Abstract: SA-PO536
Determinants of Renal Progenitor Cell Responsiveness to the Inductive Wnt9b Signal from the Ureteric Bud
Session Information
- Developmental Biology
November 04, 2017 | Location: Hall H, Morial Convention Center
Abstract Time: 10:00 AM - 10:00 AM
Category: Developmental Biology and Inherited Kidney Diseases
- 401 Developmental Biology
Authors
- Dickinson, Kyle, McGill University , Montreal, Quebec, Canada
- Carroll, Thomas J., UTSW Medical Center, Dallas, Texas, United States
- Goodyer, Paul R., McGill University, Montreal, Quebec, Canada
Background
The canonical Wnt-signalling pathway is essential for kidney development as fully primed renal progenitor cells (RPC) appear in the metanephric mesenchyme. RPCs receive inductive Wnt9b signals from the adjacent ureteric bud to initiate a proliferative program. Specificity of Wnt ligand binding is determined by the co-receptor complex, consisting of a Frizzled (Fzd1-10) and Lipoprotein related receptor protein (Lrp5/6), however, the specific molecular components conferring responsiveness in RPCs have yet to be identified. The receptor complex is stabilized by R-spondin1 and R-spondin3 (Rspo1 and Rspo3), amplifying the Wnt signal.
Methods
We obtained M15 cells, derived form E10.5 mesonephric mesenchyme (Hastie et al, 1999) and systematically analyzed Wnt receptor/signalling components required for a canonical Wnt response. To measure activation of the canonical Wnt pathway, we transfected our M15 cells with reporter plasmid 8X TOPFlash and measured luciferase activity using a luminometer. RNA was analyzed by qRT-PCR.
Results
Exposing M15 cells to external Wnt9b resulted in minimal luciferase activity suggesting a signalling component is missing. We analyzed M15 cells for components of the β-catenin/TCF pathway and found mRNA expression of Fzd1-6, Lrp6 but neither Rspo1/3. To ascertain whether absence of R-spondin accounts for the lack of response, we transfected M15/TOPFlash cells with Wnt9b and added recombinant Rspo1 or Rspo3 and observed a 4.8-fold and 7.8-fold increase in luciferase activity, respectively.
In the presence of Rspo1, we transfected the cells with Fzd1-10 and observed an additional 5-fold increase in the presence of Fzd5 but not the other Fzds. Knockdown of Lrp6 with siRNA (60% reduction in mRNA level) resulted in a 60% reduction in luciferase activity which was not rescued by Lrp5.
Conclusion
Our results suggest that early RPCs must acquire a specific receptor complex consisting of Fzd5, Lrp6 and Rspo1/3 before they can transduce an optimal β-catenin/TCF signal in response to Wnt9b during nephrogenesis. We speculate that putative RPCs lacking these components are incompetent for primary nephrogenesis and/or regeneration of damaged adult kidneys.
Funding
- Government Support - Non-U.S.