Strabismus-mediated primary archenteron invagination is uncoupled from Wnt/β-catenin-dependent endoderm cell fate specification in Nematostella vectensis (Anthozoa, Cnidaria): Implications for the evolution of gastrulation

Abstract

Background: Gastrulation is a uniquely metazoan character, and its genesis was arguably the key step that enabled the remarkable diversification within this clade. The process of gastrulation involves two tightly coupled events during embryogenesis of most metazoans. Morphogenesis produces a distinct internal epithelial layer in the embryo, and this epithelium becomes segregated as an endoderm/endomesodermal germ layer through the activation of a specific gene regulatory program. The developmental mechanisms that induced archenteron formation and led to the segregation of germ layers during metazoan evolution are unknown. But an increased understanding of development in early diverging taxa at the base of the metazoan tree may provide insights into the origins of these developmental mechanisms.

Results: In the anthozoan cnidarian Nematostella vectensis, initial archenteron formation begins with bottle cell-induced buckling of the blastula epithelium at the animal pole. Here, we show that bottle cell formation and initial gut invagination in Nematostella requires NvStrabismus (NvStbm), a maternally-expressed core component of the Wnt/Planar Cell Polarity (PCP) pathway. The NvStbm protein is localized to the animal pole of the zygote, remains asymmetrically expressed through the cleavage stages, and becomes restricted to the apical side of invaginating bottle cells at the blastopore. Antisense morpholino-mediated NvStbm-knockdown blocks bottle cell formation and initial archenteron invagination, but it has no effect on Wnt/ß-catenin signaling-mediated endoderm cell fate specification. Conversely, selectively blocking Wnt/ß-catenin signaling inhibits endoderm cell fate specification but does not affect bottle cell formation and initial archenteron invagination.

Conclusions: Our results demonstrate that Wnt/PCP-mediated initial archenteron invagination can be uncoupled from Wnt/ß-catenin-mediated endoderm cell fate specification in Nematostella, and provides evidence that these two processes could have evolved independently during metazoan evolution. We propose a two-step model for the evolution of an archenteron and the evolution of endodermal germ layer segregation. Asymmetric accumulation and activation of Wnt/PCP components at the animal pole of the last common ancestor to the eumetazoa may have induced the cell shape changes that led to the initial formation of an archenteron. Activation of Wnt/ß-catenin signaling at the animal pole may have led to the activation of a gene regulatory network that specified an endodermal cell fate in the archenteron.

Figure 1

Phylogenetic analysis of Nematostella Strabismus. A) Alignment of NvStbm sequence with Stbm homologs from 10 metazoan taxa. The alignment was generated using ClustalW. Accession numbers used to obtain sequences for the alignment: Strabismus protein from N. vectensis (XP_001630420), Homo sapiens (NP_065068- 47% identity), Mus musculus (NP_808213- 48% identity), Xenopus laevis (NP_001083892- 47% identity) Danio rerio (NP_991313- 45% identity), Branchiostoma floridae (XP_002613474- 48% identity), Caenorhabditis elegans (NP_508500- 31% identity), Drosophila melanogaster (NP_477177- 41% identity), Hydra vulgaris XP_002160166 - 47% identity), Trichoplax adhaerens (XP_002107643 - 38% identity) and Strongylocentrotus purpuratus (41% identity). The percent identities of Stbm proteins from various taxa to NvStbm are indicated. Conserved amino acid residues across all nine taxa are shown in red. The regions highlighted in grey indicate the transmembrane regions and the region highlighted in yellow indicate the peptide sequence used to generate the anti NvStbm polyclonal antibody. B) Molecular phylogeny of Strabismus proteins from various metazoan taxa. Phylogenetic analysis of the NvStbm protein was carried out to determine orthology. Amino acid alignments including NvStbm and both vertebrate and invertebrate Stbm sequences were generated using MacClade [49]. A Bayesian Phylogenetic analysis was carried out using MrBayes 3.1 [50] with a run of one million generations, sampled every 100 generations. The summary consensus tree was generated in MrBayes using the last 7,500 trees and the posterior probabilities were calculated for this consensus tree. The posterior probability values for the consensus tree are shown.

