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Biomineralization is the utilization of different minerals by a vast array of organisms to form hard tissues and shape them in various forms. Within this diversity, a common feature of all mineralized tissues is their high stiffness, implying that mechanosensing could be commonly used in biomineralization. Yet, the role of mechanosensing in biomineralization is far from clear. Here, we use the sea urchin larval skeletogenesis to investigate the role of substrate stiffness and focal adhesion kinase (FAK) in biomineralization. We demonstrate that substrate stiffness alters spicule morphology and growth, indicating a mechanosensitive response during skeletogenesis. We show that active FAK, F-actin, and vinculin are enriched around the spicules, indicating the formation of focal adhesion complexes and suggesting that the cells sense the mechanical properties of the biomineral. Furthermore, we find that FAK activity is regulated by Rho-associated protein kinase (ROCK) and is crucial for skeletal growth and normal branching. FAK and ROCK activate extracellular signal-regulated kinase (ERK), which regulates skeletogenic gene expression at the tips of the spicules. Thus, the FAK-ROCK-ERK circuit seems to provide essential mechanical feedback on spicule elongation to the skeletogenic gene regulatory network, enabling skeletal growth. Remarkably, the same factors govern mammalian osteoblast differentiation in vitro and pathological calcification in vivo. Thus, this study highlights a common mechanotransduction pathway in biomineralization that was probably independently co-opted across different organisms to shape mineralized structures in metazoans., Competing Interests: Competing interests statement:The authors declare no competing interest.

The regulation of mitotic spindle position and orientation, and consequent division plane specification, is critical for early embryo development, tissue architecture and stem cells. Accordingly, defects in spindle positioning have been associated with the emergence of tissue disorders and mis-specification of cell fates, causing the formation of tumors, for instance, in stem cell populations. In such context, methods to physically manipulate mitotic spindle position or orientation in living cells bear the promise of dissecting the causal impact of spindle mis-positioning on cell or tissue behavior. Here, we describe a method to directly modulate mitotic spindle position and orientation with in vivo magnetic tweezers in developing sea urchin embryos. This method allows for monitoring the impact of spindle orientation on embryo development and quantifying forces and torques needed to move or rotate spindles. It may be transposable to many other cell, embryo or tissue types., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

