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
© Kumburegama et al; licensee BioMed Central Ltd. 2011
Received: 22 August 2010
Accepted: 21 January 2011
Published: 21 January 2011
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.
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.
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.
The origin of metazoans from a choanoflagellate-like protist and the vast diversification of this clade is a remarkable evolutionary chronology in the history of life on Earth. From relatively simple origins, metazoans have radiated to produce organisms with levels of physiological and morphological complexity unmatched in other multicellular forms that have emerged in several clades [1, 2]. The unique character that enabled the evolution of complex metazoans is generally believed to be the process of gastrulation . The evolution of gastrulation produced a functionally distinct internal cell layer, and the interaction between the different tissue layers most likely led to the induction of new cell types, tissues, and organs . Beginning with the seminal observations of Ernst Haeckel, a number of models have been proposed to reconstruct the evolution of gastrulation [reviewed in 4]. Many of these models posit that activation of morphogenesis on one side of a hypothetical blastula-like "urmetazoan" enabled cells on the outside to internalize and form an archenteron . Regardless of the details of the individual models, it is likely that a crucial step in the evolution of gastrulation was the co-option of a localized molecular asymmetry that was present in ancient embryos to effect the cell shape changes that led to cell ingression and/or epithelial bending. However, the nature of the primordial anisotropy that triggered initial gastrulation movements is not known, and no existing model provides a molecular explanation for the initial evolution of a functional gut [3, 5].
One ancient polarity that is present in most metazoan eggs is the animal-vegetal (AV) or primary axis of the egg [3, 6]. The animal pole is defined by the site of polar body release during meiosis, and the AV axis is polarized by asymmetric distribution of maternal factors in the form of RNA, protein or organelles that can impart differential developmental potentials to blastomeres derived from the different poles of the egg [3, 6, 7]. In most animals, the AV axis predicts the axial properties of the embryo and adult. For example, in bilaterians, a clade that includes most animal phyla, patterns of gastrulation morphogenesis vary but a majority of these organisms develop their endomesoderm from vegetal pole-derived blastomeres [3, 7]. In Porifera and Placozoa, two animal phyla generally believed to be two of the earliest diverging clades [8, 9], little is known about the relationship between initial egg polarity and larval/adult patterning [10, 11]. Additionally, these animals lack a functional endodermal germ layer. The Cnidaria and Ctenophora are the two remaining extant basal, non-bilaterian metazoan phyla and recent phylogenomic analyses indicate that they diverged after the appearance of the Porifera and the Placozoa . In these two taxa that are clear outgroups to the bilaterians , and the only non-bilaterians to undergo true gastrulation, endoderm specification and archenteron formation are initiated at the animal pole [3, 7, 12]. These observations have led to the proposal that formation of an archenteron and segregation of an endodermal germ layer evolved at the animal pole, and the mechanisms that regulate these processes were moved to the vegetal pole sometime after the divergence of the urbilaterian from the last shared common ancestor with cnidarians and ctenophores [7, 13]. This idea is supported at the molecular level by the observation that the site of gastrulation in several bilaterians and cnidarians is marked by nuclear ß-catenin [3, 13–18]. If archenteron formation and segregation of an endodermal germ layer evolved at the animal pole, then cnidarians and ctenophores are key taxa for gaining insights into the developmental mechanisms that led to the evolution of these processes.
