Christine E. Schnitzler

The genome of the ctenophore Mnemiopsis leidyi and its implications for cell type evolution

Introduction An understanding of ctenophore biology is critical for reconstructing events that occurred early in animal evolution. The phylogenetic relationship of ctenophores (comb jellies) to other animals has been a source of long-standing debate. Until recently, it was thought that Porifera (sponges) was the earliest diverging animal lineage, but recent reports have instead suggested Ctenophora as the earliest diverging animal lineage. Because ctenophores share some of the same complex cell types with bilaterians (such as neural and mesodermal cells), the phylogenetic position of ctenophores affects how we think about the early evolution of these cell types. The phylogenetic position of the ctenophore Mnemiopsis leidyi and its implications regarding the origin of mesodermal cell types. (A) Adult M. leidyi. (B) Summary of the relationships of the five main branches of animals and the outgroup Choanoflagellata

A Tyrosine-sulfated Peptide Based on the N Terminus of CCR5 Interacts with a CD4-enhanced Epitope of the HIV-1 gp120 Envelope Glycoprotein and Inhibits HIV-1 Entry

The sequential association of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp120 with CD4 and a seven-transmembrane segment coreceptor such as CCR5 or CXCR4 initiates entry of the virus into its target cell. The N terminus of CCR5, which contains several sulfated tyrosines, plays a critical role in the CD4-dependent association of gp120 with CCR5 and in viral entry. Here we demonstrate that a tyrosine-sulfated peptide based on the N terminus of CCR5, but not its unsulfated analogue, inhibits infection of macrophages and peripheral blood mononuclear cells by CCR5-dependent, but not CXCR4-dependent, HIV-1 isolates. The sulfated peptide also inhibited the association of CCR5-expressing cells with gp120-soluble CD4 complexes and, less efficiently, with MIP-1α. Moreover, this peptide inhibited the precipitation of gp120 by 48d and 23e antibodies, which recognize CD4-inducible gp120 epitopes, but not by several other antibodies that recognize proximal epitopes. The ability of the sulfated peptide to block 48d association with gp120 was dependent in part on seven tropism-determining residues in the third variable (V3) and fourth conserved (C4) domains of gp120. These data underscore the important role of the N-terminal sulfate moieties of CCR5 in the entry of R5 HIV-1 isolates and localize a critical contact between gp120 and CCR5.

Apoptosis and autophagy as mechanisms of dinoflagellate symbiont release during cnidarian bleaching: every which way you lose.

Cnidarian bleaching results from the breakdown in the symbiosis between the host cnidarian and its dinoflagellate symbiont. Coral bleaching in recent years has increasingly caused degradation and mortality of coral reefs on a global scale. Although much is understood about the environmental causes of bleaching, the underlying cellular mechanisms of symbiont release that drive the process are just beginning to be described. In this study, we investigated the roles of two cellular pathways, host cell apoptosis and autophagy, in the bleaching process of the symbiotic anemone Aiptasia pallida. Host cell apoptosis was experimentally manipulated using gene knockdown of an anemone caspase by RNA interference, chemical inhibition of caspase using ZVAD-fmk and an apoptosis-inducer wortmannin. Autophagy was manipulated by chemical inhibition using wortmannin or induction using rapamycin. The applications of multiple single treatments resulted in some increased bleaching in anemones under control conditions but no significant drop in bleaching in individuals subjected to a hyperthermic stress. These results indicated that no single pathway is responsible for symbiont release during bleaching. However, when multiple inhibitors were applied simultaneously to block both apoptosis and autophagy, there was a significant reduction in bleaching in heat-stressed anemones. Our results allow us to formulate a model for cellular processes involved in the control of cnidarian bleaching where apoptosis and autophagy act together in a see-saw mechanism such that if one is inhibited the other is induced. Similar interconnectivity between apoptosis and autophagy has previously been shown in vertebrates including involvement in an innate immune response to pathogens and parasites. This suggests that the bleaching response could be a modified immune response that recognizes and removes dysfunctional symbionts.

