Par system components are asymmetrically localized in ectodermal epithelia, but not during early development in the sea anemone Nematostella vectensis

Abstract

Background: 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.

Results: 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.

Conclusions: 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.

Keywords: Bilateria; Cnidaria; Nematostella vectensis; Par proteins; Polarity.

Figure 1

Components of the Par system are conserved across metazoans. (A) The ‘Par system’ is a set of regulatory proteins that direct the polarity of the cell. For the examined bilaterian animals, the Par system is influenced by the interaction between cell-cell junctions, cytoskeletal elements, and components of the Wnt signaling pathways. (B) In bilaterian animals, Par-3, Par-6, and aPKC form a bi/tripartite complex that localizes to apical regions of the cell, binding to CDC42 and CRUMBS. In contrast, both Lgl and Par-1 localize at the basolateral cortex of the cell. Mutual antagonism by phosphorylation has been proposed as the mechanism that controls the segregation of these two distinct cortical domains. (C) Par proteins are present in the genome of sequenced Metazoa, including Cnidaria, Ctenophores, Porifera, and Placozoa. However, the function of Par proteins, in epithelial (blue branches) and early embryogenesis (red asterisk), has only been described for some bilaterian animals, and there are no descriptions available for non-bilaterian animals (black branches).

Figure 2

Identification of mRNA distribution by whole-mount in situ hybridization. Maternal mRNA of NvaPKC, NvPar-3, NvPar-6, NvPar-1, and NvLgl are asymmetrically distributed during the early development of N. vectensis. In blastula and gastrula stages, these genes are uniformly distributed in the ectoderm. The distribution of all five genes is not consistent during early stages, suggesting no association with the animal-vegetal axis of the embryo; therefore, the protein localization is required.

Figure 3

Specificity of N. vectensis antibodies tested by pre-adsorption experiments. (A) Western blots of N. vectensis gastrula extracts using specific antibodies against NvaPKC (a′), NvLgl (b′), NvPar-1 (c′), and NvPar-6 (d′). Pre-adsorption of the antibodies with a tenfold excess of the antigen peptide resulted in the elimination of the staining of a single band for NvaPKC and NvLgl (a′ and b′, respectively) and the titration of anti-NvPar-1(c′). Arrowheads indicate the molecular weight in KD. A red arrow indicates the band recognized by the antibody for each protein. (B) Whole-mount immunohistochemistry pre-adsorption experiments show that the staining pattern was eliminated in early embryos when pre-incubated antibodies against NvaPKC and NvLgl with the respective peptide. (C) shows the negative control when the primary antibody was not added.

Figure 4

Two phases of N. vectensis aPKC (NvaPKC) and Par-6 (NvPar-6) localization. Immunostaining against NvaPKC (A) and NvPar-6 (B) during different stages of N. vectensis development. These proteins do not localize asymmetrically in unfertilized eggs and in the cells of cleavage stages; however, they do localize asymmetrically in the epithelial cells of blastula and gastrula stage embryos. Similar to some bilaterians, NvaPKC and NvPar-6 localize at the apical zone of the cell (insets in blastula and gastrula). Both proteins distribute throughout the microtubule cytoskeleton (labeled with tubulin antibody staining), and labeling was not observed in the endoderm/gastrodermis of gastrula and polyps. White arrows indicate astral/centrosomal and (pro)nuclear structures. White arrowheads indicate the endodermal layer in gastrula and polyp stages. Actin cytoskeleton is shown in gray. Animal/vegetal axis (A/V) and apical/basal axis are indicated. Insets shown in blastula, gastrula, and polyp stages are higher magnification of the zone indicated with the dashed lines. Images ranging from unfertilized to gastrula and insets in polyp stages correspond to a single slice from z-stack series. Image of the whole polyps corresponds to a 3D reconstruction from z-stack series.

