Par system components are asymmetrically localized in ectodermal epithelia, but not during early development in the sea anemone Nematostella vectensis
© Salinas-Saavedra et al. 2015
Received: 20 December 2014
Accepted: 12 March 2015
Published: 9 May 2015
The evolutionary origins of cell polarity in metazoan embryos are unclear. In most bilaterian animals, embryonic and cell polarity are set up during embryogenesis with the same molecules being utilized to regulate tissue polarity at different life stages. Atypical protein kinase C (aPKC), lethal giant larvae (Lgl), and Partitioning-defective (Par) proteins are conserved components of cellular polarization, and their role in establishing embryonic asymmetry and tissue polarity have been widely studied in model bilaterian groups. However, the deployment and role of these proteins in animals outside Bilateria has not been studied. We address this by characterizing the localization of different components of the Par system during early development of the sea anemone Nematostella vectensis, a member of the clade Cnidaria, the sister group to bilaterian animals.
Immunostaining using specific N. vectensis antibodies and the overexpression of mRNA-reporter constructs show that components of the N. vectensis Par system (NvPar-1, NvPar-3, NvPar-6, NvaPKC, and NvLgl) distribute throughout the microtubule cytoskeleton of eggs and early embryos without clear polarization along any embryonic axis. However, they become asymmetrically distributed at later stages, when the embryo forms an ectodermal epithelial layer. NvLgl and NvPar-1 localize in the basolateral cortex, and NvaPKC, NvPar-6, and NvPar-3 at the apical zone of the cell in a manner seen in bilaterian animals.
The cnidarian N. vectensis exhibits clear polarity at all stages of early embryonic development, which appears to be established independent of the Par system reported in many bilaterian embryos. However, in N. vectensis, using multiple immunohistochemical and fluorescently labeled markers in vivo, components of this system are deployed to organize epithelial cell polarity at later stages of development. This suggests that Par system proteins were co-opted to organize early embryonic cell polarity at the base of the Bilateria and that, therefore, different molecular mechanisms operate in early cnidarian embryogenesis.
KeywordsCnidaria Bilateria Polarity Nematostella vectensis Par proteins
Embryonic cell polarity arises during early development and is critical for axial organization (for example, the oral-aboral, the anterior-posterior, or the dorso-ventral axis). In most bilaterian animals, the oocyte already possesses an internal polarity that is set up maternally called the animal-vegetal axis (A/V axis). Rearrangements of the egg’s cytoplasm and cortical domains in different embryos further polarize the embryo, ensuring proper partitioning of maternal determinants into distinct daughter cells during the cleavage program. The A/V polarity of the embryo and the apico-basal (A/B) polarity of epithelial tissue, in the bilaterian animals examined (including vertebrates, nematode worms, and insects; Figure 1C), have been described as the products of the interaction between the same set of Par system proteins [4-8,11,20-29].
How this complex pattern of molecular interactions arose in animal evolution is unclear. In particular, it is not yet known whether the Par system first served to polarize cells within tissues and was later co-opted to polarize embryos, or, evolved to polarize embryos, and then maintained to polarize cells/tissues during later development. The resolution of this question requires the study of non-bilaterians like cnidarians. Cnidarians (sea anemones, corals, hydroids, and ‘jellyfish’) are composed of polarized outer (epidermal) and inner (gastrodermal) tissue and are the sister group to the group of Bilateria. This phylogenetic position makes them pivotal for reconstructing the evolutionary history of the Par system. Interest in the molecular mechanisms regulating cnidarian development has grown significantly in the past decade, due, in part, to the existence of genomic sequence information. However, while there are some studies that have focused on Wnt signaling pathways [30-35], cytoskeleton [36,37], and cell junctions , there is no published data about the localization and function of Par proteins relative to embryonic and tissue cell polarity in cnidarians. In addition, cnidarians undergo gastrulation at the animal pole of the egg, not the vegetal pole like bilaterian embryos [39-43], so the establishment of embryonic polarity in cnidarians is even more interesting. Par proteins are highly conserved proteins in the genome of Metazoa (including Cnidaria) (Figure 1C) , which raises the question: are the same molecular mechanisms conserved for specifying A/V and epithelial cell polarity in Cnidaria as in Bilateria?
