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

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 veg2 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 β-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

Interaction of TLR2 and TLR4 ligands with the N-terminal domain of Gp96 amplifies : innate and adaptive immune responses

Activation of dendritic cells by ligands for Toll-like receptors (TLR) is a crucial event in the initiation of innate and adaptive immune responses. Several classes of TLR ligands have been identified that interact with distinct members of the TLR-family. TLR4 ligands include lipopolysaccharide derived from different Gram-negative bacteria and viral proteins. Recent reports have demonstrated the TLR-mediated activation of dendritic cells by heat shock proteins (HSPs). However, doubts were raised as to what extent this effect was due to lipopolysaccharide contaminations of the HSP preparations. We re-examined this phenomenon using Gp96 or its N-terminal domain, nominally endotoxin-free ( or =50 microg/ml) but not at lower concentrations. However, preincubation of low amounts of Gp96 with TLR2 and TLR4 ligands at concentrations unable to activate dendritic cells by themselves results in the production of high levels of proinflammatory cytokines, up-regulation of activation markers, and amplification of T cell activation. Our results provide significant new insights into the mechanism of HSP-mediated dendritic cell activation and present a new function of HSPs in the amplification of dendritic cell activation by bacterial products and induction of adaptive immune responses