Heather Q. Marlow

The onset of microbial associations in the coral Pocillopora meandrina

Associations between healthy adult reef-building corals and bacteria and archaea have been observed in many coral species, but the initiation of their association is not understood. We investigated the onset of association between microorganisms and Pocillopora meandrina, a coral that vertically seeds its eggs with symbiotic dinoflagellates before spawning. We compared the bacterial communities associated with prespawned oocyte bundles, spawned eggs, and week old planulae using multivariate analyses of terminal restriction fragment length polymorphisms of SSU rRNA genes, which revealed that the composition of bacteria differed between these life stages. Additionally, planulae raised in ambient seawater and seawater filtered to reduce the microbial cell density harbored dissimilar bacterial communities, though SSU rRNA gene clone libraries showed that planulae raised in both treatments were primarily associated with different members of the Roseobacter clade of Alphaproteobacteria. Fluorescent in situ hybridization with an oligonucleotide probe suite targeting all bacteria and one oligonucleotide probe targeting members of the Roseobacter clade was used to localize the bacterial cells. Only planulae greater than 3 days old were observed to contain internalized bacterial cells, and members of the Roseobacter clade were detected in high abundance within planula tissues exposed to the ambient seawater treatment. We conclude that the onset of association between microorganisms and the coral P. meandrina appears to occur through horizontal uptake by planulae older than 79 h, and that uptake is preferential to members of the Roseobacter clade and potentially sensitive to the ambient seawater microbial community.

Functional roles of Notch signaling in the cnidarian Nematostella vectensis

Notch signaling is among the oldest of known Metazoan signaling pathways and is used in a multitude of developmental contexts to effect cellular differentiation, specification and the maintenance of stem cell state. Here we report the isolation and expression of the canonical Notch signaling pathway in the early branching metazoan Nematostella vectensis (Anthozoa, Cnidaria) during embryonic and larval development. We have used pharmacological treatment, morpholino knockdown, and dominant negative misexpression experiments to demonstrate that Notch signaling acts to mediate cnidogenesis, the development of cnidarian-specific neural effecter cells. Notch signaling often results in the transcriptional activation of NvHes genes, a conserved family of bHLH transcription factors. A loss of Notch signaling through use of pharmacological inhibition or knock-down of the Notch effecter gene Suppressor of Hairless Su(H) similarly results in a loss of cnidocyte cell fate. We also provide evidence that Notch signaling is responsible for certain aspects of neurogenesis in developing N. vectensis planula in which disruption of Notch cleavage via the pharmacological agent DAPT results in increased expression of neural marker genes in vivo. This data suggests that Notch signaling acting on components of the developing nervous system is an ancient role of this pathway. The shared requirement of Notch signaling for the development of both cnidocytes and neurons further supports the hypothesis that cnidocytes and neurons share common origins as multifunctional sensory-effecter cells.

Embryonic development in two species of scleractinian coral embryos: Symbiodinium localization and mode of gastrulation

SUMMARY Reef‐building scleractinian corals widely engage in symbiotic relationships with Symbiodinium dinoflagellates (zooxanthellae), which reside inside cells of the gastrodermis. In most cases, sexually produced larvae acquire their symbionts from the environment in the early developmental stages preceding settlement; however, some scleractinian corals maternally “seed” their oocytes with symbionts, and these symbionts are reported to be restricted to the gastrodermis at the time of its formation (gastrulation). A precise mechanism for how Symbiodinium are translocated to endoderm in these seeded species was previously unknown. In order to examine the process of endoderm formation and Symbiodinium localization during gastrulation, we have examined two species of “robust” clade scleractinians: Fungia scutaria (nonseeded) and Pocillopora meandrina (maternally seeded). We determined that both species, independent of whether or not they are seeded, undergo a “nutritive” stage before gastrulation, wherein lipid‐rich cells (F. scutaria) or membrane‐bound cellular fragments (P. meandrina) are passed to the blastocoel where they are subsequently taken up by the definitive endoderm. This emergent property of anthozoan development has been co‐opted to facilitate the movement of Symbiodinium to the blastocoel (future site of endoderm), in the seeded species, where they are later phagocytosed by the newly formed definitive endoderm. Additionally, both species of robust clade scleractinians examined gastrulate by way of invagination, as do the majority of anthozoans. This invagination differs from the prawn chip‐type gastrulation seen in the complex clade corals and provides evidence for a possible linkage between gastrulation type and phylogenetic history.

Ectopic activation of the canonical wnt signaling pathway affects ectodermal patterning along the primary axis during larval development in the anthozoan Nematostella vectensis.