Functional disruption of the Par system in bilaterian embryos leads to failures in the segregation of maternal components and disruptions in the orientation of cell divisions, directly affecting the polarity of the cell [28,29,45,46]. Interestingly, under normal conditions, the early embryos of the sea anemone, Nematostella vectensis, show similar characteristics to Par system mutants of some bilaterian animals. The early embryos of N. vectensis develop with a series of random, asymmetric, and non-synchronous cleavages without clear polarity [32,47]. They possess weak cell junctions, and it is practically impossible to determine the number of cells or cell cycles without any markers due to the lack of characteristic cell boundaries (Additional file 1). In spite of this ‘chaotic cleavage program,’ N. vectensis embryos successfully give rise to an organized and polarized monolayer of blastula epithelia on their way to polyp formation.
Are Par system proteins localized in N. vectensis, and if so, when? Does their distribution suggest that the Par system plays a role in embryonic polarity, tissue polarity, or both? This study attempts to address these questions by giving a general characterization of the most common proteins of the Par system during the early development of the starlet sea anemone N. vectensis. Results reported here will provide a framework for future functional experiments to get a better understanding on the evolution of cell polarity in Metazoa.
In situ hybridization
List of primer sequences used to clone the genes used in this study
NvaPKC (full length)
NvPar-6 (full length)
NvPar-3 (full length)
NvLgl (full length)
Embryos of different stages were fixed at room temperature in fresh 3.4% formaldehyde, HEPES 0.1 M (pH 6.9), EGTA 0.05 M (pH 8 to 9), MgSO4 0.005 M, NaCl 0.2 M, glutaraldehyde 0.2%, Triton X-100 0.2%, phosphate-buffered saline (PBS) 1×, and pure water for 1 to 2 h at room temperature (von Dassow fixative). Fixed embryos were rinsed 5× in PBTB (PBS buffer plus 1% BSA and 0.1% Triton X-100). To visualize F-actin, embryos were incubated in Biodipy-FL phallacidin (Life Technologies, Carlsbad, CA, USA) diluted 1:200 in PBTB. Tubulin was visualized by incubation in anti-alpha tubulin (Sigma T9026, Sigma-Aldrich, St. Louis, MO, USA). Affinity-purified N. vectensis anti-aPKC (anti-NvaPKC) and anti-Lgl (anti-NvLgl) peptide antibodies were raised against a selected amino acid region of the NvaPKC protein (PTNEDLGPKRKP; Bethyl Inc., Montgomery, TX, USA) and NvLgl protein (GNFDPFSDDPR; Bethyl Inc., Montgomery, TX, USA), respectively. Affinity-purified N. vectensis anti-Par-1 (anti-NvPar1) and anti-Par-6 (anti-Par6) peptide antibodies were previously raised by the same company (Bethyl Inc., Montgomery, TX, USA) and were kept under storage conditions in the lab. The selected amino acid regions used for NvPar-1 and NvPar-6 protein were CRSTFHSGERPRDRQRDE and CENPTVDNETGILSI, respectively. Blast searches against the N. vectensis genome sequences showed that the amino acid sequences were not present in any predicted N. vectensis proteins other than the expected protein. Primary antibody incubations 1:100 were carried out in blocking buffer (5% normal goat serum in PBTB) at 4°C overnight. After incubation with the primary antibodies, animals were washed with PBTB (5×) for 10 min each wash. Secondary antibodies (Alexa 594-conjugated anti-mouse and Alexa 647-conjugated anti-Rabbit, Invitrogen A21203 and A21245 (Invitrogen, Grand Island, NY, USA), respectively) were used at 1:500 to allow for visualization. All incubations were conducted over night at 4°C. Stained embryos were rinsed again in PBS (5×) and dehydrated into isopropanol using the gradient 50%, 75%, 90%, and 100% and mounted in Murray’s mounting media (MMM; 1:2 benzyl benzoate:benzyl alcohol) for visualization. We scored more than 1,000 embryos per each antibody staining and confocal imaged more than 50 embryos at each stage.
The coding region for each gene of interest was PCR-amplified. The PCR product was then cloned into pSPE3-mVenus or pSPE3-mCherry using the Gateway system . Eggs were injected directly after fertilization as previously described [48,57] with the mRNA encoding one or more Par complex proteins fused in frame with reporter fluorescent proteins (N-terminal tag) using final concentrations between 400 and 750 ng/μl. Live embryos were kept at 16°C and visualized after the mRNA of the FP was translated into protein. Par proteins mRNAs were co-injected with Lifeact::mTurquoise2 (Lifeact::mTq2) mRNA to visualize the cortex of the cells. We injected and recorded more than 500 embryos for each experiment and confocal imaged approximately 10 specimens for each stage. Live embryos were mounted in one-third sea water for visualization. Images were documented at cleavage stages (3 to 4 hpf and 9 to 10 hpf), blastula (12 to 15 hpf), and gastrula (24 to 30 hpf) stages. The cloned genes are listed in Table 1.
