作，对四种 Muv 突变体进行了分子鉴定，这些突变体定义了一种新的表型类别，称为“不适当的外阴增殖 (ivp)”。这些基因是 Cbr-htz-1 (H2A.z)、Cbr-spr-4 (REST/NRSF)、Cbr-gon-14（hAT 转座子家族）和 Cbr-ivp-3（核糖核酸酶 H 样超家族）。尽管所有四个基因在秀丽隐杆线虫中都是保守的，但存在重大功能差异。虽然任何线虫直系同源基因（gon-14 除外）的突变都会导致正常的外阴表型，但 C.briggsae 突变体几乎完全渗透性 Muv。 为了了解ivp基因的遗传途径，进行了RNA-seq实验。结果表明，C. briggsae Muv 突变体表现出种系基因的失调，使我们提出 Muv 基因可能起到促进体细胞分化的作用。未来的实验将集中于了解 ivp 基因在 C. briggsae 中的作用机制及其与 C. elegans 的功能差异。为此，我们计划通过正向和反向遗传（如 CRISPR 和 RNAi）方法相结合来表征相互作用的基因和下游靶标。这些实验应该揭示 C. briggsae Muv 基因是否通过改变已知的秀丽隐杆线虫直系同源物的调节来定义新的途径，或者是否以不同的方式使用现有的发育途径。最终，这些发现将揭示后生动物发育机制的保守性和差异性。