<i>Kif3a</i> acts upstream of <i>Fgf8</i>.

<p>(A–D) Analysis of primary cilia (arrows) in metanephric mesenchyme cells derived from <i>WT</i> and <i>Kif3a<sup>−/−MM</sup></i> 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 <i>Kif3a</i>-deficient cells were transfected with a plasmid encoding <i>Kif3a</i> fused to GFP. Cilia in <i>Kif3a</i>-deficient mesenchyme cells (B, B’) are vestigial in comparison to cilia on WT cells (A, A’). Transfection with <i>Kif3a</i> results in localization of GFP to the cilium in each treatment group (C, D) and lengthening of the cilium in <i>Kif3a<sup>−/−MM</sup></i> cells (D versus B). (E) Quantitation of cilia length in untransfected and transfected WT and <i>Kif3a<sup>−/−MM</sup></i> cells. Expression of <i>Kif3a</i> partially rescues cilia length in <i>Kif3a-</i>deficient cells. (F) Quantitation of <i>Ptc1</i> and <i>Gli1</i> mRNA expression, measured by quantitative RT-PCR in untransfected and transfected WT and <i>Kif3a<sup>−/−MM</sup></i> cells. <i>Ptc1</i> and <i>Gli1</i> mRNA expression is not affected by <i>Kif3a</i> deficiency or transfection with <i>Kif3a.</i> (G) Expression of <i>Fgf8</i>, <i>Ptc1</i>, and <i>Gli1</i> 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 <i>Fgf8</i> 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.</p>