Frank Bollig

Identification of adult nephron progenitors capable of kidney regeneration in zebrafish

Loss of kidney function underlies many renal diseases. Mammals can partly repair their nephrons (the functional units of the kidney), but cannot form new ones. By contrast, fish add nephrons throughout their lifespan and regenerate nephrons de novo after injury, providing a model for understanding how mammalian renal regeneration may be therapeutically activated. Here we trace the source of new nephrons in the adult zebrafish to small cellular aggregates containing nephron progenitors. Transplantation of single aggregates comprising 10–30 cells is sufficient to engraft adults and generate multiple nephrons. Serial transplantation experiments to test self-renewal revealed that nephron progenitors are long-lived and possess significant replicative potential, consistent with stem-cell activity. Transplantation of mixed nephron progenitors tagged with either green or red fluorescent proteins yielded some mosaic nephrons, indicating that multiple nephron progenitors contribute to a single nephron. Consistent with this, live imaging of nephron formation in transparent larvae showed that nephrogenic aggregates form by the coalescence of multiple cells and then differentiate into nephrons. Taken together, these data demonstrate that the zebrafish kidney probably contains self-renewing nephron stem/progenitor cells. The identification of these cells paves the way to isolating or engineering the equivalent cells in mammals and developing novel renal regenerative therapies.

Scribble participates in Hippo signaling and is required for normal zebrafish pronephros development

Spatial organization of cells and their appendages is controlled by the planar cell polarity pathway, a signaling cascade initiated by the protocadherin Fat in Drosophila. Vertebrates express 4 Fat molecules, Fat1–4. We found that depletion of Fat1 caused cyst formation in the zebrafish pronephros. Knockdown of the PDZ domain containing the adaptor protein Scribble intensified the cyst-promoting phenotype of Fat1 depletion, suggesting that Fat1 and Scribble act in overlapping signaling cascades during zebrafish pronephros development. Supporting the genetic interaction with Fat1, Scribble recognized the PDZ-binding site of Fat1. Depletion of Yes-associated protein 1 (YAP1), a transcriptional co-activator inhibited by Hippo signaling, ameliorated the cyst formation in Fat1-deficient zebrafish, whereas Scribble inhibited the YAP1-induced cyst formation. Thus, reduced Hippo signaling and subsequent YAP1 disinhibition seem to play a role in the development of pronephric cysts after depletion of Fat1 or Scribble. We hypothesize that Hippo signaling is required for normal pronephros development in zebrafish and that Scribble is a candidate link between Fat and the Hippo signaling cascade in vertebrates.

Characterization of mesonephric development and regeneration using transgenic zebrafish

The zebrafish is a valuable vertebrate model for kidney research. The majority of previous studies focused on the zebrafish pronephros, which comprises only two nephrons and is structurally simpler than the mesonephros of adult fish and the metanephros of mammals. To evaluate the zebrafish system for more complex studies of kidney development and regeneration, we investigated the development and postinjury regeneration of the mesonephros in adult zebrafish. Utilizing two transgenic zebrafish lines (wt1b::GFP and pod::NTR-mCherry), we characterized the developmental stages of individual mesonephric nephrons and the temporal-spatial pattern of mesonephrogenesis. We found that mesonephrogenesis continues throughout the life of zebrafish, with a rapid growth phase during the juvenile period and a slower phase in adulthood such that the total nephron number of juvenile and adult fish linearly correlates with body mass. Following gentamicin-induced renal injury, the zebrafish mesonephros can undergo de novo regeneration of mesonephric nephrons, a process known as neonephrogenesis. We found that wt1b expression was induced in individually dispersed cells in the mesonephric interstitium as early as 48 h following injury. These wt1b-expressing cells formed aggregates by 72-96 h following injury which proceeded to form nephrons. This suggests that wt1b may serve as an early marker of fated renal progenitor cells. The synchronous nature of regenerative neonephrogenesis suggests that this process may be useful for studies of nephron development.