A profound collaboration between the germline and somatic cells of an organism is the creation of a functional gonad. Here we establish a foundation for studying molecular gonadogenesis in the sea urchin by use of RNA-seq, quantitative mRNA measurements, and in-situ hybridizations throughout the life cycle of the variegated sea urchin, Lytechinus variegatus (Lv). We found through three distinct analyses that the ovary and testis of this echinoderm expresses unique transcripts involved in gametogenesis, and also discovered uncharacterized gene products unique to each gonad. We further developed a pipeline integrating timepoint RNA-seq data throughout development to identify hallmark gene expression in gonads. We found that meiotic and candidate genes involved in sex determination are first expressed surprisingly early during larval growth, and well before metamorphosis. We further discovered that individual larvae express varying amounts of male- or female-hallmarks before metamorphosis, including germline, oocyte, sperm, and meiotic related genes. These distinct male- or female-gonad gene profiles may indicate the onset of, and commitment to, development of a bipotential gonad primordium, and may include metabolic differences, supported by the observation that transcripts involved in glycolysis are highly enriched in the ovary compared to the testis. Together these data support a hypothesis that sex determination is initiated prior to metamorphosis in the sea urchin and that the many uncharacterized genes unique to each gonad type characterized herein may reveal unique pathways and mechanisms in echinoderm reproduction., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Growing evidence suggests that metabolic regulation directly influences cellular function and development and thus may be more dynamic than previously expected. In vivo and in real-time analysis of metabolite activities during development is crucial to test this idea directly. In this study, we employ two metabolic biosensors to track the dynamics of pyruvate and oxidative phosphorylation (Oxphos) during the early embryogenesis of the sea urchin. A pyruvate sensor, PyronicSF, shows the signal enrichment on the mitotic apparatus, which is consistent with the localization patterns of the corresponding enzyme, pyruvate kinase (PKM). The addition of pyruvate increases the PyronicSF signal, while PKM knockdown decreases its signal, responding to the pyruvate level in the cell. Similarly, a ratio-metric sensor, Grx-roGFP, that reads the redox potential of the cell responds to DTT and H 2 O 2 , the known reducer and inducer of Oxphos. These observations suggest that these metabolic biosensors faithfully reflect the metabolic status in the cell during embryogenesis. The time-lapse imaging of these biosensors suggests that pyruvate and Oxphos levels change both spatially and temporarily during embryonic development. Pyruvate level is increased first in micromeres compared to other blastomeres at the 16-cell stage and remains high in ectoderm while decreasing in endomesoderm during gastrulation. In contrast, the Oxphos signal first decreases in micromeres at the 16-cell stage, while it increases in the endomesoderm during gastrulation, showing the opposite trend of the pyruvate signal. These results suggest that metabolic regulation is indeed both temporally and spatially dynamic during embryogenesis, and these biosensors are a valuable tool to monitor metabolic activities in real-time in developing embryos., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: Understanding the dynamics of the gut microbiota in sea urchins is crucial for comprehending the ecological balance in marine ecosystems. The gut microbiota plays a vital role in nutrient metabolism, immune system modulation, and pathogen protection. The microbial composition and dynamics of naturally occurring sea urchin Tripneustes ventricosus have yet to be thoroughly investigated. We hypothesized the gut microbiota of T. ventricosus in the Caribbean, varies across life stages and seasons., Methods: Thirty-six naturally occurring large individuals and six small individuals (42 animals) were collected from shallow waters on the northeastern coast of Puerto Rico in February and August of 2019. The fecal pellet's microbiota was characterized by sequencing V4 region of the 16S rRNA gene., Results: We found significant differences in the composition of fecal pellet microbiota between seasons and life stages. Phylum Bacteroidota had greater relative abundance in August, while Firmicutes was more dominant in February. Propionigenium and Roseimarinus had greater relative abundance in August, while Candidatus Hepatoplasma, and Kistimonas had greater relative abundance in February. Differences in the gut digest microbiota were not found between small and large urchins, but small urchins displayed a slightly higher diversity and dominance of Bacteroidota and Proteobacteria, while large urchins exhibited a greater relative abundance of Fusobacteria and Desulfobacterota. However, the genera Ferrominas and Propionigenium counts were significantly lower in small individuals., Discussion: This is the first report for this species in the Caribbean region and adds to our comprehension of the microbiota of the white sea urchin across collection periods and size classes, highlighting the dynamic nature of the gut microbiota., Competing Interests: The authors declare that they have no competing interests., (© 2024 Rodríguez-Barreras et al.)

The Antarctic sea urchin Sterechinus neumayeri (Echinoida; Echinidae) is routinely used as a model organism for Antarctic biology. Here, we present a high-quality genome of S. neumayeri. This chromosomal-level assembly was generated using PacBio long-read sequencing and Hi-C chromatin conformation capture sequencing. This 885.3-Mb assembly exhibits high contiguity with a scaffold length N50 of 36.7 Mb assembled into 20 chromosomal length scaffolds. These putative chromosomes exhibit a high degree of synteny compared to other sea urchin models. We used transcript evidence gene modeling combined with sequence homology to identify 21,638 gene models that capture 97.4% of BUSCO orthologs. Among these, we were able to identify and annotate conserved developmental gene regulatory network orthologs, positioning S. neumayeri as a tractable model for comparative studies on evolution and development., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