The anthozoan cnidarian Nematostella vectensis has emerged as an important model organism for comparative molecular studies of embryonic development. Recent studies have identified several key cellular and molecular processes involved in initial germ layer segregation and archenteron formation in this animal [13, 14, 19–21]. Gastrulation is initiated in Nematostella by primary archenteron invagination at the animal pole of the embryo [20, 21]. This primary invagination is induced by an apical constriction of cells at the future blastopore, leading to the formation of bottle cells and a subsequent buckling of the blastula wall [20, 21]. It is well established that bottle cells play an important role in initiating gastrulation in embryos of diverse taxa, and components of the ß-catenin-independent Wnt/PCP pathway appear to play a particularly important role in regulating apical constriction of these cells during initial archenteron formation [22–26]. There are several "core" PCP genes that are conserved between vertebrates and Drosophila, and we have initiated studies to determine the roles of these genes in regulating early polarity events in Nematostella. Here, we focus on the core Wnt/PCP gene NvStrabismus (NvStbm). We show that NvStbm is maternally expressed, and the NvStbm protein becomes localized to the animal pole of the zygote, and remains asymmetrically localized to the apical end of blastomeres at one side of the embryo during the early cleavage stages. At the initiation of primary invagination, NvStbm becomes restricted to the apical end of cells at the blastopore. Knockdown of NvStbm using antisense morpholinos blocks bottle cell formation and archenteron invagination but has no effect on cell fate specification. Strikingly, blocking the Wnt/ß-catenin pathway, which is required for endoderm specification in Nematostella[13, 14] has no effect on primary archenteron invagination indicating that initial gastrulation morphogenesis can be uncoupled from endoderm cell fate specification in this embryo. We propose a novel model for the evolution of gastrulation in metazoans that is based on a deeply conserved organismic polarity, and evolutionarily conserved signal transduction pathways.
Cloning and phylogenetic analysis of an Stbm ortholog from Nematostella
Expression of NvStbm during Nematostella development
NvStbm knockdown blocks primary invagination
NvStbm knockdown does not affect ß-catenin nuclearization and endoderm specification
In bilaterians endoderm specification and morphogenesis are tightly coupled, and usually cell fate specification precedes morphogenesis [30–32]. Endoderm cell fate specification in Nematostella requires Wnt/ß-catenin signaling [13, 14], and studies have shown that there is crosstalk between the Wnt/ß-catenin and the Wnt/PCP pathways . This raised the possibility that NvStbm knockdown may affect Wnt/ß-catenin signaling and thereby block gastrulation by disrupting endoderm specification. To examine this we first analyzed embryos co-injected with Nvß-catenin::RFP mRNA (0.3 μg/μl) and NvStbm-MO or Control-MO to determine if NvStbm-knockdown affected the nuclearization of ß-catenin in blastomeres at the animal pole [13, 14]. Analysis of these embryos showed that there was no difference in the Nvß-catenin::RFP expression pattern between NvStbm morphants, and control embryos (Figure 4C-F). Consistent with this observation, WMISH using several endoderm- and ectoderm-specific markers showed normal gene expression patterns in NvStbm morphants compared to controls (Figure 4G-L). These results indicated that failure of primary archenteron invagination in NvStbm morphant embryos was most likely not due to disruption of ß-catenin-dependent endoderm specification.
Nematostella embryos undergo primary archenteron invagination in the absence of endoderm cell fate specification
We had previously reported that LvCadherin and Xß-catEn mRNA-injected embryos failed to gastrulate based on the lack of an archenteron . In the experiments where we blocked nuclear ß-catenin by overexpressing cadherin, we consistently saw loss of endoderm gene expression and a failure of gastrulation in Nematostella, phenotypes we have reproduced in this study (Figure 5F, L). However, if primary archenteron invagination is uncoupled from endodermal cell fate specification, then LvCadherin-overexpressing embryos should produce bottle cells and consequently undergo primary invagination, but we have never observed this (Figure 5F, L, M). A possible explanation for this discrepancy comes from recent work that has shown that certain cadherins can affect the actin cytoskeleton . Hence, it is possible that the lack of bottle cells and primary invagination in cadherin-overexpressing embryos is due to LvCadherin disrupting the cortical actin in bottle cells, thereby blocking apical constriction of these cells.
Our observations in the current study that Xß-catEn-overexpressing embryos consistently form a primary invagination contradict a result from our previous report . In the previous study we used light microscopy to analyze the morphology of Nematostella embryos. These embryos are relatively opaque. Hence it is possible that the discrepancy between the results of the previous study and the current study is because when we previously examined Xß-catEn-overexpressing embryos the blastocoel appeared hollow because the invaginated archenteron had lost its epithelial integrity, and this unusual phenotype was undetectable using light microscopy. In the current study, examination of many Xß-catEn mRNA-injected embryos using scanning confocal microscopy clearly showed that these embryos formed a primary invagination, but these archentera lost their epithelial integrity by the late gastrula stage (Figure 5E, K). By using the Dsh-DIX dominant-negative, Axin and Xß-catEn to downregulate nuclear ß-catenin signaling, and through careful analysis of these phenotypes using scanning confocal microscopy and molecular markers, we show here that endoderm specification is not required for primary archenteron invagination in Nematostella, and moreover that the epithelium of this initial archenteron loses its integrity in the absence of nuclear ß-catenin.
