Investigating the Role of Shroom3 in Kidney Development

Abstract

Nephrons develop from a specialized group of mesenchyme cells known as the nephron progenitors. Nephron progenitors can very dynamic as they can self-renew, migrate, and change their cell morphology. These alterations are essential for orientating and organizing select cells for progression through various stages of nephrogenesis. However, the underlying mechanisms that drive these dynamic morphological changes are not fully understood. Shroom3 is an actin-binding protein that regulates cell shape changes by modulating the actin cytoskeleton. In mice and humans, mutations in Shroom3 are associated with poor nephron function and chronic kidney disease. Despite these findings, the underlying mechanisms of Shroom3 function and how genetic mutations contribute to abnormal nephron formation are unclear. Here, we investigated functional roles for Shroom3 in the nephron progenitor population by analyzing E13.5 and E18.5 Wildtype and Shroom3 deficient mice (termed Shroom3-/-). First, using in-situ hybridization (ISH) and immunofluorescence (IF), we confirm Shroom3 expression in select nephron progenitors. Next, we demonstrated abnormal cell shape and abnormal nephron progenitor cell clustering using H&E staining and Pax2 immunofluorescence. We showed a reduction in nephron progenitor cell numbers and decreased cell length in E13.5 Shroom3-/- kidneys. Using markers of cell orientation, we discovered altered cell orientation in some but not all nephron progenitor cells. While analyzing the cell cytoskeleton, we also demonstrated the abnormal distribution of F-actin in Shroom-/- nephron progenitors. Lastly, immunofluorescence and transmission electron microscopy analysis of Shroom3-/- nephron progenitors confirmed the abnormal shape and reduced filopodia-like thin actin-based membrane protrusions. Our findings conclude that Shroom3 is essential for maintaining and regulating nephron progenitor cell morphology. Taken together, these findings could help explain why Shroom3 mutations are highly associated with kidney disease.