In the framework of a safe-by-design approach, we previously assessed the eco-safety of nanostructured cellulose sponge (CNS) leachate on sea urchin reproduction. It impaired gamete quality, gamete fertilization competence, and embryo development possibly due to the leaching of chemical additives used during the CNS synthesis process. To extend this observation and identify the component(s) that contribute to CNS ecotoxicity, in the present study, we individually screened the cytotoxic effects on sea urchin Arbacia lixula and Paracentrotus lividus gametes and embryos of the three main constituents of CNS, namely cellulose nanofibers, citric acid, and branched polyethylenimine. The study aimed to minimize any potential safety risk of these components and to obtain an eco-safe CNS. Among the three CNS constituents, branched polyethylenimine resulted in the most toxic agent. Indeed, it affected the physiology and fertilization competence of male and female gametes as well as embryo development in both sea urchin species. These results are consistent with those previously reported for CNS leachate. Moreover, the characterisation of CNS leachate confirmed the presence of detectable branched polyethylenimine in the conditioned seawater even though in a very limited amount. Altogether, these data indicate that the presence of branched polyethylenimine is a cause-effect associated with a significant risk in CNS formulations due to its leaching upon contact with seawater. Nevertheless, the suggested safety protocol consisting of consecutive leaching treatments and conditioning of CNS in seawater can successfully ameliorate the CNS ecotoxicity while maintaining the efficacy of its sorbent properties supporting potential environmental applications., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

Calcium carbonate is present in many biominerals, including in the exoskeletons of crustaceans and shells of mollusks. High Mg-containing calcium carbonate was synthesized by high temperatures, high pressures or high molecular organic matter. For example, biogenic high Mg-containing calcite is synthesized under strictly controlled Mg concentration at ambient temperature and pressure. The spines of sea urchins consist of calcite, which contain a high percentage of magnesium. In this study, we investigated the factors that increase the magnesium content in calcite from the spines of the sea urchin, Heliocidaris crassispina. X-ray diffraction and inductively coupled plasma mass spectrometry analyses showed that sea urchin spines contain about 4.8% Mg. The organic matrix extracted from the H. crassispina spines induced the crystallization of amorphous phase and synthesis of magnesium-containing calcite, while amorphous was synthesized without SUE (sea urchin extract). In addition, aragonite was synthesized by SUE treated with protease-K. HC tropomyosin was specifically incorporated into Mg precipitates. Recombinant HC-tropomyosin induced calcite contained 0.1-2.5% Mg synthesis. Western blotting of sea urchin spine extracts confirmed that HC tropomyosin was present in the purple sea urchin spines at a protein weight ratio of 1.5%. These results show that HC tropomyosin is one factor that increases the magnesium concentration in the calcite of H. crassispina spines., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The mass mortality event of the herbivorous sea urchin Diadema antillarum in 1983-1984 has been a major contributor to the diminished resilience of coral reefs throughout the Caribbean. The reduction in grazing pressure resulted in algae proliferation, which inhibited coral recruitment after disturbances such as disease, hurricanes, pollution and climatic change induced marine heat waves. Natural recovery of D. antillarum after the 1983-1984 die-off has been slow. However, the few locations with recovered populations exhibit signs of improvement in coral reef health, prompting interest in D. antillarum restoration. Current restoration strategies include translocation of wild individuals, the restocking of juveniles that are either cultured from gametes or collected as settlers and head-started in a nursery, and assisted natural recovery by providing suitable settlement substrate. Both the collection of wild settlers and assisted natural recovery necessitate an understanding of the local, spatiotemporal trends in settlement. In this study, which was carried out on the Dutch Caribbean Island of Saba, artificial turf settlement collectors were deployed at nine locations around the island and monitored from June 2019 till July 2020 (13 months). The primary objective was to identify trends in larval settlement in space and time, to be able to optimize restoration efforts. Additionally, the small size of Saba allowed us to deploy settlement collectors around the island and compare D. antillarum settlement between windward and leeward sides. Our study showed that on Saba, D. antillarum settlement peaked in June and July, following similar seasonal trends observed around other islands in the Northeastern Caribbean. By far the most settlement occurred at the leeward side of the island, suggesting that hydrodynamic forces entrained D. antillarum larvae in the lee of Saba and/or calmer waters facilitated settlement. Limited settlement occurred on the more exposed windward locations. The identified high settlement locations are candidates for settler collection and restoration attempts. Continued monitoring of D. antillarum settlement, especially in light of the 2022 D. antillarum die-off, holds significance as it can provide insights into the potential of natural recovery., Competing Interests: The authors declare that they have no competing interests., (© 2024 Klokman and Hylkema.)

