Sea Urchin Embryos Research Articles

A Survey on Tubulin and Arginine Methyltransferase Families Sheds Light on P. lividus Embryo as Model System for Antiproliferative Drug Development.

Tubulins and microtubules (MTs) represent targets for taxane-based chemotherapy. To date, several lines of evidence suggest that effectiveness of compounds binding tubulin often relies on different post-translational modifications on tubulins. Among them, methylation was recently associated to drug resistance mechanisms impairing taxanes binding. The sea urchin is recognized as a research model in several fields including fertilization, embryo development and toxicology. To date, some α- and β-tubulin genes have been identified in P. lividus, while no data are available in echinoderms for arginine methyl transferases (PRMT). To evaluate the exploiting of the sea urchin embryo in the field of antiproliferative drug development, we carried out a survey of the expressed α- and β-tubulin gene sets, together with a comprehensive analysis of the PRMT gene family and of the methylable arginine residues in P. lividus tubulins. Because of their specificities, the sea urchin embryo may represent an interesting tool for dissecting mechanisms of tubulin targeting drug action. Therefore, results herein reported provide evidences supporting the P. lividus embryo as animal system for testing antiproliferative drugs.

Cis-Regulatory analysis for later phase of anterior neuroectoderm-specific foxQ2 expression in sea urchin embryos.

The specification of anterior neuroectoderm is controlled by a highly conserved molecular mechanism in bilaterians. A forkhead family gene, foxQ2, is known to be one of the pivotal regulators, which is zygotically expressed in this region during embryogenesis of a broad range of bilaterians. However, what controls the expression of this essential factor has remained unclear to date. To reveal the regulatory mechanism of foxQ2, we performed cis-regulatory analysis of two foxQ2 genes, foxQ2a and foxQ2b, in a sea urchin Hemicentrotus pulcherrimus. In sea urchin embryos, foxQ2 is initially expressed in the entire animal hemisphere and subsequently shows narrower expression restricted to the anterior pole region. In this study, as a first step to understand the foxQ2 regulation, we focused on the later restricted expression and analyzed the upstream cis-regulatory sequences of foxQ2a and foxQ2b by using the constructs fused to short half-life green fluorescent protein. Based on deletion and mutation analyses of both foxQ2, we identified each of the five regulatory sequences, which were 4-9 bp long. Neither of the regulatory sequences contains any motifs for ectopic activation or spatial repression, suggesting that later mRNA localization is regulated in situ. We also suggest that the three amino acid loop extension-class homeobox gene Meis is involved in the maintenance of foxQ2b, the expression of which is dominant during embryogenesis.

Molecular and Morphological Toxicity of Diatom-Derived Hydroxyacid Mixtures to Sea Urchin Paracentrotus lividus Embryos.

Oxylipins such as polyunsaturated aldehydes (PUAs) and hydroxyacids (HEPEs) are signaling molecules derived from the oxidation of polyunsaturated fatty acids. They are common in diatoms that constitute a major group of microalgae in freshwater and oceanic ecosystems. Although HEPEs represent the most common oxylipins produced by diatoms, little information is available on their effects on marine invertebrates, and most of the information has been obtained by testing individual HEPEs. Our previous studies reported that four hydroxyacids, i.e., 5-, 9-, 11-, and 15-HEPE, were able to induce malformations and a marked developmental delay in sea urchin Paracentrotus lividus embryos, which had not been reported for other oxylipins. Here, we tested a mixture of 5-, 9-, 11-, and 15-HEPE at different concentrations for the first time. The results showed that mixtures of HEPEs have synergistic effects that are much more severe compared to those of individual HEPEs: The HEPE mixtures induced malformations in sea urchin embryos at lower concentrations. Increasing HEPE mixture concentrations induced a marked increase in the number of delayed embryos, until all embryos were delayed at the highest concentration tested. At the molecular level, the HEPE mixtures induced variations in the expression of 50 genes involved in different functional processes, mainly down-regulating these genes at the earliest stages of embryonic development. These findings are ecologically significant, considering that during diatom blooms, sea urchins could accumulate HEPEs in concentrations comparable to those tested in the present study.

