A Framework for the Establishment of a Cnidarian Gene Regulatory Network for "endomesoderm" Specification: the Inputs of ß-catenin/TCF Signaling

Understanding the functional relationship between intracellular factors and extracellular signals is required for reconstructing gene regulatory networks (GRN) involved in complex biological processes. One of the best-studied bilaterian GRNs describes endomesoderm specification and predicts that both mesoderm and endoderm arose from a common GRN early in animal evolution. Compelling molecular, genomic, developmental, and evolutionary evidence supports the hypothesis that the bifunctional gastrodermis of the cnidarian-bilaterian ancestor is derived from the same evolutionary precursor of both endodermal and mesodermal germ layers in all other triploblastic bilaterian animals. We have begun to establish the framework of a provisional cnidarian "endomesodermal" gene regulatory network in the sea anemone, Nematostella vectensis, by using a genome-wide microarray analysis on embryos in which the canonical Wnt/ß-catenin pathway was ectopically targeted for activation by two distinct pharmaceutical agents (lithium chloride and 1-azakenpaullone) to identify potential targets of endomesoderm specification. We characterized 51 endomesodermally expressed transcription factors and signaling molecule genes (including 18 newly identified) with fine-scale temporal (qPCR) and spatial (in situ) analysis to define distinct co-expression domains within the animal plate of the embryo and clustered genes based on their earliest zygotic expression. Finally, we determined the input of the canonical Wnt/ß-catenin pathway into the cnidarian endomesodermal GRN using morpholino and mRNA overexpression experiments to show that NvTcf/canonical Wnt signaling is required to pattern both the future endomesodermal and ectodermal domains prior to gastrulation, and that both BMP and FGF (but not Notch) pathways play important roles in germ layer specification in this animal. We show both evolutionary conserved as well as profound differences in endomesodermal GRN structure compared to bilaterians that may provide fundamental insight into how GRN subcircuits have been adopted, rewired, or co-opted in various animal lineages that give rise to specialized endomesodermal cell types.