Modelling human lower urinary tract malformations in zebrafish

Abstract

Advances in molecular biology are improving our understanding of the genetic causes underlying human congenital lower urinary tract (i.e., bladder and urethral) malformations. This has recently led to the identification of the first disease-causing variants in the gene BNC2 for isolated lower urinary tract anatomical obstruction (LUTO), and of WNT3 and SLC20A1 as genes implicated in the pathogenesis of the group of conditions called bladder-exstrophy-epispadias complex (BEEC). Implicating candidate genes from human genetic data requires evidence of their influence on lower urinary tract development and evidence of the found genetic variants' pathogenicity. The zebrafish (Danio rerio) has many advantages for use as a vertebrate model organism for the lower urinary tract. Rapid reproduction with numerous offspring, comparable anatomical kidney and lower urinary tract homology, and easy genetic manipulability by Morpholino®-based knockdown or CRISPR/Cas editing are among its advantages. In addition, established marker staining for well-known molecules involved in urinary tract development using whole-mount in situ hybridization (WISH) and the usage of transgenic lines expressing fluorescent protein under a tissue-specific promoter allow easy visualization of phenotypic abnormalities of genetically modified zebrafish. Assays to examine the functionality of the excretory organs can also be modeled in vivo with the zebrafish. The approach of using these multiple techniques in zebrafish not only enables rapid and efficient investigation of candidate genes for lower urinary tract malformations derived from human data, but also cautiously allows transferability of causality from a non-mammalian vertebrate to humans.

Fig. 1

Schematic overview of human and zebrafish urinary tract. A Schematics of human urinary system. Topological subdivisions of the phenotypic complexity of BEEC (red) and LUTO (blue) are depicted. B Overview of zebrafish urinary system. It is composed of 2 nephrons with a pair of glomeruli (bright red) and tubules that can be divided in a proximal and distal segment. The proximal part is subdivided into neck (orange), proximal convoluted (yellow) and proximal straight tubule (green). The distal portion is divided in distal early (blue), corpuscle of Stannius (brown) and distal late (purple). The last segment depicts the pronephric duct (pink) that distally fuses with the cloaca (dark red). C Schematic depiction of cloacal region at 120 hpf. The hindgut (green) opens to the exterior, adjacent to the pronephric duct (pink), both fusing with the cloaca (dark red). Created with BioRender.com

Fig. 2

Zebrafish as a model for developmental lower urinary tract defects. A, B Zebrafish injected with bnc2 Morpholino (MO) frequently develop a pronephric distal outlet obstruction (‘vesicle’; enlargement in B) at 33 hpf compared with controls. C WISH with a pax2a probe relates the bnc2 MO induced pronephric outlet obstruction to distal parts of the pronephric ducts and the cloaca. D At 48 hpf bnc2 was expressed in the terminal section of the pronephric ducts. E Staining for actin (red) and DAPI (blue) allows the visualization of the hindgut (white arrow) and distal pronephric duct (white arrow head) in wild-type zebrafish larvae. F, G Tg(HGj4A) zfl in dorsal view at 33 hpf showing distal pronephric outlet obstruction (asterix) with resulting dilatation of pronephric ducts (indicated by arrow heads) in bnc2 MO zfl (G) compared to zfl injected with control (Ctrl) MO. H, I Proximal pronephric region of Tg(wt1b:GFP) in dorsal view with cystic dilation of glomeruli (arrow heads) and pronephric ducts (asterisks) in bnc2 MO zfl, which recapitulate human hydronephrosis (I). J–M Zfl are depicted after the ingestion of sulforhodamine. Pictures are shown in brightfield (J, K) and fluorescent red channel (L, M). The intestine and cloaca appear regular and ensure excretion of the red dye (asterisk in L) in controls. slc20a1a knockdown impairs excretory function of the cloaca (black arrow in K) and leads to distension of the hindgut. Scale bars: A, B 500 μm (100 μm magnification in B), C, D; F–I 100 μm, E 30 μm, J–M: 50 μm. A–C; F–I Modified from Kolvenbach et al. [15]. J–M Modified from Rieke et al. [17]