Light-induced, spatiotemporal control of protein in the developing embryo of the sea urchin.

Differential protein regulation is a critical biological process that regulates cellular activity and controls cell fate determination. It is especially important during early embryogenesis when post-transcriptional events predominate differential fate specification in many organisms. Light-induced approaches have been a powerful technology to interrogate protein functions with temporal and spatial precision, even at subcellular levels within a cell by controlling laser irradiation on the confocal microscope. However, application and efficacy of these tools need to be tested for each model system or for the cell type of interest because of the complex nature of each system. Here, we introduce two types of light-induced approaches to track and control proteins at a subcellular level in the developing embryo of the sea urchin. We found that the photoconvertible fluorescent protein Kaede is highly efficient to distinguish pre-existing and newly synthesized proteins with no apparent phototoxicity, even when interrogating proteins associated with the mitotic spindle. Further, chromophore-assisted light inactivation (CALI) using miniSOG successfully inactivated target proteins of interest in the vegetal cortex and selectively delayed or inhibited asymmetric cell division. Overall, these light-induced manipulations serve as important molecular tools to identify protein function for for subcellular interrogations in developing embryos.

Modularity and hierarchy in biological systems: Using gene regulatory networks to understand evolutionary change.

Modularity and hierarchy are important theoretical concepts in biology, and both are useful frameworks to understand the evolution of complex systems. Gene regulatory networks (GRNs) provide a powerful mechanistic model for modularity in animal development, as they are made up of modular (or self-contained) circuits, which are deployed in a hierarchical manner over time. Over the years, studies in the sea urchin, Strongylocentrotus purpuratus, have provided an illustrative example of how these regulatory circuits are responsible for processes such as cell differentiation and cell state specificity. However, GRNs are themselves made up of a nested series of interactions, as each gene can be regulated by multiple cis-regulatory elements, which can be further broken down into distinct transcription factor binding sites (TFBS). As a result, modularity can be applied to each "level" of this complex hierarchy. Throughout the literature, there is considerable discussion about the roles modular circuits, modular enhancers, and modular TFBS play in evolution, yet there is little discussion about how these nested interactions operate as a whole. In this chapter, we discuss how modular changes at different levels of the GRN hierarchy affect animal development and aim to provide a unified framework to understand the role of modularity in evolution.

Cellular pathways of calcium transport and concentration toward mineral formation in sea urchin larvae.

Sea urchin larvae have an endoskeleton consisting of two calcitic spicules. The primary mesenchyme cells (PMCs) are the cells that are responsible for spicule formation. PMCs endocytose sea water from the larval internal body cavity into a network of vacuoles and vesicles, where calcium ions are concentrated until they precipitate in the form of amorphous calcium carbonate (ACC). The mineral is subsequently transferred to the syncytium, where the spicule forms. Using cryo-soft X-ray microscopy we imaged intracellular calcium-containing particles in the PMCs and acquired Ca-L2,3 X-ray absorption near-edge spectra of these Ca-rich particles. Using the prepeak/main peak (L2'/ L2) intensity ratio, which reflects the atomic order in the first Ca coordination shell, we determined the state of the calcium ions in each particle. The concentration of Ca in each of the particles was also determined by the integrated area in the main Ca absorption peak. We observed about 700 Ca-rich particles with order parameters, L2'/ L2, ranging from solution to hydrated and anhydrous ACC, and with concentrations ranging between 1 and 15 M. We conclude that in each cell the calcium ions exist in a continuum of states. This implies that most, but not all, water is expelled from the particles. This cellular process of calcium concentration may represent a widespread pathway in mineralizing organisms.

A Peak of H3T3 Phosphorylation Occurs in Synchrony with Mitosis in Sea Urchin Early Embryos.

The sea urchin embryo provides a valuable system to analyse the molecular mechanisms orchestrating cell cycle progression and mitosis in a developmental context. However, although it is known that the regulation of histone activity by post-translational modification plays an important role during cell division, the dynamics and the impact of these modifications have not been characterised in detail in a developing embryo. Using different immuno-detection techniques, we show that the levels of Histone 3 phosphorylation at Threonine 3 oscillate in synchrony with mitosis in Sphaerechinus granularis early embryos. We present, in addition, the results of a pharmacological study aimed at analysing the role of this key histone post-translational modification during sea urchin early development.

