Molecular determinants of WNT9b responsiveness in nephron progenitor cells

Abstract

Primed nephron progenitor cells (NPCs) appear in metanephric mesenchyme by Ell.5 and differentiate in response to the inductive WNT9b signal from the ureteric bud. However, the NPC WNT-receptor complex is unknown. We obtained M15 cells from E10.5 mesonephric mesenchyme and systematically analyzed components required for canonical WNT9b-responsiveness. When M15 cells were transfected with a (β-catenin luciferase reporter plasmid, exposure to recombinant WNT9b resulted in minimal luciferase activity. We then analyzed mRNA-expression of WNT-pathway components and identified Fzdl-6 and Lrp6 transcripts but not RSPO1. When M15 cells were treated with recombinant RSPO1 the response to transfected WNT9b was augmented 4.8-fold. Co-transfection of M15 cells with Fzd5 (but no other Fzd family member) further increased the WNT9b signal to 16.8-fold and siRNA knockdown of Fzd5 reduced the signal by 52%. Knockdown of Lrp6 resulted in 60% WNT9b signal reduction. We confirmed Fzd5, Lrp6 and RSPO1 rrtRNA expression in CITED1(+) NPCs from E15.5 embryonic mouse kidney. Thus, while many WNT signaling-pathway components are present by E10.5, optimum responsiveness of Ell.5 cap mesenchyme requires that NPCs acquire RSPO1, FZD5 and LRP6.Summary StatementResponsiveness to the inductive WMT9b signal from ureteric bud is crucial for nephrogenesis. Here we analyze the molecules needed to prime nephron progenitor cells in embryonic mouse kidney.

R-spondin signalling is essential for the maintenance and differentiation of mouse nephron progenitors

“…These findings are consistent with recent in vitro data that described Rspo1 to enhance canonical signalling upon treatment of a mesonephric cell line (M15) with WNT9b (Ref. 33 ). However, at present we can not exclude that R-spondins may also modulate non-canonical WNT signalling, which might affect cell adhesion and migration of NPCs.…”

Paracrine and autocrine R-spondin signalling is essential for the maintenance and differentiation of renal stem cells

“…These findings are consistent with recent in vitro data that described Rspo1 to enhance canonical signalling upon treatment of a mesonephric cell line (M15) with WNT9b (Ref. 31 ).…”

Age-Dependent Changes in the Progenitor Translatome Coordinated in part byTsc1Increase Perception of Signaling Inputs to End Nephrogenesis

“…It has been appreciated for over a decade that a UB-derived Wnt9b canonical signal is required for both maintenance of self-renewal capacity of NPC as well as for their differentiation (Carroll et al, 2005;Karner et al, 2011). Recently, work in cells derived from E10.5 mesonephric mesenchyme identified a role for Rspo1, Lrp6 and Fzd5 in enabling Wnt9b responsiveness as cells transitioned from the intermediate mesoderm to the metanephric mesenchyme identity (Dickinson et al, 2019). It was further demonstrated that low signaling input promotes selfrenewal and high signaling input promotes differentiation (Ramalingam et al, 2018).…”