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Supplementary Figure 3: Phenotypic analysis of ift80 and ift172 targeted embryos.
(a, b) Expression analysis of ift80 and ift172 by whole mount in situ hybridization in st. 26/27 embryos. The magnified images show a spotty pattern on the epidermis. (c, d) Expression analysis of aplnr, nkx2.5, prox1, nkcc2 and sglt1 of X. tropicalis (c) and X. laevis (d) unilaterally CRISPR targeted tadpoles. The bar graphs indicate a stronger (gray) or weaker (black) signal on the injected side. (e, f) Tomato-Lectin stain visualizes the pronephric tubules in unilaterally CRISPR targeted stage 39 tadpoles. Measurements of the bounding box area of the proximal tubule was not different between the injected side of the embryo and the wildtype half in each case. (g) Quantification of centrioles of epidermal MCCs after injection of centrin-RFP mRNA together with sgRNAs and Cas9. There was no significant difference in centriole number in CRISPR targeted cells. Error bars indicate SEM. p>0.05 ns (not significant); p<0.05 *; p<0.01 **; p<0.001 ***; Scale bars represent 0.5mm |
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fig. 2. Cystic kidney disease and cilia defects in ift80 and ift172 CRISPR–targeted X. tropicalis. (A–C) CRISPR/Cas9 targeting of slc45a2, ift80, and ift172 were performed unilaterally in two-cell–stage embryos. Mesonephroi of stage 61 to 63 froglets were analyzed by microCT scans and kidneys and cysts (red arrowheads in B and C) were segmented for 3D volumetric analysis; red: cysts on the injected side; green: uninjected side). (D) The number of cysts (>0.2mm), (E) the ratio of total cyst volume to kidney volume, and (F) the kidney volume (excluding cysts) was calculated and compared with kidneys of control injected animals. (G) Whole-mount in situ hybridization detects ift80 and ift172 in the multiciliated nephrostomes of the pronephros in stage 36 to 38 X. laevis. (H) Schematic depiction of the embryonic renal system of Xenopus. (I and J) Excretion assay with fluorescein-dextran at stage 38 to 40. Blue arrowheads point to the proximal part of the pronephros. Yellow arrowheads indicate fluorescence signal in the distal tubule, lacking on the injected side (J). A: anterior, P: posterior; excr: excreting; and emb: embryos. (K) Confocal images of multiciliated epidermal cells (MCCs) stained against acetylated tubulin (cyan). Centrin-RFP fusion protein served as a lineage marker (red arrowheads) and indicates CRISPR-targeted MCCs. Blue arrowheads point to nontargeted (wild type) cells. (L) The ciliated area was determined for each cell. Error bars indicate SEM. P > 0.05 ns (not significant); *P P P A–C) 1 mm, (G and I) 0.5 mm, and (L) 10 µm.] |
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ift172 (intraflagellar transport 80) gene expression in a X. laevis embryo, assayed via in situ hybridization NF stage 35-38, lateral view, anterior left, dorsal up. |
