A cell-based model of Nematostella vectensis gastrulation including bottle cell formation, invagination and zippering

AbstractThe gastrulation of Nematostella vectensis, the starlet sea anemone, is morphologically simple yet involves many conserved cell behaviors such as apical constriction, invagination, bottle cell formation, cell migration and zippering found during gastrulation in a wide range of more morphologically complex animals.In this article we study Nematostella gastrulation using a combination of morphometrics and computational modeling. Through this analysis we frame gastrulation as a non-trivial problem, in which two distinct cell domains must change shape to match each other geometrically, while maintaining the integrity of the embryo. Using a detailed cell-based model capable of representing arbitrary cell-shapes such as bottle cells, as well as filopodia, localized adhesion and constriction, we are able to simulate gastrulation and associate emergent macroscopic changes in embryo shape to individual cell behaviors.We have developed a number of testable hypotheses based on the model. First, we hypothesize that the blastomeres need to be stiffer at their apical ends, relative to the rest of the cell perimeter, in order to be able to hold their wedge shape and the dimensions of the blastula, regardless of whether the blastula is sealed or leaky. We also postulate that bottle cells are a consequence of cell strain and low cell–cell adhesion, and can be produced within an epithelium even without apical constriction. Finally, we postulate that apical constriction, filopodia and de-epithelialization are necessary and sufficient for gastrulation based on parameter variation studies

Asymmetric developmental potential along the animal–vegetal axis in the anthozoan cnidarian, Nematostella vectensis, is mediated by Dishevelled

The relationship between egg polarity and the adult body plan is well understood in many bilaterians . However, the evolutionary origins of embryonic polarity are not known. Insight into the evolution of polarity will come from understanding the ontogeny of polarity in non-bilaterian forms, such as cnidarians . We examined how the axial properties of the starlet sea anemone, Nematostella vectensis (Anthozoa, Cnidaria), are established during embryogenesis . Egg-cutting experiments and sperm localization show that Nematostella eggs are only fertilized at the animal pole . Vital marking experiments demonstrate that the egg animal pole corresponds to the sites of first cleavage and gastrulation, and the oral pole of the adult . Embryo separation experiments demonstrate an asymmetric segregation of developmental potential along the animal–vegetal axis prior to the 8-cell stage . We demonstrate that Dishevelled (Dsh) plays an important role in mediating this asymmetric segregation of developmental fate . Although NvDsh mRNA is ubiquitously expressed during embryogenesis, the protein is associated with the female pronucleus at the animal pole in the unfertilized egg, becomes associated with the unipolar first cleavage furrow, and remains enriched in animal pole blastomeres. Our results suggest that at least one mechanism for Dsh enrichment at the animal pole is through its degradation at the vegetal pole. Functional studies reveal that NvDsh is required for specifying embryonic polarity and endoderm by stabilizing β-catenin in the canonical Wnt signaling pathway . The localization of Dsh to the animal pole in Nematostella and two other anthozoan cnidarians (scleractinian corals) provides a possible explanation for how the site of gastrulation has changed in bilaterian evolution while other axial components of development have remained the same and demonstrates that modifications of the Wnt signaling pathway have been used to pattern a wide variety of metazoan embryos

Expanded Functional Diversity of Shaker K⁺ Channels in Cnidarians Is Driven by Gene Expansion

<div><p>The genome of the cnidarian <i>Nematostella vectensis</i> (starlet sea anemone) provides a molecular genetic view into the first nervous systems, which appeared in a late common ancestor of cnidarians and bilaterians. <i>Nematostella</i> has a surprisingly large and diverse set of neuronal signaling genes including paralogs of most neuronal signaling molecules found in higher metazoans. Several ion channel gene families are highly expanded in the sea anemone, including three subfamilies of the Shaker K⁺ channel gene family: Shaker (Kv1), Shaw (Kv3) and Shal (Kv4). In order to better understand the physiological significance of these voltage-gated K⁺ channel expansions, we analyzed the function of 18 members of the 20 gene Shaker subfamily in <i>Nematostella</i>. Six of the Nematostella Shaker genes express functional homotetrameric K⁺ channels <i>in vitro</i>. These include functional orthologs of bilaterian Shakers and channels with an unusually high threshold for voltage activation. We identified 11 Nematostella Shaker genes with a distinct “silent” or “regulatory” phenotype; these encode subunits that function only in heteromeric channels and serve to further diversify Nematostella Shaker channel gating properties. Subunits with the regulatory phenotype have not previously been found in the Shaker subfamily, but have evolved independently in the Shab (Kv2) family in vertebrates and the Shal family in a cnidarian. Phylogenetic analysis indicates that regulatory subunits were present in ancestral cnidarians, but have continued to diversity at a high rate after the split between anthozoans and hydrozoans. Comparison of Shaker family gene complements from diverse metazoan species reveals frequent, large scale duplication has produced highly unique sets of Shaker channels in the major metazoan lineages.</p></div

Comparison of inactivation in NvShak channels.