A Novel Wilms Tumor 1 (WT1) Target Gene Negatively Regulates the WNT Signaling Pathway

Mammalian kidney development requires the functions of the Wilms tumor gene WT1 and the WNT/β-catenin signaling pathway. Recent studies have shown that WT1 negatively regulates WNT/β-catenin signaling, but the molecular mechanisms by which WT1 inhibits WNT/β-catenin signaling are not completely understood. In this study, we identified a gene, CXXC5, which we have renamed WID (WT1-induced Inhibitor of Dishevelled), as a novel WT1 transcriptional target that negatively regulates WNT/β-catenin signaling. WT1 activates WID transcription through the upstream enhancer region. In the developing kidney, Wid and Wt1 are coexpressed in podocytes of maturing nephrons. Structure-function analysis demonstrated that WID interacts with Dishevelled via its C-terminal CXXC zinc finger and Dishevelled binding domains and potently inhibits WNT/β-catenin signaling in vitro and in vivo. WID is evolutionarily conserved, and ablation of wid in zebrafish embryos with antisense morpholino oligonucleotides perturbs embryonic kidney development. Taken together, our results demonstrate that the WT1 negatively regulates WNT/β-catenin pathway via its target gene WID and further suggest a role for WID in nephrogenesis.

Cofilin-1 Inactivation Leads to Proteinuria – Studies in Zebrafish, Mice and Humans

Background Podocytes are highly specialized epithelial cells on the visceral side of the glomerulus. Their interdigitating primary and secondary foot processes contain an actin based contractile apparatus that can adjust to changes in the glomerular perfusion pressure. Thus, the dynamic regulation of actin bundles in the foot processes is critical for maintenance of a well functioning glomerular filtration barrier. Since the actin binding protein, cofilin-1, plays a significant role in the regulation of actin dynamics, we examined its role in podocytes to determine the impact of cofilin-1 dysfunction on glomerular filtration. Methods and Findings We evaluated zebrafish pronephros function by dextran clearance and structure by TEM in cofilin-1 morphant and mutant zebrafish and we found that cofilin-1 deficiency led to foot process effacement and proteinuria. In vitro studies in murine and human podocytes revealed that PMA stimulation induced activation of cofilin-1, whereas treatment with TGF-β resulted in cofilin-1 inactivation. Silencing of cofilin-1 led to an accumulation of F-actin fibers and significantly decreased podocyte migration ability. When we analyzed normal and diseased murine and human glomerular tissues to determine cofilin-1 localization and activity in podocytes, we found that in normal kidney tissues unphosphorylated, active cofilin-1 was distributed throughout the cell. However, in glomerular diseases that affect podocytes, cofilin-1 was inactivated by phosphorylation and observed in the nucleus. Conclusions Based on these in vitro and in vivo studies we concluded cofilin-1 is an essential regulator for actin filament recycling that is required for the dynamic nature of podocyte foot processes. Therefore, we describe a novel pathomechanism of proteinuria development.

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

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.

Sipl1 and Rbck1 Are Novel Eya1-Binding Proteins with a Role in Craniofacial Development

ABSTRACT The eyes absent 1 protein (Eya1) plays an essential role in the development of various organs in both invertebrates and vertebrates. Mutations in the human EYA1 gene are linked to BOR (branchio-oto-renal) syndrome, characterized by kidney defects, hearing loss, and branchial arch anomalies. For a better understanding of Eya1s function, we have set out to identify new Eya1-interacting proteins. Here we report the identification of the related proteins Sipl1 (Shank-interacting protein-like 1) and Rbck1 (RBCC protein interacting with PKC1) as novel interaction partners of Eya1. We confirmed the interactions by glutathione S-transferase (GST) pulldown analysis and coimmunoprecipitation. A first mechanistic insight is provided by the demonstration that Sipl1 and Rbck1 enhance the function of Eya proteins to act as coactivators for the Six transcription factors. Using reverse transcriptase PCR (RT-PCR) and in situ hybridization, we show that Sipl1 and Rbck1 are coexpressed with Eya1 in several organs during embryogenesis of both the mouse and zebrafish. By morpholino-mediated knockdown, we demonstrate that the Sipl1 and Rbck1 orthologs are involved in different aspects of zebrafish development. In particular, knockdown of one Sipl1 ortholog as well as one Rbck1 ortholog led to a BOR syndrome-like phenotype, with characteristic defects in ear and branchial arch formation.

