Two alleles of zebrafish bmp4. st37 (A–F) is an insertion allele of bmp4 isolated in a screen for dominant enhancers of vox and vent; st72 (G, H) is a null allele identified in a reverse genetic screen with a yeast-based truncation assay. (A) Physical map of an approximately 31 kb region on LG17 that contains the four known exons of bmp4. Noncoding exons are purple, coding exons are yellow. The location and number of recombinants in 7475 meioses is shown for the SNP markers used in high resolution mapping of st37. The red arrowhead denotes the insertion site. (B) RT-PCR on total RNA from 70% epiboly embryos, 24 hpf embryos, and dissected ovaries with primers in exon 1 and exon 4 (32 of the insertion) and primers in exon 3 and exon 4 (coding region) of bmp4. bmp4 mRNA is significantly reduced in st37 homozygous mutants at both 70% epiboly and 24 hpf as compared to wild-type and heterozygous embryos. The reduction of bmp4 mRNA is less extreme in st37 mutant ovaries when assayed with primers within the coding region of bmp4, suggesting the existence of bmp4 isoforms with alternate 5′ UTR in mutant ovaries. (C–F) bmp4 expression in bmp4^st37 homozygotes (D, F) and wild-type siblings (C, E). (C, D) Shield stage, animal pole view, dorsal to the right. (E, F) Bud stage, lateral view, dorsal to the right. (G) A sequencing trace from the st72 F1 heterozygous male identifies a G to T mutation in the fourth exon of bmp4 that changes Glu(209) to a stop. (H) Schematic representation of the BMP4 protein and the location of the st72 lesion. The bmp4^st72 lesion causes the BMP4 protein to truncate in the prodomain.

Tail patterning is affected in bmp4 mutants. dlx3 (A ,B), flk1 (C, D), myod (E, F), pax2a (G, H), evx1 (I, J) and prdm1 (K, L) expression in 26 hpf wild-type (A, C, E, G, I, K) and bmp4^st72 mutant (B, D, F, H, J, L) embryos. Gaps in dlx3 and prdm1 expression in the tail fin (arrow) and reduction of staining in the cloacal region (arrowhead) are apparent in bmp4^st72 mutants (B, L). There is also a reduction in the extent of the flk1 expression domain in the tail of bmp4 mutants (D, bracket). Tail somites are fused across the ventral midline (F, arrowhead, inset) and the pronephric terminus is slightly altered in shape and location (H, arrowhead) in bmp4^st72 mutants. Expression of evx1 is reduced and more internal in bmp4^st72 mutants (J) than in wild-type embryos (I). All views are lateral with anterior to the left except the inset in panel F, which is a dorsal view.

Expansion of blood fates in bmp4^st72 mutants. (A, B) Expression of pax2a in bmp^st72 mutants (B) and wild-type siblings (A) at the 10-somite stage. The expression of pax2a appears slightly upregulated in the mesoderm below the tailbud. (C, D) gata1 expression in bmp^st72 mutants (D) and wild-type siblings (C) at the 10-somite stage. In bmp^st72 mutants, but not in their wild-type siblings, gata1 expression extends all the way around the tailbud.

Morphology of the tail of live 28 hpf embryos from crosses with swr^ta72, snh^ty68a and bmp4^st72 or bmp4^st37. (A) C2 phenotype. (B) C1b phenotype with a bifurcated tail (arrowhead). (C) Strong C1 (Cls) phenotype in which more than half of the tail fin is lost (arrowhead). Cloacal defects are also apparent (arrow). (D) Weak C1 (Clw) phenotype displaying a slight reduction in ventral tail fin (arrowhead). (E) Wild-type phenotype. Anterior is to the left.

Blood fate is first expanded and then reduced upon progressive loss of BMP activity. gata1 expression at 10 somites in embryos from a cross between a triple bmp heterozygote and a bmp4^st72 heterozygote. (A) Wild-type phenotype exhibited by a bmp4^st72 heterozygote. Expansion of the gata1 expression domain ventral to the tailbud is evident in a bmp4^st72 homozygote (B) and a bmp4^st72/bmp4^st72; snh^ty68a/+ embryo (C). Reduction of the gata1 expression domain is observed upon further reduction of BMP, as in these bmp4^st72/bmp4^st72; swr^ta72/+ (D) and bmp4^st72/bmp4^st72; snh^ty68a/+; swr^ta72/+ (E) embryos, although ectopic gata1 expression ventral to the tailbud is still apparent.

bmp expression is unaffected in bmp4 mutants. bmp4 (A, B, E, F) and bmp2b (C, D, G, H) expression in bmp4^st72 mutants and wild-type siblings at bud stage (A–D) and 24 hpf (E–H). bmp4 and bmp2b expression in bmp4^st72 mutants (B, D) is similar to that seen in their wild-type siblings (A, C) at bud stage. At 24 hpf, a gap in the bmp4 and bmp2b expression patterns is apparent in the ventral tail fin (arrowheads) in bmp4^st72 mutants (F, H). However, expression in other regions is unaffected, suggesting that the absence of staining is more likely due to the lack of ventral tail fin fate than a direct effect on expression levels. Panels A–D are lateral views with dorsal to the right; panels E–H are lateral views with anterior to the left.

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.