Global warming is causing ocean warming and acidification. The distribution of Heliocidaris erythrogramma coincides with the eastern Australia climate change hot spot, where disproportionate warming ...makes marine biota particularly vulnerable to climate change. In keeping with near-future climate change scenarios, we determined the interactive effects of warming and acidification on fertilization and development of this echinoid. Experimental treatments (20-26°C, pH 7.6-8.2) were tested in all combinations for the 'business-as-usual' scenario, with 20°C/pH 8.2 being ambient. Percentage of fertilization was high (>89%) across all treatments. There was no difference in percentage of normal development in any pH treatment. In elevated temperature conditions, +4°C reduced cleavage by 40 per cent and +6°C by a further 20 per cent. Normal gastrulation fell below 4 per cent at +6°C. At 26°C, development was impaired. As the first study of interactive effects of temperature and pH on sea urchin development, we confirm the thermotolerance and pH resilience of fertilization and embryogenesis within predicted climate change scenarios, with negative effects at upper limits of ocean warming. Our findings place single stressor studies in context and emphasize the need for experiments that address ocean warming and acidification concurrently. Although ocean acidification research has focused on impaired calcification, embryos may not reach the skeletogenic stage in a warm ocean.
In the sea urchin, entry of β-catenin into the nuclei of the vegetal cells at 4th and 5th cleavages is necessary for activation of the endomesoderm gene regulatory network. Beyond that, little is ...known about how the embryo uses maternal information to initiate specification. Here, experiments establish that of the three maternal Wnts in the egg, Wnt6 is necessary for activation of endodermal genes in the endomesoderm GRN. A small region of the vegetal cortex is shown to be necessary for activation of the endomesoderm GRN. If that cortical region of the egg is removed, addition of Wnt6 rescues endoderm. At a molecular level, the vegetal cortex region contains a localized concentration of Dishevelled (Dsh) protein, a transducer of the canonical Wnt pathway; however, Wnt6 mRNA is not similarly localized. Ectopic activation of the Wnt pathway, through the expression of an activated form of β-catenin, of a dominant-negative variant of GSK-3β or of Dsh itself, rescues endomesoderm specification in eggs depleted of the vegetal cortex. Knockdown experiments in whole embryos show that absence of Wnt6 produces embryos that lack endoderm, but those embryos continue to express a number of mesoderm markers. Thus, maternal Wnt6 plus a localized vegetal cortical molecule, possibly Dsh, is necessary for endoderm specification; this has been verified in two species of sea urchin. The data also show that Wnt6 is only one of what are likely to be multiple components that are necessary for activation of the entire endomesoderm gene regulatory network.
Background
Sea urchins are model organisms for studying the spatial‐temporal control of gene activity during development. The Southern California species, Lytechinus pictus, has a sequenced genome ...and can be raised in the laboratory from egg to egg in 4 to 5 months.
Results
Here, we present new techniques for generating parthenogenetic larvae of this species and include a gallery of photomicrographs of morphologically abnormal larvae that could be used for transcriptomic analysis.
Conclusions
Comparison of gene expression in parthenogenotes to larvae produced by fertilization could provide novel insights into gene expression controls contributed by sperm in this important model organism. Knowledge gained from transcriptomics of sea urchin parthenogenotes could contribute to parthenogenetic studies of mammalian embryos.
Key Findings
Our paper reintroduces the potential value of studying parthenogenotes since the last such paper was published 46 years ago, before transcriptomics existed.
This paper describes the simple culture of Lytechinus pictus in petri plates in the absence of large quantities of seawater and continuous stirring motors.
This paper provides the first photomicrographs of parthenogenetic sea urchin larvae in the developmental biology literature.
Studying transcriptomics in these parthenogenotes might illuminate controls the sperm genome contributes to gene activation during development.
Processes such as the activation of protein and DNA synthesis would also be interesting to study in parthenogenote sea urchin embryos and larvae.