Imaging of N. vectensis embryos
Images of live and fixed embryos were taken using a confocal Zeiss LSM 710 microscope (Carl Zeiss SMT Inc., Peabody, MA, USA) using a Zeiss C-Apochromat 40× water immersion objective (N.A. 1.20). Pinhole settings varied between 1.2 and 1.6 A.U. pinhole. z-stack images were processed using Imaris 7.6.4 (Bitplane Co., Belfast, UK) software for three-dimensional reconstructions and ImageJ for single slice and movies. Final figures were assembled using Adobe Illustrator and Adobe Photoshop (Adobe Systems, Mountain View, CA, USA).
Identification of maternal and zygotic mRNA distribution by whole-mount in situ hybridization
Two phases of Par protein localization during the early development of N. vectensis
N. vectensis aPKC (NvaPKC) and Par-6 (NvPar-6)
N. vectensis Lgl (NvLgl) and Par-1 (NvPar-1)
Polarization of N. vectensis Par-3 (NvPar-3) in vivo
Defining apical and basolateral limits using multiple fluorescent protein reporters
There has been great interest in the development and evolution of cell polarity in metazoan lineages. To date, these processes have been thoroughly described in the Bilateria, the clade that includes all the best studied animal model systems. During bilaterian development, the same set of protein interactions, described here as the Par system, is utilized to organize the polarity of both the embryonic cells and epithelial cells. As would be expected, these proteins are also present in Cnidaria [44,59], the sister group of Bilateria, and their role in establishing cell polarity is investigated here for the first time.
Comparison of the Par system between Cnidaria and Bilateria
Polarization of the oocyte during oogenesis is a critical step for the proper embryonic development of many animals. Cnidarian eggs and embryos have been shown to be highly polarized; the animal pole always gives rise to the site of gastrulation and gives rise to the oral pole [32,47]. This polarization is generated during oogenesis  where several maternal determinants such as organelles, mRNAs, and proteins are localized to the animal pole. For example, Dishevelled, Flamingo, and Strabismus (components of the Wnt signaling pathway) are asymmetrically localized to the animal pole prior to the first cleavage of N. vectensis egg [31,32,61], and these proteins also serve to polarize the early embryo, have essential roles specifying the site of gastrulation [31,32], and polarize epithelial cells . A similar situation has been described in the vegetal pole of bilaterian embryos, suggesting that bilaterian and cnidarian animals share conserved mechanisms to polarize their cells. Thus, we expected to find evidence that the Par system would also play a role in establishing the polarity of N. vectensis embryos as they do in bilaterian embryos. However, this does not appear to be the case.
The ectodermal epithelium formed during embryogenesis in N. vectensis is composed of columnar cells, which are joined together by a belt of adherens junctions [38,47,62]. In contrast, following gastrulation, endodermal cells lose their columnar shape and become disorganized, resulting in fewer (or none) and shorter adherens junctions, leaving more spaces between cells . Curiously, our results, either by immunohistochemistry or using living embryos, indicate that no components of the Par system are expressed in endodermal tissue, even at later stages (Figures 4 and 7). Since Par proteins are expressed throughout the blastula, it might be possible that loss of cell-cell adhesion in endodermal cells induces the degradation of Par proteins. During the formation of bilaterian epithelia, the Par system is essential for the formation and maintenance of cell-cell junctions (tight junctions in vertebrates and adherent junctions in invertebrates) [63-67]. In addition, the adhesions between cells become necessary to maintain A/B polarity and Par protein asymmetry [5,16,68]. Therefore, the disruptions of junctional complexes may lead the disruption of the A/B polarity and Par proteins asymmetry. Similar interdependency has been described in different bilaterian animals [10,16], suggesting the conservation of some mechanisms involved in epithelia formation. The absence of Par protein expression has been reported previously in the immature endoderm of fish , and in mesodermally derived MDCK cells, where Snail expression disrupts the localization of the Par complexes and A/B polarity by repression of Crumbs3 transcription . Interestingly, gastrodermal cells of N. vectensis exhibit molecular profiles that are similar to, and arguably synonymous with, both bilaterian endoderm and mesoderm (including Snail of ). It is therefore possible that the absence (or degradation) of Par system components in the gastrodermis of N. vectensis is a reflection of its dual functional specification. While theoretically interesting, this point, and its associated biological implications, must be vetted by extensive functional analysis.