Figure 5

In vivo localization of NvaPKC::mVenus during different stages of N. vectensis development. The overexpression of NvaPKC::mVenus (A) protein, by mRNA microinjection, displays similar patterns observed with the antibody staining against the same protein, and no signal was observed in the endoderm. This messenger was co-injected with Lifeact::mTq2 mRNA (B) to visualize the shape of the cells and the apical cortex. As is seen in (C), NvaPKC::mVenus is below the cortical actin during all observed stages. White arrows indicate astral/centrosomal and (pro)nuclear structures. White arrowheads indicate the locations adjacent to other cells (cell contacts) where NvaPKC::mVenus is localized. Images of unfertilized, cleavage, and side panels in gastrula stages correspond to a single slice from z-stack series. Image of the whole blastula and gastrula corresponds to a 3D reconstruction from z-stack series. Side panels in blastula and gastrula stages are a mid section from the z-stack series to show the apical distribution of NvaPKC::mVenus along the A/B axis. See Additional files 5, 6, 7, 8, and 9 for more details of every stage presented here in C.

Figure 6

In vivo localization of NvPar-6::mVenus during different stages of N. vectensis development. The overexpressed NvPar-6::mVenus protein (A) displays similar patterns observed with the antibody staining against the same protein, and no signal was observed in the endoderm. Co-injection with Lifeact::mTq2 mRNA (B) shows the shape of the cells and their apical cortex. Similar to NvaPKC::mVenus, NvPar-6::mVenus is below the cortical actin during all observed stages (C). White arrows indicate astral/centrosomal and (pro)nuclear structures. White arrowheads indicate the locations adjacent to other cells (cell contacts) where NvPar-6::mVenus is localized. Images of unfertilized, cleavage, and side panels in gastrula stages correspond to a single slice from z-stack series. Image of the whole blastula and gastrula corresponds to a 3D reconstruction from z-stack series. Side panels in blastula and gastrula stages are a mid section from the z-stack series to show the apical distribution of NvPar-6::mVenus along the A/B axis. Additional files 10, 11, 12, 13, and 14 for more details of every stage presented here in C.

Figure 7

Two phases of N. vectensis Lgl (NvLgl) and Par-1 (NvPar-1) localization. No asymmetric localization of NvLgl (A) and NvPar-1 (B) occurs during cleavage stages; however, they localize asymmetrically in the epithelial cells at later stages (insets on blastula and gastrula). Similar to some bilaterians, NvLgl and NvPar-1 extend throughout basolateral cortex of the cell. Both proteins distribute throughout the microtubule cytoskeleton (labeled with tubulin antibody staining), and no labeling was observed in the endoderm/gastrodermis (gastrula and polyps were observed). White arrows indicate astral/centrosomal and (pro)nuclear structures. White arrowheads indicate the endodermal layer in gastrula and polyp stages. Actin cytoskeleton is shown as gray. Animal/vegetal axis (A/V) and apical/basal axis are indicated. Insets shown in blastula, gastrula, and polyp stages are higher magnification of the zone indicated with the dashed lines. Images ranging from unfertilized to gastrula and insets in polyp stages correspond to a single slice from z-stack series. Image of the whole polyps correspond to a 3D reconstruction from z-stack series.

Figure 8

In vivo localization of NvLgl::mCherry during different stages of N. vectensis development. The overexpression of NvLgl::mCherry protein (A) displays similar patterns observed with the antibody staining against the same protein, and no signal was observed in the endoderm. Lifeact::mTq2 mRNA (B) was co-injected to visualize the shape of the cells and the apical cortex. In (C), NvLgl::mCherry is always expressed below the cortical actin and localized in the lateral cortex during all observed stages. White arrows indicate astral/centrosomal and (pro)nuclear structures. White arrowheads indicate the locations adjacent to other cells (cell contacts) where NvLgl::mCherry is localized. Images of unfertilized, cleavage, and side panels in gastrula stages correspond to a single slice from z-stack series. Image of the whole blastula and gastrula corresponds to a 3D reconstruction from z-stack series. Side panels in blastula and gastrula stages are a mid section from the z-stack series to show the basolateral distribution of NvLgl::mCherry along the A/B axis. See Additional files 15, 16, 17, 18, and 19 for more details of every stage presented here in C.

