Figure 3

PmAlx1 is expressed in the sea star epithelial mesoderm, but induces skeletogenesis in sea urchins. WMISH using a probe against PmAlx1 demonstrates that transcripts are localized to the central vegetal pole mesoderm of sea star blastulae (A); in later development, transcripts are detected in the coelomic mesoderm, but never in the mesenchyme, as seen by two-color in situ hybridization with PmEts1 (B). In larvae, staining is detected in the lateral aspects of the anterior coeloms (filled arrows in C) as well as in the posterior coelom (arrow in C). To confirm that the absence of a larval skeleton in sea stars is not due to a difference in the sea star PmAlx1 protein, we overexpressed PmAlx1 in sea urchins. (D-F) Ectopic expression of PmAlx1 mRNA in sea urchin embryos results in increased skeletogenesis, as has been observed for overexpression of sea urchin alx1 mRNA [41]. Control embryos were injected with mRNA encoding the RFP variant mCherry. Representative sea urchin gastrula are shown (D-F). Ectopic skeletal spicules were observed upon overexpression of PmAlx1 (E, F). Numbers of embryos showing the illustrated phenotype is shown in the lower right corner (from a total of 40 embryos for the control RFP, and 80 embryos in the PmAlx1 injected embryos); D shows normal skeletal formation, E shows increase in number of skeletal forming centers, and F and dramatic increase in skeleton.