The evolution of cell polarity and the Par system
Our findings indicate that the bilaterian Par system shares a role in polarizing epithelial cells in cnidarians, but not in the cells of early embryo. If the situation in N. vectensis is representative of other prebilaterians, it would suggest that the polarization of early embryonic cells by the Par system could have arisen at the base of the Bilateria. The most parsimonious evolutionary explanation for this pattern is that the Par system played a role in establishing the epithelial cell polarity in the most recent common ancestor of Cnidaria and Bilateria and that the Par system was then co-opted into an early role in bilaterian embryogenesis.
The asymmetric localization of the proteins involved in the Par system is a developmental process that seems to be conserved (Figure 1); however, there is some evidence in other bilaterian systems that these genes are integrated into the embryonic system at different developmental stages. In Caenorhabditis elegans, the first cue of asymmetry arises after fertilization triggered by cytoskeleton reorganization, induced by the sperm centrosome . Similar events have been described for the leech Helobdella robusta, with the exception that the asymmetrical localization of Par proteins is observable at two-cell stage . On the other hand, in Drosophila, asymmetric localization of Par proteins takes place during oogenesis and is induced by some signals from the follicle cells that surround the vegetal pole [3,29]. During mouse embryogenesis, the first clues of polarity are observed during oogenesis, when Par-6 localizes at the animal pole of the oocyte [11,27,70]. In Xenopus, membrane polarization occurs during oogenesis and an asymmetric localization of Par system components is observed when the oocyte is completely mature [4,10,71]. In a different way, sea urchin embryos show asymmetry of Par proteins along the A/V axis at the 16-cell stage ; however, aPKC and Par6 are already polarized in blastomeres at the two-cell stage . In all of these bilaterian organisms, the Par system appears to be causally involved in establishing cellular polarity, and functional disruption of the Par system components gives rise to similar phenotype when inactive in early bilaterian embryos: when disrupted, Par proteins distribute throughout the embryo without clear polarization and associate with cortical and cytoplasmic components such as centrosomes and nuclei . Similar distributions were observed in equivalent stages for all proteins studied in this work (NvaPKC, NvPar-3, NvPar-6, NvPar-1, and NvLgl) during early embryogenesis of N. vectensis (see Figures 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13; cleavage stages). Furthermore, the overexpression of any of the Par system components by mRNA injection did not disrupt the formation of polarized epithelia, and the overexpressed protein displayed the same distribution observed with the antibody staining. This suggests that Par system might be inactive in N. vectensis blastomeres.
One thing of interest is that when par complex proteins are mutated/knocked down in bilaterians, it affects the organization of the cleavage program and the segregation of maternal components [28,29,45,46]. Most cnidarians, including N. vectensis, have a ‘chaotic’ cleavage program in which cells divide asynchronously and asymmetrically in a unique way from embryo to embryo. Our results raise the possibility that this chaotic development observed in N. vectensis embryogenesis is correlated to the symmetric distribution of Par proteins and that they were not related to the processes regulating early cleavages. Perhaps the co-option of Par complex proteins facilitated the more stereotyped cleavage patterns seen in many bilaterian forms.
Early embryogenesis in many bilaterians is characterized by a stereotypical pattern of cell divisions directed by the association of different molecular pathways (Figure 1A). These interactions act to polarize the egg and embryonic cells and are used later to polarize the cells of epithelial tissues (Figure 14). Here, we report an initial characterization of the spatial and temporal dynamics of the Par system proteins in a non-bilaterian metazoan, the cnidarian N. vectensis. Similar to the situation seen in bilaterian animals, proteins of the Par system are localized asymmetrically in the ectodermal epithelium of N. vectensis, but asymmetric localization was not observed during cleavage stages (Figure 14). These results suggest that different mechanisms establish cellular polarity in N. vectensis embryos and demonstrate that although these proteins are used to organize epithelial cell polarity later in development, they are not utilized to organize early embryonic cells in the same way they do in later evolving animals. We hypothesize that the ancestral Par system could have functioned to maintain cell-cell contacts and generate polarized epithelia. Further, it is likely that currently unidentified components, which are necessary to direct Par system polarization (Figure 1A), are not present or non-functional during early embryogenesis of N. vectensis. Collectively, this data suggests that the molecular regulators of cell polarity in tissues were co-opted into early embryonic cells at the base of the Bilateria (Figure 14). In this scenario, an activated Par system could have shifted to earlier stages directing the polarization, plane of cell division, and the partitioning of the maternal components in the cytoplasm. Interestingly, the same polarity system that operates in the epidermis of N. vectensis does not seem to be involved with gastrodermal polarity. Future work will be directed to assess the evolutionary significance of this, and the function of Par system and its interaction with other polarity pathways during the early development of N. vectensis.