Sea urchin embryos are a useful model system for investigating early developmental processes and the underlying gene regulatory networks. Most functional studies using sea urchin embryos rely on ...antisense morpholino oligonucleotides to knockdown gene functions. However, major concerns related to this technique include off-target effects, variations in morpholino efficiency, and potential morpholino toxicity; furthermore, such problems are difficult to discern. Recent advances in genome editing technologies have introduced the prospect of not only generating sequence-specific knockouts, but also providing genome-engineering applications. Two genome editing tools, zinc-finger nuclease (ZFN) and transcription activator-like effector nucleases (TALENs), have been utilized in sea urchin embryos, but the resulting efficiencies are far from satisfactory. The CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 (CRISPR-associated nuclease 9) system serves as an easy and efficient method with which to edit the genomes of several established and emerging model organisms in the field of developmental biology. Here, we apply the CRISPR/Cas9 system to the sea urchin embryo. We designed six guide RNAs (gRNAs) against the well-studied nodal gene and discovered that five of the gRNAs induced the expected phenotype in 60–80% of the injected embryos. In addition, we developed a simple method for isolating genomic DNA from individual embryos, enabling phenotype to be precisely linked to genotype, and revealed that the mutation rates were 67–100% among the sequenced clones. Of the two potential off-target sites we examined, no off-target effects were observed. The detailed procedures described herein promise to accelerate the usage of CRISPR/Cas9 system for genome editing in sea urchin embryos.
•The CRISPR/Cas9 system is an effective tool for genome editing in sea urchin embryos.•Genotyping of individual embryos enables phenotype to be linked with genotype.•The CRISPR/Cas9 system induced phenotype and mutation efficiently.
Echinoidea is a clade of marine animals including sea urchins, heart urchins, sand dollars and sea biscuits. Found in benthic habitats across all latitudes, echinoids are key components of marine ...communities such as coral reefs and kelp forests. A little over 1000 species inhabit the oceans today, a diversity that traces its roots back at least to the Permian. Although much effort has been devoted to elucidating the echinoid tree of life using a variety of morphological data, molecular attempts have relied on only a handful of genes. Both of these approaches have had limited success at resolving the deepest nodes of the tree, and their disagreement over the positions of a number of clades remains unresolved.
We performed de novo sequencing and assembly of 17 transcriptomes to complement available genomic resources of sea urchins and produce the first phylogenomic analysis of the clade. Multiple methods of probabilistic inference recovered identical topologies, with virtually all nodes showing maximum support. In contrast, the coalescent-based method ASTRAL-II resolved one node differently, a result apparently driven by gene tree error induced by evolutionary rate heterogeneity. Regardless of the method employed, our phylogenetic structure deviates from the currently accepted classification of echinoids, with neither Acroechinoidea (all euechinoids except echinothurioids), nor Clypeasteroida (sand dollars and sea biscuits) being monophyletic as currently defined. We show that phylogenetic signal for novel resolutions of these lineages is strong and distributed throughout the genome, and fail to recover systematic biases as drivers of our results.
Our investigation substantially augments the molecular resources available for sea urchins, providing the first transcriptomes for many of its main lineages. Using this expanded genomic dataset, we resolve the position of several clades in agreement with early molecular analyses but in disagreement with morphological data. Our efforts settle multiple phylogenetic uncertainties, including the position of the enigmatic deep-sea echinothurioids and the identity of the sister clade to sand dollars. We offer a detailed assessment of evolutionary scenarios that could reconcile our findings with morphological evidence, opening up new lines of research into the development and evolutionary history of this ancient clade.
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•The sea urchin skeleton is generated by a sequence of morphogenetic processes.•Each morphogenetic process is controlled by a specific regulatory sub-circuit.•Mineral bearing vesicles ...perform an active diffusion motion in the skeletogenic cells.•Spicule elongation at the tips of the rods is driven by tips-specific regulatory states.•Possible common origin of sea urchin skeletogenesis and vertebrates’ vascularization.
Biomineralization is the process in which soft organic tissues use minerals to produce shells, skeletons and teeth for various functions such as protection and physical support. The ability of the cells to control the time and place of crystal nucleation as well as crystal orientation and stiffness is far beyond the state-of-the art of human technologies. Thus, understanding the biological control of biomineralization will promote our understanding of embryo development as well as provide novel approaches for material engineering. Sea urchin larval skeletogenesis offers an excellent platform for functional analyses of both the molecular control system and mineral uptake and deposition. Here we describe the current understanding of the genetic, molecular and cellular processes that underlie sea urchin larval skeletogenesis. We portray the regulatory genes that define the specification of the skeletogenic cells and drive the various morphogenetic processes that occur in the skeletogenic lineage, including: epithelial to mesenchymal transition, cell migration, spicule cavity formation and mineral deposition into the spicule cavity. We describe recent characterizations of the size, motion and mineral concentration of the calcium-bearing vesicles in the skeletogenic cells. We review the distinct specification states within the skeletogenic lineage that drive localized skeletal growth at the tips of the spicules. Finally, we discuss the surprising similarity between the regulatory network and cellular processes that drive sea urchin skeletogenesis and those that control vertebrate vascularization. Overall, we illustrate the novel insights on the biological regulation and evolution of biomineralization, gained from studies of the sea urchin larval skeletogenesis.
