Fig 4. Expression patterns of putative skeletogenic genes in embryos expressing alx1.

Solutions 0.5 mg/ml in mRNA for Hpalx1 (A-D), 1 mg/ml in mRNA for Apalx1 (E-G) or EGFP (I-L) were injected into starfish eggs, which were next reared for 15 h (to the early gastrula stage) or 24 h (to the mid-gastrula stage). (A) App19 was detected in the presumptive mesoderm. (B) App19 expression was retained by mesoderm cells at the tip of the archenteron. (C) ApCA1 mRNA was also detected in the presumptive mesoderm. (D) ApCA1 expression was retained in the mesoderm of gastrulae. (E-H) The similar expression patterns to the sea urchin Hpalx1 overexpression were observed in the starfish Apalx1 overexpressed embryos. (I-L) Neither App19 nor ApCA1 was detectable in control embryos. Scale bars: 50μm. (M) Changes in the expression levels of skeletogenic gene orthologs upon injection of Hpalx1 mRNA into starfish. Relative expression levels in the 24-h-old embryos were measured via real-time PCR. The data show -ΔΔCt values relative to those of (control) EGFP-expressing embryos. The expression level of each gene was normalized to that of the ApEF1a gene. Three distinct trials with different batches of eggs were run. Asterisks indicate significant difference between ΔCt values of control and Hpalx1 overexpression (paired Student’s t-test, p < 0.05). Endogenous Apalx1 was highly upregulated upon ectopic Hpalx1 expression.