Fig 1: nup133 knockdown induces moderate foot processes effacement. (a,d) Transmission electron micrographs showing the glomerular capillary wall of 5 dpf uninjected control (a) and nup133 MO injected larvae (d). (b,e) Two-fold magnification of the areas indicated in (a,d), and (c,f,h) representative images from distinct larvae. Images were pseudocolored to better highlight the glomerular filtration barrier components (podocyte foot processes in green, fenestrated endothelium in blue). Arrows point to irregular-shaped foot processes that are more frequently found in nup133 MO larvae. (g) Quantification of foot processes (FP) density along the basement membrane, presented as average number of FP/µm, was performed as described under materials and methods. The distinct labels reflect values measured on the 3 wt or morphant embryos. Statistical analyses were performed using Wilcoxon-Mann-Whitney Rank Sum Test. **P = 0.01 (p = 0.002486). Image (h) corresponds to a basement membrane surrounding a large cyst (presented in Supplemental Fig. S6e). Note that statistical analyses performed without the latter nup133 morphant larva (values indicated by black squares on the graph) still revealed a significant difference between the control and the nup133 morphants (p = 0.01855). Abbreviations: BS: bowman space; C: capillary lumen; EC: endothelial cells; FP: foot processes. Scale bars 1 µm.
Fig 2: Partial knockdown of nup133 causes glomerular expansion in zebrafish. (a) Gross morphology of 3 dpf control and nup133 MO embryos (left panels: lateral view; middle panel: dorso-lateral view from two distinct embryos). Scale bar, 500 µm. Two-fold magnification of the indicated area is shown in the rightmost panels. Arrows indicate the pronephric cysts detected in the nup133 MO embryos. (b) Dorsal view of 3dpf Tg(wt1b:EGFP) embryos uninjected (control, top panels), injected with nup133 MO (middle panels), or sequentially injected with 3xHA-mCherry-Dr nup133 mRNA (wt mRNA) and nup133 MO (bottom panels). Overlays of transmission (gray) and GFP-signal (green) images reveal the glomerulus, proximal tubules, and exocrine pancreas. Scale bar, 500 µm. Two-fold magnification of the indicated area and of the same area from a distinct larvae are shown in the right and rightmost panels, respectively. The glomerular structure is indicated in brackets. Asterisks point to cystic dilations of the pronephros in nup133 MO (middle panels). Note that the nup133 MO + wt mRNA embryo displays a left-sided exocrine pancreas (arrowhead) (see also Supplemental Fig. S5). (c) Relative proportion of embryos showing or not kidney cysts at 3 dpf. For each condition, the total number of embryos analyzed is indicated (n=, quantified in 2 distinct experiments for control MO injections and 5 experiments for nup133 MO injections. See also Supplementary Fig. S3). Unlike the embryos injected with control MO, those injected with nup133 MO frequently feature kidney cysts. On the other hand nup133 MO + wt mRNA showed significantly fewer cysts than nup133 MO alone. ***P < 0.0001 using a Fisher exact probability test.
Fig 3: Glomerular filtration is impaired in nup133 morphants. Representative images of cross sections of 4 dpf control (left panels) and nup133 morphants larvae (right panels) fixed 20 minutes after injection of Alexa Fluor™ 647 conjugated-BSA into the common cardinal vein. Lower panels represent a higher magnification view of the proximal tubule region (dotted lines). Note the uptake of fluorescent BSA in the apical endosomes of the proximal tubules of the Nup133-depleted larva (arrows). Scale bars 50 µm.
Fig 4: Nup133 depletion does not result in major nuclear pore complex assembly defects. (a) Representative transverse sections of the glomerulus of 3 dpf Tg(wt1b:EGFP) embryos treated with either control or nup133 morpholinos (MO) and stained with mAb414 (not shown in these panels) and DAPI. In these sections, GFP-positive cells mark the glomerulus and the neck region of the proximal tubules. Scale bars, 100 µm. (b) Fivefold magnification of the areas indicated in (a) encompassing the glomerulus (G, indicated by the dashed lines) and the liver (L). mAb414 antibody, that recognizes a subset of FG-nucleoporins, shows specific staining around the nucleus of all cells (visualized with DAPI), with however cell-type-dependent variations in intensity. Note for instance the more prominent staining of liver as compared to glomerular cells. In contrast, no major difference can be seen between control and nup133 MO-treated embryos. Insets show a fourfold magnification of representative liver nuclei, revealing the punctate staining typical of NPC staining (mAb414). Scale bars, 20 µm.
Fig 5: Normal development of the pronephric tubules and glomerulus in nup133 morphants. (a) Lateral view of pax2a mRNA expression in pronephric tubules at 24 hpf in uninjected (control) and nup133 MO-injected embryos reveal that the developmental expression of pax2a is not altered in nup133 morphants. Two-fold magnification of the pronephric tubules is also shown. (b) Pronephros marker cdh17 mRNA expression in nup133 MO is comparable to that of uninjected controls at 24 and 72 hpf (dorsal view). (c) Glomerular development in control and nup133 MO embryos visualized using the podocyte differentiation marker wt1a (dorsal view). At 30 hpf (upper panels), wt1a marks future podocytes with two distinct domains in both control and nup133 MO. At 48 hpf (middle panels), the glomerular primordia merge to the midline to form a single glomerulus. At these stages, mRNA expression does not differ between control and nup133 MO (middle panels). At 58 hpf (lower panels), after the onset of glomerular filtration, the increased area labeled by the wt1a probe (arrow) reflects the glomerular expansion observed in 7 out of 13 nup133 morphants analyzed. Scale bars, 500 µm.
Supplier Page from Abcam for Anti-NUP133 antibody [EPR10809]