Non-muscle myosin IIA is required for the development of the zebrafish glomerulus

Abstract

Mutations in the MYH9 gene, coding for the non-muscle myosin heavy chain IIA (NMHC-IIA), are responsible for syndromes characterized by macrothrombocytopenia associated with deafness, cataracts, and severe glomerular disease. Electron microscopy of renal biopsies from these patients found glomerular abnormalities characterized by alterations in mesangial cells, podocytes, and thickening of the glomerular basement membrane. Knockout of NMHC-IIA in mice is lethal, and therefore little is known about the glomerular-related functions of Myh9. Here, we use zebrafish as a model to study the role and function of zNMHC-IIA in the glomerulus. Knockdown of zNMHC-IIA resulted in malformation of the glomerular capillary tuft characterized by few and dilated capillaries of the pronephros. In zNMHC-IIA morphants, endothelial cells failed to develop fenestrations, mesangial cells were absent or reduced, and the glomerular basement membrane appeared nonuniformly thickened. Knockdown of zNMHC-IIA did not impair the formation of podocyte foot processes or slit diaphragms; however, podocyte processes were less uniform in these morphants compared to controls. In vivo clearance of fluorescent dextran indicated that the glomerular barrier function was not compromised by zNMHC-IIA knockdown; however, glomerular filtration was significantly reduced. Thus, our results demonstrate an important role of zNMHC-IIA for the proper formation and function of the glomerulus in zebrafish.

Simultaneous assessment of glomerular filtration and barrier function in live zebrafish

“…Furthermore, attention has been focused on glomerular barrier function so far, whereas glomerular filtration in zebrafish as a phenotype after gene knockdown has remained largely unexplored. The clearance of low-molecular-weight dextran from the circulation appears to be well suited for the assessment of glomerular filtration (7). In the present study, we demonstrate that quantification of fluorescence intensities by confocal microscopy in the cardinal vein allowed us to determine the clearance of injected 10-and 500-kDa dextrans from the circulation to simultaneously assess glomerular filtration and barrier function in live zebrafish larvae.…”

“…Zebrafish were grown, mated, and maintained at 28.5°C, according to standard protocols (9) and as previously described (7). Zebrafish husbandry and experiments on larvae were performed in accordance with German animal protection law overseen by the agencies of the Federal State of Mecklenburg-Pomerania.…”

Developmental Localization of Nephrin in Zebrafish and Medaka Pronephric Glomerulus

“…Flattened type, which is interpreted as a hypoplastic glomerulus consisting of a glomerular capillary and mesangium, as found in some nephrin knockdown models and in the zebrafish mosaic eye (moe) mutant (Kramer-Zucker et al 2005) 3. Vesicular type, in which the glomerulus has retained a primitive vesicular structure, coupled with agenesis of the glomerular capillary and mesangium, and is recognized as paired vesicles (Serluca et al 2002;Müller et al 2011; The normal glomerular architecture is remarkably perturbed, particularly in the septal and vesicular types and, in such a disorganized glomerulus, it is difficult to identify the podocytes by conventional light microscopy. Fortunately, the anti-Nephrin antibody developed in this study was able to clearly visualize the podocytes and readily determine their position in the mpp5a m520 mutant, in which the pronephric glomerular architecture is disrupted and exhibits vesiculartype disorganization .…”

Murine Double Minute-2 Prevents p53-Overactivation-Related Cell Death (Podoptosis) of Podocytes

“…32 Embryos were kept and handled in E3 solution. Two transgene zebrafish strains were used (ET and CADE).…”