Vázquez-Manrique Rafael P, Nagy Anikó I, Legg James C, Bales Olivia A M, Ly Sung, Baylis Howard A
Department of Zoology, University of Cambridge, Cambridge, United Kingdom.
PLoS Genet. 2008 Mar 28;4(3):e1000043. doi: 10.1371/journal.pgen.1000043.
Migration of cells within epithelial sheets is an important feature of embryogenesis and other biological processes. Previous work has demonstrated a role for inositol 1,4,5-trisphosphate (IP(3))-mediated calcium signalling in the rearrangement of epidermal cells (also known as hypodermal cells) during embryonic morphogenesis in Caenorhabditis elegans. However the mechanism by which IP(3) production is stimulated is unknown. IP(3) is produced by the action of phospholipase C (PLC). We therefore surveyed the PLC family of C. elegans using RNAi and mutant strains, and found that depletion of PLC-1/PLC-epsilon produced substantial embryonic lethality. We used the epithelial cell marker ajm-1::gfp to follow the behaviour of epidermal cells and found that 96% of the arrested embryos have morphogenetic defects. These defects include defective ventral enclosure and aberrant dorsal intercalation. Using time-lapse confocal microscopy we show that the migration of the ventral epidermal cells, especially of the leading cells, is slower and often fails in plc-1(tm753) embryos. As a consequence plc-1 loss of function results in ruptured embryos with a Gex phenotype (gut on exterior) and lumpy larvae. Thus PLC-1 is involved in the regulation of morphogenesis. Genetic studies using gain- and loss-of-function alleles of itr-1, the gene encoding the IP(3) receptor in C. elegans, demonstrate that PLC-1 acts through ITR-1. Using RNAi and double mutants to deplete the other PLCs in a plc-1 background, we show that PLC-3/PLC-gamma and EGL-8/PLC-beta can compensate for reduced PLC-1 activity. Our work places PLC-epsilon into a pathway controlling epidermal cell migration, thus establishing a novel role for PLC-epsilon.
上皮细胞层内细胞的迁移是胚胎发育及其他生物学过程的一个重要特征。先前的研究表明，在秀丽隐杆线虫胚胎形态发生过程中，1,4,5-三磷酸肌醇（IP(3)）介导的钙信号传导在表皮细胞（也称为皮下细胞）重排中发挥作用。然而，刺激IP(3)产生的机制尚不清楚。IP(3)由磷脂酶C（PLC）作用产生。因此，我们利用RNA干扰和突变菌株对线虫的PLC家族进行了研究，发现PLC-1/PLC-ε缺失会导致大量胚胎致死。我们使用上皮细胞标记物ajm-1::gfp来追踪表皮细胞的行为，发现96%的停滞胚胎存在形态发生缺陷。这些缺陷包括腹侧包被缺陷和背侧插入异常。通过延时共聚焦显微镜观察，我们发现腹侧表皮细胞，尤其是领头细胞的迁移在plc-1(tm753)胚胎中较慢且常出现迁移失败的情况。因此，plc-1功能丧失会导致胚胎破裂，出现肠道外露（Gex）表型和体表不平的幼虫。由此可见，PLC-1参与形态发生的调控。利用线虫中编码IP(3)受体的itr-1基因的功能获得和功能缺失等位基因进行的遗传学研究表明，PLC-1通过ITR-1发挥作用。我们利用RNA干扰和双突变体在plc-1背景下耗尽其他PLC，结果表明PLC-3/PLC-γ和EGL-8/PLC-β可以补偿PLC-1活性的降低。我们的研究将PLC-ε置于控制表皮细胞迁移的信号通路中，从而确立了PLC-ε的新作用。
