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

我的nserc资助的研究项目重点了解遗传网络，通过解剖线虫基因功能的异同分子机制，控制真核生物器官形成。为此，我们正在进行生殖系统发育的比较分析，特别是雌雄同体的外阴，在线虫（蠕虫）秀丽隐杆线虫和它的姐妹物种C. briggsae。之前在我们实验室通过前向基因筛选分离了大量的突变体。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基因进行了一些详细的研究。其中10种被克隆并进行了详细的功能研究（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阶段发挥作用，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同源物相比在细胞诱导模式上表现出差异的Muv基因。在与Chamberlin博士的实验室（俄亥俄州立大学）的合作中，我们对四种Muv突变体进行了分子表征，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）”。这些基因分别是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）。尽管这四个基因在秀丽隐杆线虫中都是保守的，但它们在功能上存在主要差异。除了gon-14外，秀丽隐杆线虫的任何同源基因突变都会导致正常的外阴表型，而秀丽隐杆线虫的突变几乎完全渗透Muv。为了了解ivp基因的遗传途径，我们进行了RNA-seq实验。结果表明，C. briggsae Muv突变体表现出生殖系基因的错误调控，这使我们提出Muv基因可能促进体细胞分化。未来的实验将集中在了解ivp基因在秀丽隐杆线虫中的作用机制及其与秀丽隐杆线虫的功能差异。为此，我们计划通过结合正向和反向遗传（如CRISPR和RNAi）方法来表征相互作用的基因和下游靶点。这些实验将揭示C. briggsae Muv基因是否通过改变已知秀丽隐杆线虫同源物的调节来定义新的途径，或者是否现有的发育途径被不同地使用。最终，这些发现将揭示后生动物发育机制的守恒和分化。

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.