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Cited by 140Developmental Biology and Gene RegulationMarine Invertebrate Physiology and EcologyWnt/β-catenin signaling in development and cancer

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

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Röttinger, Eric, Dahlin, Paul, Martindale, Mark Q.·2012·PLoS Genetics
DOI 10.1371/journal.pgen.1003164PMC PMC3531958
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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.

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Figures

Figure 1 Dose-dependent effects of the Gsk3ß inhibitors, lithium chloride (LiCl), and 1-azakenpaullone (AZ) on embryonic gene expression. 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). In situ hybridization on blastula stages using NvfoxB (A–F, M–R) or NvfgfA1 (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.
Figure 1 Dose-dependent effects of the Gsk3ß inhibitors, lithium chloride (LiCl), and 1-azakenpaullone (AZ) on embryonic gene expression. 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). In situ hybridization on blastula stages using NvfoxB (A–F, M–R) or NvfgfA1 (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.CC BY · PubMed Central
Figure 2 Ectopic activation of canonical Wnt signaling. (A–D) Control, (E–H) lithium chloride (LiCl) treated or (I–L) 1-azakenpaullone (AZ) treated embryos. (A,E,I) Embryos injected with mRNA encoding Nvß-catenin:GFP. The insets correspond to the details of the dashed squares to show the green nuclear ßcatenin (nß-catenin) localization in ectopic domains. Red (rhodamine) dextran was co-injected with Nvß-catenin:GFP mRNA and the merged images are shown in (A,E,I, see Figure S2 for the individual images). The dashed line in E indicates the absence of nß-catenin at the vegetal pole. Confocal z-sections using phalloidin (green) to show f-actin filaments and propidium iodide (red) to visualize the nuclei. (B,F,J) blastula stages (24 hpf), (C,G,K) late gastrula stages (48 hpf), (D,H) early planula (96 hpf) or (L) 10 day old planula larvae (see Figure S3 for better temporal resolution of the AZ phenotype). (hpf) hours post fertilization. All images are lateral views with the animal/oral pole (indicated by *) to the top.
Figure 2 Ectopic activation of canonical Wnt signaling. (A–D) Control, (E–H) lithium chloride (LiCl) treated or (I–L) 1-azakenpaullone (AZ) treated embryos. (A,E,I) Embryos injected with mRNA encoding Nvß-catenin:GFP. The insets correspond to the details of the dashed squares to show the green nuclear ßcatenin (nß-catenin) localization in ectopic domains. Red (rhodamine) dextran was co-injected with Nvß-catenin:GFP mRNA and the merged images are shown in (A,E,I, see Figure S2 for the individual images). The dashed line in E indicates the absence of nß-catenin at the vegetal pole. Confocal z-sections using phalloidin (green) to show f-actin filaments and propidium iodide (red) to visualize the nuclei. (B,F,J) blastula stages (24 hpf), (C,G,K) late gastrula stages (48 hpf), (D,H) early planula (96 hpf) or (L) 10 day old planula larvae (see Figure S3 for better temporal resolution of the AZ phenotype). (hpf) hours post fertilization. All images are lateral views with the animal/oral pole (indicated by *) to the top.CC BY · PubMed Central
Figure 3 The effects of AZ and LiCl on global gene expression and their specificity in activating canonical Wnt signaling. (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). In situ hybridization on Xßcat69:GFP injected blastula stages using (G) NvfoxB or (H) NvfgfA1 antisense probes. Controls are the same as in Figure 1 and Figure 2 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.
Figure 3 The effects of AZ and LiCl on global gene expression and their specificity in activating canonical Wnt signaling. (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). In situ hybridization on Xßcat69:GFP injected blastula stages using (G) NvfoxB or (H) NvfgfA1 antisense probes. Controls are the same as in Figure 1 and Figure 2 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.CC BY · PubMed Central
Figure 4 Spatial expression analysis on untreated embryos of 18 genes upregulated by LiCl or AZ treatments. Wild type gene expression analysis by in situ hybridization of genes upregulated by LiCl or AZ treatments. (A–R) All animals are blastula stages (24 hpf). All images are lateral views with the animal pole (presumptive endomesoderm) to the top and the insets correspond to animal pole views. Antisense probes used as indicated.
