Kif3a acts upstream of Fgf8.
(A–D) Analysis of primary cilia (arrows) in metanephric mesenchyme cells derived from WT and Kif3a−/−MM metanephroi dissected free of ureteric bud. Cilia are identified by expression of α-AcT. (A’–D’) Higher magnification of images in A–D, respectively. WT and Kif3a-deficient cells were transfected with a plasmid encoding Kif3a fused to GFP. Cilia in Kif3a-deficient mesenchyme cells (B, B’) are vestigial in comparison to cilia on WT cells (A, A’). Transfection with Kif3a results in localization of GFP to the cilium in each treatment group (C, D) and lengthening of the cilium in Kif3a−/−MM cells (D versus B). (E) Quantitation of cilia length in untransfected and transfected WT and Kif3a−/−MM cells. Expression of Kif3a partially rescues cilia length in Kif3a-deficient cells. (F) Quantitation of Ptc1 and Gli1 mRNA expression, measured by quantitative RT-PCR in untransfected and transfected WT and Kif3a−/−MM cells. Ptc1 and Gli1 mRNA expression is not affected by Kif3a deficiency or transfection with Kif3a. (G) Expression of Fgf8, Ptc1, and Gli1 mRNA, measured by real time RT-PCR using RNA isolated from kidney explants and from untransfected and transfected cultured metanephric mesenchyme cells. (H) Quantitation of Fgf8 mRNA levels measured by quantitiative RT-PCR as in panel G. MM, metanephric mesenchyme; UB, ureteric bud; WT, wild-type. (***, P<0.001; **, P<0.01; *, P<0.05), Scale bars: (A–D) 25 micrometer, (I–L) 50 micrometer.