atypical protein kinase C
hours post fertilization
lethal giant larvae
Murray’s mounting media
- N. vectensis :
Nv, Nematostella vectensis
- Par proteins:
We thank Tim Q. DuBuc for providing Lifeact::mTurquoise2. We thank Michael S. Layden, Barbara Battelle, and Leslie S. Babonis for the technical and confocal microscopy assistance. We thank Elaine C. Seaver, reviewers, and the members of Martindale’s lab for the helpful discussion. This research was supported by the NSF.
- Johnston DS, Sanson B. Epithelial polarity and morphogenesis. Curr Opin Cell Biol. 2011;23:540–6.View ArticleGoogle Scholar
- Thompson BJ. Cell polarity: models and mechanisms from yeast, worms and flies. Development. 2012;140:13–21.View ArticleGoogle Scholar
- St Johnston D, Ahringer J. Cell polarity in eggs and epithelia: parallels and diversity. Cell. 2010;141:757–74.PubMedView ArticleGoogle Scholar
- Cha S-W, Tadjuidje E, Wylie C, Heasman J. The roles of maternal Vangl2 and aPKC in Xenopus oocyte and embryo patterning. Development. 2011;138:3989–4000.PubMed CentralPubMedView ArticleGoogle Scholar
- Chan E, Nance J. Mechanisms of CDC-42 activation during contact-induced cell polarization. J Cell Sci. 2013;126:1692–702.PubMed CentralPubMedView ArticleGoogle Scholar
- Dollar GL, Weber U, Mlodzik M, Sokol SY. Regulation of lethal giant larvae by dishevelled. Nature. 2005;437:1376–80.PubMedView ArticleGoogle Scholar
- Nance J, Zallen JA. Elaborating polarity: PAR proteins and the cytoskeleton. Development. 2011;138:799–809.PubMed CentralPubMedView ArticleGoogle Scholar
- Ossipova O, Dhawan S, Sokol S, Green JBA. Distinct PAR-1 proteins function in different branches of Wnt signaling during vertebrate development. Dev Cell. 2005;8:829–41.PubMedView ArticleGoogle Scholar
- Morais-de-Sa E, Mukherjee A, Lowe N. Slmb antagonises the aPKC/Par-6 complex to control oocyte and epithelial polarity. Development. 2014;141:2984–92.PubMed CentralPubMedView ArticleGoogle Scholar
- Etienne-Manneville S, Hall A. Cell polarity: Par6, aPKC and cytoskeletal crosstalk. Curr Opin Cell Biol. 2003;15:67–72.PubMedView ArticleGoogle Scholar
- Munro EM. PAR proteins and the cytoskeleton: a marriage of equals. Curr Opin Cell Biol. 2006;18:86–94.PubMedView ArticleGoogle Scholar
- Joberty G, Petersen C, Gao L, Macara IG. The cell-polarity protein Par6 links Par3 and atypical protein kinase C to Cdc42. Nat Cell Biol. 2000;2:531–9.PubMedView ArticleGoogle Scholar
- Lin D, Edwards AS, Fawcett JP, Mbamalu G, Scott JD, Pawson T. A mammalian PAR-3-PAR-6 complex implicated in Cdc42/Rac1 and aPKC signalling and cell polarity. Nat Cell Biol. 2000;2:540–7.PubMedView ArticleGoogle Scholar
- Hutterer A, Betschinger J, Petronczki M, Knoblich JA. Sequential roles of Cdc42, Par-6, aPKC, and Lgl in the establishment of epithelial polarity during Drosophila embryogenesis. Dev Cell. 2004;6:845–54.PubMedView ArticleGoogle Scholar
- Chen J, Zhang M. The Par3/Par6/aPKC complex and epithelial cell polarity. Exp Cell Res. 2013;319:1357–64.PubMedView ArticleGoogle Scholar
- Ohno S. Intercellular junctions and cellular polarity: the PAR–aPKC complex, a conserved core cassette playing fundamental roles in cell polarity. Curr Opin Cell Biol. 2001;13:641–8.PubMedView ArticleGoogle Scholar
- Sun TQ, Lu B, Feng JJ, Reinhard C, Jan YN, Fantl WJ, et al. PAR-1 is a dishevelled-associated kinase and a positive regulator of Wnt signalling. Nat Cell Biol. 2001;3:628–36.PubMedView ArticleGoogle Scholar
- Walston T, Tuskey C, Edgar L, Hawkins N, Ellis G, Bowerman B, et al. Multiple Wnt signaling pathways converge to orient the mitotic spindle in early C. elegans embryos. Dev Cell. 2004;7:831–41.PubMedView ArticleGoogle Scholar