Genomic organization, evolution, and expression of photoprotein and opsin genes in Mnemiopsis leidyi: a new view of ctenophore photocytes

BackgroundCalcium-activated photoproteins are luciferase variants found in photocyte cells of bioluminescent jellyfish (Phylum Cnidaria) and comb jellies (Phylum Ctenophora). The complete genomic sequence from the ctenophore Mnemiopsis leidyi, a representative of the earliest branch of animals that emit light, provided an opportunity to examine the genome of an organism that uses this class of luciferase for bioluminescence and to look for genes involved in light reception. To determine when photoprotein genes first arose, we examined the genomic sequence from other early-branching taxa. We combined our genomic survey with gene trees, developmental expression patterns, and functional protein assays of photoproteins and opsins to provide a comprehensive view of light production and light reception in Mnemiopsis.ResultsThe Mnemiopsis genome has 10 full-length photoprotein genes situated within two genomic clusters with high sequence conservation that are maintained due to strong purifying selection and concerted evolution. Photoprotein-like genes were also identified in the genomes of the non-luminescent sponge Amphimedon queenslandica and the non-luminescent cnidarian Nematostella vectensis, and phylogenomic analysis demonstrated that photoprotein genes arose at the base of all animals. Photoprotein gene expression in Mnemiopsis embryos begins during gastrulation in migrating precursors to photocytes and persists throughout development in the canals where photocytes reside. We identified three putative opsin genes in the Mnemiopsis genome and show that they do not group with well-known bilaterian opsin subfamilies. Interestingly, photoprotein transcripts are co-expressed with two of the putative opsins in developing photocytes. Opsin expression is also seen in the apical sensory organ. We present evidence that one opsin functions as a photopigment in vitro, absorbing light at wavelengths that overlap with peak photoprotein light emission, raising the hypothesis that light production and light reception may be functionally connected in ctenophore photocytes. We also present genomic evidence of a complete ciliary phototransduction cascade in Mnemiopsis.ConclusionsThis study elucidates the genomic organization, evolutionary history, and developmental expression of photoprotein and opsin genes in the ctenophore Mnemiopsis leidyi, introduces a novel dual role for ctenophore photocytes in both bioluminescence and phototransduction, and raises the possibility that light production and light reception are linked in this early-branching non-bilaterian animal.

Tyrosine-sulfated Peptides Functionally Reconstitute a CCR5 Variant Lacking a Critical Amino-terminal Region

Entry of most primary human immunodeficiency virus, type 1 (HIV-1) isolates into their target cells requires the cellular receptor CD4 and the G protein-coupled chemokine coreceptor CCR5. An acidic, tyrosine-rich, and tyrosine-sulfated domain of the CCR5 amino terminus plays a critical role in the ability of CCR5 to serve as an HIV-1 coreceptor, and tyrosine-sulfated peptides based on this region physically associate with the HIV-1 envelope glycoprotein gp120 and slow HIV-1 entry into CCR5-expressing cells. Here we show that the same tyrosine-sulfated peptides, but not their unsulfated analogs, can restore the HIV-1 coreceptor activity of a CCR5 variant lacking residues 2–17 of its amino terminus. Additionally, these sulfated peptides restored the ability of this CCR5 variant to mobilize calcium in response to the chemokines macrophage inflammatory factors 1α and 1β. These observations show that a tyrosine-sulfated region of the CCR5 amino terminus can function independently to mediate association of chemokines and the HIV-1 envelope glycoprotein with the remaining domains of CCR5.

