Dose-dependent responses to canonical Wnt transcriptional complexes in the regulation of mammalian nephron progenitors
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1.
University of Southern California
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2.
Icahn School of Medicine at Mount Sinai
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3.
Semmelweis University
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4.
Amgen (United States)
Abstract
In vivo and in vitro studies argue that concentration-dependent Wnt signaling regulates mammalian nephron progenitor cell (NPC) programs. Canonical Wnt signaling is regulated through the stabilization of β-catenin, a transcriptional co-activator when complexed with Lef/Tcf DNA-binding partners. Using the GSK3β inhibitor CHIR99021 (CHIR) to block GSK3β-dependent destruction of β-catenin, we examined dose-dependent responses to β-catenin in mouse NPCs, using mRNA transduction to modify gene expression. Low CHIR-dependent proliferation of NPCs was blocked on β-catenin removal, with evidence of NPCs arresting at the G2-M transition. While NPC identity was maintained following β-catenin removal, mRNA-seq identified low CHIR and β-catenin dependent genes. High CHIR activated nephrogenesis. Nephrogenic programming was dependent on Lef/Tcf factors and β-catenin transcriptional activity. Molecular and cellular features of early nephrogenesis were driven in the absence of CHIR by a mutated stabilized form of β-catenin. Chromatin association studies indicate low and high CHIR response genes are likely direct targets of canonical Wnt transcriptional complexes. Together, these studies provide evidence for concentration-dependent Wnt signaling in the regulation of NPCs and provide new insight into Wnt targets initiating mammalian nephrogenesis.
Copyright and License
© 2024. Published by The Company of Biologists Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.
Acknowledgement
We thank former and current members of the McMahon laboratory for helpful comments, technical assistance and useful discussion, especially Jill McMahon, JinJin Guo, Muskaan Singh, Dr Tracy Tran and Dr Kevin Peterson. We thank Dr Christopher Garcia and Dr Yi Miao for sharing the Wnt mimetic bi-specific antibody. We are grateful for the assistance of Dr Seth Ruffins in the biological imaging core facility and Bernadette Masinsin in the flow core facility, and thank our colleagues Dr Yulia Schwartz and Dr Unmesh Jadhav for illuminating discussions.
Funding
Work in A.P.M.’s laboratory was supported by the National Institutes of Health (R01 DK054364 to A.P.M. and F31 DK122777 to H.B.). Open access funding provided by the University of Southern California. Deposited in PMC for immediate release.
Contributions
Conceptualization: H.B., A.P.M.; Methodology: H.B., S.K.; Validation: H.B., S.K., B.D., B.-M.B.; Formal analysis: H.B., S.K., N.O.L., Q.G.; Investigation: H.B., A.P.M., N.O.L., B.D., B.-M.B.; Resources: H.B., A.P.M.; Data curation: H.B., N.O.L., B.D., B.-M.B.; Writing - original draft: H.B.; Writing - review & editing: H.B., A.P.M., B.D.; Visualization: H.B., S.K., B.D.; Supervision: H.B., A.P.M., N.O.L.; Project administration: A.P.M.; Funding acquisition: H.B., A.P.M.
Data Availability
The bulk mRNA-seq data have been deposited in GEO under accession number GSE231753. The mouse embryonic kidney single cell RNA-seq data have deposited at GEO under accession number GSE232482.
Conflict of Interest
A.P.M. is a consultant or scientific advisor to Novartis, eGENESIS, Trestle Biotherapeutics and IVIVA Medical.
Supplemental Material
- Supplementary information- pdf file
- Table S1. Data table including bulk mRNA Seq, sc-RNA-seq, ChIP analyses and intersections- xlsx file
- Table S2. ChIP binding of indicated transcription (co)factors binding sites in E16.5 mouse nephron progenitor cells in the specified CHIR conditions- xlsx file
- Table S3. Methods Table including primer sequences, antibodies and sgRNA sequences and RNA scope probes- xlsx file
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Additional details
- National Institutes of Health
- R01 DK054364
- National Institutes of Health
- DK122777
- Accepted
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2024-09-02Accepted
- Caltech groups
- Division of Biology and Biological Engineering
- Publication Status
- Published