- Nance J. Cell biology in development: getting to know your neighbor: cell polarization in early embryos. J Cell Biol. 2014;206:823–32.PubMed CentralPubMedView ArticleGoogle Scholar
- Tepass U. The apical polarity protein network in Drosophila epithelial cells: regulation of polarity, junctions, morphogenesis, cell growth, and survival. Annu Rev Cell Dev Biol. 2012;28:655–85.PubMedView ArticleGoogle Scholar
- Patalano S, Pruliere G, Prodon F, Paix A, Dru P, Sardet C, et al. The aPKC-PAR-6-PAR-3 cell polarity complex localizes to the centrosome attracting body, a macroscopic cortical structure responsible for asymmetric divisions in the early ascidian embryo. J Cell Sci. 2006;119:1592–603.PubMedView ArticleGoogle Scholar
- Weisblat DA. Asymmetric cell divisions in the early embryo of the leech Helobdella robusta. Prog Mol Subcell Biol. 2007;45:79–95.PubMedGoogle Scholar
- Munro E, Bowerman B. Cellular symmetry breaking during Caenorhabditis elegans development. Cold Spring Harb Perspect Biol. 2009;1:a003400.PubMed CentralPubMedView ArticleGoogle Scholar
- Schneider SQ, Bowerman B. Cell polarity and the cytoskeleton in the Caenorhabditis elegans zygote. Annu Rev Genet. 2003;37:221–49.PubMedView ArticleGoogle Scholar
- Macara IG. Parsing the polarity code. Nat Rev Mol Cell Biol. 2004;5:220–31.PubMedView ArticleGoogle Scholar
- Goldstein B, Macara IG. The PAR proteins: fundamental players in animal cell polarization. Dev Cell. 2007;13:609–22.PubMed CentralPubMedView ArticleGoogle Scholar
- Vinot S, Le T, Maro B, Louvet-Vallée S. Two PAR6 proteins become asymmetrically localized during establishment of polarity in mouse oocytes. Curr Biol. 2004;14:520–5.PubMedView ArticleGoogle Scholar
- Alford LM, Ng MM, Burgess DR. Cell polarity emerges at first cleavage in sea urchin embryos. Dev Biol. 2009;330:12–20.PubMedView ArticleGoogle Scholar
- Doerflinger H, Vogt N, Torres IL, Mirouse V, Koch I, Nüsslein-Volhard C, et al. Bazooka is required for polarisation of the Drosophila anterior-posterior axis. Development. 2010;137:1765–73.PubMed CentralPubMedView ArticleGoogle Scholar
- Wikramanayake AH, Hong M, Lee PN, Pang K, Byrum CA, Bince JM, et al. An ancient role for nuclear catenin in the evolution of axial polarity and germ layer segregation. Nature. 2003;426:446–50.PubMedView ArticleGoogle Scholar
- Kumburegama S, Wijesena N, Xu R, Wikramanayake AH. 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. EvoDevo. 2011;2:2.PubMed CentralPubMedView ArticleGoogle Scholar
- Lee PN, Kumburegama S, Marlow HQ, Martindale MQ, Wikramanayake AH. Asymmetric developmental potential along the animal-vegetal axis in the anthozoan cnidarian, Nematostella vectensis, is mediated by Dishevelled. Dev Biol. 2007;310:169–86.PubMedView ArticleGoogle Scholar
- Momose T, Kraus Y, Houliston E. A conserved function for Strabismus in establishing planar cell polarity in the ciliated ectoderm during cnidarian larval development. Development. 2012;139:4374–82.PubMedView ArticleGoogle Scholar
- Momose T, Derelle R, Houliston E. A maternally localised Wnt ligand required for axial patterning in the cnidarian Clytia hemisphaerica. Development. 2008;135:2105–13.PubMedView ArticleGoogle Scholar
- Röttinger E, Dahlin P, Martindale MQ. A framework for the establishment of a cnidarian gene regulatory network for “endomesoderm” specification: the inputs of ß-catenin/TCF signaling. PLoS Genet. 2012;8:e1003164.PubMed CentralPubMedView ArticleGoogle Scholar
- Amiel A, Houliston E. Three distinct RNA localization mechanisms contribute to oocyte polarity establishment in the cnidarian Clytia hemisphaerica. Dev Biol. 2009;327:191–203.PubMedView ArticleGoogle Scholar
