Functional analysis of genes in the developing zebrafish pronephros and vasculature

Abstract

During glomerulogenesis, the recruitment and assembly of visceral epithelial cells (podocytes), endothelial capillary cells, and smooth muscle pericytes (mesangial cells) result in the formation of the glomerular tuft. The mature glomerulus functions by passage of plasma under hemodynamic pressure across a filter, the glomerular filtration barrier, and the integrity of the barrier is crucial for proper function. A common pathology shared by virtually all glomerular diseases is the loss of filtration barrier function leading to proteinuria, the leakage of protein into the urine, and further glomerular and tubular damage. Thus, the podocyte slit diaphragm and its associated proteins have been the focus of intense research in the filtration barrier field. Even so, the understanding of how the podocytes, endothelia, and mesangial cells function together and communicate with each other within the mature glomerulus is at an early stage. With this question in mind, GlomBase, a bioinformatics database that describes the mammalian glomerular transcriptome, was created as a foundation from which to explore new aspects of glomerular biology.

We have applied the zebrafish pronephric glomerulus as a model system to study novel aspects of glomerular biology. Our approach takes advantage of the rapid development and genetic accessibility of the renal system in combination with GlomBase to conduct a highthroughput functional analysis. We reasoned that if a gene is important for glomerular function in the zebrafish it might also be important in mammalian glomerular function. In this novel genetic screen, we have coupled gene knockdown using morpholinos with a physiological glomerular dye filtration assay to test for selective glomerular permeability in living zebrafish larvae. We identified the crb2b gene as a regulator of podocyte foot process formation. We found that Nephrin, a major slit diaphragm component, is apically mis-localized in podocytes lacking crb2b function. These observations suggest that Crb proteins may regulate protein trafficking and provide a way of understanding foot process formation within the larger context of apical-basal cell differentiation.

The Angiomotin (Amot) family of proteins plays roles in endothelial migration, cell shape, and tube formation and members of this family are present within GlomBase. As a first step towards functionally characterizing Angiomotin family members in the zebrafish, we inactivated the amot gene in zebrafish using morpholinos within the Tg (fli1:EGFP)y1 transgenic line which expresses GFP within the developing vasculature. Zebrafish lacking amot function showed a clear, specific, and quantifiable defect in the formation of intersegmental vessels (ISVs) and this arose from a defect in endothelial cell migration and filopodia formation. These studies identified an evolutionarily conserved function for amot in blood vessel formation and paved the way for future studies of Amot family members within the glomerular vasculature.

We then applied the zebrafish pronephros to the study of the third cell type of the glomerulus, the mesangial cell. Mesangial cells are specialized vascular pericytes within the glomerulus and associate intimately with the glomerular capillaries. However, a pericyte population within the zebrafish has not been thus far described. We found cells that express the early pericyte marker pdgfrb and these cells were also closely associated with vasculature in the eye, brain, and glomerulus. Morpholino knockdown of pdgfrb resulted in dilation of the glomerular capillaries phenocopying mouse knock out data and arguing for a conserved role for Pdgfrb signaling in recruiting mesangial cells during maturation of the glomerular tuft. These studies establish the zebrafish as a system for studying pericyte development.