Short- and long-term impacts of variable hypoxia exposures on kelp forest sea urchins.

Climate change is altering the intensity and variability of environmental stress that organisms and ecosystems experience, but effects of changing stress regimes are not well understood. We examined impacts of constant and variable sublethal hypoxia exposures on multiple biological processes in the sea urchin Strongylocentrotus purpuratus, a key grazer in California Current kelp forests, which experience high variability in physical conditions. We quantified metabolic rates, grazing, growth, calcification, spine regeneration, and gonad production under constant, 3-hour variable, and 6-hour variable exposures to sublethal hypoxia, and compared responses for each hypoxia regime to normoxic conditions. Sea urchins in constant hypoxia maintained baseline metabolic rates, but had lower grazing, gonad development, and calcification rates than those in ambient conditions. The sublethal impacts of variable hypoxia differed among biological processes. Spine regrowth was reduced under all hypoxia treatments, calcification rates under variable hypoxia were intermediate between normoxia and constant hypoxia, and gonad production correlated negatively with continuous time under hypoxia. Therefore, exposure variability can differentially modulate the impacts of sublethal hypoxia, and may impact sea urchin populations and ecosystems via reduced feeding and reproduction. Addressing realistic, multifaceted stressor exposures and multiple biological responses is crucial for understanding climate change impacts on species and ecosystems.

Single cell RNA-seq in the sea urchin embryo show marked cell-type specificity in the Delta/Notch pathway.

Sea urchin embryos are excellent for in vivo functional studies because of their transparency and tractability in manipulation. They are also favorites for pharmacological approaches since they develop in an aquatic environment and addition of test substances is straightforward. A concern in many pharmacological tests though is the potential for pleiotropic effects that confound the conclusions drawn from the results. Precise cellular interpretations are often not feasible because the impact of the perturbant is not known. Here we use single-cell mRNA (messenger RNA) sequencing as a metric of cell types in the embryo and to determine the selectivity of two commonly used inhibitors, one each for the Wnt and the Delta-Notch pathways, on these nascent cell types. We identified 11 distinct cell types based on mRNA profiling, and that the cell lineages affected by Wnt and Delta/Notch inhibition were distinct from each other. These data support specificity and distinct effects of these signaling pathways in the embryo and illuminate how these conserved pathways selectively regulate cell lineages at a single cell level. Overall, we conclude that single cell RNA-seq analysis in this embryo is revealing of the cell types present during development, of the changes in the gene regulatory network resulting from inhibition of various signaling pathways, and of the selectivity of these pathways in influencing developmental trajectories.

Microsphere packages of carotenoids: intact sea urchin eggs tracked by Raman spectroscopy tools.

Although natural exposure to ambient UV radiation in oligotrophic seawater at small depths can reach the levels responsible for cellular damage, the sea urchin Paracentrotus lividus is frequently in such sites, particularly on the southern Adriatic Sea shore. Spawning their eggs and spending their early life stage in rocky shores at depths of 0.5-2 m are the results of their successful adaptation strategies, although adults may dwell at greater depths. Surprisingly, there is a paucity of reports regarding the carotenoid content in sea urchin eggs. Beyond their important role in photoprotection against high UV exposure, cell division and early development, the content and distribution of carotenoids contribute to the successful survival of sea urchins and also determine the color of their gonads (roe), which is of commercial importance as a delicacy. Herein, for the first time, we have described the carotenoid content and distribution in intact, freshly released eggs of P. lividus species, non-destructively employing resonance Raman spectroscopy and imaging; near-infrared Raman spectroscopy revealed additional molecular carotenoid content. Echinenone and ß-carotene resonance Raman signals were the most intense, and they were identified as the principal carotenoids that are preferentially accumulated in eggs rather than in gonads. Raman imaging in confocal mode revealed the uniform distribution of the carotenoid signal over the whole eggs, while the distribution of proteins appeared spotted. Egg carotenoids generally maintained their identity after 2 months of dry storage, with slight signs of C[double bond, length as m-dash]C bond oxidation. The potential utilization of P. lividus sea urchin eggs as valuable microsphere packages of native carotenoids is discussed.

Cell rearrangement induced by filopodial tension accounts for the late phase of convergent extension in the sea urchin archenteron.

