Fig. S1

Fig. S1 st37 is an insertion allele of bmp4. (A) Physical map of an approximately 31 kb region on LG17 that contains the four reported exons of bmp4. RT-PCR on RNA from wild-type ovaries identified two novel bmp4 isoforms. These isoforms contain one of two new alternative first exons (designated E1B and E1C) spliced to the 2nd, 3rd, and 4th exons of bmp4. The locations of the two new exons are depicted with arrows. Noncoding exons are purple, coding exons are yellow. The red arrowhead denotes the st37 insertion site. (B) Expanded view of the genomic region containing the first noncoding exon of bmp4. The locations of probes used in Southern blot analyses are shown along, with BstXI restriction sites. Sequence from the boxed region is presented in E. (C) Schematic of the LG8 insertion. Arrowheads denote alternative first exons identified in bmp4 transcripts from bmp4^st37 mutant ovaries. (D) Southern blots on genomic DNA from st37 mutants, st37 heterozygotes and wild-type siblings with probes at the 5′ and 3′ ends of the first exon of bmp4 demonstrate that the bmp4 locus is altered by the st37 mutation. When the DNA is digested with the restriction enzyme BstXI, both probes recognize the expected 3.4kb restriction fragment in the DNA from wild-type and heterozygous embryos. Probe 1 detects an -10kb fragment in the DNA from homozygous st37 embryos whereas Probe 2 fails to recognize a fragment in the mutant DNA. These data suggest that the bmp4 locus in st37 mutants contains an insertion larger than 6.5kb with at least one BstXI restriction site. Differences in the bmp4 locus are also apparent in Southern blots with DNA digested by PsiI and HpyCHIV as well as NheI, SmlI, SfcI, MmeI, BspMI, and BssSI (data not shown). (E) Sequence of the st37 insertion. The insertion occurs in the 5′ UTR of bmp4, 47 bp from the 3′ end of exon 1. Black sequence denotes wild-type bmp4 genomic sequence with exon 1 sequence in capital letters. Sequence from the insert is shown in red. The underlined sequences were spliced to bmp4 exon 2 sequence in mRNA from st37 mutants. (F) Sequences of the alternative first exons identified within the bmp4 locus and within the st37 LG8 insertion. 3′ intronic sequence is shown in lowercase.