Figure 5.

Expression of endomesoderm markers in normal, lithium-treated and zinc-treated embryos. Shown are whole-mount in situ hybridizations (WISHs) of endomesodermal marker genes on blastula stage (columns 1, 3, and 5) and gastrula stage (columns 2, 4, and 6) sea urchin embryos. The genes under considerations are indicated on the right hand side. Endo16, FoxA, and GataE are known, and Smip is a new gene that is expressed in the endoderm. The expression is strongly expanded in lithium-treated embryos (columns 3 and 4), whereas only at the most animal pole are ectodermal tissues left in the embryo. Blastula stage zinc-treated embryos do not exhibit any expression of endodermal markers (column 5). Gastrula stage zinc-treated embryos (column 6) do occasionally begin to express early endomesodermal markers as they recover from treatment (see Materials and methods). Hex is a transcription factor that is expressed at low levels in primary mesenchyme cells (PMCs) and predominantly in secondary mesenchyme cell (SMC) cells. Expression is upregulated in lithium-treated embryos, as determined by quantitative real-time polymerase chain reaction (Q-PCR; columns 3 and 4; compare with Table 1) but seems unchanged as determined by WISH and is eliminated in blastula stage zinc-treated embryos. P19 is a PMC-specific gene identified in the screen. Although its expression appears to be quantitatively upregulated in lithium-treated and zinc-treated embryos (see Table 2), WISH analysis indicates that the number of PMC cells forming is normal in lithium-treated or zinc-treated embryos, but that the PMCs migrate to the animal pole in lithium-treated embryos and to the vegetal pole in zinc-treated embryos. In neither case does a skeleton form.