Ret and Etv4 Promote Directed Movements of Progenitor Cells during Renal Branching Morphogenesis.

Paul Riccio,Simon Hippenmeyer,Hui Zong,Cristina Cebrian,Frank Costantini,Emma Rawlins

doi:10.1371/journal.pbio.1002382

Abstract

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.

Highlights

Kidney development begins with the outgrowth of the primary ureteric bud (UB) from the nephric duct into the adjacent metanephric mesenchyme
By time-lapse microscopy of mouse fetal kidneys developing in culture, in which individual ureteric bud cells are fluorescently labeled, we show that most cells at the tips behave as progenitors, some of whose daughters remain at the tips while others populate the tubular uretic bud trunks
We use Mosaic Analysis with Double Markers, a genetic method that generates pairs of mutant and wild type cells, each labeled with a different fluorescent protein

Summary

Introduction

Kidney development begins with the outgrowth of the primary ureteric bud (UB) from the nephric duct into the adjacent metanephric mesenchyme. In mice lacking any of these genes, the UB usually fails to form, causing renal agenesis, or else branches minimally, causing severe renal hypoplasia [5,16]. Mutations in these genes are sometimes associated with renal agenesis or other congenital defects of the kidney or urinary tract in humans [6,17]. While the requirement for GDNF, and for several other signals (including fibroblast growth factors [FGFs], Wnts, and bone morphogenetic proteins [BMPs]), to achieve the normal extent and pattern of renal branching (and, indirectly, nephron number) is well established [2,17,18], the specific cellular behaviors controlled by these signals, and how these behaviors contribute to the branching morphogenesis of the UB epithelium, remain unclear

Methods

Results

Discussion

Conclusion