CA impairs embryonic renal development. (A) H&E staining of kidney sections from E13.5 to E17.5 Hoxb7-Plk4, Six2-Plk4, and control embryos. (B) Immunofluorescence images of kidney sections from E15.5 mice stained with antibodies against Six2 (to identify MM cells) and WT1 (glomerular precursors). Nuclei are stained with DAPI. There was a significant decrease in the density of both Six2-positive progenitors and WT1-positive structures compared with control samples. (C) Higher magnification images of the distribution of Six2-positive MM cells in Hoxb7-Plk4, Six2-Plk4, and control embryos at E15.5, highlighting the reduced concentration of mesenchymal progenitors. (D) Quantification of the number of Six2-positive progenitors and total MM cell density (DAPI-positive nuclei) per UB tip structure from E15.5 to E17.5. Data represent 25 UB tip structures scored from five mice of each genotype at each time point. Two-tailed unpaired t test was used for statistical analysis (*, P < 0.05). (E) Representative images from live-cell imaging of kidney explant cultures of Six2-Cre^GFP-Plk4, Hoxb7-Cre^GFP-Plk4, and relevant controls. We note a progressive decrease in the density of the MM in Six2-Cre^GFP-Plk4 kidneys, and reduced number of ureteric branches in Hoxb7-Cre^GFP-Plk4 compared with control mice. (F) Quantification of the number of UB tips in Hoxb7-Cre^GFP-Plk4 and control kidneys, as shown in E. n = 4 kidneys analyzed for each genotype. A two-tailed unpaired t test was used for statistical analysis (*, P < 0.05). Error bars denote SD. (G) Quantification of the number of WT1-positive structures per kidney section from E15.5 to E17.5, examples of which are shown in B. n = 5 kidneys analyzed for each genotype (E15.5), and n = 4 for each genotype (E17.5). A two-tailed unpaired t test was used for statistical analysis (*, P < 0.05).