A convenient synthesis of cis-restricted combretastatin analogues with pyrazole and isoxazole cores

A series of combretastatin analogues, diarylpyrazoles and diarylisoxazoles, have been synthesized and evaluated for their antimitotic tubulin-binding activity using the phenotypic sea urchin (Paracentrotus lividus) embryo assay. One pyrazole analogue and four isoxazole analogues have been identified as potent antimitotic agents comparable with combretastatins A-2 and A-4, with the lowest observable effective concentration of 1–10 nmol dm−3 for cleavage alteration of the test embryos.

Spatial and temporal patterns of gene expression during neurogenesis in the sea urchin Lytechinus variegatus

BackgroundThe sea urchin is a basal deuterostome that is more closely related to vertebrates than many organisms traditionally used to study neurogenesis. This phylogenetic position means that the sea urchin can provide insights into the evolution of the nervous system by helping resolve which developmental processes are deuterostome innovations, which are innovations in other clades, and which are ancestral. However, the nervous system of echinoderms is one of the least understood of all major metazoan phyla. To gain insights into echinoderm neurogenesis, spatial and temporal gene expression data are essential. Then, functional data will enable the building of a detailed gene regulatory network for neurogenesis in the sea urchin that can be compared across metazoans to resolve questions about how nervous systems evolved.ResultsHere, we analyze spatiotemporal gene expression during sea urchin neurogenesis for genes that have been shown to be neurogenic in one or more species. We report the expression of 21 genes expressed in areas of neurogenesis in the sea urchin embryo from blastula stage (just before neural progenitors begin their specification sequence) through pluteus larval stage (when much of the nervous system has been patterned). Among those 21 gene expression patterns, we report expression of 11 transcription factors and 2 axon guidance genes, each expressed in discrete domains in the neuroectoderm or in the endoderm. Most of these genes are expressed in and around the ciliary band. Some including the transcription factors Lv-mbx, Lv-dmrt, Lv-islet, and Lv-atbf1, the nuclear protein Lv-prohibitin, and the guidance molecule Lv-semaa are expressed in the endoderm where they are presumably involved in neurogenesis in the gut.ConclusionsThis study builds a foundation to study how neurons are specified and evolved by analyzing spatial and temporal gene expression during neurogenesis in a basal deuterostome. With these expression patterns, we will be able to understand what genes are required for neural development in the sea urchin. These data can be used as a starting point to (1) build a spatial gene regulatory network for sea urchin neurogenesis, (2) identify how subtypes of neurons are specified, (3) perform comparative studies with the sea urchin, protostome, and vertebrate organisms.

Leachates of micronized plastic toys provoke embryotoxic effects upon sea urchin Paracentrotus lividus

Microplastics are defined as plastic fragments <5 mm, and they are found in the ocean where they can impact on the ecosystem. Once released in seawater, microplastics can be internalized by organisms due to their small size, moreover they can also leach out several additives used in plastic manufacturing, such as plasticizers, flame retardants, etc., resulting toxic for biota. The aim of this study was to test the toxicity of micronized PVC products with three different colors, upon Paracentrotus lividus embryos. In particular, we assessed the effects of micronized plastics and microplastic leachates. Results showed a decrease of larval length in plutei exposed to low concentrations of micronized plastics, and a block of larval development in sea urchin embryos exposed to the highest dose. Virgin PVC polymer did not result toxic on P. lividus embryos, while an evident toxic effect due to leached substances in the medium was observed. In particular, the exposure to leachates induced a development arrest immediately after fertilization or morphological alterations in plutei. Finally, PVC products with different colors showed different toxicity, probably due to a different content and/or combination of heavy metals present in coloring agents.

Generation, expression and utilization of single-domain antibodies for in vivo protein localization and manipulation in sea urchin embryos.

Single-domain antibodies, also known as nanobodies, are small antigen-binding fragments (~15kDa) that are derived from heavy chain only antibodies present in camelids (VHH, from camels and llamas), and cartilaginous fishes (VNAR, from sharks). Nanobody V-like domains are useful alternatives to conventional antibodies due to their small size, and high solubility and stability across many applications. In addition, phage display, ribosome display, and mRNA/cDNA display methods can be used for the efficient generation and optimization of binders in vitro. The resulting nanobodies can be genetically encoded, tagged, and expressed in cells for in vivo localization and functional studies of target proteins. Collectively, these properties make nanobodies ideal for use within echinoderm embryos. This chapter describes the optimization and imaging of genetically encoded nanobodies in the sea urchin embryo. Examples of live-cell antigen tagging (LCAT) and the manipulation of green fluorescent protein (GFP) are shown. We discuss the potentially transformative applications of nanobody technology for probing membrane protein trafficking, cytoskeleton re-organization, receptor signaling events, and gene regulation during echinoderm development.