Figure 9

In vivo localization of NvPar-3::mVenus during different stages of N. vectensis development. The overexpression of NvPar3::mVenus (A) protein, by mRNA microinjection, displays two phases of distribution, similar to what was observed in NvaPKC and NvPar-6: no asymmetric localization during earlier cleavage stages but differential localization along the A/B axis of the cells during later stages (blastula and gastrula). Interestingly, NvPar3::mVenus localizes only in the apico-lateral zone of the cells, presumably at the cell-cell junctions complex and centrosomes (arrowhead), and no signal was observed in the endoderm. NvPar3::mVenus was co-injected with Lifeact::mTq2 mRNA (B) to visualize the shape of the cells and the apical cortex. As is seen in (C), NvPar3::mVenus is below the cortical actin during all observed stages. White arrows indicate astral/centrosomal and (pro)nuclear structures. Images of unfertilized, cleavage, and side panels in gastrula stages correspond to a single slice from z-stack series. Image of the whole blastula and gastrula corresponds to a 3D reconstruction from z-stack series. Side panels in blastula and gastrula stages are a mid section from the z-stack series to show the apico-lateral distribution of NvPar3::mVenus along the A/B axis. See Additional files 21, 22, 23, 24, and 25 for more details of every stage presented here in C.

Figure 10

In vivo embryonic localization of NvPar-6::mVenus and NvLgl::mCherry during different stages of N. vectensis development. Co-injection of NvPar-6::mVenus (A) and NvLgl::mCherry (B) confirms our observations for the localization of both proteins through separate experimental approaches. Throughout N. vectensis development, both proteins separate into distinct domains (C). Lifeact::mTq2 mRNA (D) was used to visualize the shape of the cells and the apical cortex. NvPar-6::mVenus and NvLgl::mCherry were always observed below the cortical actin during all observed stages (E). Images of cleavage and blastula stages correspond to a single slice from z-stack series. Images of the gastrula stage correspond to a 3D reconstruction from z-stack series. See Additional files 27, 28, 29, and 30 and Figure 11 for more details of every stage presented here.

Figure 11

In vivo cortical localization of NvPar-6::mVenus and NvLgl::mCherry during different stages of N. vectensis development. z-stack sections from the stages are shown in Figure 10. Beginning at later cleavage stages, both NvPar-6::mVenus (A) and NvLgl::mCherry (B) began to distribute in different regions inside the cells (C) below the cortical actin labeled with Lifeact::mTq2 (D): NvPar-6::mVenus was always observed towards the apical side while NvLgl::mCherry moved basolaterally in non-overlapping domains.

Figure 12

In vivo embryonic localization of NvPar-3::mVenus and NvLgl::mCherry during different stages of N. vectensis development. Co-injection of NvPar-3::mVenus (A) and NvLgl::mCherry (B) displays the same distribution observed when we overexpressed each protein separately: co-distribution during earlier stages but asymmetric localization in the cells of later stages (C). Lifeact::mTq2 mRNA (D) was used to visualize the shape of the cells and the apical cortex. NvPar-3::mVenus and NvLgl::mCherry were observed below the cortical actin during all observed stages (E). White arrowheads indicate the localization of NvLgl::mCherry at the cell contacts. All images correspond to a 3D reconstruction from z-stack series. See Additional files 31, 32, 33, and 34 and Figure 13 for more details of every stage presented here.

Figure 13

In vivo cortical localization of NvPar-3::mVenus and NvLgl::mCherry during different stages of N. vectensis development. z-stack sections from the stages are shown in Figure 12. NvPar-3::mVenus (A) and NvLgl::mCherry (B) distributed in different regions inside the cells (C) below the cortical actin labeled with Lifeact::mTq2 (D): NvPar-3::mVenus was always observed towards the apico-lateral membrane while NvLgl::mCherry moved basolaterally. NvPar-3::mVenus is localized at the cell-cell contacts where the separation between apical and basolateral regions is clearly defined (C, gastrula insets). Insets show the asymmetric distribution of NvPar-3::mVenus and NvLgl::mCherry in the cells of ectodermal epithelium at the gastrula stage. The magnified region was selected to represent the asymmetric distribution of those proteins and is not intended for function implication.

Figure 14

Par system co-opted into early embryonic stages at the base of the Bilateria. The Par system described for bilaterian animals is deployed during early stages to establish cell polarity, maintained during development, and later used to polarize the cells of epithelial tissues. A different scenario was observed for the embryonic and tissue cell polarity in Cnidaria: Par system proteins are not asymmetrically distributed during early development of N. vectensis, but they later become polarized in the cells of the epithelia. This suggests that the Par system, a critical regulator of tissue cell polarity, was co-opted into establishing early embryonic polarity at the base of the Bilateria (yellow star).