Visualizing gene expression during zebrafish pronephros development and regeneration.

The vertebrate kidney is comprised of functional units known as nephrons. Defects in nephron development or activity are a common feature of kidney disease. Current medical treatments are unable to ameliorate the dire consequences of nephron deficit or injury. Although there have been tremendous advancements in our understanding of nephron ontogeny and the response to damage, many significant knowledge gaps still remain. The zebrafish embryo kidney, or pronephros, is an ideal model for many renal development and regeneration studies because it is comprised of nephrons that share conserved features with the nephron units that comprise the mammalian metanephric kidney. In this chapter, we provide an overview about the benefits of using the zebrafish pronephros to study the mechanisms underlying nephrogenesis as well as epithelial repair and regeneration. We subsequently detail methods for the spatiotemporal assessment of gene and protein expression in zebrafish embryos that can be used to extend the understanding of nephron development and disease, and thereby create new opportunities to identify therapeutic strategies for regenerative medicine.

Tfap2a is a novel gatekeeper of nephron differentiation during kidney development.

Renal functional units known as nephrons undergo patterning events during development that create a segmental array of cellular compartments with discrete physiological identities. Here, from a forward genetic screen using zebrafish, we report the discovery that transcription factor AP-2 alpha (tfap2a) coordinates a gene regulatory network that activates the terminal differentiation program of distal segments in the pronephros. We found that tfap2a acts downstream of Iroquois homeobox 3b (irx3b), a distal lineage transcription factor, to operate a circuit consisting of tfap2b, irx1a and genes encoding solute transporters that dictate the specialized metabolic functions of distal nephron segments. Interestingly, this regulatory node is distinct from other checkpoints of differentiation, such as polarity establishment and ciliogenesis. Thus, our studies reveal insights into the genetic control of differentiation, where tfap2a is essential for regulating a suite of segment transporter traits at the final tier of zebrafish pronephros ontogeny. These findings have relevance for understanding renal birth defects, as well as efforts to recapitulate nephrogenesis in vivo to facilitate drug discovery and regenerative therapies.

EpCAM controls morphogenetic programs during zebrafish pronephros development.

Epithelial cell adhesion molecule EpCAM is a transmembrane glycoprotein that is dynamically expressed in human and murine renal epithelia during development. The levels of EpCAM in the renal epithelium are upregulated both during regeneration after ischemia/reperfusion injury and in renal-derived carcinomas. The role of EpCAM in early kidney development, however, has remained unclear. The zebrafish pronephros shows a similar segmentation pattern to the mammalian metanephric nephron, and has recently emerged as a tractable model to study the regulatory programs governing early nephrogenesis. Since EpCAM shows persistent expression in the pronephros throughout early development, we developed a method to study the global changes in gene expression in specific pronephric segments of wild type and EpCAM-deficient zebrafish embryos. In epcam mutants, we found 379 differentially expressed genes. Gene ontology analysis revealed that EpCAM controls various developmental programs, including uretric bud development, morphogenesis of branching epithelium, regulation of cell differentiation and cilium morphogenesis.

ISL1 is a major susceptibility gene for classic bladder exstrophy and a regulator of urinary tract development.

Previously genome-wide association methods in patients with classic bladder exstrophy (CBE) found association with ISL1, a master control gene expressed in pericloacal mesenchyme. This study sought to further explore the genetics in a larger set of patients following-up on the most promising genomic regions previously reported. Genotypes of 12 markers obtained from 268 CBE patients of Australian, British, German Italian, Spanish and Swedish origin and 1,354 ethnically matched controls and from 92 CBE case-parent trios from North America were analysed. Only marker rs6874700 at the ISL1 locus showed association (p = 2.22 × 10-08). A meta-analysis of rs6874700 of our previous and present study showed a p value of 9.2 × 10-19. Developmental biology models were used to clarify the location of ISL1 activity in the forming urinary tract. Genetic lineage analysis of Isl1-expressing cells by the lineage tracer mouse model showed Isl1-expressing cells in the urinary tract of mouse embryos at E10.5 and distributed in the bladder at E15.5. Expression of isl1 in zebrafish larvae staged 48 hpf was detected in a small region of the developing pronephros. Our study supports ISL1 as a major susceptibility gene for CBE and as a regulator of urinary tract development.

Gulo Acts as a de novo Marker for Pronephric Tubules in Xenopus laevis.

BACKGROUNDS/AIMS: Vitamin C is an antioxidant and acts as a cofactor for several key enzymatic catalytic reactions in animals. Amphibians produce vitamin C in their kidneys, as opposed to mammals that produce vitamin C in their liver. Gulo serves as a crucial enzyme for vitamin C synthesis in mammals, but the characteristics and localization of its homologous genes during kidney development in Xenopus laevis, an amphibian, remains unknown. METHODS: We aligned amino acid sequences of Gulo across different species by using bioinformatics methods and detected patterns of expression for Gulo during kidney development by using RT-PCR and in situ hybridization. RESULTS: We identified a new site on the X. laevis genome, LOC495407. Sequence alignment analysis indicated this fragment is highly conserved and homologous to gulo genes in mammals. RT-PCR and in situ hybridization results reveal that X. laevis gulo is maternally expressed during the early stages of embryonic development, particularly, in the tubules of the pronephros from the middle tail-bud stage and onward in embryos. CONCLUSION: Gulo is a novel specific marker for pronephros tubules in X. laevis, and may be used as a potential marker for kidney development studies and disease diagnosis in mammals.

Function and Regulation of the Wilms' Tumor Suppressor 1 (WT1) Gene in Fish.

