More than a colour; how pigment influences colourblind microbes.

Many animals have pigments when they themselves cannot see colour. Perhaps those pigments enable the animal to avoid predators, or to attract mates. Maybe even those pigmented surfaces are hosts for microbes, even when the microbes do not see colour. Do some pigments then serve as a chemical signal for a good or bad microbial substrate? Maybe pigments attract or repel various microbe types? Echinoderms serve as an important model to test the mechanisms of pigment-based microbial interactions. Echinoderms are marine benthic organisms, ranging from intertidal habitats to depths of thousands of metres and are exposed to large varieties of microbes. They are also highly pigmented, with a diverse variety of colours between and even within species. Here we focus on one type of pigment (naphthoquinones) made by polyketide synthase, modified by flavin-dependent monoxygenases, and on one type of function, microbial interaction. Recent successes in targeted gene inactivation by CRISPR/Cas9 in sea urchins supports the contention that colour is more than it seems. Here we dissect the players, and their interactions to better understand how such host factors influence a microbial colonization. This article is part of the theme issue 'Sculpting the microbiome: how host factors determine and respond to microbial colonization'.

Conservation and contrast in cell states of echinoderm ovaries.

Echinoderms produce functional gametes throughout their lifespan, in some cases exceeding 200 years. The histology and ultrastructure of echinoderm ovaries has been described but how these ovaries function and maintain the production of high-quality gametes remains a mystery. Here, we present the first single cell RNA sequencing data sets of mature ovaries from two sea urchin species (Strongylocentrotus purpuratus [Sp] and Lytechinus variegatus [Lv]), and one sea star species (Patiria miniata [Pm]). We find 14 cell states in the Sp ovary, 16 cell states in the Lv ovary and 13 cell states in the ovary of the sea star. This resource is essential to understand the structure and functional biology of the ovary in echinoderms, and better informs decisions in the utilization of in situ RNA hybridization probes selective for various cell types. We link key genes with cell clusters in validation of this approach. This resource also aids in the identification of the stem cells for prolonged and continuous gamete production, is a foundation for testing changes in the annual reproductive cycle, and is essential for understanding the evolution of reproduction of this important phylum.

Single-Cell Transcriptome and Pigment Biochemistry Analysis Reveals the Potential for the High Nutritional and Medicinal Value of Purple Sea Cucumbers.

The sea cucumber Apostichopus japonicus has important nutritional and medicinal value. Unfortunately, we know little of the source of active chemicals in this animal, but the plentiful pigments of these animals are thought to function in intriguing ways for translation into clinical and food chemistry usage. Here, we found key cell groups with the gene activity predicted for the color morphology of sea cucumber body using single-cell RNA-seq. We refer to these cell populations as melanocytes and quinocytes, which are responsible for the synthesis of melanin and quinone pigments, respectively. We integrated analysis of pigment biochemistry with the transcript profiles to illuminate the molecular mechanisms regulating distinct pigment formation in echinoderms. In concert with the correlated pigment analysis from each color morph, this study expands our understanding of medically important pigment production, as well as the genetic mechanisms for color morphs, and provides deep datasets for exploring advancements in the fields of bioactives and nutraceuticals.

Molecular mechanisms of tubulogenesis revealed in the sea star hydro-vascular organ.

A fundamental goal in the organogenesis field is to understand how cells organize into tubular shapes. Toward this aim, we have established the hydro-vascular organ in the sea star Patiria miniata as a model for tubulogenesis. In this animal, bilateral tubes grow out from the tip of the developing gut, and precisely extend to specific sites in the larva. This growth involves cell migration coupled with mitosis in distinct zones. Cell proliferation requires FGF signaling, whereas the three-dimensional orientation of the organ depends on Wnt signaling. Specification and maintenance of tube cell fate requires Delta/Notch signaling. Moreover, we identify target genes of the FGF pathway that contribute to tube morphology, revealing molecular mechanisms for tube outgrowth. Finally, we report that FGF activates the Six1/2 transcription factor, which serves as an evolutionarily ancient regulator of branching morphogenesis. This study uncovers distinct mechanisms of tubulogenesis in vivo and we propose that cellular dynamics in the sea star hydro-vascular organ represents a key comparison for understanding the evolution of vertebrate organs.

