The sphk2 gene is maternally expressed. Shown are representative wildtype embryos analyzed for sphk2 transcripts by in situ hybridization at the stages indicated: (A) one-cell stage, (B) 1,000-cell stage (3 hpf), (C) sphere stage (blastula, 4 hpf), and (D) shield stage (gastrulation, 6 hpf). (E) At 24 hpf, sphk2 is expressed ubiquitously. In contrast (F–J), sphk1 transcripts are not detected above background by in situ hybridization until somitogenesis.

The sphk2^MZ mutant phenocopies the mil zygotic mutant. Shown are representative embryos at 32 hr post fertilization imaged in brightfield (column 1: lateral view, anterior to the left), (column 2: ventral view, anterior to the left), or analyzed by in situ hybridization for expression of the myl7 gene (column 3, ventral view, anterior to the left). Column 4 shows representative images of tails at this stage. Embryos were derived from crosses as indicated on the left: (A) Wildtype (WT), (B) heterozygous mil adults, (C) female and male sphk2^wc1/wc1 mutants, (D) female sphk2 homozygous mutant and male sphk2 heterozygous mutant and (E) female sphk2 heterozygous mutant and male sphk2 homozygous mutant. (F) Female sphk2 homozygous mutant crossed to a wildtype male. Note that only the homozygous mil mutant embryos (25% of the embryos from the heterozygote parental cross) display pericardial edema (columns 1, 2), cardia bifida (column 3), and tail blisters (column 4), as do 100% (200/200) of the sphk2MZ mutant embryos. These phenotypes are rescued in embryos that have a single wildtype allele (72/149, 48% of the embryos in D), if embryos inherit maternal message from a wildtype maternal allele (50% of the embryos in E, which are zygotically null but phenotypically normal), or if embryos inherit a single wildtype allele from the male parent (64/64, 100% of the embryos in F).

Expression of Sphk2 in YSL can rescue normal cardiogenesis. Shown are representative embryos following in situ hybridization for myl7 transcripts at 32 hpf. (A) Wildtype (WT) embryos (25/25, 100%) display normal cardiac development. (B) Injection of 250 pg of sphk2 mRNA into the single cell (65/65, 100%) or (C) targeted to the YSL (51/51, 100%) does not alter cardiac development. (D) All maternal/zygotic sphk2^MZ embryos show the cardia bifid phenotype (0/40 embryos are normal, 0%). (E) Injection of 250 pg sphk2 mRNA into the 1-cell stage embryos was able to rescue the bifid phenotype as shown by the presence of cardiac tissue at the midline (7/44 embryos, 16%). (F) Targeting 250 pg sphk2 mRNA specifically into the YSL (confirmed by co-injection of rhodamine dextran tracer, as documented in G) was able to rescue the bifid phenotype (19/88 embryos, 22%). (H) In contrast, injection of mRNA encoding a predicted kinase dead Sphk2 (KD) into the fertilized egg (56/56, 100%) or (I) targeted to the YSL (43/43, 100%) failed to rescue the bifid phenotype. (J) Injection of 1 ng S1P in the fertilized egg (47/47, 100%) or YSL (47/47, 100%, not shown) did not alter cardiac development of wildtype embryos, whereas (K) injection of 1 ng S1P into the fertilized egg (15/51, 29%) or (L) targeted to the YSL (9/32, 28%) was able to rescue the bifid phenotype of sphk2^MZ embryos.