The Wilms' tumor suppressor gene Wt1 is highly conserved among vertebrates. In contrast to mammals, most fish species possess two wt1 paralogs that have been named wt1a and wt1b. Concerning wt1 in fish, most work so far has been done using zebrafish, focusing on the embryonic kidney, the pronephros. In this chapter we will describe the structure and development of the pronephros as well as the role that the wt1 genes play in the embryonic zebrafish kidney. We also discuss Wt1 target genes and describe the potential function of the Wt1 proteins in the adult kidney. Finally we will summarize data on the role of Wt1 outside of the kidney.

Zebrafish kidney development.

The kidney of the zebrafish shares many features with other vertebrate kidneys including the human kidney. Similar cell types and shared developmental and patterning mechanisms make the zebrafish pronephros a valuable model for kidney organogenesis. Here we review recent advances in studies of zebrafish pronephric development and provide experimental protocols to analyze kidney cell types and structures, measure nephron function, live image kidney cells in vivo, and probe mechanisms of kidney regeneration after injury.

The Rho-GTPase binding protein IQGAP2 is required for the glomerular filtration barrier.

Podocyte dysfunction impairs the size selectivity of the glomerular filter, leading to proteinuria, hypoalbuminuria, and edema, clinically defined as nephrotic syndrome. Hereditary forms of nephrotic syndrome are linked to mutations in podocyte-specific genes. To identify genes contributing to podocyte dysfunction in acquired nephrotic syndrome, we studied human glomerular gene expression data sets for glomerular-enriched gene transcripts differentially regulated between pretransplant biopsy samples and biopsies from patients with nephrotic syndrome. Candidate genes were screened by in situ hybridization for expression in the zebrafish pronephros, an easy-to-use in vivo assay system to assess podocyte function. One glomerulus-enriched product was the Rho-GTPase binding protein, IQGAP2. Immunohistochemistry found a strong presence of IQGAP2 in normal human and zebrafish podocytes. In zebrafish larvae, morpholino-based knockdown of iqgap2 caused a mild foot process effacement of zebrafish podocytes and a cystic dilation of the urinary space of Bowman's capsule upon onset of urinary filtration. Moreover, the glomerulus of zebrafish morphants showed a glomerular permeability for injected high-molecular-weight dextrans, indicating an impaired size selectivity of the glomerular filter. Thus, IQGAP2 is a Rho-GTPase binding protein, highly abundant in human and zebrafish podocytes, which controls normal podocyte structure and function as evidenced in the zebrafish pronephros.

Heat shock 70-kDa protein 5 (Hspa5) is essential for pronephros formation by mediating retinoic acid signaling.

Heat shock 70-kDa protein 5 (Hspa5), also known as binding immunoglobulin protein (Bip) or glucose-regulated protein 78 (Grp78), belongs to the heat shock protein 70 kDa family. As a multifunctional protein, it participates in protein folding and calcium homeostasis and serves as an essential regulator of the endoplasmic reticulum (ER) stress response. It has also been implicated in signal transduction by acting as a receptor or co-receptor residing at the plasma membrane. Its function during embryonic development, however, remains largely elusive. In this study, we used morpholino antisense oligonucleotides (MOs) to knock down Hspa5 activity in Xenopus embryos. In Hspa5 morphants, pronephros formation was strongly inhibited with the reduction of pronephric marker genes Lim homeobox protein 1 (lhx1), pax2, and ß1 subunit of Na/K-ATPase (atp1b1). Pronephros tissue was induced in vitro by treating animal caps with all-trans-retinoic acid and activin. Depletion of Hspa5 in animal caps, however, blocked the induction of pronephros as well as reduced the expression of retinoic acid (RA)-responsive genes, suggesting that knockdown of Hspa5 attenuated RA signaling. Knockdown of Hspa5 in animal caps resulted in decreased expression of lhx1, a transcription factor directly regulated by RA signaling and essential for pronephros specification. Co-injection of Hspa5MO with lhx1 mRNA partially rescued the phenotype induced by Hspa5MO. These results suggest that the RA-Lhx1 signaling cascade is involved in Hspa5MO-induced pronephros malformation. This study shows that Hspa5, a key regulator of the unfolded protein response, plays an essential role in pronephros formation, which is mediated in part through RA signaling during early embryonic development.

Neph3 associates with regulation of glomerular and neural development in zebrafish.

Neph3 (filtrin) is a membrane protein expressed in the glomerular epithelial cells (podocytes), but its role in the glomerulus is still largely unknown. To characterize the function of Neph3 in the glomerulus, we employed the zebrafish as a model system. Here we show that the expression of neph3 in pronephros starts before the onset of nephrin and podocin expression, peaks when the nephron primordium differentiates into glomerulus and tubulus, and is then downregulated upon glomerular maturation. By histology, we found that neph3 is specifically expressed in pronephric podocytes at 36hpf. Furthermore, disruption of neph3 expression by antisense morpholino oligonucleotides results in distorted body curvature and transient pericardial edema, the latter likely reflecting perturbation of glomerular osmoregulatory function. Histological analysis of neph3 morphants reveals altered glomerular morphology and dilated pronephric tubules. The phenotype of neph3 morphants, curved body and pericardial edema, is rescued by wild-type zebrafish neph3 mRNA. In addition to glomerulus, neph3 is highly expressed in the developing brain and specific regions of mature midbrain and hindbrain. In line with this, neph3 morphants show aberrant brain morphology. Collectively, the expression of neph3 in glomerulus and brain together with the morphant phenotype imply that neph3 is a pleiotropic gene active during distinct stages of tissue differentiation and associates directly in the regulation of both glomerular and neural development.