Functional annotation of a hugely expanded nanos repertoire in Lytechinus variegatus, the green sea urchin.

Nanos genes encode essential RNA-binding proteins involved in germline determination and germline stem cell maintenance. When examining diverse classes of echinoderms, typically three, sometimes four, nanos genes are present. In this analysis, we identify and annotate nine nanos orthologs in the green sea urchin, Lytechinus variegatus (Lv). All nine genes are transcribed and grouped into three distinct classes. Class one includes the germline Nanos, with one member: Nanos2. Class two includes Nanos3-like genes, with significant sequence similarity to Nanos3 in the purple sea urchin, Strongylocentrotus purpuratus (Sp), but with wildly variable expression patterns. The third class includes several previously undescribed nanos zinc-finger genes that may be the result of duplications of Nanos2. All nine nanos transcripts occupy unique genomic loci and are expressed with unique temporal profiles during development. Importantly, here we describe and characterize the unique genomic location, conservation, and phylogeny of the Lv ortholog of the well-studied Sp Nanos2. However, in addition to the conserved germline functioning Nanos2, the green sea urchin appears to be an outlier in the echinoderm phyla with eight additional nanos genes. We hypothesize that this expansion of nanos gene members may be the result of a previously uncharacterized L1-class transposon encoded on the opposite strand of a nanos2 pseudogene present on chromosome 12 in this species. The expansion of nanos genes described here represents intriguing insights into germline specification and nanos evolution in this species of sea urchin.

Elements of divergence in germline determination in closely related species.

Evolutionary transitions are particularly important in development of the germ line, cells which directly impact sexual reproduction. Differences in the primordial germ cells (PGCs) of two sea urchin species were examined here by stage-matched, integrated, single cell RNA-seq (scRNA-seq) datasets. Even though both species rely on inherited mechanisms to specify their germ line, this analysis revealed a variety of differences in germline gene expression, including a broader expression of the germline factor Nanos2 (Nan2) in Lytechinus variegatus (Lv) compared to Strongylocentrotus purpuratus (Sp). In Sp, Nan2 mRNA expression is highly restricted to the PGCs by a lability element in its 3'UTR, which is lacking in the mRNA of Lv-Nan2, thus explaining the difference. We discovered that the Lv-Nan2 3'UTR instead leads to its specific translation in the PGCs. The results emphasize that regulatory mechanisms resulting in germline specification rely greatly on post-transcriptional restrictions of key gene products.

A case of hermaphroditism in the gonochoristic sea urchin, Strongylocentrotus purpuratus, reveals key mechanisms of sex determination†.

Sea urchins are usually gonochoristic, with all of their five gonads either testes or ovaries. Here, we report an unusual case of hermaphroditism in the purple sea urchin, Strongylocentrotus purpuratus. The hermaphrodite is self-fertile, and one of the gonads is an ovotestis; it is largely an ovary with a small segment containing fully mature sperm. Molecular analysis demonstrated that each gonad producedviable gametes, and we identified for the first time a somatic sex-specific marker in this phylum: Doublesex and mab-3 related transcription factor 1 (DMRT1). This finding also enabled us to analyze the somatic tissues of the hermaphrodite, and we found that the oral tissues (including gut) were out of register with the aboral tissues (including tube feet) enabling a genetic lineage analysis. Results from this study support a genetic basis of sex determination in sea urchins, the viability of hermaphroditism, and distinguish gonad determination from somatic tissue organization in the adult.

Identification of the genes encoding candidate septate junction components expressed during early development of the sea urchin, Strongylocentrotus purpuratus, and evidence of a role for Mesh in the formation of the gut barrier.

