A bipolar role of the transcription factor ERG for cnidarian germ layer formation and apical domain patterning

Abstract

Germ layer formation and axial patterning are biological processes that are tightly linked during embryonic development of most metazoans. In addition to canonical WNT, it has been proposed that ERK-MAPK signaling is involved in specifying oral as well as aboral territories in cnidarians. However, the effector and the molecular mechanism underlying latter phenomenon is unknown. By screening for potential effectors of ERK-MAPK signaling in both domains, we identified a member of the ETS family of transcription factors, Nverg that is bi-polarily expressed prior to gastrulation. We further describe the crucial role of NvERG for gastrulation, endomesoderm as well as apical domain formation. The molecular characterization of the obtained NvERG knock-down phenotype using previously described as well as novel potential downstream targets, provides evidence that a single transcription factor, NvERG, simultaneously controls expression of two different sets of downstream targets, leading to two different embryonic gene regulatory networks (GRNs) in opposite poles of the developing embryo. We also highlight the molecular interaction of cWNT and MEK/ERK/ERG signaling that provides novel insight into the embryonic axial organization of Nematostella, and show a cWNT repressive role of MEK/ERK/ERG signaling in segregating the endomesoderm in two sub-domains, while a common input of both pathways is required for proper apical domain formation. Taking together, we build the first blueprint for a global cnidarian embryonic GRN that is the foundation for additional gene specific studies addressing the evolution of embryonic and larval development.

Keywords: Apical organ; Cnidarian; ERG; ERK signaling; ETS transcription factor; Embryonic development; Endomesoderm; Evolution; Gastrulation; Gene expression; Gene regulatory network; Nematostella vectensis; Sea anemone.

Figure 1. NvErg is required for endomesoderm formation, gastrulation and participates in apical tuft development

(A) Identification of NvErg and analysis of its spatiotemporal expression. (Aa) Excerpt of the phylogenetic analysis of the ETS transcription factor complement in Nematostella, indicating the existence of NvERG and NvETS1 orthologs in cnidarians. The full analysis can be found in Fig. S1. To the right of the tree, the bars indicated the protein domain organization of either the human representative (Hs, greyed out rectangles) of the subfamily as well as the protein domain organization of the Nematostella ortholog (Nv). Green rectangles indicate the Pointed domain, black rectangles the ETS domain. (Ab) Temporal expression of Nverg analyzed by qPCR during embryonic development of the first 48 hours post fertilization. The y-axis indicates relative fold changes compared to fertilized eggs. (Ac–Af) Spatial (in situ hybridization) expression of Nverg at early (Ac) and late (Ad) blastula, mid (Ae) and late (Af) gastrula stages. Orientation of blastula stages was determined by the thickening of the animal pole prior to its invagination that was observable in certain embryos of a given batch at the analyzed time point. Animal pole to the top and vegetal pole to the bottom. The black bar in the upper right corner of Ac–Af indicates the scale bars: 50μm (Ba) Schematic representation of the genomic organization of NvERG, and the recognition site of MO-NvERG and the position of the PCR primers to verify the efficiency of the splice blocking morpholino. (Bb) Splice blocking efficiency of MO-NvERG analyzed by RT-PCR at increasing concentrations of MO-NvERG injected embryos. (Bc) Schematic representation of the protein structure of the various tools used in Fig. 1C and Fig. S2. (C) Morphological effects of inhibiting NvERG function during early Nematostella development. (Ca–Cd) Control embryos injected with MO-CTRL, (Ce–Ch) embryos injected with MO-NvERG or (Ci–Cl) mRNA encoding a dominant negative version of NvERG (NvERG-DB1) at 24 (Ca,Ce,Ci, late blastula), 48 (Cb,Cf,Cj, late gastrula), 72 (Cc,Cg,Ck, early planula) and 96 (Cd,Ch,Cl, late planula) hpf. All images are lateral views with the animal/oral pole to the top and confocal z-sections using phalloidin (green) to show f-actin filaments and propidium iodide (red) to visualize the nuclei. The insets in (Cd, Ch, Ci) correspond to lateral views of embryos stained with acetylated tubulin to visualize the presence or absence of the apical tuft (yellow arrow in Cd). The numbers in the insets indicate the number or embryos with the represented phenotype/total amount of analyzed animals.

Figure 2. MEK/ERK signaling targets expressed in the presumptive endomesoderm

Wild-type endomesodermal gene expression analysis by in situ hybridization of genes differentially regulated by U0126 treatments. All animals are either blastula (24hpf - A–F, M–R, Y–Zd, Zk–Zp) or gastrula (48hpf – G–L, S–X, Ze–Zj, Zq–Zv) stages. All images are lateral views with the animal pole (presumptive endomesoderm) to the top. The insets correspond to animal pole views (A–G, I, K, M–R, T, Y–Zd, Zh, Zv, Zk–Zp) or optical cross-sections. Antisense probes used as indicated at the top of each pair of embryos. Green stars in N, T and R, X indicate that these genes were upregulated under U0126 conditions. All other genes were downregulated.

