Isthmin-1 is required for branching morphogenesis during early kidney development

Abstract

Isthmin-1 (Ism1) is a secreted protein characterized by two functional domains: a central TSR and a C-terminal AMOP domain. It was first identified in Xenopus to be synexpressed with Fgf8 at the mid-hind brain boundary. Ism1 is reported to be involved in several physiological processes including angiogenesis inhibition, left-right asymmetry, glucose tolerance and lung homeostasis. However, little is known about its receptor and correlated signaling transduction. Ism1-deficient mice exhibited a spectrum of renal abnormalities including renal agenesis and hypoplasia.

The defective renal genesis in Ism1 deficient mice was traced back to early stages. Both ureteric bud (UB) branching morphogenesis and metanephric mesenchyme (MM) condensation were disrupted in the absence of Ism1 at E11.5. MM without UB invasion ultimately underwent apoptosis at E12.5. The aim of current study is to investigate the biological function of Ism1 in early kidney development.

Ism1 was expressed prominently in MM in E11.5 kidney rudiments. Ex vivo experiments indicated that Ism1 created an environment conducive to morphogenetic events in both wild-type and Ism1 mutant kidney, suggesting the positive role of Ism1 in renal branching morphogenesis. At E11.5, Ism1-/- kidneys exhibited downregulated Gdnf expression and Ret signaling downstream (Etv5 and p-Erk), suggesting that loss of Ism1 attenuates Gdnf/Ret signaling. The involvement of Ism1 in the Gdnf/Ret signaling were further supported by the interaction between Ism1 and Gdnf, as well as the co-receptor, Gfrα1, suggesting that Ism1 regulates early kidney development by promoting Gdnf recruitment to Ret/Gfrα1.

To construct a transcriptomic mapping of the initiation of kidney development, we conducted 10x Genome sequencing of E10.75 (budding) and E11.5 (the first branching) kidneys. Different from the nephrogenesis (from E12.5 onwards) which has been intensively explored, single-cell analysis for the first branching morphogenesis is less understood. Hence, we delineate the initiation of kidney development stepwise by which the nephric progenitor at the origin gives rise to the following differentiated functional cell types. Renal branching morphogenesis is a complex process largely dependent on epithelium and mesenchyme interaction, thus crosstalks between cell types were examined. Through co-expression analysis, Ism1 was found to be highly correlated with Gdnf (R2=0.93) spatiotemporally, consistent with our molecular analyses that Ism1 directly participated in Ret signaling.

Furthermore, by comparing transcriptome and communication divergence between wild-type and Ism1-/- mice, we found that the first branching in Ism1-/- kidney was blocked by downregulated cell motility in UE (ureteric epithelium) lineage. In addition, the niche for nephric progenitor in NPC (nephrogenic progenitor cell) lineage was also defective as early as E10.75, well before any defect can be observed. Cross-talk analysis revealed the attenuation of Ret/Gdnf signaling in Ism1-/- mice, in line with our findings in molecular experiments.

In summary, this study revealed a critical role for Ism1 in renal branching morphogenesis through regulating Gdnf/Ret/Gfrα1 signaling during early kidney development, with both molecular and cellular explanation. However, whether Ism1 is required for other signaling pathways, directly or indirectly, remains to be further investigated and verified.