Understanding how growth trajectories of calcifying invertebrates are affected by changing climate requires acclimation experiments that follow development across life-history transitions. In a ...long-term acclimation study, the effects of increased acidification and temperature on survival and growth of the tropical sea urchin Tripneustes gratilla from the early juvenile (5 mm test diameter—TD) through the developmental transition to the mature adult (60 mm TD) were investigated. Juveniles were reared in a combination of three temperature and three pH/pCO2 treatments, including treatments commensurate with global change projections. Elevated temperature and pCO2/pH both affected growth, but there was no interaction between these factors. The urchins grew more slowly at pH 7.6, but not at pH 7.8. Slow growth may be influenced by the inability to compensate coelomic fluid acid–base balance at pH 7.6. Growth was faster at +3 and +6°C compared to that in ambient temperature. Acidification and warming had strong and interactive effects on reproductive potential. Warming increased the gonad index, but acidification decreased it. At pH 7.6 there were virtually no gonads in any urchins regardless of temperature. The T. gratilla were larger at maturity under combined near-future warming and acidification scenarios (+3°C/pH 7.8). Although the juveniles grew and survived in near-future warming and acidification conditions, chronic exposure to these stressors from an early stage altered allocation to somatic and gonad growth. In the absence of phenotypic adjustment, the interactive effects of warming and acidification on the benthic life phases of sea urchins may compromise reproductive fitness and population maintenance as global climatic change unfolds.
The spectacular escalation in complexity in early bilaterian evolution correlates with a strong increase in the number of microRNAs. To explore the link between the birth of ancient microRNAs and ...body plan evolution, we set out to determine the ancient sites of activity of conserved bilaterian microRNA families in a comparative approach. We reason that any specific localization shared between protostomes and deuterostomes (the two major superphyla of bilaterian animals) should probably reflect an ancient specificity of that microRNA in their last common ancestor. Here, we investigate the expression of conserved bilaterian microRNAs in Platynereis dumerilii, a protostome retaining ancestral bilaterian features, in Capitella, another marine annelid, in the sea urchin Strongylocentrotus, a deuterostome, and in sea anemone Nematostella, representing an outgroup to the bilaterians. Our comparative data indicate that the oldest known animal microRNA, miR-100, and the related miR-125 and let-7 were initially active in neurosecretory cells located around the mouth. Other sets of ancient microRNAs were first present in locomotor ciliated cells, specific brain centres, or, more broadly, one of four major organ systems: central nervous system, sensory tissue, musculature and gut. These findings reveal that microRNA evolution and the establishment of tissue identities were closely coupled in bilaterian evolution. Also, they outline a minimum set of cell types and tissues that existed in the protostome-deuterostome ancestor.
The ecologically significant shift in developmental strategy from planktotrophic (feeding) to lecithotrophic (nonfeeding) development in the sea urchin genus Heliocidaris is one of the most ...comprehensively studied life history transitions in any animal. Although the evolution of lecithotrophy involved substantial changes to larval development and morphology, it is not known to what extent changes in gene expression underlie the developmental differences between species, nor do we understand how these changes evolved within the context of the well-defined gene regulatory network (GRN) underlying sea urchin development. To address these questions, we used RNA-seq to measure expression dynamics across development in three species: the lecithotroph Heliocidaris erythrogramma, the closely related planktotroph H. tuberculata, and an outgroup planktotroph Lytechinus variegatus. Using well-established statistical methods, we developed a novel framework for identifying, quantifying, and polarizing evolutionary changes in gene expression profiles across the transcriptome and within the GRN. We found that major changes in gene expression profiles were more numerous during the evolution of lecithotrophy than during the persistence of planktotrophy, and that genes with derived expression profiles in the lecithotroph displayed specific characteristics as a group that are consistent with the dramatically altered developmental program in this species. Compared to the transcriptome, changes in gene expression profiles within the GRN were even more pronounced in the lecithotroph. We found evidence for conservation and likely divergence of particular GRN regulatory interactions in the lecithotroph, as well as significant changes in the expression of genes with known roles in larval skeletogenesis. We further use coexpression analysis to identify genes of unknown function that may contribute to both conserved and derived developmental traits between species. Collectively, our results indicate that distinct evolutionary processes operate on gene expression during periods of life history conservation and periods of life history divergence, and that this contrast is even more pronounced within the GRN than across the transcriptome as a whole.
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