Figure 3. MEK/ERK signaling targets expressed in broad ectodermal domains

Wild type ectoderm gene expression analysis by in situ hybridization of genes downregulated by U0126 treatments. All animals are either blastula (24hpf - A–F, M–R, Y–Za) or gastrula (48hpf – G–L, S–X, Zb–Zd) stages. All images are lateral views with the animal pole to the top. All insets correspond to vegetal pole/aboral views. Antisense probes used as indicated at the top of each pair of embryos.

Figure 4. High density gene expression profiling

(A, B) Schematic representation of stage-specific expression domains prior to, and after gastrulation movements. (C, D) Summarized results of the temporal high density profiling (qPCR) used to determine the presence of maternal transcripts and significant zygotic up-regulation of a given gene expressed in (C) endomesodermal, or (D) broad ectodermal domains (see Fig. S3 for details). The Cp value corresponds to the cycle number at detection threshold (crossing point). (hpf) hours post fertilization. Visual keys used to describe the spatial expression domain determined by in situ hybridization at 24hpf or 48hpf same as in A,B. (n.d.) Not determined. (*) Indicate genes that have been identified and their spatial blastula and gastrula expression patterns characterized elsewhere (see Tables S4 and S5 in [35] for references). However, to include them into our GRNs we performed qPCRs also for these genes (i.e. fgfA1, fgfA2, ax1 etc…). (**) qPCR value from [24].

Figure 5. Molecular phenotype analysis of Mo-NvERG injected embryos on genes expressed in the endomesoderm

(A) Changes in gene expression of 66 potential components of the cnidarian endomesoderm GRN within the animal hemisphere after NvERG knock-down compared to control embryos analyzed by qPCR. Changes in gene expression are indicated as relative fold changes compared to MO-CTRL injected embryos (x ± sem, n = 3 per gene). The grey bar indicates no significant change in gene expression (−1.5,1.5). Information on the iconography (stars and circles) are indicated below the graph. Gene expression domains at the blastula stage are the same as Figure 4A. (B) Analysis of the molecular effects of NvERG inhibition (G–K, Q–U, ZA–ZE) compared to control injections (B–F, L–P, V–Z) on endomesodermal gene expression analyzed by in situ hybridization. Antisense probes used as indicated in the bottom left corner of each image (also valid for the corresponding inset). The red dashed circle in (V,Z) indicates the central ring expression of Nvbra showing extension of its expression domain into the central domain. 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. Insets are animal pole views.

Figure 6. Molecular phenotype analysis of MO-NvERG, MO-NvFGFRA and NvSix3/6 injected embryos on genes expressed in the apical domain

(A) Changes in gene expression of 15 potential components of the cnidarian apical domain GRN within the vegetal hemisphere after NvERG (blue), NvFGFRA (green) or NvSix3/6 (yellow) knock-downs compared to control embryos analyzed by qPCR. Changes in gene expression are indicated as relative fold changes compared to MO-CTRL injected control embryos (x ± sem, n = 3 per gene). The grey bar indicates no significant change in gene expression (−1.5,1.5). Stars below the bars indicate significant variation. Analysis of the molecular effects of NvERG (G–K), NvFGFRA (Q–U) and NvSix3/6 (Y–ZA) inhibition compared to control injections (B–F, L–P, V–X) on apical domain gene expression analyzed by in situ hybridization. Antisense probes used as indicated in the bottom left corner of each image (also valid for the corresponding inset). 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 7. Updated gene regulatory network orchestrating embryonic development in the cnidarian N. vectensis

Enhanced Biotapestry diagram [86] of the gene regulatory network describing the gene deployment at 24hpf and regulatory interactions of endomesodermal, ectodermal and neuronal genes identified in previous studies [24,31,35,39,74]. 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. The colored boxes represent the spatial domains as described in Figure 4A. Genes inactivated by repression in a given territory are represented in light grey. Controversial results [24,76] about the role of cWnt/TCF signaling on NvsnailA expression is indicated by a red dashed arrow. The same GRN, including non-connected genes that are expressed within the specific territories is provided in Figure S5. A first draft of the global GRN framework for body wall endomesoderm, pharynx (endomesoderm and ectoderm), mouth, body wall ectoderm, sub-apical and apical domain including components of the Nematostella nervous system at the end of gastrulation (48 hpf) is provided in Figure S6.