The primary axis of cnidarians runs from the oral pole to the apical tuft and defines the major body axis of both the planula larva and adult polyp. In the anthozoan cnidarian Nematostella vectensis, the primary oral-aboral (O-Ab) axis first develops during the early embryonic stage. Here, we present evidence that pharmaceutical activators of canonical wnt signaling affect molecular patterning along the primary axis of Nematostella. Although not overtly morphologically complex, molecular investigations in Nematostella reveal that the O-Ab axis is demarcated by the expression of differentially localized signaling molecules and transcription factors that may serve roles in establishing distinct ectodermal domains. We have further characterized the larval epithelium by determining the position of a nested set of molecular boundaries, utilizing several newly characterized as well as previously reported epithelial markers along the primary axis. We have assayed shifts in their position in control embryos and in embryos treated with the pharmacological agents alsterpaullone and azakenpaullone, Gsk3β inhibitors that act as canonical wnt agonists, and the Wnt antagonist iCRT14, following gastrulation. Agonist drug treatments result in an absence of aboral markers, a shift in the expression boundaries of oral markers toward the aboral pole, and changes in the position of differentially localized populations of neurons in a dose-dependent manner, while antagonist treatment had the opposite effect. These experiments are consistent with canonical wnt signaling playing a role in an orally localized wnt signaling center. These findings suggest that in Nematostella, wnt signaling mediates O-Ab ectodermal patterning across a surprisingly complex epithelium in planula stages following gastrulation in addition to previously described roles for the wnt signaling pathway in endomesoderm specification during gastrulation and overall animal-vegetal patterning at earlier stages of anthozoan development.

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

The 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.

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

The genome of the cnidarian Nematostella vectensis (starlet sea anemone) provides a molecular genetic view into the first nervous systems, which appeared in a late common ancestor of cnidarians and bilaterians. Nematostella 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 Nematostella. Six of the Nematostella Shaker genes express functional homotetrameric K+ channels in vitro. 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.

Specificity of Associations between Bacteria and the Coral Pocillopora meandrina during Early Development

ABSTRACT Relationships between corals and specific bacterial associates are thought to play an important role in coral health. In this study, the specificity of bacteria associating with the coral Pocillopora meandrina was investigated by exposing coral embryos to various strains of cultured marine bacteria, sterile seawater, or raw seawater and examining the identity, density, and location of incorporated cells. The isolates utilized in this experiment included members of the Roseobacter and SAR11 clades of the Alphaproteobacteria, a Pseudoalteromonas species of the Gammaproteobacteria, and a Synechococcus species of the Cyanobacteria phylum. Based on terminal restriction fragment length polymorphism analysis of small-subunit rRNA genes, similarities in bacterial communities associated with 170-h-old planulae were observed regardless of treatment, suggesting that bacteria may have been externally associated from the outset of the experiment. Microscopic examination of P. meandrina planulae by fluorescence in situ hybridization with bacterial and Roseobacter clade-specific oligonucleotide probes revealed differences in the densities and locations of planulae-associated cells. Planulae exposed to either raw seawater or strains of Pseudoalteromonas and Roseobacter harbored the highest densities of internally associated cells, of which 20 to 100% belonged to the Roseobacter clade. Planulae exposed to sterile seawater or strains of the SAR11 clade and Synechococcus did not show evidence of prominent bacterial associations. Additional analysis of the raw-seawater-exposed planulae via electron microscopy confirmed the presence of internally associated prokaryotic cells, as well as virus-like particles. These results suggest that the availability of specific microorganisms may be an important factor in the establishment of coral-bacterial relationships.

13-P034 Notch signaling in the Anthozoan Cnidarian Nematostella vectensis

organization is its bilateral symmetry, most evident at the level of the vertebrae and skeletal muscles. Here, we show that Rere (Atrophin2)-deficient mouse embryos form asymmetrical somites in a temporally defined window. During the time period spanning the formation of somites 7 to 13 in Rere mutants, there is a lack of left–right coordination of the oscillatory behavior of the cyclic genes and of determination front regression. The somite laterality defect in the mutant is controlled by the left–right signaling machinery. Rere mutants are similar to embryos deprived of retinoic acid (RA). Rere controls RA signaling, which is required to maintain somite symmetry by buffering Fgf8 action in the left– right signaling pathway. Rere is recruited to the promoter of RA targets (e.g., RAR-beta) but does not bind to the RAR–RXR complex. Rere binds to the nuclear receptor NR2F2 (COUP-TF2), which is also recruited to the RAR-beta promoter. Asymmetrical expression of NR2F2 in the presomitic mesoderm overlaps with the asymmetry of the RA signaling response, supporting a role for NR2F2 and Rere in the control of somite symmetry and of the RA pathway. In humans, major defects of the bilateral symmetry of somite derivatives are observed at the spine level in a class of diseases called scoliosis. A better understanding of the Reredependent, RA pathway described in this work, which affects the symmetry of vertebral precursors, could be clinically relevant to human spine pathologies.