In this study, we have presented results that implicate the core Wnt/PCP protein NvStbm in regulating primary archenteron invagination in Nematostella. We also show that in this cnidarian, NvStbm-regulated primary archenteron invagination is independent of Wnt/ß-catenin signaling-dependent endoderm cell fate specification, demonstrating an uncoupling of initial gut morphogenesis from endoderm cell fate specification. Our results support a model for the evolution of an archenteron in the last common ancestor to the eumetazoa where asymmetric localization and activation of Wnt/PCP signaling components at the animal pole induced the cell shape changes that led to epithelial buckling and morphogenesis of the first gut. Concomitant or subsequent activation of Wnt/ß-catenin signaling in animal pole blastomeres may have activated a gene regulatory network that specified endodermal cell fates in the archenteron.
Strabismus and regulation of primary archenteron invagination in Nematostella
Cnidarians display more diverse gastrulation mechanisms than any other metazoan phylum . However, a majority of examined anthozoan (and scyphozoan) species gastrulate by invagination, and it has been suggested that this mechanism was the primitive mode of gastrulation in cnidarians . Typical of other anthozoans, archenteron formation in Nematostella is initiated by primary invagination at the animal pole of the embryo. Our results indicate that NvStbm plays a critical role in regulating primary invagination, and moreover, that this protein regulates this process by mediating the formation of bottle cells. NvStbm is expressed maternally and the protein becomes highly restricted to the apical end of bottle cells at the blastopore in Nematostella embryos. Knockdown of NvStbm protein levels results in the absence of apical constriction of cells at the animal pole, failure of bottle cell formation, and inhibition of primary archenteron invagination. To the best of our knowledge, Stbm has not been directly implicated in bottle cell formation in other metazoans, but this protein plays a critical role in regulating cell polarity and cell shape changes in many other cellular contexts [37–40]. Of relevance to the current study is that Stbm has been implicated in regulating the cell polarity mediating convergence and extension (CE) movements in vertebrates [37, 38, 40]. The mechanism of cell polarity regulation by Stbm during CE is not well understood, but it is believed to involve proteins in the Wnt/PCP pathway including Dsh, Prickle, RhoA, ROCK and Rac . In many developmental contexts, apical constriction of bottle cells is regulated by RhoA-mediated activation of myosin II at the apical cortex . We currently do not know how NvStbm regulates apical constriction in Nematostella through the Wnt/PCP pathway, but some observations suggest a role for NvDsh in this process. Dsh has been shown to regulate bottle cell formation in Xenopus embryos, and it is one of a relatively small number of proteins known to directly interact with Stbm [39, 40]. NvDsh shows an expression pattern that closely matches the expression of NvStbm during early embryogenesis . Moreover, blocking Wnt/PCP signaling in Nematostella by overexpressing Dsh-DEP, a Dsh dominant-negative in the Wnt/PCP pathway, also leads to failure of bottle cell formation and inhibition of initial archenteron invagination without affecting the Wnt/ß-catenin pathway . Collectively these results suggest that an NvStbm/Dsh pathway may mediate myosin activation and regulate apical constriction and primary archenteron invagination in Nematostella. However, these possibilities and the role for other Wnt/PCP components in early gastrulation events in this cnidarian need to be addressed in future studies.