Figure 2

Expression of NvStbm in Nematostella eggs and embryos.(A-E) Whole mount RNA in situ hybridization for NvStbm. (A) Unfertilized egg. (B) Zygote. (C) 8-cell stage. (D) Blastula stage. (E) Gastrula stage. (F-J) NvStbm immunofluorescence in eggs and embryos (red). (F) Unfertilized egg. The female pronucleus at the animal pole (arrow) is labeled with anti-histone antibodies (green). (G) Zygote. (H) 8-cell. (I) Blastula. (J) Gastrula. The blastula and gastrula stages are also stained with phalloidin (green). At the gastrula stage the NvStbm is highly localized to the apical end of cells at the blastopore. The basal ends of these cells are deep inside the blastocoel at this time. (K) Western blot analysis of NvStvbm during early embryogenesis. The anti-NvStbm antiserum recognizes a single band at the expected size of 80 kD. The NvStbm protein is maternally expressed and is expressed at all the examined stages. Anti ß-tubulin was used as a loading control. (L) Preadsoption test of the specificity of the affinity purified anti NvStbm antibodies. Preadsorption of the affinity-purified anti-NvStbm polyclonal antibodies with ten fold molar excess of the NvStbm peptide used as the antigen results in elimination of staining of the 80 kD protein band observed in the control lane incubated with the anti-NvStbm antibodies. Anti ß-tubulin was used as a loading control.

Figure 3

Analysis of the efficacy of NvStbm knockdown using antisense NvStbm morpholinos. (A) Western blot analysis was used to determine the efficacy of the NvStbm-MO. Individual protein band pixel intensities were measured using the Odyssey software (LI-COR, Lincoln, NE, USA). These intensities were used to calculate the ratios of NvStbm/ß-tubulin and normalized to determine the fold difference of each NvStbm band. This analysis showed that there is approximately a five-fold decrease of the endogenous Stbm protein in NvStbm-MO-injected (650 μM) embryos compared to embryos injected with the Control-MO (650 μM). (B, C) Scanning confocal microscopical analysis of Control- and NvStbm-MO injected embryos immunostained with affinity-purified NvStbm antibodies. (B) NvStbm antibody stained Control- MO-injected (650 μM) embryos showed strong NvStbm expression at the apical end of invaginating cells at the blastopore while (C) a majority of NvStbm-MO (650 μM) injected embryos did not show NvStbm expression. Also, NvStbm-MO injected embryos did not undergo initial archenteron invagination. The numbers in (B) represent the number of cases with NvStbm staining, and the numbers in (C) represent the number of cases without NvStbm staining.

Figure 4

The effect of NvStbm knockdown on endoderm specification and primary invagination in Nematostella embryos. (A) Control-MO injected gastrula at 12 to 14 hpf. (B) NvStbm-MO injected embryo at 12-14 hpf. Embryos are stained with phalloidin (green) and propidium iodide (red). (C, D, E, F) Embryos co-injected with Nvß-catenin::RFP RNA and with either Cont-MO or NvStbm-MO. (C) In embryos injected with the Cont-MO, initially Nvß-catenin::RFP is expressed in all blastomeres as previously shown. (D) By the blastula stage Nvß-catenin::RFP is stabilized in animal-half derived blastomeres and enters the nuclei in these cells. Similar expression dynamics are seen in NvStbm-MO and Nvß-catenin::RFP RNA injected embryos at the early cleavage stage (E) and blastula stage (F), indicating that knockdown of NvStbm does not affect Wnt/ß-catenin signaling. (G-L) Analysis of gene expression in Control- and NvStbm-MO injected embryos. Similar to Cont-MO injected embryos that express NvSnail (G) and NvFz10 (I) in the endoderm, NvStbm-MO injected embryos also express NvSnail (H) and NvFz10 (J) but show no signs of archenteron invagination. NvFz5 expression in Cont-MO (K) and NvStbm-MO (L) injected embryos. (M) Quantification of the gastrulation phenotype in embryos using confocal microscopy shows that in contrast to Uninjected- (94.6%, n = 37) and Control-MO injected (96.8%, n = 31) embryos which gastrulated normally, very few NvStbm-MO injected embryos displayed an archenteron (9%, n = 66).

