A Regulatory Gene Network That Directs Micromere Specification in the Sea Urchin Embryo

Abstract

Micromeres and their immediate descendants have three known developmental functions in regularly developing sea urchins: immediately after their initial segregation, they are the source of an unidentified signal to the adjacent veg(2) cells that is required for normal endomesodermal specification; a few cleavages later, they express Delta, a Notch ligand which triggers the conditional specification of the central mesodermal domain of the vegetal plate; and they exclusively give rise to the skeletogenic mesenchyme of the postgastrular embryo. We demonstrate the key components of the zygotic regulatory gene network that accounts for micromere specificity. This network is a subelement of the overall endomesoderm specification network of the Strongylocentrotus purpuratus embryo. A central role is played by a newly discovered gene encoding a paired class homeodomain transcription factor which in micromeres acts as a repressor of a repressor: the gene is named pmar1 (paired-class micromere anti-repressor). pmar1 is expressed only during cleavage and early blastula stages, and exclusively in micromeres. It is initially activated as soon as the micromeres are formed, in response to Otx and beta-Catenin/Tcf inputs. The repressive nature of the interactions mediated by the pmar1 gene product was shown by the identical effect of introducing mRNA encoding the Pmar1 factor, and mRNA encoding an Engrailed-Pmar1 (En-Pmar1) repressor domain fusion. In both cases, the effects are derepression: of the delta gene; and of skeletogenic genes, including several transcription factors normally expressed only in micromere descendants, and also a set of downstream skeletogenic differentiation genes. The spatial phenotype of embryos bearing exogenous mRNA encoding Pmar1 factor or En-Pmar1 is expansion of the domains of expression of the downstream genes over most or all of the embryo. This results in transformation of much of the embryo into skeletogenic mesenchyme cells that express skeletogenic markers. The normal role of pmarl is to prevent, exclusively in the micromeres, the expression of a repressor that is otherwise operative throughout the embryo. This function accounts for the localization of delta transcription in micromeres, and thereby for the conditional specification of the vegetal plate mesoderm. It also explains why skeletogenic differentiation gene batteries normally function only in micromere descendants. More generally, the regulatory network subelement emerging from this work shows how the specificity of micromere function depends on continuing global regulatory interactions, as well as on early localized inputs.

Global regulatory logic for specification of an embryonic cell lineage

“…Furthermore, if pmar1 mRNA is made to be present in all cells at the same levels as normally in the micromeres, the whole embryo turns into skeletogenic mesenchyme (ref. 25 and Fig. 2E).…”

“…All such genes, for reasons that will become apparent in the next section, (i) must be shut down by interference with nuclearization of ␤-catenin [by injection of a truncated dominant negative form of cadherin mRNA, ⌬-cadherin (24)]; and (ii) must be ectopically activated by injection of mRNA encoding the Pmar1 repressor (25). Both methods were used in screens for regulatory genes that contribute to micromere lineage specification.…”

“…The first genes in the sequence are the pmar1 genes (there is a cluster of several very similar pmar1 genes). The pmar1 genes are activated by the ␤-catenin/ Tcf transcription complex plus Otx, i.e., by two of the micromerespecific inputs just enumerated (25,30). Expression of pmar1 in the micromere lineage (Fig.…”

“…The factor encoded by the pmar1 genes is a transcriptional repressor (25), and the second element in the subcircuit is the immediate target gene of this repressor. This gene encodes a second repressor, HesC (32).…”

Molecular basis of lithium action: integration of lithium-responsive signaling and gene expression networks

“…Recently, a regulatory gene network that directs specific developmental events has been identified in developing sea urchin embryo. 114,115 This provides a heuristic model for constructing a lithium-responsive gene network as a means to identify signature genes that direct the therapeutic or nontherapeutic action of lithium. While chromosome immunoprecipitation could start with antibodies against several known lithiumresponsive transcription factors such as AP-1, CREB, NF-kB, LEF/TCF, these are general transcription Figure 3 Model for a gene network for lithium-responsive genes: defining the therapeutic effect of chronic lithium.…”