Multiplex cis-regulatory analysis.

Recent progress in multiplex cis-regulatory analysis has increased the speed of identifying enhancers and promoters, and enabled efficient incorporation of cis-regulatory information into gene regulatory network models. Three types of barcode reporters have been developed for multiplex reporter assays in sea urchin embryos: 13-tags and 129-tags for QPCR, 130 Nanotags for NanoString, and 100 million N25-tags for next-generation sequencing. In this chapter, to facilitate adoption of high-throughput cis-regulatory analysis in sea urchin embryos, I provide practical guidelines to best utilize barcode reporters that are compatible with either QPCR or next-generation sequencing. I expect that the guidelines are also applicable to other invertebrate embryos.

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.

Whole mount in situ hybridization techniques for analysis of the spatial distribution of mRNAs in sea urchin embryos and early larvae.

A critical process in embryonic development is the activation and spatial localization of mRNAs to specific cells and territories of the embryo. Revealing the spatial distribution of mRNAs and how it changes during development is a vital piece of information that aids in understanding the signaling and regulatory genes driving specific gene regulatory networks. In the laboratory, a cost-efficient, reliable method to determine the spatial distribution of mRNAs in embryos is in situ hybridization. This sensitive and straightforward method employs exogenous antisense RNA probes to find specific and complementary sequences in fixed embryos. Antigenic moieties conjugated to the ribonucleotides incorporated in the probe cross-react with antibodies, and numerous staining methods can be subsequently employed to reveal the spatial distribution of the targeted mRNA. The quality of the data produced by this method is equivalent to the experience of the researcher, and thus a thorough understanding of the numerous steps comprising this method is important for obtaining high quality data. Here we compile and summarize several protocols that have been employed chiefly on five sea urchin species in numerous laboratories around the world. Whereas the protocols can vary for the different species, the overarching steps are similar and can be readily mastered. When properly and carefully undertaken, in situ hybridization is a powerful tool providing unambiguous data for which there currently is no comparable substitute and will continue to be an important method in the era of big data and beyond.

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.

Measuring voltage and ion concentrations in live embryos.

Abstract Homeostasis of charged particles in biological systems is fundamental for life. Indeed, the efficient synthesis of ATP is predicated on an electrochemical gradient. Ion pumps and channels act as conduits that regulate membrane potential by controlling ion flux. This phenomenon is critical for the generation of action potentials in excitable cells such as neurons and muscle fibers, and for acidification of lysosomes in all cells. However, the production of action potentials or pH differentials is merely one facet of bioelectricity. Increasing evidence has shown that ion channels and pumps also play critical roles in other cellular processes: cell cycle regulation, wound healing, regeneration, and symmetry breaking during development. In recent years, the functional roles of ion channels have been explored in echinoderm development. The application of fluorescence-based ion- and voltage-sensitive dyes allows for live measurements of ion concentrations with both spatial and temporal resolution. In this chapter, we describe the use of such dyes for interrogating and visualizing ion and voltage gradients in sea urchin embryos.

A Report on the Fertilisation and Early Development in the Sea Urchin

The primary objective of this study was to investigate fertilisation, cleavage and gastrulation in sea urchin embryos. It was found that a difference between room and 4°C stored samples were obtained, with only 55% of the embryos reaching the plateus larvae stage. After 72h, cell death occurred in the 4°C stored samples. The parthenogenic embryos died, and didn't pass the blastula stage of early gastrulation. This paper addresses 4 main topics of sea urchin embryo development, including parthenogenesis and the interference of fertilisation.

High resolution imaging of the cortex isolated from sea urchin eggs and embryos.