George Oster was a pioneer in using mechanical models to interrogate morphogenesis in animal embryos. Convergent extension is a particularly important morphogenetic process to which George Oster gave significant attention. Late elongation of the sea urchin archenteron is a classic example of convergent extension in a monolayered tube, which has been proposed to be driven by extrinsic axial tension due to the activity of secondary mesenchyme cells. Using a vertex-based mechanical model, we show that key features of archenteron elongation can be accounted for by passive cell rearrangement due to applied tension. The model mimics the cell elongation and the Poisson effect (necking) that occur in actual archenterons. We also show that, as predicted by the model, ablation of secondary mesenchyme cells late in archenteron elongation does not result in extensive elastic recoil. Moreover, blocking the addition of cells to the base of the archenteron late in archenteron elongation leads to excessive cell rearrangement consistent with tension-induced rearrangement of a smaller cohort of cells. Our mechanical simulation suggests that responsive rearrangement can account for key features of archenteron elongation and provides a useful starting point for designing future experiments to examine the mechanical properties of the archenteron.

The painted sea urchin, Lytechinus pictus, as a genetically-enabled developmental model.

Although sea urchins are one of the oldest and most widely used marine model systems, few species have been routinely kept in culture through multiple generations. The workhorse of the field is the purple urchin Strongylocentrotus purpuratus. However, one disadvantage of S. purpuratus is its long generation time, making it impractical as a model for generating and maintaining transgenic lines. In an effort to develop a sea urchin that is suitable for transgenerational experiments and the generation of transgenic lines, we have focused on development of updated culturing methods and genomic resources for the painted sea urchin, Lytechinus pictus. Compared to S. purpuratus, L. pictus have relatively large eggs, develop into optically clear embryos, and the smaller adults can become gravid in under a year. Fifty years ago, Hinegardner developed culturing methods for raising L. pictus through metamorphosis. Here, we provide an updated protocol for establishing and maintaining L. pictus in the laboratory, and describe a new genome resource for this urchin. In our hands, L. pictus reach the 4-armed pluteus stage at 4 days; become competent to metamorphosis at 24 days; and are gravid by 6 months. Plutei and juveniles are fed on a diet of algae and diatoms, and adults are fed on kelp. We also make available a L. pictus transcriptome generated from developmental stages (eggs to 2-day-old plutei) to support the annotation of our genome sequencing project, and to enhance the utility of this species for molecular studies and transgenesis.

Microinjection methods for sea urchin eggs and blastomeres.

Methods for microinjection into sea urchin eggs have become relatively easier because of the technical improvements by a number of researchers in the past decades. However, the size and the characteristics, such as the elasticity and toughness, of the eggs and embryos differ in species, so that we still need to modify the details of methods to adapt to each target. In this section, I list microinjection methods for three species: Hemicentrotus pulcherrimus, which has relatively tough eggs, Temnopleurus reevesii, which has slightly weak eggs, and Strongylocentrotus purpuratus, which is the most used species in sea urchin biology. In addition, I describe the methods for co-injection of morpholino anti-sense oligonucleotides and mRNAs, as well as the method for microinjection into blastomeres.

Methods for transplantation of sea urchin blastomeres.

The stereotypic cleavage pattern of sea urchin embryos provides a platform for dissection of early lineage decisions that lead to cell diversification. Cell transplantation provides a useful tool for understanding those decisions. The methods described in this paper provide a guide for how to produce embryonic mosaics in which either one cell is transplanted or an entire tier of cells are transplanted to a host embryo. Although the results of such a cut and paste experiment can be documented in many ways, one of the most useful approaches follows progeny of the transplanted cell as they go through morphogenesis using time-lapse imaging. Methods for mounting and imaging the embryos are provided.

Sea urchin embryonic cilia.

Cilia are exceptionally complicated subcellular structures involved in swimming and developmental signaling, including induction of left-right asymmetry in larval stages. We summarize the history of research on sea urchin embryonic cilia. The high salt method to isolate cilia is presented first; methods to block cilia formation and to lengthen cilia are presented in the appendix. Evidence suggests that regenerated cilia may not be as physiologically perfect as those formed normally during embryogenesis. Sea urchin embryonic cilia are valuable models for studying molecular details of cilia assembly and differentiation as well as gene activation, cell signaling, and pattern formation during development.