Figure 4 Spatial expression analysis on untreated embryos of 18 genes upregulated by LiCl or AZ treatments. Wild type gene expression analysis by in situ hybridization of genes upregulated by LiCl or AZ treatments. (A–R) All animals are blastula stages (24 hpf). All images are lateral views with the animal pole (presumptive endomesoderm) to the top and the insets correspond to animal pole views. Antisense probes used as indicated.CC BY · PubMed Central
Figure 5 Gene expression re-analysis of previously published genes involved in endomesoderm development. Wild type gene expression analysis by in situ hybridization of previously published genes (for original publication, see Table S3). (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.
Figure 5 Gene expression re-analysis of previously published genes involved in endomesoderm development. Wild type gene expression analysis by in situ hybridization of previously published genes (for original publication, see Table S3). (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.CC BY · PubMed Central
Figure 6 Co-expression domains in the N. vectensis blastula and high-density gene expression profiling. (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 Figure S5 for details). Visual keys used to describe the spatial expression domain determined by in situ 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 in situ hybridization at 24 hpf are shown in Figure S6.
Figure 6 Co-expression domains in the N. vectensis blastula and high-density gene expression profiling. (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 Figure S5 for details). Visual keys used to describe the spatial expression domain determined by in situ 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 in situ hybridization at 24 hpf are shown in Figure S6.CC BY · PubMed Central
Figure 7 Preliminary spatial and temporal co-expression map. Biotapestry diagram of a preliminary spatial and temporal co-expression map describing the expression data identified in this study. Genes were placed based on their maternal or temporal zygotic appearance as indicated on the y-axis (see arrow on right) and spatial expression domains identified in Figure 6A, 6B as indicated on the x-axis. The dashed lines virtually separate the animal hemisphere prior to the blastula stage into three presumptive domains based on the spatial expression at 24 hpf of the given gene. The * next to gene names indicates that no clear zygotic upregulation was detected prior to the onset of gastrulation (Figure 6) and we therefore placed the genes at 24 hpf.
Figure 7 Preliminary spatial and temporal co-expression map. Biotapestry diagram of a preliminary spatial and temporal co-expression map describing the expression data identified in this study. Genes were placed based on their maternal or temporal zygotic appearance as indicated on the y-axis (see arrow on right) and spatial expression domains identified in Figure 6A, 6B as indicated on the x-axis. The dashed lines virtually separate the animal hemisphere prior to the blastula stage into three presumptive domains based on the spatial expression at 24 hpf of the given gene. The * next to gene names indicates that no clear zygotic upregulation was detected prior to the onset of gastrulation (Figure 6) and we therefore placed the genes at 24 hpf.CC BY · PubMed Central
Figure 8 Inhibition of NvTcf prevents pharynx formation. (A) Schematic presentation of the ß-catenin/Tcf interaction for transcriptional activity: the morpholino oligonucleotide MoTcf_trans (red) targeting the translation initiation site of NvTcf 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) Nvdntcf:Venus 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.
Figure 8 Inhibition of NvTcf prevents pharynx formation. (A) Schematic presentation of the ß-catenin/Tcf interaction for transcriptional activity: the morpholino oligonucleotide MoTcf_trans (red) targeting the translation initiation site of NvTcf 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) Nvdntcf:Venus 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.CC BY · PubMed Central
Figure 9 Overexpression of NvTcf:Venus can reverse effects of MoTcf. (A) Sequence information for NvTcf (wild-type), NvTcf:Venus (only ORF), NvTcf5′:Venus (containing part of the 5′UTR) and the target sequence for MoTcf_trans. (B) Overexpression of Nvtcf:Venus or (D) Nvtcf5′:Venus alone or in presence of MoTcf_trans (C,E) showing that MoTcf has no effect on Nv-tcf:Venus translation (C), while MoTcf inhibits translation of Nv-tcf5′:Venus (E). Note the red color in (E) indicating the dextran that was used for microinjection. The red chanel has been omitted in B,C,D for a better visualization of nuclear Tcf:Venus. (F) Effects on gene expression after injection of Nvtcf:Venus (blue), MoTcf_trans (orange) or Nvtcf:Venus and MoTcf (light blue). NvTcf:Venus has the capacity to revert the effects of MoTcf_trans supporting the idea that MoTcf specifically targets endogenous Nvtcf in the injected embryos.
