Antagonistic BMP-cWNT signaling in the cnidarian Nematostella vectensis reveals insight into the evolution of mesoderm

Abstract

Gastrulation was arguably the key evolutionary innovation that enabled metazoan diversification, leading to the formation of distinct germ layers and specialized tissues. Differential gene expression specifying cell fate is governed by the inputs of intracellular and/or extracellular signals. Beta-catenin/Tcf and the TGF-beta bone morphogenetic protein (BMP) provide critical molecular signaling inputs during germ layer specification in bilaterian metazoans, but there has been no direct experimental evidence for a specific role for BMP signaling during endomesoderm specification in the early branching metazoan Nematostella vectensis (an anthozoan cnidarian). Using forward transcriptomics, we show that beta-catenin/Tcf signaling and BMP2/4 signaling provide differential inputs into the cnidarian endomesodermal gene regulatory network (GRN) at the onset of gastrulation (24 h postfertilization) in N. vectensis Surprisingly, beta-catenin/Tcf signaling and BMP2/4 signaling regulate a subset of common downstream target genes in the GRN in opposite ways, leading to the spatial and temporal differentiation of fields of cells in the developing embryo. Thus, we show that regulatory interactions between beta-catenin/Tcf signaling and BMP2/4 signaling are required for the specification and determination of different embryonic regions and the patterning of the oral-aboral axis in Nematostella We also show functionally that the conserved "kernel" of the bilaterian heart mesoderm GRN is operational in N. vectensis, which reinforces the hypothesis that the endoderm and mesoderm in triploblastic bilaterians evolved from the bifunctional endomesoderm (gastrodermis) of a diploblastic ancestor, and that slow rhythmic contractions might have been one of the earliest functions of mesodermal tissue.

Keywords: cell fate; cell signaling; evodevo; gene regulatory network; heart kernel.

Fig. 1.

Gastrulation and germ layer specification in Nematostella. (A) cWnt signaling provides inputs into the endomesodermal GRN in Nematostella to activate endomesodermal gene expression in the animal half blastomeres. BMP2/4 expession during early and later development in Nematostella suggests that it might be playing different regulatory roles during development. (B) Phylogenetic relationships among major metazoan lineages showing the position of Cnidaria as the sister taxon to all bilaterians (61). (C) At 24 hpf (late blastula stage) the animal hemisphere contains at least four domains defined by differential gene expression (25).

Fig. 2.

Differential inputs from cWnt and BMP2/4 signaling. (A) Effect of NvBMP2/4 knockdown and NvTcf knockdown on germ layer specification in Nematostella embryos. (A′) Control-MO–injected embryo at 48 hpf. (B′) NvBMP2/4-MO–injected embryo at 48 hpf. Embryos are stained with phalloidin (green) and propidium iodide (red). Note how disorganized the internal issue is and the lack of endomesodermal epithelialization. (B) Effects on gene expression after BMP MO injection (E′–H′ and M′–P′) compared with control embryos (A′–D′ and I′–L′) analyzed by in situ hybridization. Insets show oral views of the embryo. (C) Venn diagram depicting unique and shared sets of down-regulated and up-regulated genes between dnTCF-injected embryos vs. dextran-injected control embryos and BMP2/4 morpholino-injected embryos vs. dextran-injected control embryos at 24 hpf. (D) Model GRN circuit that would potentially result in separating the central ring domain into an inner and an outer ring. (E) At 24 hpf, the animal hemisphere contains at least four domains defined by differential gene expression as demonstrated by Röttinger et al. (25): the central domain, the central ring, the central ring + ring domain, and the external ring. Based on the model proposed in this study, integrated inputs from canonical Wnt signaling and BMP2/4 signaling splits the central ring domain into two: an outer central ring coexpressing NvTcf and NvBMP2/4 and an inner central ring expressing NvBra, NvFoxA, and NvWntA.

Fig. 3.

BMP2/4-regulated components of heart-field specification GRN kernel. (A) BMP2/4-deficient embryos lack NvNkx2.5 and NvEhand expression compared with control-MO–injected embryos at 24 hpf. (B) Potential heart-field specification GRN kernel assembled from experimental data from Drosophila and vertebrates [adapted from Davidson and Erwin (4)].

Fig. 4.

Ancestral heart-field specification GRN kernel is present in Nematostella endoderm. (A) Components of the bilaterian heart-field specification GRN, NvEhand, and NvGata are lost in the endoderm (black arrowheads) and NvMef2 of NvNkx2.5-deficient embryos compared with their normal expression in control-MO–injected embryos. Ectodermal expression of NvGata is not affected by knockdown of NvNkx2.5 (white arrowheads). (B) An ancestral heart-field specification GRN kernel is present in Nematostella where NvNkx2.5 is activated by BMP2/4, which in turn activates NvEhand, NvGata, and NvMef2 in the endoderm. Note the absence of the Gata lockdown feedback loop.

Fig. S1.

Morpholino mediated knock down and CRISPR/Cas9 mediated knock out of NvNkx2.5 in Nematostella. (A) A splice blocking anti-sense morpholino oligo was designed targeting the splice site between intron1 and exon2. (B) PCR showing an amplified DNA fragment of 880bp from cDNA from control embryos but absent in cDNA from NvNkx2.5 MO injected embryos. NvNkx2.5 gene deletion efficiency was determined by amplifying the NvNkx2.5 gene fragment.

Fig. S2.

CRISPR/Cas9 mediated knock out of NvNkx2.5 in Nematostella. (A) gRNA sequences and primers used for the PCR assay are shown on the genomic sequence of NvNkx 2.5. (B) Note the truncated fragments of NvNkx2.5 with a smaller size than the wild type resulting from CRISPR/Cas9 mediated mutagenesis. (C) NvNkx2.5 knockout embryos (Cas9 and gRNAs) lack NvNkx2.5 expression compared with control embryos (Cas9 only) embryos at 24 hpf.

Fig. S3.

NvGata does not provide regulatory inputs into the heart-field specification GRN kernel in Nematostella. NvNkx2.5 and NvEhand expression is not affected in NvGata MO-injected embryos compared with control MO-injected embryos at 24 hpf.