Septate junctions (SJs) evolved as cell-cell junctions that regulate the paracellular barrier and integrity of epithelia in invertebrates. Multiple morphological variants of SJs exist specific to different epithelia and/or phyla but the biological significance of varied SJ morphology is unclear because the knowledge of the SJ associated proteins and their functions in non-insect invertebrates remains largely unknown. Here we report cell-specific expression of nine candidate SJ genes in the early life stages of the sea urchin Strongylocentrotus purpuratus. By use of in situ RNA hybridization and single cell RNA-seq we found that the expression of selected genes encoding putatively SJ associated transmembrane and cytoplasmic scaffold molecules was dynamically regulated during epithelial development in the embryos and larvae with different epithelia expressing different cohorts of SJ genes. We focused a functional analysis on SpMesh, a homolog of the Drosophila smooth SJ component Mesh, which was highly enriched in the endodermal epithelium of the mid- and hindgut. Functional perturbation of SpMesh by both CRISPR/Cas9 mutagenesis and vivo morpholino-mediated knockdown shows that loss of SpMesh does not disrupt the formation of the gut epithelium during gastrulation. However, loss of SpMesh resulted in a severely reduced gut-paracellular barrier as quantitated by increased permeability to 3-5 â€‹kDa FITC-dextran. Together, these studies provide a first look at the molecular SJ physiology during the development of a marine organism and suggest a shared role for Mesh-homologous proteins in forming an intestinal barrier in invertebrates. Results have implications for consideration of the traits underlying species-specific sensitivity of marine larvae to climate driven ocean change.

Gene regulatory divergence amongst echinoderms underlies appearance of pigment cells in sea urchin development.

Larvae of the sea urchin, Strongylocentrotus purpuratus, have pigmented migratory cells implicated in immune defense and gut patterning. The transcription factor SpGcm activates the expression of many pigment cell-specific genes, including those involved in pigment biosynthesis (SpPks1 and SpFmo3) and immune related genes (e.g. SpMif5). Despite the importance of this cell type in sea urchins, pigmented cells are absent in larvae of the sea star, Patiria miniata. In this study, we tested the premises that sea stars lack genes to synthesize echinochrome pigment, that the genes are present but are not expressed in the larvae, or rather that the homologous gene expression does not contribute to echinochrome synthesis. Our results show that orthologs of sea urchin pigment cell-specific genes (PmPks1, PmFmo3-1 and PmMifL1-2) are present in the sea star genome and expressed in the larvae. Although no cell lineage homologous to migratory sea urchin pigment cells is present, dynamic gene activation accomplishes a similar spatial and temporal expression profile. The mechanisms regulating the expression of these genes, though, is highly divergent. In sea stars, PmGcm lacks the central role in pigment gene expression since it is not expressed in PmPks1 and PmFmo3-1-positive cells, and knockdown of Gcm does not abrogate pigment gene expression. Pigment genes are instead expressed in the coelomic mesoderm early in development before later being expressed in the ectoderm. These findings were supported by in situ RNA hybridization and comparative scRNA-seq analyses. We conclude that simply the coexpression of Pks1 and Fmo3 orthologs in cells of the sea star is not sufficient to underlie the emergence of the larval pigment cell in the sea urchin.

Single-cell RNA-sequencing analysis of early sea star development.

Echinoderms represent a broad phylum with many tractable features to test evolutionary changes and constraints. Here, we present a single-cell RNA-sequencing analysis of early development in the sea star Patiria miniata, to complement the recent analysis of two sea urchin species. We identified 20 cell states across six developmental stages from 8 hpf to mid-gastrula stage, using the analysis of 25,703 cells. The clusters were assigned cell states based on known marker gene expression and by in situ RNA hybridization. We found that early (morula, 8-14 hpf) and late (blastula-to-mid-gastrula) cell states are transcriptionally distinct. Cells surrounding the blastopore undergo rapid cell state changes that include endomesoderm diversification. Of particular import to understanding germ cell specification is that we never see Nodal pathway members within Nanos/Vasa-positive cells in the region known to give rise to the primordial germ cells (PGCs). The results from this work contrast the results of PGC specification in the sea urchin, and the dataset presented here enables deeper comparative studies in tractable developmental models for testing a variety of developmental mechanisms.

