Data from: Ret and Etv4 promote directed movements of progenitor cells during renal branching morphogenesis

Branching morphogenesis of the epithelial ureteric bud forms the renal collecting duct system and is critical for normal nephron number, while low nephron number is implicated in hypertension and renal disease. Ureteric bud growth and branching requires GDNF signaling from the surrounding mesenchyme to cells at the ureteric bud tips, via the Ret receptor tyrosine kinase and coreceptor Gfrα1; Ret signaling up-regulates transcription factors Etv4 and Etv5, which are also critical for branching. Despite extensive knowledge of the genetic control of these events, it is not understood, at the cellular level, how renal branching morphogenesis is achieved or how Ret signaling influences epithelial cell behaviors to promote this process. Analysis of chimeric embryos previously suggested a role for Ret signaling in promoting cell rearrangements in the nephric duct, but this method was unsuited to study individual cell behaviors during ureteric bud branching. Here, we use Mosaic Analysis with Double Markers (MADM), combined with organ culture and time-lapse imaging, to trace the movements and divisions of individual ureteric bud tip cells. We first examine wild-type clones and then Ret or Etv4 mutant/wild-type clones in which the mutant and wild-type sister cells are differentially and heritably marked by green and red fluorescent proteins. We find that, in normal kidneys, most individual tip cells behave as self-renewing progenitors, some of whose progeny remain at the tips while others populate the growing UB trunks. In Ret or Etv4 MADM clones, the wild-type cells generated at a UB tip are much more likely to remain at, or move to, the new tips during branching and elongation, while their Ret−/− or Etv4−/− sister cells tend to lag behind and contribute only to the trunks. By tracking successive mitoses in a cell lineage, we find that Ret signaling has little effect on proliferation, in contrast to its effects on cell movement. Our results show that Ret/Etv4 signaling promotes directed cell movements in the ureteric bud tips, and suggest a model in which these cell movements mediate branching morphogenesis.上皮性输尿管芽（epithelial ureteric bud）的分支形态发生形成肾收集管系统，对维持正常肾单位数量至关重要；而肾单位数量不足与高血压及肾脏疾病密切相关。输尿管芽的生长与分支依赖于周围间充质通过Ret受体酪氨酸激酶（Ret receptor tyrosine kinase）和共受体Gfrα1，向输尿管芽尖端细胞传递胶质细胞源性神经营养因子（GDNF）信号；Ret信号可上调转录因子Etv4与Etv5的表达，二者同样对分支过程不可或缺。尽管目前已对上述事件的遗传调控机制有了较为充分的认知，但在细胞层面，肾分支形态发生的具体实现路径，以及Ret信号如何调控上皮细胞行为以促进该过程，仍未被阐明。此前针对嵌合胚胎（chimeric embryos）的研究曾提示，Ret信号参与生肾管（nephric duct）内的细胞重排，但该方法并不适用于探究输尿管芽分支过程中的单个细胞行为。本研究借助双标记嵌合分析（Mosaic Analysis with Double Markers，MADM），结合器官培养与延时成像（time-lapse imaging）技术，追踪单个输尿管芽尖端细胞的运动与分裂过程。我们首先分析了野生型克隆（wild-type clones），随后对Ret或Etv4突变型/野生型克隆（mutant/wild-type clones）开展研究——此类克隆中，突变型与野生型姐妹细胞可通过绿色与红色荧光蛋白（fluorescent proteins）实现差异化且可遗传的标记。我们发现，在正常肾脏中，多数单个尖端细胞表现为自我更新祖细胞（self-renewing progenitors），其部分子代仍留存于尖端区域，其余子代则参与构成不断生长的输尿管芽干（UB trunks）。在Ret或Etv4的MADM克隆中，输尿管芽尖端产生的野生型细胞更倾向于留存于或迁移至新形成的尖端区域，而其Ret⁻/⁻或Etv4⁻/⁻的姐妹细胞则往往滞后，仅能参与构成芽干。通过追踪细胞谱系（cell lineage）中的连续有丝分裂（mitoses）过程，我们发现Ret信号对细胞增殖几乎无影响，这与其对细胞运动的调控作用形成鲜明对比。本研究结果表明，Ret/Etv4信号可促进输尿管芽尖端的定向细胞运动，并据此提出模型：此类细胞运动介导了分支形态发生过程。