Inhibition of NvTcf prevents pharynx formation.

<p>(A) Schematic presentation of the ß-catenin/Tcf interaction for transcriptional activity: the morpholino oligonucleotide MoTcf_trans (red) targeting the translation initiation site of <i>NvTcf</i> and the NvdnTcf protein lacking the ß-catenin binding domain which prevents transcriptional activation of NvTcf by ß-catenin. (B) Overexpression of NvdnTcf:Venus is detected in the nuclei of all blastomeres at the blastula stage showing that the nuclear localization of NvdnTcf is not affected by deletion of the ß-catenin binding domain. (C–K) Confocal z-sections using phalloidin (green) to stain f-actin filaments and propidium iodide (red) to visualize the nuclei. (C–E) Control, (F–H) MoTcf_trans injected and (I–K) Nv<i>dntcf:Venus</i> injected embryos. (C,F,I) blastula (24 hpf), (D,G,J) late gastrula (48 hpf), (E,H,K) early planula larva (96 hpf). The numbers in the upper right corner indicate the ratio of embryos with the indicated phenotype to the total number of analyzed embryos. The arrows in E indicate the position of the pharynx. All images are lateral views with oral (indicated by *) to the top.</p

Gene expression re-analysis of previously published genes involved in endomesoderm development.

<p>Wild type gene expression analysis by <i>in situ</i> hybridization of previously published genes (for original publication, see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003164#pgen.1003164.s011" target="_blank">Table S3</a>). (A–Zg) All animals are blastula stages and the inset corresponds to animal views. Antisense probes used as indicated. All images are lateral views with the presumptive endomesoderm (future oral pole) to the top. The white stars (Q–Zb, Zd) indicate genes positively affected by LiCl or AZ treatments as determined by array experiments.</p

Selection of 104 genes upregulated after LiCl or AZ treatments.

<p>Selected transcription factors and signaling molecules that were significantly (P<0.05) at least 2-fold upregulated by AZ and LiCl treatments. SpotID: genome protein model ID (JGI) used for the array design. The gene name is based on the best blast hit (see Material and Methods) and if available the previously published name(s) is used. Color code and abbreviations are indicated in the table legend at the bottom.</p

Analysis of NvTcf inhibition by qPCR.

<p>Changes in gene expression after NvTcf knock-down (<i>Nvdntcf:Venus</i>) compared to control embryos shown by qPCR. Effects of <i>Nvdntcf:Venus</i> (blue) overexpression on transcriptional control of 50 potential components of the cnidarian endomesoderm GRN. Changes in gene expression are indicated as relative fold changes compared to dextran injected control embryos ( ± sem, n = 3 per gene). The grey bar indicates no significant change in gene expression (−1,1). Below each analyzed gene the star indicates the effects of LiCl or AZ treatments. Gene expression domains at the blastula stage are the same as <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003164#pgen-1003164-g006" target="_blank">Figure 6A, 6B</a>. The effects of <i>Nvdntcf:Venus</i> and MoTcf_trans injection show an overall similar effect (represented in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003164#pgen.1003164.s008" target="_blank">Figure S8</a>).</p

Provisional gene regulatory network orchestrating endomesoderm formation in the cnidarian <i>N. vectensis</i>.

<p>Biotapestry diagram of the provisional gene regulatory network describing the regulatory interactions of endomesodermal genes identified in this study at 24 hpf. No assumption on whether these interactions are direct or indirect is made. Solid lines indicate functional evidence obtained by qPCR as well as <i>in situ</i> hybridization, dashed lines indicate evidence obtained only by qPCR or hypothetical linkages. The colored boxes represent the spatial domains as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003164#pgen-1003164-g006" target="_blank">Figure 6A, 6B</a>. The genes that are inactive (repressed in that territory by NvTcf) are represented in light grey. Evidence for NvLhx1/5 controlling <i>Nv-chordin</i> expression obtained from Yasuoka et al. 2009.</p

A Framework for the Establishment of a Cnidarian Gene Regulatory Network for “Endomesoderm” Specification: The Inputs of ß-Catenin/TCF Signaling

<div><p>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, <em>Nematostella vectensis</em>, 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 (<em>in situ</em>) 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.</p> </div

Dose dependent effects of LiCl and AZ on <i>Nv-foxB</i> and <i>Nv-fgfA1</i> expression.