A Review of Asteroid Biology in the Context of Sea Star Wasting: Possible Causes and Consequences.

AbstractSea star wasting-marked in a variety of sea star species as varying degrees of skin lesions followed by disintegration-recently caused one of the largest marine die-offs ever recorded on the west coast of North America, killing billions of sea stars. Despite the important ramifications this mortality had for coastal benthic ecosystems, such as increased abundance of prey, little is known about the causes of the disease or the mechanisms of its progression. Although there have been studies indicating a range of causal mechanisms, including viruses and environmental effects, the broad spatial and depth range of affected populations leaves many questions remaining about either infectious or non-infectious mechanisms. Wasting appears to start with degradation of mutable connective tissue in the body wall, leading to disintegration of the epidermis. Here, we briefly review basic sea star biology in the context of sea star wasting and present our current knowledge and hypotheses related to the symptoms, the microbiome, the viruses, and the associated environmental stressors. We also highlight throughout the article knowledge gaps and the data needed to better understand sea star wasting mechanistically, its causes, and potential management.

Pigmentation biosynthesis influences the microbiome in sea urchins.

Organisms living on the seafloor are subject to encrustations by a wide variety of animals, plants and microbes. Sea urchins, however, thwart this covering. Despite having a sophisticated immune system, there is no clear molecular mechanism that allows sea urchins to remain free of epibiotic microorganisms. Here, we test the hypothesis that pigmentation biosynthesis in sea urchin spines influences their interactions with microbes in vivo using CRISPR/Cas9. We report three primary findings. First, the microbiome of sea urchin spines is species-specific and much of this community is lost in captivity. Second, different colour morphs associate with bacterial communities that are similar in taxonomic composition, diversity and evenness. Lastly, loss of the pigmentation biosynthesis genes polyketide synthase and flavin-dependent monooxygenase induces a shift in which bacterial taxa colonize sea urchin spines. Therefore, our results are consistent with the hypothesis that host pigmentation biosynthesis can, but may not always, influence the microbiome in sea urchin spines.

Optimizing CRISPR/Cas9-based gene manipulation in echinoderms.

The impact of new technology can be appreciated by how broadly it is used. Investigators that previously relied only on pharmacological approaches or the use of morpholino antisense oligonucleotide (MASO) technologies are now able to apply CRISPR-Cas9 to study biological problems in their model organism of choice much more effectively. The transitions to new CRISPR-based approaches could be enhanced, first, by standardized protocols and education in their applications. Here we summarize our results for optimizing the CRISPR-Cas9 technology in a sea urchin and a sea star, and provide advice on how to set up CRISPR-Cas9 experiments and interpret the results in echinoderms. Our goal through these protocols and sharing examples of success by other labs is to lower the activation barrier so that more laboratories can apply CRISPR-Cas9 technologies in these important animals.

Post-transcriptional regulation of factors important for the germ line.

Echinoderms are a major model system for many general aspects of biology, including mechanisms of gene regulation. Analysis of transcriptional regulation (Gene regulatory networks, direct DNA-binding of proteins to specific cis-elements, and transgenesis) has contributed to our understanding of how an embryo works. This chapter looks at post-transcriptional gene regulation in the context of how the primordial germ cells are formed, and how the factors essential for this process are regulated. Important in echinoderms, as in many embryos, is that key steps of fate determination are made post-transcriptionally. This chapter highlights these steps uncovered in sea urchins and sea stars, and links them to a general theme of how the germ line may regulate its fate differently than many of the embryo's somatic cell lineages.

A single-cell RNA-seq analysis of Brachyury-expressing cell clusters suggests a morphogenesis-associated signal center of oral ectoderm in sea urchin embryos.