Response of the symbiotic cnidarian Anthopleura elegantissima transcriptome to temperature and UV increase

Elevated temperature and solar radiation, including ultraviolet radiation, are now recognized as the primary environmental stresses that lead to mass cnidarian bleaching. This study takes a functional genomics approach to identifying genes that change expression soon after exposure to these stressors in the temperate sea anemone Anthopleura elegantissima that harbors Symbiodinium, the same genus of symbionts found in reef-building corals. Symbiotic anemones were subjected to elevated temperature or UV over a 24 h period. cDNA from these animals was hybridized to a 10,000-feature cDNA microarray of A. elegantissima. Overall 2.7% of the 10,000 features were found to be differentially expressed as a function of temperature or UV stress. Of the 86 features sequenced, 45% displayed significant homology to sequences in GenBank. There are 27 features that were differentially expressed in both stress conditions. Gene ontology analysis placed the differentially expressed genes in a wide range of categories including cytoskeleton organization and biogenesis, protein biosynthesis, cell proliferation, apoptosis and transport. This suggests that the early stress response to elevated temperature and UV involves essentially all aspects of host cellular regulation and machinery and that downstream cnidarian bleaching is a complex cellular response in host tissues.

Regulation of cnidarian–dinoflagellate mutualisms: Evidence that activation of a host TGFβ innate immune pathway promotes tolerance of the symbiont

Animals must manage interactions with beneficial as well as detrimental microbes. Immunity therefore includes strategies for both resistance to and tolerance of microbial invaders. Transforming growth factor beta (TGFβ) cytokines have many functions in animals including a tolerance-promoting (tolerogenic) role in immunity in vertebrates. TGFβ pathways are present in basal metazoans such as cnidarians but their potential role in immunity has never been explored. This study takes a two-part approach to examining an immune function for TGFβ in cnidarians. First bioinformatic analyses of the model anemone Aiptasia pallida were used to identify TGFβ pathway components and explore the hypothesis that an immune function for TGFβs existed prior to the evolution of vertebrates. A TGFβ ligand from A. pallida was identified as one that groups closely with vertebrate TGFβs that have an immune function. Second, cellular analyses of A. pallida were used to examine a role for a TGFβ pathway in the regulation of cnidarian-dinoflagellate mutualisms. These interactions are stable under ambient conditions but collapse under elevated temperature, a phenomenon called cnidarian bleaching. Addition of exogenous human TGFβ suppressed an immune response measured as LPS-induced nitric oxide (NO) production by the host. Addition of anti-TGFβ to block a putative TGFβ pathway resulted in immune stimulation and a failure of the symbionts to successfully colonize the host. Finally, addition of exogenous TGFβ suppressed immune stimulation in heat-stressed animals and partially abolished a bleaching response. These findings suggest that the dinoflagellate symbionts somehow promote host tolerance through activation of tolerogenic host immune pathways, a strategy employed by some intracellular protozoan parasites during their invasion of vertebrates. Insight into the ancient, conserved nature of host-microbe interactions gained from this cnidarian-dinoflagellate model is valuable to understanding the evolution of immunity and its role in the regulation of both beneficial and detrimental associations.

MicroRNAs and essential components of the microRNA processing machinery are not encoded in the genome of the ctenophore Mnemiopsis leidyi

BackgroundMicroRNAs play a vital role in the regulation of gene expression and have been identified in every animal with a sequenced genome examined thus far, except for the placozoan Trichoplax. The genomic repertoires of metazoan microRNAs have become increasingly endorsed as phylogenetic characters and drivers of biological complexity.ResultsIn this study, we report the first investigation of microRNAs in a species from the phylum Ctenophora. We use short RNA sequencing and the assembled genome of the lobate ctenophore Mnemiopsis leidyi to show that this species appears to lack any recognizable microRNAs, as well as the nuclear proteins Drosha and Pasha, which are critical to canonical microRNA biogenesis. This finding represents the first reported case of a metazoan lacking a Drosha protein.ConclusionsRecent phylogenomic analyses suggest that Mnemiopsis may be the earliest branching metazoan lineage. If this is true, then the origins of canonical microRNA biogenesis and microRNA-mediated gene regulation may postdate the last common metazoan ancestor. Alternatively, canonical microRNA functionality may have been lost independently in the lineages leading to both Mnemiopsis and the placozoan Trichoplax, suggesting that microRNA functionality was not critical until much later in metazoan evolution.