Figure 9 Overexpression of NvTcf:Venus can reverse effects of MoTcf. (A) Sequence information for NvTcf (wild-type), NvTcf:Venus (only ORF), NvTcf5′:Venus (containing part of the 5′UTR) and the target sequence for MoTcf_trans. (B) Overexpression of Nvtcf:Venus or (D) Nvtcf5′:Venus alone or in presence of MoTcf_trans (C,E) showing that MoTcf has no effect on Nv-tcf:Venus translation (C), while MoTcf inhibits translation of Nv-tcf5′:Venus (E). Note the red color in (E) indicating the dextran that was used for microinjection. The red chanel has been omitted in B,C,D for a better visualization of nuclear Tcf:Venus. (F) Effects on gene expression after injection of Nvtcf:Venus (blue), MoTcf_trans (orange) or Nvtcf:Venus and MoTcf (light blue). NvTcf:Venus has the capacity to revert the effects of MoTcf_trans supporting the idea that MoTcf specifically targets endogenous Nvtcf in the injected embryos.CC BY · PubMed Central
Figure 10 Analysis of NvTcf inhibition by qPCR. Changes in gene expression after NvTcf knock-down (Nvdntcf:Venus) compared to control embryos shown by qPCR. Effects of Nvdntcf:Venus (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 Figure 6A, 6B. The effects of Nvdntcf:Venus and MoTcf_trans injection show an overall similar effect (represented in Figure S8).
Figure 10 Analysis of NvTcf inhibition by qPCR. Changes in gene expression after NvTcf knock-down (Nvdntcf:Venus) compared to control embryos shown by qPCR. Effects of Nvdntcf:Venus (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 Figure 6A, 6B. The effects of Nvdntcf:Venus and MoTcf_trans injection show an overall similar effect (represented in Figure S8).CC BY · PubMed Central
Figure 11 Analysis of Nv-Tcf inhibition by in situ hybridization. (A–Zj) Effects on gene expression after MoTcf_trans (G–L) or Nvdntcf:Venus (M–R, Y–Zj) injection compared to control embryos (A–F, S–Zg) analyzed by in situ hybridization. Antisense probes used as indicated. The black dashed circle in (T,U) indicates the central domain expression of NvduxABC and Nvfgf8A and the white dashed circle in (Z,Za) the central domain showing extension of its expression domain into the central ring. The numbers in the upper right corner indicates the ratio of embryos with perturbed gene expression to the total number of analyzed embryos. All images are lateral views with the presumptive endomesoderm (animal pole) to the top.
Figure 11 Analysis of Nv-Tcf inhibition by in situ hybridization. (A–Zj) Effects on gene expression after MoTcf_trans (G–L) or Nvdntcf:Venus (M–R, Y–Zj) injection compared to control embryos (A–F, S–Zg) analyzed by in situ hybridization. Antisense probes used as indicated. The black dashed circle in (T,U) indicates the central domain expression of NvduxABC and Nvfgf8A and the white dashed circle in (Z,Za) the central domain showing extension of its expression domain into the central ring. The numbers in the upper right corner indicates the ratio of embryos with perturbed gene expression to the total number of analyzed embryos. All images are lateral views with the presumptive endomesoderm (animal pole) to the top.CC BY · PubMed Central
Figure 12 Provisional gene regulatory network orchestrating endomesoderm formation in the cnidarian N. vectensis. 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 in situ hybridization, dashed lines indicate evidence obtained only by qPCR or hypothetical linkages. The colored boxes represent the spatial domains as described in Figure 6A, 6B. The genes that are inactive (repressed in that territory by NvTcf) are represented in light grey. Evidence for NvLhx1/5 controlling Nv-chordin expression obtained from Yasuoka et al. 2009.
Figure 12 Provisional gene regulatory network orchestrating endomesoderm formation in the cnidarian N. vectensis. 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 in situ hybridization, dashed lines indicate evidence obtained only by qPCR or hypothetical linkages. The colored boxes represent the spatial domains as described in Figure 6A, 6B. The genes that are inactive (repressed in that territory by NvTcf) are represented in light grey. Evidence for NvLhx1/5 controlling Nv-chordin expression obtained from Yasuoka et al. 2009.CC BY · PubMed Central

