Loss of FAT1 causes a renal tubular phenotype via defective RHO GTPase signalling.(a) IMCD3 cells ciliated apically and formed a spheroid containing a central lumen (asterisk) when grown in 3D matrigel culture for 3 days; lumens were perturbed on Fat1 knockdown (shRNA#1 and shRNA#3). Cells were stained for acetylated α-tubulin (red), β-catenin (green) and DAPI (blue). Scale bars, 10 µm. (b) Percentage of abnormal spheroids. More than 50 spheroids were examined in each experiment. Data represent the mean+s.d. of three independent experiments in b,c. *P<0.05, t-test. (c) The effect of Fat1 knockdown on cell migration. Bar graphs represent the area under curves of d. (d) The effect of Fat1 knockdown on cell migration. Compared with baseline (dashed black line), addition of serum strongly increases migration rate in IMCD3 cells with scrambled shRNA (Scr) (solid black line). In contrast, IMCD3 cells stably transfected with Fat1 shRNAs #1 (red continuous line) or #3 (red dashed line) exhibited slower rate of migration. Each cell index value corresponds to the average of more than triplicates and s.d. is in only one direction for clarity. (e) Active GTP-bound RHOA precipitated from IMCD3. Cells transfected with scrambled control siRNA versus Fat1 shRNA exhibited no significant difference in relative RHOA activity. This is representative of three experiments. (f) Active GTP-bound CDC42 or RAC1 using a GST-PAK1 (CRIB) pull-down assay. Note that Fat1 knockdown leads to a significant decrease in relative CDC42 and RAC1 (31% and 44%, respectively) compared with Scr control cells. This is representative of four experiments. Quantification of e and f is presented in Supplementary Fig. 7. (g-h) fat1 morpholino-oligonucelotide (MO) was injected to Wt1b::GFP transgenic zebrafish. Zebrafish injected with control MO did not produce any phenotype. Depletion of fat1 by a fat1 MO targeting the translation initiation site of zebrafish fat1 caused pronephric cysts (asterisks) in 78 of 325 zebrafish embryos (24%). Scale bars, 100 µm. (i) Activator I (Rho/Rac/Cdc42 activator I) reduced cyst formation to 9.7% (26 of 268 embryos), and activator II (Rac/Cdc42 activator II) to 12.0% (59 of 490 embryos). *P<0.001, χ²-test.