Abstract
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.
Highlights
During metazoan development one cell gives rise to thousands of daughter cells, each acquiring a particular fate depending on their temporal and spatial coordinates within the organism
While Nv-fgfA1 expression was undetectable in AZ treated embryos at 10 mM and 30 mM (Figure 1W, 1X) its expression appeared only slightly reduced in lithium chloride (LiCl) treated embryos at the highest concentrations (Figure 1J, 1K)
In this study we took advantage of the growing number of molecular and functional resources in the cnidarian sea anemone N. vectensis to establish the framework for the first provisional gene regulatory networks (GRN) underlying endomesoderm (EM) formation in a non-bilaterian metazoan
Summary
During metazoan development one cell gives rise to thousands of daughter cells, each acquiring a particular fate depending on their temporal and spatial coordinates within the organism. The fate of each cell is determined by its set of expressed genes and controlled by the action of transcriptional activators and/or repressors whose activity is governed by intracellular (e.g. localized cytoplasmic factors, RNA binding proteins), or extracellular signals (e.g. endocrine or exocrine signaling pathways) All together, these components form gene regulatory networks that underlie the formation of distinct cell types or germ layers. Ectoderm gives rise to skin and nervous system, endoderm gives rise to the derivatives of the digestive tract including the intestine and digestive glands, and mesodermal derivatives include muscle, connective tissue, blood, coelomic cavities, kidneys/nephridia, somatic portions of the gonad, and skeletal elements Both classic descriptions as well as modern molecular analyses of germ layer formation in bilaterian organisms as diverse as nematodes, sea urchins, and vertebrates have indicated that these decisions are largely made in a two steps: ectodermal fates first separate from a Author Summary
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