- Tamulonis C, Postma M, Marlow HQ, Magie CR, de Jong J, Kaandorp J. A cell-based model of Nematostella vectensis gastrulation including bottle cell formation, invagination and zippering. Dev Biol. 2011;351:217–28.PubMedView ArticleGoogle Scholar
- Magie CR, Martindale MQ. Cell-cell adhesion in the Cnidaria: insights into the evolution of tissue morphogenesis. Biol Bull. 2008;214:218–32.PubMedView ArticleGoogle Scholar
- Martindale MQ. Evolution of development: the details are in the entrails. Curr Biol. 2013;23:R25–8.PubMedView ArticleGoogle Scholar
- Martindale M. The evolution of metazoan axial properties. Nat Rev Genet. 2005;6:917–27.PubMedView ArticleGoogle Scholar
- Martindale MQ, Hejnol A. A developmental perspective: changes in the position of the blastopore during bilaterian evolution. Dev Cell. 2009;17:162–74.PubMedView ArticleGoogle Scholar
- Freeman G. The cleavage initiation site establishes the posterior pole of the hydrozoan embryo. Wilhelm Roux’s Arch Dev Biol. 1981;190:123–5.View ArticleGoogle Scholar
- Teissier G. Etude experimentale du developpement de quelques hydraires, par Georges Teissier, de sciences, vol. No 2164. Paris: Doctorat es sciences naturelles; 1931.Google Scholar
- Fahey B, Degnan BM. Origin of animal epithelia: insights from the sponge genome. Evol Dev. 2010;12:601–17.PubMedView ArticleGoogle Scholar
- Kemphues KJ, Priess JR, Morton DG. Cheng NS. Identification of genes required for cytoplasmic localization in early C elegans embryos Cell. 1988;52:311–20.Google Scholar
- Lu MS, Johnston CA. Molecular pathways regulating mitotic spindle orientation in animal cells. Development. 2013;140:1843–56.PubMed CentralPubMedView ArticleGoogle Scholar
- Fritzenwanker JH, Genikhovich G, Kraus Y, Technau U. Early development and axis specification in the sea anemone Nematostella vectensis. Dev Biol. 2007;310:264–79.PubMedView ArticleGoogle Scholar
- Layden MJ, Röttinger E, Wolenski FS, Gilmore TD, Martindale MQ. Microinjection of mRNA or morpholinos for reverse genetic analysis in the starlet sea anemone. Nematostella vectensis Nat Protoc. 2013;8:924–34.View ArticleGoogle Scholar
- Hand C, Uhlinger KR. The culture, sexual and asexual reproduction, and growth of the sea anemone Nematostella vectensis. Biol Bull. 1992;182:169–76.View ArticleGoogle Scholar
- Stefanik DJ, Friedman LE, Finnerty JR. Collecting, rearing, spawning and inducing regeneration of the starlet sea anemone. Nematostella vectensis Nat Protoc. 2013;8:916–23.View ArticleGoogle Scholar
- Wolenski FS, Layden MJ, Martindale MQ, Gilmore TD, Finnerty JR. Characterizing the spatiotemporal expression of RNAs and proteins in the starlet sea anemone. Nematostella vectensis Nat Protoc. 2013;8:900–15.View ArticleGoogle Scholar
- Battelle BA, Andrews AW, Kempler KE, Edwards SC, Smith WC. Visual arrestin in Limulus is phosphorylated at multiple sites in the light and in the dark. Vis Neurosci. 2000;17:813–22.PubMedView ArticleGoogle Scholar
- Babonis LS, Hyndman KA, Lillywhite HB, Evans DH. Immunolocalization of Na+/K + −ATPase and Na+/K+/2Cl − cotransporter in the tubular epithelia of sea snake salt glands. Comp Biochem Physiol, Part A Mol Integr Physiol. 2009;154:535–40.PubMedView ArticleGoogle Scholar
- Piermarini PM, Verlander JW, Royaux IE, Evans DH. Pendrin immunoreactivity in the gill epithelium of a euryhaline elasmobranch. Am J Physiol Regul Integr Comp Physiol. 2002;283:R983–92.PubMedView ArticleGoogle Scholar
- Wolenski F, Finnerty J, Gilmore T. Preparation of antiserum and detection of proteins by Western blotting using the starlet sea anemone, Nematostella vectensis. 2012.Google Scholar