The cellular cortex-consisting of the plasma membrane and the adjacent outer few microns of the cytoplasm-is a critically important, dynamic and complex region in the sea urchin egg and embryo. Some 40 years ago it was discovered that isolated cortices could be obtained from eggs adhered to glass coverslips and since that time this preparation has been used in a wide range of studies, including seminal research on fertilization, exocytosis, the cytoskeleton, and cytokinesis. In this chapter, we discuss methods for isolating cortices from eggs and embryos, including those undergoing cell division. We also provide protocols for analyzing cortical architecture and dynamics using specific localization methods combined with super-resolution Structured Illumination and Stimulated Emission Depletion light microscopy and platinum replica transmission electron microscopy.

Trapping, tagging and tracking: Tools for the study of proteins during early development of the sea urchin.

The exquisite synchronicity of sea urchin development provides a reliable model for studying maternal proteins in the haploid egg as well as those involved in egg activation, fertilization and early development. Sea urchin eggs are released by the millions, enabling the quantitative evaluation of maternally stored and newly synthesized proteins over a range of time (seconds to hours post fertilization). During this window of development exist many hallmark and unique biochemical interactions that can be investigated for the purpose of characterizing profiles of kinases and other signaling proteins, manipulated using pharmacology to test sufficiency and necessity, for identification of post translational modifications, and for capturing protein-protein interactions. Coupled with the fact that sea urchin eggs and embryos are transparent, this synchronicity also results in large populations of cells that can be evaluated for newly synthesized protein localization and identification through use of the Click-iT technology. We provide basic protocols for these approaches and direct readers to the appropriate literature for variations and examples.

3D+time imaging of normal and twin sea urchin embryos for the reconstruction of their cell lineage.

The Mediterranean sea urchin, Paracentrotus lividus, has been a powerful model to study embryonic development since the late 1800s. As a model, it has the advantage of having external fertilization, it can easily be manipulated experimentally, and it has semi-transparent embryonic stages, which makes it ideal for live imaging. Embryogenesis is a highly dynamic process with intrinsic variability. The reconstruction of cell dynamics and an assessment of such variability from in vivo observations has proven to be a challenge. Here, we provide an innovative methodology for manipulation and immobilization of embryos and their long-term 3D+time imaging. We then describe the twinning procedure that allows us to assess the variability and robustness of developmental processes. We demonstrate the reconstruction of cell lineages based on automated image processing and cell tracking using the BioEmergences workflow as well as the use of interactive visualization tools (Mov-IT software) for lineage validation, correction and analysis.

Transcriptome analysis of regeneration during Xenopus laevis experimental twinning

Animal embryos have the remarkable property of self-organization. Over 125 years ago, Hans Driesch separated the two blastomeres of sea urchin embryos and obtained twins, in what was the foundation of experimental embryology. Since then, embryonic twinning has been obtained experimentally in many animals. In a recent study, we developed bisection methods that generate identical twins reliably from Xenopus blastula embryos. In the present study, we have investigated the transcriptome of regenerating half-embryos after sagittal and dorsal-ventral (D-V) bisections. Individual embryos were operated at midblastula (stage 8) with an eyelash hair and cultured until early gastrula (stage 10.5) or late gastrula (stage 12) and the transcriptome of both halves were analyzed by RNA-seq. Since many genes are activated by wound healing in Xenopus embryos, we resorted to stringent sequence analyses and identified genes up-regulated in identical twins but not in either dorsal or ventral fragments. At early gastrula, cell division-related transcripts such as histones were elevated, whereas at late gastrula, pluripotency genes (such as sox2) and germ layer determination genes (such as eomesodermin, ripply2 and activin receptor ACVRI) were identified. Among the down-regulated transcripts, sizzled, a regulator of Chordin stability, was prominent. These findings are consistent with a model in which cell division is required to heal damage, while maintaining pluripotency to allow formation of the organizer with a displacement of 90 0 from its original site. The extensive transcriptomic data presented here provides a valuable resource for data mining of gene expression during early vertebrate development.

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.

Unlocking mechanisms of development through advances in tools.

This perspective describes how our understanding of sea urchin development has been enabled by advances in technology. The early conceptual discoveries that put the sea urchin embryo on the research map had to wait until technologies were available to explain how those concepts worked. The explanatory phase continues as a number of mechanisms continue to be understood in ever greater detail, all made possible by further technical advances.

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.