Uncoupling of primary archenteron invagination and endoderm specification in Nematostella
Our observations in Nematostella raise the possibility that during evolution of gastrulation, initial morphogenesis of the archenteron could have occurred without prior segregation of an endodermal germ layer. The data presented here suggest that a maternally expressed Wnt/PCP pathway component localized to the animal pole in Nematostella embryos regulates primary archenteron invagination, while Wnt/ß-catenin signaling mediates endoderm specification. An important question, however, is whether the mechanisms we observe in Nematostella are restricted to anthozoans, or if they are more generally seen in the Cnidaria. There is no other anthozoan model system where mechanisms of endoderm specification and gastrulation are understood at the molecular level. Similarly, to the best of our knowledge, functional molecular analysis of early embryonic development has not been done in cubozoans, or in scyphozoans. The fourth cnidarian class comprises the hydrozoans, and from this group Hydra has proved to be an important cnidarian model for studying axial patterning in the adult. However, due to technical difficulties of obtaining eggs and embryos, Hydra is not easily amenable to molecular manipulation during the embryonic stages . Over the past several years, the hydrozoan Clytia hemisphaerica has emerged as an important model system for studying hydrozoan embryogenesis . Elegant work done in Clytia has established the importance of Wnt/ß-catenin signaling in endoderm specification in this organism [15, 43]. In this hydrozoan, oral/endoderm specification is regulated by CheWnt3, which is localized to the animal pole of the unfertilized egg. Blocking CheWnt3 using an antisense morpholino eliminated nuclearization of ß-catenin and the expression of endodermal markers. However, while these embryos were delayed in gastrulation, by the planula stage they had what appeared to be a complete endodermal epithelium . Similar to what we observed in Nematostella [13, this study], in Clytia, the archentera of embryos lacking nuclear ß-catenin did not express any markers of endoderm differentiation . Interestingly, when the translation of CheFz1, a Frizzled homolog localized to the animal pole of the unfertilized Clytia egg, was blocked using an antisense morpholino, these embryos did not nuclearize ß-catenin or express markers for endoderm, and moreover, they did not show any signs of gastrulation . Based on the effect of CheFz1 knockdown on the polarity of the embryo, these authors suggested that this Frizzled receptor was involved in endoderm specification through Wnt/ß-catenin signaling, as well as signaling through a Wnt/PCP pathway. In sum, these results suggest that morphogenesis of the gut and cell fate specification are also uncoupled in Clytia, and in this case as well, the activities are regulated by the two Wnt pathways. Gastrulation in Clytia and most hydrozoans involves ingression rather than invagination, but this process also involves bottle cell formation by apical constriction of cells at the blastopore [12, 43]. It would be interesting to determine if Clytia Stbm orthologs and other components of Wnt/PCP signaling affect the formation of bottle cells in this embryo as seen in Nematostella.
Localization of Wnt/PCP components to the animal pole, and the evolution of gastrulation
Bottle cell formation is a common strategy that is used to initiate epithelial bending in metazoan embryos . In particular, bottle cell formation via apical constriction is widely used to initiate archenteron invagination in diverse taxa . This highly conserved mechanism for initiating gastrulation has led to the suggestion that localized induction of bottle cells could have led to the initial formation of an archenteron . However, a molecular mechanism for how the coordinated and spatially localized apical constriction of a group of cells could have occurred, that is based on deeply conserved organismic underpinnings has heretofore been missing. Recent studies have implicated Wnt/PCP pathway components in bottle cell formation during gastrulation initiation in C. elegans, sea urchins and Xenopus[22–25]. In this study, our data implicate a core component of the Wnt/PCP pathway in the formation of bottle cells in embryos of a modern descendant of one of the earliest diverging metazoan phyla, indicating a possible ancient co-option of this pathway for mediating apical constriction at one end of the embryo. Other studies have shown that there is an enrichment of Wnt pathway components at the animal pole of cnidarian eggs [15, 43] raising an intriguing possibility for the initial evolution of the archenteron. There is increasing evidence that Wnt pathway components accumulate at centrosomes and cilia found in many epithelial cell types [44–46]. The animal pole is defined by the site of polar body release [6, 7], hence oocytes of all metazoans should have centrosomes localized at this pole during meiosis. Interestingly, oocytes of some invertebrate deuterostomes have a flagellum at the animal pole, and it has been shown that the AV axis in these animals corresponds to the apical-basal polarity of the germinal epithelium, suggesting that the AV axis may be homologous to the apical basal polarity of epithelial cells [47, 48]. The prevalence of this character in other taxa is unknown, but it is likely that cilia/flagella and the centrosomes associated with this organelle are homologous to the flagellar apparatus in the unicellular protist ancestor of metazoans. Thus, we propose that the centrosomes and flagellum of the protist last common ancestor of metazoans was the original polarity that was co-opted during metazoan evolution to enrich components of the Wnt pathway to one end of the eumetazoan oocyte (Figure 7B). Cell autonomous activation of Wnt/PCP signaling may have led to apical constriction-mediated cell shape change and initial archenteron invagination. Nuclearization of ß-catenin at the animal pole by these localized Wnt pathway components may have activated the expression of gene products that led to the segregation of a novel germ layer, and the evolution of a functional gut.