Visualizing egg and embryonic polarity.

During development metazoan embryos have to establish the molecular coordinates for elaboration of the embryonic body plan. Typically, bilaterian (bilaterally symmetric animals) embryos establish anterior-posterior (AP) and dorsal-ventral (DV) axes, and in most cases the AP axis is established first. For over a century it has been known that formation of the AP axis is strongly influenced by the primary axis of the egg, the animal-vegetal (AV) axis. The molecular basis for how the AV axis influences AP polarity remains poorly understood, but sea urchins have proven to be important for elucidating the molecular basis for this process. In fact, it is the first model system where a critical role for Wnt signaling in specification and patterning the AV and AP axis was first established. One current area of research is focused on identifying the maternal factors that regulate localized activation of Wnt/ß-catenin signaling at the vegetal pole during development. An essential tool for this work is the means to identify the AV polarity in oocytes and eggs. This permits investigation into how polarity is established and allows development of experimental strategies to identify maternal factors that contribute to and control axial polarity. This chapter provides protocols to accomplish this in sea urchin eggs and early embryos. We describe simple methods to visualize polarity including direct observation of eggs and oocytes, using a microscope for overt morphological signs of polarity, and more extensive methods involving localization of known factors indicative of inherent embryonic polarity, such as the upstream regulators of the Wnt/ß-catenin pathway.

Methods to label, isolate, and image sea urchin small micromeres, the primordial germ cells (PGCs).

Small micromeres of the sea urchin are believed to be primordial germ cells (PGCs), fated to give rise to sperm or eggs in the adult. Sea urchin PGCs are formed at the fifth cleavage, undergo one additional division during blastulation, and migrate to the coelomic pouches of the pluteus larva. The goal of this chapter is to detail classical and modern techniques used to analyze primordial germ cell specification, gene expression programs, and cell behaviors in fixed and live embryos. The transparency of the sea urchin embryo enables both live imaging techniques and in situ RNA hybridization and immunolabeling for a detailed molecular characterization of these cells. Four approaches are presented to highlight small micromeres with fluorescent molecules for analysis by live and fixed cell microscopy: (1) small molecule dye accumulation during cleavage and blastula stages, (2) primordial germ cell targeted RNA expression using the Nanos untranslated regions, (3) fusing genes of interest with a Nanos2 targeting peptide, and (4) EdU and BrdU labeling. Applications of the live labeling techniques are discussed, including sorting by fluorescence-activated cell sorting for transcriptomic analysis, and, methods to image small micromere behavior in whole and dissociated embryos by live confocal microscopy. Finally, summary table of antibody and RNA probes as well as small molecule dyes to label small micromeres at a variety of developmental stages is provided.

Culture of and experiments with sea urchin embryo primary mesenchyme cells.

Skeletogenesis in the sea urchin embryo gives rise to a pair of intricate endoskeletal spicules. Deposition of these skeletal elements in the early larva is the outcome of a morphogenetic program that begins with maternal inputs in the early zygote and results in the specification of the large micromere-primary mesenchyme cell (PMC) lineage. PMCs are of considerable interest as a model system, not only to dissect the mechanism of specific developmental processes, but also to investigate their evolution and the unrivaled level of control over the formation of a graded, mechanically robust, yet single crystalline biomineral. The ability to study gene regulatory circuits, cellular behavior, signaling pathways, and molecular players involved in biomineralization is significantly boosted by the high level of autonomy of PMCs. In fact, in the presence of horse serum, micromeres differentiate into PMCs and produce spicules in vitro, separated from the embryonic milieu. PMC culture eliminates indirect effects that can complicate the interpretation of experiments in vivo, offers superior spatiotemporal control, enables PMC-specific readouts, and is compatible with most imaging and characterization techniques. In this chapter, we provide an updated protocol, based on the pioneering work by Okazaki and Wilt, for the isolation of micromeres and subsequent culture of PMCs, as well as protocols for fixation and staining for fluorescent microscopy, preparation of cell cultures for electron microscopy, and the isolation of RNA.

Methods for collection, handling, and analysis of sea urchin coelomocytes.