- Roure A, Rothbächer U, Robin F, Kalmar E, Ferone G, Lamy C, et al. A multicassette Gateway vector set for high throughput and comparative analyses in ciona and vertebrate embryos. PLoS ONE. 2007;2:e916.PubMed CentralPubMedView ArticleGoogle Scholar
- DuBuc TQ, Dattoli A, Babonis LS, Salinas Saavedra M, Röttinger E, Martindale MQ, et al. In vivo imaging of Nematostella vectensis embryogenesis and late development using fluorescent probes. BMC Cell Biol. 2014;15:44.PubMed CentralPubMedView ArticleGoogle Scholar
- Feldman JL, Priess JR. A role for the centrosome and PAR-3 in the hand-Off of MTOC function during epithelial polarization. Curr Biol. 2012;22:575–82.PubMed CentralPubMedView ArticleGoogle Scholar
- Putnam NH, Srivastava M, Hellsten U, Dirks B, Chapman J, Salamov A, et al. Sea anemone genome reveals ancestral eumetazoan gene repertoire and genomic organization. Sup Sci. 2007;317:86–94.Google Scholar
- Eckelbarger KJ, Hand C, Uhlinger KR. Ultrastructural features of the trophonema and oogenesis in the starlet sea anemone, Nematostella vectensis (Edwardsiidae). Invertebr Biol. 2008;127:381–95.View ArticleGoogle Scholar
- Wijesena NM. Wnt Signaling in the Cnidarian Nematostella vectensis: insights into the evolution of gastrulation. Open Access Dissertations. Paper 906. http://scholarlyrepository.miami.edu/oa_dissertations/906 (2012)
- Magie CR, Daly M, Martindale MQ. Gastrulation in the cnidarian Nematostella vectensis occurs via invagination not ingression. Dev Biol. 2007;305:483–97.PubMedView ArticleGoogle Scholar
- Hirose T, Izumi Y, Nagashima Y, Tamai-Nagai Y, Kurihara H, Sakai T, et al. Involvement of ASIP/PAR-3 in the promotion of epithelial tight junction formation. J Cell Sci. 2002;115:2485–95.PubMedGoogle Scholar
- Achilleos A, Wehman A, Nance J. PAR-3 mediates the initial clustering and apical localization of junction and polarity proteins during C. elegans intestinal epithelial cell polarization. Development. 2010;137:1833.PubMed CentralPubMedView ArticleGoogle Scholar
- Sonawane M, Martin-Maischein H, Schwarz H, Nüsslein-Volhard C. Lgl2 and E-cadherin act antagonistically to regulate hemidesmosome formation during epidermal development in zebrafish. Development. 2009;136:1231–40.PubMedView ArticleGoogle Scholar
- Afonso C, Henrique D. PAR3 acts as a molecular organizer to define the apical domain of chick neuroepithelial cells. J Cell Sci. 2006;119:4293–304.PubMedView ArticleGoogle Scholar
- Harris TJC, Peifer M. The positioning and segregation of apical cues during epithelial polarity establishment in Drosophila. J Cell Biol. 2005;170:813–23.PubMed CentralPubMedView ArticleGoogle Scholar
- Karner C, Wharton KA, Carroll TJ. Apical-basal polarity, Wnt signaling and vertebrate organogenesis. Semin Cell Dev Biol. 2006;17:214–22.PubMedView ArticleGoogle Scholar
- Whiteman EL, Liu C-J, Fearon ER, Margolis B. The transcription factor snail represses Crumbs3 expression and disrupts apico-basal polarity complexes. Oncogene. 2008;27:3875–9.PubMed CentralPubMedView ArticleGoogle Scholar
- Vinot S, Le T, Ohno S, Pawson T, Maro B, Louvet-Vallée S. Asymmetric distribution of PAR proteins in the mouse embryo begins at the 8-cell stage during compaction. Dev Biol. 2005;282:307–19.PubMedView ArticleGoogle Scholar
- M-a N, Fukui A, Izumi Y, Akimoto K, Asashima M, Ohno S. Meiotic maturation induces animal-vegetal asymmetric distribution of aPKC and ASIP/PAR-3 in Xenopus oocytes. Development. 2000;127:5021–31.Google Scholar
- Pruliere G, Cosson J, Chevalier S, Sardet C, Chênevert J. Atypical protein kinase C controls sea urchin ciliogenesis. Mol Biol Cell. 2011;22:2042–53.PubMed CentralPubMedView ArticleGoogle Scholar
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.