Figure 5

Nematostella embryos inhibited in ß-catenin signaling fail to specify endoderm, but undergo initial archenteron invagination. (A-L) Morphological analysis of early (A-F) and late (G-L) gastrula stage embryos that are uninjected controls (A, G), or injected with GFP (B, H), NvDsh-DIX::GFP (C, I), SpAxin (D, J), Xß-cat-Eng (E,K), or LvCadherin (F, L) mRNA. Nuclei are stained with propidium iodide (red), and F-actin with phalloidin (green). Similar to controls (A, B), NvDsh-DIX (C), SpAxin (D) and Xß-cat-Eng (E) overexpressing embryos develop a primary invagination unlike LvCadherin (F) overexpressing embryos. However, while controls develop a normal endodermal epithelium at the late gastrula stage (G, H), NvDsh-DIX (I), SpAxin (J) and Xß-cat-Eng (K) overexpressing embryos have compacted coelenterons. Cadherin overexpressing embryos do not show any primary invagination and never develop an endodermal epithelium (L). (M) Quantification of the different phenotypes in mid and late gastrula stage embryos observed using confocal imaging clearly show that 91.1% (n = 45) of NvDsh-DIX RNA-injected embryos, 88.4% (n = 43) of SpAxin RNA-injected embryos and 85.7% (n = 49) of ß-catEn RNA-injected embryos cannot maintain the gut epithelium in contrast to 90.9% (n = 33) of uninjected and 83% (n = 59) of GFP RNA-injected embryos which show a normal endodermal epithelium. None of the Lvcadherin RNA injected embryos gastrulated (n = 23).

Figure 6

Expression of endodermal and ectodermal genes in control and ß-catenin signaling-disrupted Nematostella embryos. (A-O) WMISH for endodermal and ectodermal markers in uninjected, GFP mRNA- and NvDsh-DIX::GFP mRNA-injected embryos. Control uninjected (A, D, G, J) and GFP mRNA injected (B, E, H, K) embryos show expression of NvSnail and NvFz10 in the early A, B, G, H) and mid (D, E, J, K) gastrula stages. In contrast, NvDsh-DIX::GFP mRNA-injected embryos do not express these markers at the early (C, I) or mid (F, L) gastrula stages. Normal expression of the ectodermal marker NvFz5 is seen in uninjected (M), GFP mRNA injected (N), and NvDsh-DIX::GFP mRNA (O) injected embryos.

Figure 7

Wnt signaling and the evolution of a functional gut. (A) In Nematostella, initial archenteron invagination is regulated by bottle cell induction by NvStbm, and this can occur independently of endoderm cell fate specification mediated by Wnt/ß-catenin signaling. Both Wnt pathways are required for completion of gastrulation. It is possible that a single upstream ligand (X) or receptor (Fz) coordinates the activation of both branches of Wnt signaling during embryogenesis. (B) Model indicating the co-option of a polarity found in the oocytes of the last common ancestor to the eumetazoa to locally activate Wnt signaling at the animal pole. The centrosomes associated with the oocyte nucleus at meiosis served as a scaffold to localize critical Wnt pathway components to the apical pole (a). These components would be inherited by blastomeres at the animal pole (b), and their activation would drive apical constriction of these cells to form bottle cells (c), leading to the initial invagination of an archenteron (d). Endoderm specification mediated by Wnt/ß-catenin could have been coordinated with primary archenteron invagination by a single upstream ligand (A). Alternatively, endoderm specification could have occurred after the evolution of the primary invagination by the localized activation of the Wnt/ß-catenin pathway at the animal pole.