Modelling human lower urinary tract malformations in zebrafish

The zebrafish has emerged as a promising non-mammalian vertebrate model for studying LUTMs. Key advantages include large numbers of offspring with rapid ex utero development and anatomic structures with similarity to the human developing urinary tract. The embryonic kidney, also called pronephros, forms by patterning of intermediate mesoderm [15, 16]. The anterior intermediate mesoderm develops into two filtering glomeruli fused at the midline (Fig. 1B) [15, 16]. Each is linked to a pronephric tubule, connecting the glomeruli to the pronephric ducts, that distally fuse at the cloaca opening, forming around 24–30 h post-fertilization (hpf) (Fig. 1B-C). At 48 hpf, the hindgut also inserts into the cloaca, which only opens for excretion at around 120 hpf (Fig. 1C) [15, 16]. Zebrafish do not have a urinary bladder for urine storage, but similar anatomic excretion surrogates, such as the distal pronephric ducts and cloaca, that allow the analysis of the developing lower urinary tract (Fig. 1B-C) [14, 18]. For instance, the terminal end of the pronephros represents a putative homolog to the human Wolffian duct that inserts into the bladder [14, 18]. Similarly, the cloaca ensures urine outflow corresponding to the human urethra. Defects of the cloaca have been previously successfully modeled in zebrafish [19‐22]. BEEC and LUTO likely arise from abnormal development of the cloaca, making zebrafish a suitable model for analyzing pathomechanisms underlying the diseases. Another key advantage of zebrafish includes easy and fast genetic modulation. There is evidence for extensive evolutionary conservation between human and zebrafish genomes with 84% of human genes associated with human disease having a zebrafish ortholog [23]. Similarly, Drosophila (D.) melanogaster and Caenorhabditis (C.) elegans, two other valuable non-mammalian model organisms are also characterized by simple genetic manipulation, but only share 77% (D. melanogaster) [24] and 65% (C. elegans) [25] of matching disease genes. Modeling of human genetic disease in zebrafish can be achieved by using Morpholino®-based knockdown and CRISPR/Cas genome editing. The term Morpholino refers to an oligomer molecule, inhibiting formation of the translation-initiation or splicing complex, thereby knocking down gene function [26]. CRISPR/Cas uses guide RNA that recognizes complementary DNA sequences, signaling the Cas9 to cut the double-stranded DNA at the targeted location [27]. Non-homologous end joining cellular repair mechanisms introduce insertions or deletions, resulting in gene knock-out through genomic disruption [27]. Previous studies have shown the utility of transient suppression models or stable mutants for testing disease relevance in a very short time period as compared with mammalian vertebrate models [15, 28]. In our recent study, we showed that CRISPR/Cas F0 mosaic mutants or Morpholino knockdown of bnc2 in zebrafish results in a distal pronephric outlet obstruction, illustrated as a ‘vesicle’, phenocopying the human anatomical LUTO phenotype (Fig. 2A-B) [15]. Furthermore, Morpholino-based knockdown of the zebrafish orthologs wnt3 and slc20a1a caused expansion of the cloacal lumen, implicating their important role in urinary tract development and potential involvement in BEEC formation [14, 17]. In addition, we used the zebrafish model to determine the pathogenicity of variants identified in our LUTO cases with variants in BNC2 [15]. Together with bnc2 Morpholino, knocking down endogenous bnc2 zebrafish RNA, human BNC2 RNA bearing identified variants was co-injected into zebrafish embryos in one-cell stage. Human BNC2 RNA lacks the Morpholino-binding site and is thereby not detected by the Morpholino. No amelioration of the described LUTO-like phenotype was observed in contrast to co-injections of wild-type human BNC2 RNA together with bnc2 Morpholino [15]. The pathogenicity of the tested variants can therefore be assumed. Alternatively, patient-specific variants can be introduced applying CRISPR/Cas9 (knock-in) requiring a donor template for homology-directed repair in addition to the Cas9 enzyme and guide RNA [29]. The emergence of a phenotype corroborates the pathogenicity of the targeted variant.