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Cites

Show all 149 references

Research works printed but not linked: 4

  1. A Rodaway, 2001, Mesendoderm. an ancient germ layer?, Cell, 105, 169
  2. CA Byrum, 2003, Gastrulation in the Cnidara and Ctenophora, Gastrulation, From Cells to Embryo CSHL Press, 33
  3. Fautin DG, Mariscal R.N. (1991) Cnidaria: Anthozoa. New York: Wiley-Liss.
  4. U Technau, 2011, Evolutionary crossroads in developmental biology: Cnidaria, Development, 138, 1447, 10.1242/dev.048959 · DOI · Paper page

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Metadata

Authors
Röttinger, E., Dahlin, P., & Martindale, M. Q.
Affiliations
University of Hawaiʻi at Mānoa (US)
Corresponding author
Martindale, Mark Q.
ORCID
Venue
PLoS Genetics
Publisher
Public Library of Science (PLoS)
Published
2012-12-27
Volume
8
Issue
12
Pages
e1003164
ISSN
  • 1553-7390 (ISSN-L)
  • 1553-7404
DOI
10.1371/journal.pgen.1003164
PMC
PMC3531958
OpenAlex
W1989889624
Cited by
140
References
149
Citations by year
2026: 3 · 2025: 14 · 2024: 10 · 2023: 3 · 2022: 10 · 2021: 5 · 2020: 5 · 2019: 12 · 2018: 12 · 2017: 13 · 2016: 16 · 2015: 13 · 2014: 15 · 2013: 9
FWCI
4.75
Type
journal article
Language
en
Open access
Licence
Locations
  • PLoS Genetics (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • HAL (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • Figshare (submittedVersion, cc-by)
Funders
  • National Institutes of Health
  • National Institute of General Medical Sciences (R01GM093116, #GM093116)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Developmental Biology and Gene Regulation
  • Life Sciences › Agricultural and Biological Sciences › Animal Science and Zoology › Marine Invertebrate Physiology and Ecology
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Wnt/β-catenin signaling in development and cancer
Keywords
gene regulatory networks, endomesoderm specification, Wnt/β-catenin signaling, Nematostella vectensis, germ layer specification, BMP signaling, FGF signaling, Notch signaling, transcription factors, zygotic gene expression, morpholino knockdown
MeSH terms
Amino Acid Sequence, Animals, Cell Differentiation, Cnidaria, Embryo, Nonmammalian, Endoderm, Mesoderm, Sea Anemones, Transcription Factors, Signal Transduction, Lithium Chloride, Gene Expression Regulation, Developmental, Cell Lineage, Evolution, Molecular, beta Catenin, Gene Regulatory Networks, Wnt Signaling Pathway

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