Materials and methods
Spawning and gamete handling
Spawning, gamete preparation and embryo culturing was done as previously described .
Primer sequences used to clone various cDNAs used in this study
Gene or Construct
NvStbm (in situ hybridization probe)
Forward 5' CGACAAAGCGAACCAATGTTTACTCT 3'
Reverse 5' CCGACATCATGTAAGGGTTATACTCA 3'
Mixed stage N.vectensis cDNA
Forward 5'GCCGGAATTCATGCCGCAATATCGTACCAAAGAC 3'
Reverse 5' GCCTAGGCCTGACGGAGGTCTCCGAATTTAACTTG 3'
Mixed stage N.vectensis cDNA
Forward 5' AGTACGATCAGTGCGTCCCCCAATG 3'
Reverse 5' CCGCCACGACTTGAATGTTTTTGTAG 3'
Mixed stage N.vectensis cDNA
Forward 5' GCGAAGAGATCACCATTCCCATGTGC 3'
Reverse 5' ACCCACCAAACAGAGGAAGCCATTC 3'
Mixed stage N.vectensis cDNA
Forward 5' GCCGGAATTCATGAGTCTAGAAGTGTATAGGTTC 3'
Reverse 5' GCCGAGGCCTGTGATCATCGACAGATTCCACCTG 3'
Blastula stage S. purpuratus cDNA
mRNA synthesis, morpholinos and microinjection
Synthesis of mRNA was done as previously described [13, 14]. Anti-sense morpholino oligonucleotides for NvStbm (NvStbm-MO; 5' GCGCTAAACTTGTTACAATCACAGC 3') and standard control morpholinos (5' CCTCTTACCTCAGTTACAATTTATA 3') were synthesized at Gene Tools (Philomath, OR, USA) (Table 1). The NvStbm MO was a translation blocking morpholino designed to bind to the 5' UTR of the NvStbm mRNA immediately upstream of the translation start site. Morpholinos and synthetic RNA were diluted in 40% glycerol, and control RNAs were injected at the same molar concentrations as the RNAs coding for the experimental constructs. Injected embryos were cultured at 25°C in 1/3X seawater.
Antibody production and Western blot analysis
Anti-NvStbm polyclonal rabbit antibodies were raised against a selected sequence corresponding to amino acids 308 to 322 of NvStbm (NH2-TASSQGRRNPGRNDR-COOH), and affinity-purified using the immunizing peptide (Bethyl Labs, Montgomery, TX, USA). Western blots were performed as previously described [13, 14]. Anti-NvStbm and anti-ß-Tubulin (DSHB, Iowa City, IA, USA) antibodies were used at 1:750 and 1:1000 dilutions, respectively. Goat anti-Rabbit IRDye 680 (1:12,000) and Goat anti-Mouse IRDye 800 (1:12,000) were used as secondary antibodies (LI-COR, Lincoln, NE, USA). Stained Western blots were analyzed using an Odyssey Infrared Imager and the Odyssey software (LI-COR).
Immunochemistry, whole mount RNA in situ hybridization and microscopy
Immunochemistry and WMISH was performed as previously described . Anti-NvStbm and mouse anti-histone antibodies (Millipore, MA, USA) were used at 1:100 and 1:250 dilutions, respectively. Immunostained embryos were examined using a Leica TCS SP5 scanning confocal microscope.
Convergence and extension
Green fluorescent protein
Hours post fertilization
Red fluorescent protein
Whole mount in situ hybridization
- Wnt/PCP pathway:
Wnt/Planar Cell Polarity pathway
- Xß-catEn Xenopus :
We thank Y. Marikawa and J. Baker for critical reading of the manuscript and suggestions, and Joanna Kobayashi for technical assistance. We also thank three anonymous reviewers whose suggestions substantially improved this manuscript. This work was supported by a grant from the National Science Foundation (IOS 0720365) to AHW.
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