Zebrafish larvae (zfl) are nearly transparent, which allows direct examination and visualization of developing structures under the microscope. Their transparency also facilitates staining of designated tissue. Whole-mount in situ hybridization (WISH) using RNA probes to detect the mRNA of interest, can be applied for locating gene expression [30]. The transcription factors pax2a and evx1 are established WISH marker molecules that highlight the developing distal pronephric and cloacal component structures, responsible for urine excretion [22]. Abnormalities during the cloaca-forming process can thus be easily detected using WISH in zebrafish. Staining against pax2a in our bnc2 knockdown zebrafish indicated the intimate relation of the ‘vesicle’ with the distal pronephric duct at the cloacal opening (Fig. 2C) [15]. Moreover, WISH serves as a powerful tool for studying the expression of potential new disease genes. Specific probes for the gene of interest can be easily invoked to depict location-specific expression at the critical time points of urinary tract development. In this manner, the expression of bnc2 was detected in developing cloacal and pronephric duct tissue (Fig. 2D) [15, 17]. Similarly, phalloidin staining can be employed to visualize cytoskeleton actin filaments and by this to analyze cloaca morphogenesis (Fig. 2E) [14, 19, 31]. This allows the evaluation of cellular organization of the cloacal epithelium and size of the lumen of the distal pronephros and hindgut inserting into the cloaca (Fig. 2E). Using this method, Baraknowska Korberg et al. [14] detected cloacal expansion and cellular disorganization potentially arising from cloacal obstruction in wnt3 knockdown zebrafish morphants. Phenotypic anomalies can be simply visualized with transgenic lines expressing fluorescent protein under a tissue-specific promoter in the nearly transparent zfl. For example, the mnr2b/hlxb9lb enhancer trap line Tg(HGj4A) expressing green fluorescent protein (GFP) in the developing distal pronephric region has been used to illustrate the distal pronephric outlet obstruction in vivo after bnc2 Morpholino knockdown (Fig. 2F–G) [15, 32]. Furthermore, Tg(wt1b:GFP) zfl harboring GFP under a Wilms Tumor 1b gene promoter can be conveniently screened for renal and tubular dilatations [33], which occurred in genetic models for bnc2 and slc20a1a, strongly supporting the hypothesis that the observed cloacal malformations lead to a blockage of urine outflow (Fig. 2H-I) [15, 17]. This observed upper urinary tract phenotype in zebrafish might resemble hydronephrosis seen in individuals with LUTMs due to obstruction.

As a useful functional assessment to determine the in vivo functionality of the hindgut and cloaca, a sulforhodamine excretion-assay can be applied [17, 31]. Here, zfl are bathed in sulforhodamine, a red fluorescent dye, for several hours at day 5 post-fertilization. They ingest and excrete it in intervals, allowing to observe normal or abnormal cloacal physiology (Fig. 2J–M). In the slc20a1a knockdown zebrafish model for BEEC, normal morphology of the intestine and regular peristalsis was observed, but cloacal opening seemed to be impaired shown by dilation of the hindgut and cloaca (Fig. 2J–M) [17].

The benefits of the zebrafish model have also been demonstrated in the evaluation of candidate genes for upper urinary tract abnormalities. For example, Brophy et al. [34] and Sanna-Cherchi et al. [35] showed that greb1l, the human ortholog GREB1L being a candidate disease gene for renal hypoplasia, is required for normal pronephros morphogenesis in zebrafish. Suppression of greb1l resulted in proximal pronephric defects, recapitulating the human phenotype. Again, this model was also successfully used, to determine the pathogenicity of discovered human missense variants, making the zebrafish a suitable model for congenital kidney and upper urinary tract malformations.

Zebrafish lack human anatomic structures such as genitalia and urinary bladder, making it a potentially distant model for a subset of human urinary tract phenotypes. Nevertheless, they have been successfully used to model several human anatomical urinary tract malformations, phenocopying human anomalies caused by genetic alterations [14, 15, 17]. However, modelling functional LUTO probably requires a mammalian model with the muscularized/innervated bladder. Other limitations may include the lack of expression of a candidate gene in the urinary tract of the zebrafish, requiring its characterization in a mammalian model. However, the zebrafish lower urinary tract, namely the distal pronephric ducts and cloaca, provides a powerful, model system to test a genetic hypothesis generated for certain candidate genes from human data so far. The approach of using these multiple techniques does not only enable rapid and efficient investigation of candidate genes for LUTMs, but also cautiously allows transferability of causality from the non-mammalian vertebrate zebrafish model to humans.