Structure–function correlation of micro1 for micromere specification in sea urchin embryos

The micromeres of sea urchin embryos have two functions: to promote the autonomous differentiation of skeletogenic cells and to induce endomesodermal tissues. Micromere specification is controlled by a double-repression gate consisting of two repressors, Pmar1 and HesC. Micro1/pmar1 encodes a transcriptional repressor with a paired-type N-terminal homeodomain and two C-terminal serine-rich repeats, each of which includes a sequence similar to engrailed homology region 1, which interacts with the co-repressor Groucho. To understand the molecular mechanisms of the double-repression gate, we examined the correlation between the structure and function of micro1. Phenotypic and gene expression pattern analyses of embryos injected with mutated micro1 mRNA revealed that micro1 consists of five functional domain and motifs; namely, a DNA-binding homeodomain, a nuclear localization signal in the C-terminal flanking region of the homeodomain, and two eh1-like motifs plus a short C-terminal stretch that together mediate transcriptional repression. Our data suggest that micro1 represses target genes, including hesC, via two redundant means: its eh1-like and C-terminal motifs. The C-terminal motif requires unidentified sequences for micro1 function; a micro1 mutant with the motif but lacking the unidentified sequences failed to trigger the double-repression gate for early micromere regulatory genes, except for delta, though it did repress hesC. Our results suggest that the spatial regulation of primary mesenchyme cell specification genes, including tbr, alx1, and ets1, may be different from that of delta.