RT-PCR analysis of Fgf8 transcripts in wild-type and acerebellar embryos. (A) Structure of wild-type cDNA and placement of primers. (B) Only transcripts containing exon 2 are detected in wild type throughout development either with primers spanning exon 2 (P5 + P6) or with one primer located in exon 2 (P7 + P6). (C) Exon 2 is missing in Fgf8 transcripts of acerebellar embryos. RT-PCR with primers spanning exon 2 (P5 + P6) yields a single band of 612 bp in wild-type embryos. In acerebellar embryos, no transcripts of wild-type size are detected; instead only transcripts without exon 2 are seen, which are shorter by 107 bases (compare lanes 2 and 4, and lane 5 and 6). In heterozygous siblings, bands of both sizes are detectable (lane 3). With one primer located in exon 2 (P5 + P8), amplification is only possible in acerebellar embryos after reamplification (compare lane 9 and 12), while in wild-type embryos and siblings one round of amplification is sufficient (lanes 7, 8), demonstrating the low abundance of exon 2 containing transcripts in acerebellar embryos. (D) acerebellar mutant embryos and their siblings were sorted by phenotype, indicated by bars. Single embryo RT-PCR with primers spanning exon 2 (P5 + P6) was performed for 101 acerebellar embryos and 13 siblings. Phenotypically wild-type siblings show either a wild-type or a heterozygous pattern. In all phenotypically acerebellar embryos tested, only exon 2-less transcript is detected, confirming genetic linkage (0 ± 0.5 cM).
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