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

我的NSERC资助的研究项目专注于通过剖析线虫基因功能相似和不同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，我们正在进行生殖系统发育的比较分析，特别是两性外阴，在线虫(蠕虫)秀丽线虫及其姊妹种布氏线虫中。早些时候，我们实验室通过正向基因筛查分离了一大批基因突变体。突变体属于三个不同的表型类型：外阴(Vul)、多外阴(Muv)和外阴突出(PVL)。到目前为止，已对17个VUL、MUV和PVL基因进行了较为详细的研究。其中10个被克隆并对其功能进行了较详细的研究(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-连环蛋白和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，这导致我们提出，这两个线虫物种之间的发育机制存在分歧。NSERC的资助将使我们能够进一步研究基因功能和途径进化变化的机制。为此，我们将重点关注与线虫同源基因相比，在细胞诱导模式上显示不同的Muv基因。在与张伯林博士的实验室(俄亥俄州立大学)的合作下，我们对四个Muv突变体进行了分子特征分析，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)。虽然这四个基因在线虫中都是保守的，但它们在功能上有很大的差异。除gon-14外，线虫的任何同源基因的突变都会导致正常的外阴表型，而布里格萨线虫的突变体几乎是完全穿透的Muv。为了解IVP基因的遗传途径，进行了RNA-seq实验。结果表明，突变株存在胚系基因的错误调控，提示突变株可能具有促进体细胞分化的作用。未来的实验将集中在了解IVP基因在桥毛虫中的作用机制以及它们与线虫的功能差异。为此，我们计划通过正向和反向遗传(如CRISPR和RNAi)相结合的方法来表征相互作用的基因和下游靶标。这些实验应该揭示，桥臂隐杆线虫Muv基因是否通过改变已知线虫同源基因的调控来定义新的途径，或者现有的发育途径是否被不同地使用。最终，这些发现将揭示后生动物在发育机制上的保守和分歧。

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.