Sea urchin coelomocytes can be collected in large numbers from adult sea urchins of the species, Strongylocentrotus purpuratus, which typically has 12-40mL of coelomic fluid. Coelomocytes are used for analysis of immune reactions and immune gene expression in addition to basic functions of cells, in particular for understanding structure and modifications of the cytoskeleton in phagocytes. The methods described here include coelomocyte isolation, blocking the clotting reaction, establishing and maintaining primary cultures, separation of different types of coelomocytes into fractions, processing live coelomocytes for light microscopy, fixation and staining for light and electron microscopy, analysis of coelomocyte populations by flow cytometry, and sorting single cells for more detailed follow-up analyses including transcriptomics or genomic characteristics. These methods are provided to make working with coelomocytes accessible to researchers who are unfamiliar with these cells and perhaps to aid others who have worked extensively with invertebrate cells.

Methods for toxicology studies in echinoderm embryos and larvae.

Sea urchin embryos have been used in toxicological studies for many decades as they are an accepted model system for investigations of chemicals that impact development. Here we describe methods for using pulse-chase experiments to study the impacts of environmental chemicals on early development as well as development of larvae. This includes the application of fluorescence plate assays with living embryos and fluorescent probes to assess cell functions (mitochondrial membrane potential, lysosome abundance, reactive oxygen species, and esterase activity) based on total cell numbers. We also describe how to use some of these fluorescent probes in embryos/larvae with confocal microscopy for the localization of cellular damage in response to toxics exposure. Finally, we assess skeleton formation in sea urchin larvae and present methods for using polarized light microscopy to examine spicule morphology.

Cryopreservation of sea urchin sperm and early life stages.

Sea urchins are excellent model organisms useful for several lines of biotechnological research. Sea urchins are typically collected from the sea and kept in research facilities all over the world for such purposes. Cryopreservation can be a very powerful tool to enhance the use of sea urchins as a model species for research. The development of cryopreservation protocols for different sea urchin gametes, embryos, and larvae allows year round access to high quality material outside the natural reproductive season. It also reduces the uncertainty and variability that may be caused by changing oceanic, meteorological, and environmental conditions. Access to cryopreserved gametes and embryos will allow using these model organisms in laboratories all around the world, regardless of their facilities or their proximity to a natural population of sea urchins. Cryopreservation is a very useful tool for aquaculture production, fisheries conservation, and wild stock enhancement allowing spat supply all year round without the need of conditioning broodstock for out of season reproduction-which is expensive, time consuming, and often unfruitful. It will also provide flexibility for selective breeding programs by allowing crossings of individuals with different reproductive seasons. Although cryopreservation protocols have been successfully developed for many valuable fisheries species such as sturgeons, salmonid fishes, and other marine invertebrates (e.g., oysters), only a small amount of research has been carried out regarding sea urchin cryopreservation. In this chapter, we outline protocols for the cryopreservation of sea urchin cells.

Temnopleurus as an emerging echinoderm model.

Sea urchins have played important roles in cell and developmental biology. They have the potential to be even more useful as models if the ability to create transgenic animals and maintain genetic lines are developed. Here, I describe the methods to produce next-generation lines using a newly introduced sea urchin model, Temnopleurus reevesii, in the laboratory. The embryos of T. reevesii have wide range of temperature tolerance between 15°C to 30°C and have high transparency, which can be a strong point in live-imaging and fluorescent immunohistochemistry. I describe how to grow and culture the embryos/larvae/juveniles/adults of T. reevesii to address the challenge of establishing inbred strains followed by introducing genetics into this species in the future.

Cidaroids, clypeasteroids, and spatangoids: Procurement, culture, and basic methods.

This chapter describes practical methods and key points for using non-camarodont echinoids including cidaroids (Order Cidaroida), clypeasteroids (also known as sand dollars; Order Clypeasteroida), and spatangoids (also known as heart urchins; Order Spatangoida) as experimental subjects for biological studies. The content described here is based on six Japanese species of echinoids (Astriclypeus manni, Clypeaster japonicus, Echinocardium cordatum, Peronella japonica, Prionocidaris baculosa, and Scaphechinus mirabilis). Specific topics addressed in this chapter include the collection and maintenance of adults, embryonic culture, and experimental procedures for micromanipulations, whole mount in situ hybridization, and immunological experiments.