Requirement for a Uroplakin 3a-Like Protein in the Development of Zebrafish Pronephric Tubule Epithelial Cell Function, Morphogenesis, and Polarity

Abstract

Uroplakin (UP)3a is critical for urinary tract development and function; however, its role in these processes is unknown. We examined the function of the UP3a-like protein Upk3l, which was expressed at the apical surfaces of the epithelial cells that line the pronephric tubules (PTs) of the zebrafish pronephros. Embryos treated with upk3l-targeted morpholinos showed decreased pronephros function, which was attributed to defects in PT epithelial cell morphogenesis and polarization including: loss of an apical brush border and associated phospho-ERM proteins, apical redistribution of the basolateral Na+/K+–ATPase, and altered or diminished expression of the apical polarity complex proteins Prkcz (atypical protein kinase C zeta) and Pard3 (Par3). Upk3l missing its C-terminal cytoplasmic domain or containing mutations in conserved tyrosine or proline residues did not rescue, or only partially rescued the effects of Upk3l depletion. Our studies indicate that Upk3l promotes epithelial polarization and morphogenesis, likely by forming or stimulating interactions with cytoplasmic signaling or polarity proteins, and that defects in this process may underlie the pathology observed in UP3a knockout mice or patients with renal abnormalities that result from altered UP3a expression.

Uroplakin 1b is critical in urinary tract development and urothelial differentiation and homeostasis

“…Upk3a is expressed in the urinary tract of Xenopus, and upk3l ( Upk3a-like ) morpholinos affect zebrafish pronephros development. 31, 32 In addition, heterozygous UPK3A mutations associate with renal adyspasia and/or vericoureteral reflux, 33 while UPK2 mutations associate with renal malformations in humans 34 and common genetic variants in UPK3A trended toward an association with duplex kidneys in a Dutch population. 35 These studies and our results support an expanded role for Upks in urinary tract development and CAKUT.…”

Zebrafish pronephros tubulogenesis and epithelial identity maintenance are reliant on the polarity proteins Prkc iota and zeta

“…The zebrafish has emerged as a valuable model for ongoing basic and translational kidney research based on the conservation of nephrons composition between zebrafish and mammalian nephrons (Poureetezadi, et al, 2013; Marra, et al, 2014). The attributes of the zebrafish embryo pronephros, in particular, have provided a novel opportunity for in vivo analysis of tubulogenesis processes (Mitra, et al, 2012; Jung, et al, 2013) and investigation of the mechanisms involved in the morphogenesis of tubular epithelial cells, such as collective cell migration (Vasilyev, et al, 2009). Our study adds to this work by providing the first spatiotemporal characterization of tubulogenesis in the zebrafish pronephros.…”

sept7b is essential for pronephric function and development of left–right asymmetry in zebrafish embryogenesis

“…The larvae were fixed with 4% PFA 1.5 h after injection, embedded in OCT compound and sectioned, and the endocytosis of fluorescein dextrans into the pronephric tubule was examined by using a Zeiss Axioplan2 microscope (Carl Zeiss Microscopy GmbH, Jena, Germany) (Kramer-Zucker et al, 2005b;Mitra et al, 2012). For the renal clearance assay, tetramethylrhodamine dextran (70,000-kDa; Invitrogen) was injected into the pericardial sac of transiently anesthetized 53-hpf zebrafish embryos, as described previously (Mitra et al, 2012;Rothschild et al, 2011). Embryos were imaged within 20 min of injection and again at 77 hpf.…”

“…Reduction of rhodamine-dextran from the pericardial sac and anterior embryo was examined by using a Zeiss Axiovert 200 inverted microscope and recorded by using the Zeiss AxioVision 3.1 software. The heartbeat of the dye-injected control and sept7b-morphant larvae was measured as previously described (Mitra et al, 2012) and reported as beats per minute.…”