Crumbs3 Is Essential for Proper Epithelial Development and Viability

Abstract

First identified in Drosophila, the Crumbs (Crb) proteins are important in epithelial polarity, apical membrane formation, and tight junction (TJ) assembly. The conserved Crb intracellular region includes a FERM (band 4.1/ezrin/radixin/moesin) binding domain (FBD) whose mammalian binding partners are not well understood and a PDZ binding motif that interacts with mammalian Pals1 (protein associated with lin seven) (also known as MPP5). Pals1 binds Patj (Pals1-associated tight-junction protein), a multi-PDZ-domain protein that associates with many tight junction proteins. The Crb complex also binds the conserved Par3/Par6/atypical protein kinase C (aPKC) polarity cassette that restricts migration of basolateral proteins through phosphorylation. Here, we describe a Crb3 knockout mouse that demonstrates extensive defects in epithelial morphogenesis. The mice die shortly after birth, with cystic kidneys and proteinaceous debris throughout the lungs. The intestines display villus fusion, apical membrane blebs, and disrupted microvilli. These intestinal defects phenocopy those of Ezrin knockout mice, and we demonstrate an interaction between Crumbs3 and ezrin. Taken together, our data indicate that Crumbs3 is crucial for epithelial morphogenesis and plays a role in linking the apical membrane to the underlying ezrin-containing cytoskeleton.

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ACKNOWLEDGMENTS

This research was supported by NIH grants DK089119 (to J.L.H.), HL079339 (to M.B.H.), DK06914 and DK073722 (to G.R.D.), DK065850 and DK089933 (to D.L.G.), and DK069605 (to B.M.).

We thank members of our outstanding core facilities at the University of Michigan: Microscopy and Image Analysis Laboratory (especially S. Almburg, J. Harrison, and J. Poore), MDRTC Morphology and Image Analysis Core (S. Lentz), Transgenic Animal Model Core (especially E. Hughes, T. Saunders, M. Schmidt, and D. Vanheyningen), and the Unit for Laboratory Animal Medicine (Managed Breeding Colony, Technical Services, and Pathology Services, especially P. Arrowsmith, I. Bergin, R. McUmber, C. Schray, and B. Popoola). We also thank K. Bentley, M. Berger, M. Green, E. Hurd, J. Kushwaha, M. Nagy, S. Patel, B. Rockich, J. Spence, U. Sajjan, and C. Tsui for reagents, technical advice, and helpful discussions.

E.L.W., S.F., J.L.H., K.D.W., C.-J.L., A.S., V.C.F., and G.H.D. performed experiments and data analysis. E.L.W., M.B.H., G.R.D., G.H.D., D.L.G., and B.M. developed the concepts and experimental approaches. E.L.W. and B.M. wrote the manuscript.