Coral larvae exhibit few measurable transcriptional changes during the onset of coral-dinoflagellate endosymbiosis.

The cellular mechanisms controlling the successful establishment of a stable mutualism between cnidarians and their dinoflagellate partners are largely unknown. The planula larva of the solitary Hawaiian scleractinian coral Fungia scutaria and its dinoflagellate symbiont Symbiodinium sp. type C1f represents an ideal model for studying the onset of cnidarian-dinoflagellate endosymbiosis due to the predictable availability of gametes, the ability to raise non-symbiotic larvae and establish the symbiosis experimentally, and the ability to precisely quantify infection success. The goal of this study was to identify genes differentially expressed in F. scutaria larvae during the initiation of endosymbiosis with Symbiodinium sp. C1f. Newly symbiotic larvae were compared to non-symbiotic larvae using a custom cDNA microarray. The 5184-feature array was constructed with cDNA libraries from newly symbiotic and non-symbiotic F. scutaria larvae, including 3072 features (60%) that were enriched for either state by subtractive hybridization. Our analyses revealed very few changes in the F. scutaria transcriptome as a result of infection with Symbiodinium sp. C1f, similar to other studies focused on the early stages of this symbiotic interaction. We suggest that these results may be due, in part, to an inability to detect the transcriptional signal from the small percentage of infected cells compared to uninfected cells. We discuss several other potential explanations for this result, including suggesting that certain types of Symbiodinium sp. may have evolved mechanisms to suppress or circumvent cnidarian host responses to infection.

Expression of multiple Sox genes through embryonic development in the ctenophore Mnemiopsis leidyi is spatially restricted to zones of cell proliferation

BackgroundThe Sox genes, a family of transcription factors characterized by the presence of a high mobility group (HMG) box domain, are among the central groups of developmental regulators in the animal kingdom. They are indispensable in progenitor cell fate determination, and various Sox family members are involved in managing the critical balance between stem cells and differentiating cells. There are 20 mammalian Sox genes that are divided into five major groups (B, C, D, E, and F). True Sox genes have been identified in all animal lineages but not outside Metazoa, indicating that this gene family arose at the origin of the animals. Whole-genome sequencing of the lobate ctenophore Mnemiopsis leidyi allowed us to examine the full complement and expression of the Sox gene family in this early-branching animal lineage.ResultsOur phylogenetic analyses of the Sox gene family were generally in agreement with previous studies and placed five of the six Mnemiopsis Sox genes into one of the major Sox groups: SoxB (MleSox1), SoxC (MleSox2), SoxE (MleSox3, MleSox4), and SoxF (MleSox5), with one unclassified gene (MleSox6). We investigated the expression of five out of six Mnemiopsis Sox genes during early development. Expression patterns determined through in situ hybridization generally revealed spatially restricted Sox expression patterns in somatic cells within zones of cell proliferation, as determined by EdU staining. These zones were located in the apical sense organ, upper tentacle bulbs, and developing comb rows in Mnemiopsis, and coincide with similar zones identified in the cydippid ctenophore Pleurobrachia.ConclusionsOur results are consistent with the established role of multiple Sox genes in the maintenance of stem cell pools. Both similarities and differences in juvenile cydippid stage expression patterns between Mnemiopsis Sox genes and their orthologs from Pleurobrachia highlight the importance of using multiple species to characterize the evolution of development within a given phylum. In light of recent phylogenetic evidence that Ctenophora is the earliest-branching animal lineage, our results are consistent with the hypothesis that the ancient primary function of Sox family genes was to regulate the maintenance of stem cells and function in cell fate determination.