Brachyury is a T-box family transcription factor and plays pivotal roles in morphogenesis. In sea urchin embryos, Brachyury is expressed in the invaginating endoderm, and in the oral ectoderm of the invaginating mouth opening. The oral ectoderm is hypothesized to serve as a signaling center for oral (ventral)-aboral (dorsal) axis formation and to function as a ventral organizer. Our previous results of a single-cell RNA-seq (scRNA-seq) atlas of early Strongylocentrotus purpuratus embryos categorized the constituent cells into 22 clusters, in which the endoderm consists of three clusters and the oral ectoderm four clusters (Foster et al., 2020). Here we examined which clusters of cells expressed Brachyury in relation to the morphogenesis and the identity of the ventral organizer. Our results showed that cells of all three endoderm clusters expressed Brachyury in blastulae. Based on expression profiles of genes involved in the gene regulatory networks (GRNs) of sea urchin embryos, the three clusters are distinguishable, two likely derived from the Veg2 tier and one from the Veg1 tier. On the other hand, of the four oral-ectoderm clusters, cells of two clusters expressed Brachyury at the gastrula stage and genes that are responsible for the ventral organizer at the late blastula stage, but the other two clusters did not. At a single-cell level, most cells of the two oral-ectoderm clusters expressed organizer-related genes, nearly a half of which coincidently expressed Brachyury. This suggests that the ventral organizer contains Brachyury-positive cells which invaginate to form the stomodeum. This scRNA-seq study therefore highlights significant roles of Brachyury-expressing cells in body-plan formation of early sea urchin embryos, though cellular and molecular mechanisms for how Brachyury functions in these processes remain to be elucidated in future studies.

A conserved node in the regulation of Vasa between an induced and an inherited program of primordial germ cell specification.

Primordial germ cells (PGCs) are specified by diverse mechanisms in early development. In some animals, PGCs are specified via inheritance of maternal determinants, while in others, in a process thought to represent the ancestral mode, PGC fate is induced by cell interactions. Although the terminal factors expressed in specified germ cells are widely conserved, the mechanisms by which these factors are regulated can be widely diverse. Here we show that a post-translational mechanism of germ cell specification is conserved between two echinoderm species thought to employ divergent germ line segregation strategies. Sea urchins segregate their germ line early by an inherited mechanism. The DEAD-box RNA - helicase Vasa, a conserved germline factor, becomes enriched in the PGCs by degradation in future somatic cells by the E3-ubiquitin-ligase Gustavus (Gustafson et al., 2011). This post-translational activity occurs early in development, substantially prior to gastrulation. Here we test this process in germ cell specification of sea star embryos, which use inductive signaling mechanisms after gastrulation for PGC fate determination. We find that Vasa-GFP protein becomes restricted to the PGCs in the sea star even though the injected mRNA is present throughout the embryo. Gustavus depletion, however, results in uniform accumulation of the protein. These data demonstrate that Gustavus-mediated Vasa turnover in somatic cells is conserved between species with otherwise divergent PGC specification mechanisms. Since Gustavus was originally identified in Drosophila melanogaster to have similar functions in Vasa regulation (Kugler et al., 2010), we conclude that this node of Vasa regulation in PGC formation is ancestral and evolutionarily transposable from the ancestral, induced PGC specification program to an inherited PGC specification mechanism.

Minor Genetic Consequences of a Major Mass Mortality: Short-Term Effects in Pisaster ochraceus.

