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

Abstract

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.

Activation of the skeletogenic gene regulatory network in the early sea urchin embryo

“…Considerable evidence indicates that pmar1 is a pivotal regulator of the micromere-PMC GRN. Misexpression of pmar1 is sufficient to activate the skeletogenic GRN in non-micromere-derived cells (Oliveri et al, 2002;Oliveri et al, 2003;Nishimura et al, 2004;Yamazaki et al, 2005;Yamazaki et al, 2009) (this study). It has been more difficult to test directly the function of pmar1 in the large micromere territory (where the gene is ordinarily expressed) owing to the difficulty in blocking the expression of multiple tandem pmar1 genes using MOs.…”

“…It should be noted that in a recent structurefunction analysis of Pmar1, one mutant construct (N-HD-A-C) that lacked a portion of the C-terminal region of the protein was reported to downregulate hesC mRNA levels throughout the embryo without expanding the expression of alx1, tbr or ets1 (Yamazaki et al, 2009). …”

From genome to anatomy: The architecture and evolution of the skeletogenic gene regulatory network of sea urchins and other echinoderms

“…Pmar1, a paired class homeodomain‐containing protein, is both necessary and sufficient for the specification of skeletogenic cells (Kitamura, Nishimura, Kubotera, Higuchi, & Yamaguchi, ; Nishimura et al, ; Oliveri et al, ; Oliveri, Davidson, & McClay, ; Yamazaki et al, ). Pmar1 functions as a repressor (Oliveri et al, ; Yamazaki, Ki, Kokubo, & Yamaguchi, ) and indirectly activates the expression of skeletogenic genes, at least in part by blocking the expression of a second repressor, hesC , a member of the HES (Hairy‐Enhancer‐of‐Split) family (Revilla‐i Domingo, Oliveri, & Davidson, ). hesC mRNA is present ubiquitously in the early embryo but is cleared from the micromere territory by the early blastula stage.…”

Paleogenomics of echinoids reveals an ancient origin for the double-negative specification of micromeres in sea urchins

“…The now-classic experiment in which ectopic overexpression of pmar1 converts all embryonic cells to a skeletogenic fate (26) has been duplicated in numerous camaradont euechinoids, including Hemicentrotus pulcherrimus (29), Paracentrotus lividus (30), and Lytechinus variegatus (31), suggesting that DNG function and circuitry are conserved in these taxa. Furthermore, whole-mount in situ hybridization data from the camarodonts H. pulcherrimus (32) and L. variegatus (33) reveal spatial distributions of hesc and its downstream targets that are consistent with the existence of the DNG in these euechinoids.…”