<p>Dose-dependent effects of LiCl and AZ analyzed by <i>in situ</i> hybridization. Analyzed AZ or LiCl concentration as indicated in Row 1 (light green) and number of embryos with phenotype scored based on expansion/reduction of the domain of expression as indicated in the column on the right. (*) under LiCl indicate a developmental delay/toxicity at that concentration.</p

Co-expression domains in the <i>N. vectensis</i> blastula and high-density gene expression profiling.

<p>(A,B) The animal hemisphere contains at least four domains defined by differential gene expression: the central domain, the central ring, the central domain+ring and the external ring. In the vegetal hemisphere, we identify only one domain, the apical domain. The gene names next to the diagram correspond to the genes expressed in each domain at the blastula stage as examined in this study. (C) Summarized results of the temporal high density profiling (qPCR) used to determine the presence of maternal transcripts and significant zygotic upregulation of a given gene expressed within the animal hemisphere (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003164#pgen.1003164.s005" target="_blank">Figure S5</a> for details). Visual keys used to describe the spatial expression domain determined by <i>in situ</i> hybridization at 24 hpf same as in A,B. Those genes (n = 19) that were positively affected by Gsk3ß inhibition as determined by our array experiments but for which no localized endomesodermal expression was observed by <i>in situ</i> hybridization at 24 hpf are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003164#pgen.1003164.s006" target="_blank">Figure S6</a>.</p

The effects of AZ and LiCl on global gene expression and their specificity in activating canonical Wnt signaling.

<p>(A,B) Predicted genome-wide microarray comparison of the effects of LiCl or AZ treatments. Genes that were significantly (P<0.05) at least 2-fold up- or downregulated were included in this analysis (C–H) Embryos injected with mRNA encoding a stabilized form of ß-catenin (Xßcat69:GFP). Red (rhodamine) dextran was coinjected with the mRNA (C). The inset in C corresponds to the detail of the dashed square to show nß-catenin localization in ectopic domains. Confocal z-sections using phalloidin (green) to stain f-actin filaments and propidium iodide (red) to visualize the nuclei in embryos of indicated stages (D–F). (C,D) blastula stages (24 hpf), (E) late gastrula stages (48 hpf), (F) planula larvae (96 hpf). <i>In situ</i> hybridization on <i>Xßcat69:GFP</i> injected blastula stages using (G) <i>NvfoxB</i> or (H) <i>NvfgfA1</i> antisense probes. Controls are the same as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003164#pgen-1003164-g001" target="_blank">Figure 1</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003164#pgen-1003164-g002" target="_blank">Figure 2</a> and all images are lateral views with the animal/oral pole (indicated by *) to the top. (hpf) hours post fertilization. Ratios in F,G,H indicate the number of embryos displaying the phenotype shown in the image to the total number of analyzed embryos.</p

Dose-dependent effects of the Gsk3ß inhibitors, lithium chloride (LiCl), and 1-azakenpaullone (AZ) on embryonic gene expression.

<p>Control blastula stages at 24 hpf (A,G,M,S) and embryos treated with increasing concentrations of LiCl (B–F, H–L) or AZ (N–R,T–X). <i>In situ</i> hybridization on blastula stages using <i>NvfoxB</i> (A–F, M–R) or <i>NvfgfA1</i> (H–L, T–X) antisense probes. All images are lateral views with the presumptive endomesoderm (animal pole, future oral pole) to the top. The insets correspond to animal pole views.</p