AbstractMass mortality events are increasing globally in frequency and magnitude, largely as a result of human-induced change. The effects of these mass mortality events, in both the long and short term, are of imminent concern because of their ecosystem impacts. Genomic data can be used to reveal some of the population-level changes associated with mass mortality events. Here, we use reduced-representation sequencing to identify potential short-term genetic impacts of a mass mortality event associated with a sea star wasting outbreak. We tested for changes in the population for genetic differentiation, diversity, and effective population size between pre-sea star wasting and post-sea star wasting populations of Pisaster ochraceus-a species that suffered high sea star wasting-associated mortality (75%-100% at 80% of sites). We detected no significant population-based genetic differentiation over the spatial scale sampled; however, the post-sea star wasting population tended toward more differentiation across sites than the pre-sea star wasting population. Genetic estimates of effective population size did not detectably change, consistent with theoretical expectations; however, rare alleles were lost. While we were unable to detect significant population-based genetic differentiation or changes in effective population size over this short time period, the genetic burden of this mass mortality event may be borne by future generations, unless widespread recruitment mitigates the population decline. Prior results from P. ochraceus indicated that natural selection played a role in altering allele frequencies following this mass mortality event. In addition to the role of selection found in a previous study on the genomic impacts of sea star wasting on P. ochraceus, our current study highlights the potential role the stochastic loss of many individuals plays in altering how genetic variation is structured across the landscape. Future genetic monitoring is needed to determine long-term genetic impacts in this long-lived species. Given the increased frequency of mass mortality events, it is important to implement demographic and genetic monitoring strategies that capture baselines and background dynamics to better contextualize species' responses to large perturbations.

Is It in the Stars? Exploring the Relationships between Species' Traits and Sea Star Wasting Disease.

AbstractAn explanation for variation in impacts of sea star wasting disease across asteroid species remains elusive. Although various traits have been suggested to play a potential role in sea star wasting susceptibility, currently we lack a thorough comparison that explores how life-history and natural history traits shape responses to mass mortality across diverse asteroid taxa. To explore how asteroid traits may relate to sea star wasting, using available data and recognizing the potential for biological correlations to be driven by phylogeny, we generated a supertree, tested traits for phylogenetic association, and evaluated associations between traits and sea star wasting impact. Our analyses show no evidence for a phylogenetic association with sea star wasting impact, but there does appear to be phylogenetic association for a subset of asteroid life-history traits, including diet, substrate, and reproductive season. We found no relationship between sea star wasting and developmental mode, diet, pelagic larval duration, or substrate but did find a relationship with minimum depth, reproductive season, and rugosity (or surface complexity). Species with the greatest sea star wasting impacts tend to have shallower minimum depth distributions, they tend to have their median reproductive period 1.5 months earlier, and they tend to have higher rugosities relative to species less affected by sea star wasting. Fully understanding sea star wasting remains challenging, in part because dramatic gaps still exist in our understanding of the basic biology and phylogeny of asteroids. Future studies would benefit from a more robust phylogenetic understanding of sea stars, as well as leveraging intra- and interspecific comparative transcriptomics and genomics to elucidate the molecular pathways responding to sea star wasting.

Derivedness Index for Estimating Degree of Phenotypic Evolution of Embryos: A Study of Comparative Transcriptomic Analyses of Chordates and Echinoderms.

Species retaining ancestral features, such as species called living fossils, are often regarded as less derived than their sister groups, but such discussions are usually based on qualitative enumeration of conserved traits. This approach creates a major barrier, especially when quantifying the degree of phenotypic evolution or degree of derivedness, since it focuses only on commonly shared traits, and newly acquired or lost traits are often overlooked. To provide a potential solution to this problem, especially for inter-species comparison of gene expression profiles, we propose a new method named "derivedness index" to quantify the degree of derivedness. In contrast to the conservation-based approach, which deals with expressions of commonly shared genes among species being compared, the derivedness index also considers those that were potentially lost or duplicated during evolution. By applying our method, we found that the gene expression profiles of penta-radial phases in echinoderm tended to be more highly derived than those of the bilateral phase. However, our results suggest that echinoderms may not have experienced much larger modifications to their developmental systems than chordates, at least at the transcriptomic level. In vertebrates, we found that the mid-embryonic and organogenesis stages were generally less derived than the earlier or later stages, indicating that the conserved phylotypic period is also less derived. We also found genes that potentially explain less derivedness, such as Hox genes. Finally, we highlight technical concerns that may influence the measured transcriptomic derivedness, such as read depth and library preparation protocols, for further improvement of our method through future studies. We anticipate that this index will serve as a quantitative guide in the search for constrained developmental phases or processes.