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Dev Biol
2009 Oct 01;3341:1-9. doi: 10.1016/j.ydbio.2009.04.017.
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A reverse genetic screen in the zebrafish identifies crb2b as a regulator of the glomerular filtration barrier.
Ebarasi L
,
He L
,
Hultenby K
,
Takemoto M
,
Betsholtz C
,
Tryggvason K
,
Majumdar A
.
Abstract
The glomerular filtration barrier is necessary for the selective passage of low molecular weight waste products and the retention of blood plasma proteins. Damage to the filter results in proteinuria. The filtration barrier is the major pathogenic site in almost all glomerular diseases and its study is therefore of clinical significance. We have taken advantage of the zebrafish pronephros as a system for studying glomerular filtration. In order to identify new regulators of filtration barrier assembly, we have performed a reverse genetic screen in the zebrafish testing a group of genes which are enriched in their expression within the mammalian glomerulus. In this novel screen, we have coupled gene knockdown using morpholinos with a physiological glomerular dye filtration assay to test for selective glomerular permeability in living zebrafish larvae. Screening 20 genes resulted in the identification of ralgps1, rapgef2, rabgef1, and crb2b. The crumbs (crb) genes encode a family of evolutionarily conserved proteins important for apical-basal polarity within epithelia. The crb2b gene is expressed in zebrafish podocytes. Electron microscopic analysis of crb2b morphants reveals a gross disorganization of podocytefoot process architecture and loss of slit diaphragms while overall polarity is maintained. Nephrin, a major component of the slit diaphragm, is apically mis-localized in podocytes from crb2b morphants suggesting that crb2b is required for the proper protein trafficking of Nephrin. This report is the first to show a role for crb function in podocyte differentiation. Furthermore, these results suggest a novel link between epithelial polarization and the maintenance of a functional filtration barrier.
Fig. 1. Glomerular dye filtration assays on morphants. (A) Control morpholino injected 4 dpf larvae show no uptake of the 500 kDa FITC dextran dye into pronephric tubules (circled by white line). Dye is present within the dorsal aorta lumen (asterisk). In the inset, a schematic of a transverse section shows the relative positions of the somites (S), notochord (N), and pronephric tubules (T). (B) In crb2b morphants, the 500 kDa FITC dye is taken up into endosomes in the pronephric tubule epithelial cells (arrowheads). Similar uptake of the 500 kDa FITC dextran tracer is observed in ralgps1(C), rabgef1 (D), rapgef2 (E) morphants, but not in ga17 morphants (F), which tested negative in this assay. Nuclei are labeled with DAPI. Scale bar, 20 μm.
Fig. 2. Morpholino knockdown. (A) Penetrance of morphant phenotypes using crb2b-ATG and crb2b-SP morpholinos. Co-injection with wildtype crb2b mRNA rescues the morphant defects. A mismatch morpholino based on crb2b-SP does not induce pericardial edema or pronephric cyst phenotypes. Sample sizes are shown above the bars. (B) Exon structure of crb2b and position of crb2b-ATG and crb2b-SP morpholinos. PCR primers are shown (arrows). (C) RTPCR of crb2b mRNA from mismatch control injected embryos yields the expected 1569 bp product. RTPCR of crb2b mRNA from crb2b-SP embryos yields an expected 1079 bp band, resulting from the deletion of Exon 9, and an additional product of 1811 bp. Glomerular α-panCrb staining observed in mismatch injected embryos (D) is lost in embryos injected with crb2b-ATG (or crb2b-SP) (E). (F, G) crb2b-ATG and crb2b-SP induce glomerular permeability defects as assayed by the re-uptake of 500 kDa FITC dextran dye into pronephric tubule endosomes. (H) The lower 1079 band results from the deletion of exon 9 and the fusion of exon 8 (black) to exon 10 (pink). The 1811 bp upper band resulted from the insertion of 242 bp of intronic sequences (blue) downstream of the morpholino target sequence. Translation of intronic sequences results in premature stop codons (asterisk).
Fig. 3. Phenotype of the crb2b morphants. (A) Control mismatch and (B) crb2b-ATG injected 2.5 dpf larvae. Note the pericardial edema (arrowhead) and pronephric cysts (arrow). (C, D) Histological sections at the level of the glomerulus in control (C) and crb2b-ATG MO (D). Note the expanded Bowman's space (asterisk) in D. (E, F) Staining with α-Na+/K+ ATPase in the pronephric tubules and ducts is not generally affected in crb2b MO. (G–L) In situ hybridization on 2 dpf wildtype (G, I, K) or crb2b-ATG (H, J, L) with wt1 (G, H), podocin (I, J), or nephrin (K, L) antisense probes. Cells expressing wt1, nephrin, and podocin are present in crb2b morphants (black arrowheads).
