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.

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).

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 ±-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).

crb2b morphants show defects in podocyte morphology. (A) Electron micrograph of a podocyte from control 4 dpf larvae shows the regular patterned array of foot processes along the GBM; GBM (black arrow) and slit diaphragms (black arrowheads). (B) In crb2b morphants, podocytes are attached to a GBM (black arrow), but the organization of regularly spaced foot processes is lost (white arrows show foot process fusion). Slit diaphragms are absent. Endothelial fenestrations are reduced in frequency. Nephrin protein is apically mis-localized in crb2b morphant podocytes. (C) Staining with α-Nephrin antibody shows that Nephrin protein is present (arrowheads) only along the surface of the GBM (visualized with WGA FITC). (D, E) In crb2b morphants, Nephrin protein is present throughout the podocytes with punctate staining (arrowheads) distal to the GBM. Nuclei visualized with DAPI; scale bar, 5 μm. (F) Immuno-electron microscopy with the α-Nephrin antibody in crb2b MO shows a podocyte with an apical membrane projection. White arrows indicate the GBM. (G) A magnification of the tip of the membrane projection indicated by the arrowhead in D. Nephrin protein is associated with the inner aspect of the plasma membrane (gold particles indicated by black arrowheads in E) within ectopic projections extending from podocyte apical membranes.

crb2b expression in the pronephric glomerulus. (A, B) Wholemount in situ hybridization reveals crb2b expression in the glomerular podocytes and retina at 4 dpf and 2 dpf (G). Arrows point to the podocyte expression. (C, D, E) DIC images of transverse sections through a 4 dpf larval glomerulus stained with the crb2b antisense in situ probe. Notice that crb2b expression is not observed in the tubular epithelia (arrowheads; n, notochord). (F) Transverse section through a 4 dpf glomerulus double stained with α-panCrb (green) and α-ZO-1 (red) antibodies. Nuclei are stained with DAPI.

Tight junctions persist in crb2b MO. (A) Electron micrographs of podocytes from control 4 dpf larvae shows the cell bodies, primary processes (asterisk), and the regular patterned array of foot processes along the GBM; GBM (black arrows). (B, C) In crb2b morphants, podocytes are attached to a GBM (black arrow in C), but the organization of regularly spaced foot processes is lost. Slit diaphragms are absent. Ectopic tight junctions are visible in morphant podocytes (white arrows in B and C). In addition, blunt ended apical membrane projections are found extending into the Bowman′s space (black arrowheads in B and C). A, B 20,000x magnification; C 40,000x magnification.

Serial reconstruction of a podocyte from a crb2b morphant. (AH) A podocyte (P) sits on top of a capillary loop (cap) and makes contact with the GBM. Apical membrane protrusions (white arrowheads) are observed which appear disorganized in structure and branch into the urinary space. When this protrusion makes contact (black arrowhead) with adjacent GBM, it appears to form foot process like structures. Ultrathin sections taken every 70 nm. (I) Higher magnification of panel F. (J) Quantification of apical projection defects in crb2b MO and controls. Scale bars, 500 nm.

Overall polarity is maintained in crb2b MO podocytes. Immunofluorescence staining of 4 dpf control (A, transverse glomerular section and A1–A4, individual podocytes) and crb2b MO (B, transverse glomerular section and B1–B4, individual podocytes) glomeruli with α-acetylated Tubulin, which labels the microtubule rich podocyte primary processes. Individual podocytes are chosen as examples. Primary processes are found lateral and basal to the podocyte nuclei, but apical to the GBM. Podocytes are identified by virtue of their unique position on the outer aspect of the capillary loops with their cell bodies in the Bowman′s space. The inset figure in A schematizes the relative positions of nucleus (blue), primary processes (red), foot processes (not stained, white), and GBM (green). Nuclei stained with DAPI. GBM stained with WGA FITC. Scale bars, 2 μM.

(A) Rabbit polyclonal antibodies directed against zebrafish Nephrin specifically recognize a single approximately protein in agreement with its predicted 140 ka size. Cell lysates from pcDNA3.1 (vector only, P) and zebrafish nephrin transfected HEK-293 cells, N. (B) Immuno-electron micrograph on wildtype podocytes with α-Nephrin antibody. Gold particles are found on the inner aspect of the slit diaphragms (arrowheads).

ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

Unillustrated author statements