An Anti-Phospholipase A2 Receptor Quantitative Immunoassay and Epitope Analysis in Membranous Nephropathy Reveals Different Antigenic Domains of the Receptor

The phospholipase A2 receptor (PLA2R) was recently discovered as a target autoantigen in patients with idiopathic membranous nephropathy (IMN). Published evidence suggests that the autoantibodies directed towards a conformation dependent epitope are currently effectively detected by a cell based assay (CBA) utilizing indirect immunofluorescence (IIF) on tissue culture cells transfected with the PLA2R cDNA. Limitations of such IIF-CBA assays include observer dependent subjective evaluation of semi-quantitative test results and the protocols are not amenable to high throughput diagnostic testing. We developed a quantitative, observer independent, high throughput capture immunoassay for detecting PLA2R autoantibodies on an addressable laser bead immunoassay (ALBIA) platform. Since reactive domains of PLA2R (i.e. epitopes) could be used to improve diagnostic tests by using small peptides in various high throughput diagnostic platforms, we identified PLA2R epitopes that bound autoantibodies of IMN patients. These studies confirmed that inter-molecular epitope spreading occurs in IMN but use of the cognate synthetic peptides in immunoassays was unable to conclusively distinguish between IMN patients and normal controls. However, combinations of these peptides were able to effectively absorb anti-PLA2R reactivity in IIF-CBA and an immunoassay that employed a lysate derived from HEK cells tranfected with and overexpressing PLA2R. While we provide evidence of intermolecular epitope spreading, our data indicates that in addition to conformational epitopes, human anti-PLA2R reactivity in a commercially available CBA and an addressable laser bead immunoassay is significantly absorbed by peptides representing epitopes of PLA2R.

Kcnh1 voltage-gated potassium channels are essential for early zebrafish development

Background: Kcnh1 is a voltage-gated potassium channel that is primarily expressed in brain. Results: Knockdown of kcnh1 in zebrafish delays neural development and causes embryonic lethality. Conclusion: Kcnh1 is involved in cell proliferation during early zebrafish development. Significance: The finding that Kcnh1 has basic functions beyond neural signaling will help to elucidate its roles in physiology and cancer formation. The Kcnh1 gene encodes a voltage-gated potassium channel highly expressed in neurons and involved in tumor cell proliferation, yet its physiological roles remain unclear. We have used the zebrafish as a model to analyze Kcnh1 function in vitro and in vivo. We found that the kcnh1 gene is duplicated in teleost fish (i.e. kcnh1a and kcnh1b) and that both genes are maternally expressed during early development. In adult zebrafish, kcnh1a and kcnh1b have distinct expression patterns but share expression in brain and testis. Heterologous expression of both genes in Xenopus oocytes revealed a strong conservation of characteristic functional properties between human and fish channels, including a unique sensitivity to intracellular Ca2+/calmodulin and modulation of voltage-dependent gating by extracellular Mg2+. Using a morpholino antisense approach, we demonstrate a strong kcnh1 loss-of-function phenotype in developing zebrafish, characterized by growth retardation, delayed hindbrain formation, and embryonic lethality. This late phenotype was preceded by transcriptional up-regulation of known cell-cycle inhibitors (p21, p27, cdh2) and down-regulation of pro-proliferative factors, including cyclin D1, at 70% epiboly. These results reveal an unanticipated basic activity of kcnh1 that is crucial for early embryonic development and patterning.

Knockdown of the Hypertension-Associated Gene NOSTRIN Alters Glomerular Barrier Function in Zebrafish (Danio rerio)

Hypertension is one of the major risk factors for chronic kidney disease. Using quantitative trait loci analysis, we identified the gene of the F-BAR protein NOSTRIN in the center of an overlapping region in rat and human quantitative trait loci that are associated with hypertension. Immunohistochemical analysis revealed a predominantly podocytic expression pattern of NOSTRIN in human and mouse glomeruli. Further, NOSTRIN colocalizes with cell–cell contact–associated proteins &bgr;-catenin and zonula occludens-1 and interacts with the slit-membrane–associated adaptor protein CD2AP. In zebrafish larvae, knockdown of nostrin alters the glomerular filtration barrier function, inducing proteinuria and leading to ultrastructural morphological changes on the endothelial and epithelial side and of the glomerular basement membrane of the glomerular capillary loop. We conclude that NOSTRIN expression is an important factor for the integrity of the glomerular filtration barrier. Disease-related alteration of NOSTRIN expression may not only affect the vascular endothelium and, therefore, contribute to endothelial cell dysfunction but might also contribute to the development of podocyte disease and proteinuria.