<p><b><i>(a)</i></b> Comparison of inactivation time course in NvShak1, NvShak4 and NvShak5 currents recorded from excised patches in response to a depolarization to +60 mV. Currents were recorded in 2 mM external K⁺ and were normalized by peak amplitude to facilitate comparison of inactivation. Scale bar indicates time. <b><i>(b)</i></b> Steady state inactivation curves are given for NvShak1, NvShak4, NvShak5 and NvShak6. Data points show mean peak current ± S.E.M. (n = 4–6) elicited during a +60 mV test pulse following a 5 s pre-pulse to the indicated voltage. Data from individual patches were normalized in amplitude prior to comparison. Curves represent single Boltzmann distribution fits; V₅₀ and slope values are reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051366#pone-0051366-t001" target="_blank">Table 1</a>. Currents elicited at +60 mV for wild type (WT, black) and N-terminal truncated (Trunc, red) versions of NvShak1, NvShak5 and NvShak4 are compared in <b><i>c-e</i></b>, respectively. Currents were normalized in amplitude for display. Truncations eliminated amino acid residues 2–30, 2–48 and 2–58 for NvShak1, NvShak4 and NvShak5, respectively. Truncation eliminated fast inactivation in NvShak1 and NvShak5, but only reduced the rate of inactivation in NvShak4. 140 mM K+ had little effect on the inactivation time course of WT NvShak4 (gray) but eliminated residual inactivation in the truncated form of NvShak4 (dark red), suggesting an unmasked C-type inactivation process in the truncated channel.</p

Summary of Novel Current Properties in NvShak3 + NvShakRx Heteromers.

<p>Summary of Novel Current Properties in NvShak3 + NvShakRx Heteromers.</p

NvShak1-6 express a functionally diverse set of Shaker currents in <i>Xenopus</i> oocytes.

<p>Families of outward K⁺ currents recorded from excised inside-out patches taken from oocytes expressing NvShak1-6 are shown in a–f. Currents were recorded in response to 100 ms depolarizations ranging from −60 to +60 mV in 10 mV increments from a holding potential of −100 mV. Scale bars are given for time and current amplitude and arrows mark the current elicited at 0 mV. Note this is the first step with significant current for NvShak2 and NvShak3. K⁺ concentration for these recordings was 2 mM in the pipette (extracellular) and 140 mM in the bath (intracellular).</p

Expression patterns of Nematostella Shaker genes determined by in situ hybridization.

<p>Expression in early stage polyps is shown for NvShak1-6 in <b><i>a,</i></b> and 7 NvShakR genes in <b><i>b</i></b>. Sagittal views are shown for all probes; coronal views are also shown for NvShak1, NvShak2, NvShak3, NvShak6, NvShakR2 and NvShakR12. Numbered arrows point to select regions of expression. (<b>1</b>) Homotetramer-competent inactivating subunits, NvShak1, NvShak4 and NvShak5, are expressed in developing tentacle bulbs, along with regulatory subunits NvShakR7, NvShakR9 and NShakR13. The same three regulatory subunits show light expression along with NvShak2 and NvShak4 in the vicinity of the developing oral nerve ring (<b>2</b>). Expression around the pharynx and pharyngeal nerve ring occurs for NvShak1, NvShak3, NvShak4, NvShak6 and the regulatory subunits NvShakR11 and NvShakR12 (<b>3</b>). All homotetramer-competent subunits (except NvShak5) and the regulatory subunits NvShakR2, NvShakR4, NvShakR11 and NvShakR12 show expression in subsets of cells in the mesenteries (<b>4</b>). NvShak6 expression is concentrated in body cells near the apical pole in early stage polyps (<b>5</b>), but transitions to mesenteric and pharyngeal expression as polyp development continues. The specific identity of Shaker-positive cells and cell-level expression overlap of Shaker transcripts remains to be determined.</p

Voltage-dependent properties of NvShak1-6 Currents.

<p>Voltage-dependent properties of NvShak1-6 Currents.</p

Shaker Family Genes in Metazoan Species.

<p>Shaker Family Genes in Metazoan Species.</p