Figure 1.

Generation of GFRa1 hypomorphic mice and analysis of Gfra1 expression. (A) The Gfra1 hypomorphic allele was generated by inserting a puΔtk cassette³¹ into intron 6 in the Gfra1 gene. (B) Representative Southern blot analysis of EcoRV-digested genomic DNA isolated from embryonic stem cells, confirming correct targeting of Gfra1 via homologous recombination (expected sizes: targeted, 7564 bp; WT, 10397 bp). (C) PCR-based genotyping of mice using the primers indicated in panel A by F and R (expected sizes: WT, 88 bp; Gfra1^{puΔtk/puΔtk}, 100 bp). (D) Gfra1^hypo/hypo mice develop megacolon, constipation, and abdominal distention, and they fail to thrive (note that the Gfra1^hypo/hypo mouse is smaller than the WT mouse). The point of dilation is indicated by black arrows. At P18–P25, 50% of Gfra1^hypo/hypo mice, sacrificed due to obvious discomfort due to abdominal swelling, have clearly visible acute erythema/hyperemia in the gastrointestinal tract (white arrows) indicative of enterocolitis; representative images are shown. (E) Relative Gfra1, Gdnf, and Ret mRNA levels at E13.5 in the indicated organs measured using qPCR (N = 4). (F and G) Western blot analysis of GFRa1 protein in the gut segment spanning from the duodenum to the ileum in E12.5 embryos. (F) Representative Western blot (samples obtained from E12.5 Gfra1 KO mice were included as a negative control); (G) normalized average GFRa1 intensity (N = 3 independent experiments). (H) At P10, the kidneys in Gfra1^hypo/hypo mice appear anatomically normal. (J) Gfra1^hypo/hypo mice are smaller than WT and heterozygous littermates at P10 (P < .001). The slight reduction in kidney size in the Gfra1^hypo/hypo mice likely reflects an overall failure of the mice to thrive. (I) Compared with WT littermates, kidney histology is grossly normal in P18 Gfra1^hypo/hypo mice. F, forward primer; R, reverse primer; EV, EcoRV site; ex 6, exon 6; puΔtk/puΔtk, Gfra1^{puΔtk/puΔtk}; KO/KO, Gfra1^KO/KO; CHO, Chinese hamster ovary cells transfected with a plasmid encoding GFP (-) or Gfra1 (+). All summary data are presented as means ± SEM. *P < .05, **P < .01, and ***P < .001. Statistical analyses were performed using the Student t test, unless indicated otherwise.