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Hedgehog/Wnt feedback supports regenerative proliferation of epithelial stem cells in bladder

Abstract

Epithelial integrity in metazoan organs is maintained through the regulated proliferation and differentiation of organ-specific stem and progenitor cells. Although the epithelia of organs such as the intestine regenerate constantly and thus remain continuously proliferative1, other organs, such as the mammalian urinary bladder, shift from near-quiescence to a highly proliferative state in response to epithelial injury2,3,4. The cellular and molecular mechanisms underlying this injury-induced mode of regenerative response are poorly defined. Here we show in mice that the proliferative response to bacterial infection or chemical injury within the bladder is regulated by signal feedback between basal cells of the urothelium and the stromal cells that underlie them. We demonstrate that these basal cells include stem cells capable of regenerating all cell types within the urothelium, and are marked by expression of the secreted protein signal Sonic hedgehog (Shh). On injury, Shh expression in these basal cells increases and elicits increased stromal expression of Wnt protein signals, which in turn stimulate the proliferation of both urothelial and stromal cells. The heightened activity of this signal feedback circuit and the associated increase in cell proliferation appear to be required for restoration of urothelial function and, in the case of bacterial injury, may help clear and prevent further spread of infection. Our findings provide a conceptual framework for injury-induced epithelial regeneration in endodermal organs, and may provide a basis for understanding the roles of signalling pathways in cancer growth and metastasis.

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Figure 1: Injury-induced proliferation and Hedgehog signalling in the bladder.
Figure 2: Shh-expressing basal cells repopulate the urothelium and form organoids in vitro.
Figure 3: Gli1 mediates injury-induced proliferation, restoration of urothelial integrity and reduction of infectious spread.
Figure 4: Hedgehog-induced expression of stromal Wnt signals mediates urothelial and stromal proliferation.

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Acknowledgements

We thank A. Oro and J. Brooks for their critical reading of the manuscript, and the Stanford Center for Digestive Diseases for help with laser capture microdissection. This research was supported in part by grants from the Department of Defense and from the National Institutes of Health (P.A.B.) and a Pathway to Independence Award (K99/R00) to I.U.M. P.A.B. is an investigator of the Howard Hughes Medical Institute.

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K.S. and P.A.B. conceived ideas and experimental design. K.S. performed the experiments. N.G. aided in immunohistochemical analysis, J.L. and J.K. helped with mouse strains, A.L. assisted with in vitro cell culture studies, L.Q. performed the genotyping of experimental mice, and I.U.M. helped analyse data. K.S. and P.A.B wrote the manuscript.

Corresponding authors

Correspondence to Kunyoo Shin or Philip A. Beachy.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Information

The file contains Supplementary Figures 1-19 with legends and Supplementary Tables 1-6. (PDF 25818 kb)

Supplementary Movie 1

The movie shows an organoid grown by culturing FACS-isolated Shh-expressing cells for 7 weeks in Matrigel was imaged and a portion was reconstructed from approximately ••• of the series of confocal EGFP images (Supplementary Fig. 11g,i). This reconstruction was rotated to display the shape and lumen of the organoid. Bladder cells were isolated directly from TM-injected ShhCreER/WT; R26mTmG/WT mice (Imaris software, Bitplane Scientific Software). (MPG 3172 kb)

Supplementary Movie 2

The movie shows organoid grown from a single cell and imaged as in Supplementary Movie 1, except that cells derived from a previously grown organoid (P1 organoid; Supplementary Fig. 11h,j; Imaris software, Bitplane Scientific Software). (MPG 2850 kb)

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Shin, K., Lee, J., Guo, N. et al. Hedgehog/Wnt feedback supports regenerative proliferation of epithelial stem cells in bladder. Nature 472, 110–114 (2011). https://doi.org/10.1038/nature09851

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