The red sea urchin (Mesocentrotus franciscanus) is one of the Earth's longest-living animals, reported to live more than 100 years with indeterminate growth, life-long reproduction, and no increase in mortality rate with age. To understand the genetic underpinnings of longevity and negligible aging, we constructed a chromosome-level assembly of the red sea urchin genome and compared it to that of short-lived sea urchin species. Genome-wide syntenic alignments identified chromosome rearrangements that distinguish short- and long-lived species. Expanded gene families in long-lived species play a role in innate immunity, sensory nervous system, and genome stability. An integrated network of genes under positive selection in the red sea urchin was involved in genomic regulation, mRNA fidelity, protein homeostasis, and mitochondrial function. Our results implicated known longevity genes in sea urchin longevity but also revealed distinct molecular signatures that may promote long-term maintenance of tissue homeostasis, disease resistance, and negligible aging., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Pervasive epizootic events have had a significant impact on marine invertebrates throughout the Caribbean, leading to severe population declines and consequential ecological implications. One such event was the regional collapse of herbivory, partly caused by the Diadema antillarum mortality event in 1983-84, resulting in a trophic cascade and altering the structure of reef communities. Consequently, there was a notable decrease in coral recruitment and an increase in the coverage of macroalgae. Nearly four decades later, in early 2022, the Caribbean basin experienced another widespread mass mortality event, further reducing the populations of D. antillarum . To assess the effects of this recent mortality event on the current demographics of D. antillarum , we surveyed eight populations along the eastern, northeastern, northern, and northwestern coast of Puerto Rico from May to July 2022, estimating their population density, size distribution, and disease prevalence. Additionally, the study compared these population parameters with data from four sites previously surveyed in 2012 and 2017 to understand the impact of the recent mortality event. The survey conducted in 2022 showed varying population densities at the surveyed reefs. Some populations exhibited mean densities of nearly one individual per square meter, while others had extremely low or no living individuals per square meter. The four populations with the highest density showed no evidence of disease, whereas the four populations with the lowest D. antillarum densities exhibited moderate to high disease prevalence. However, when considering all sites, the estimated disease prevalence remained below 5%. Nevertheless, the comparison with data from 2012 and 2017 indicated that the recent mortality event had a negative impact on D. antillarum demographics at multiple sites, as the densities in 2022 were reduced by 60.19% compared to those from the previous years. However, it is still too early to determine the severity of this new mortality event compared to the 1983-84 mortality event. Therefore, it is imperative to continue monitoring these populations., Competing Interests: The authors declare there are no competing interests., (©2023 Rodríguez-Barreras et al.)

The dissolution of anthropogenic carbon dioxide (CO 2 ) in seawater has altered its carbonate chemistry in the process of ocean acidification (OA). OA affects the viability of marine species. In particular, calcifying organisms and their early planktonic larval stages are considered vulnerable. These organisms often utilize energy reserves for metabolism rather than growth and calcification as supported by bulk RNA-sequencing (RNA-seq) experiments. Yet, transcriptomic profiling of a bulk sample reflects the average gene expression of the population, neglecting the variations between individuals, which forms the basis for natural selection. Here, we used single-embryo RNA-seq on larval sea urchin Heliocidaris crassispina, which is a commercially and ecologically valuable species in East Asia, to document gene expression changes to OA at an individual and family level. Three paternal half-sibs groups were fertilized and exposed to 3 pH conditions (ambient pH 8.0, 7.7 and 7.4) for 12 h prior to sequencing and oxygen consumption assay. The resulting transcriptomic profile of all embryos can be distinguished into four clusters, with differences in gene expressions that govern biomineralization, cell differentiation and patterning, as well as metabolism. While these responses were influenced by pH conditions, the male identities also had an effect. Specifically, a regression model and goodness of fit tests indicated a significant interaction between sire and pH on the probability of embryo membership in different clusters of gene expression. The single-embryo RNA-seq approach is promising in climate stressor research because not only does it highlight potential impacts before phenotypic changes were observed, but it also highlights variations between individuals and lineages, thus enabling a better determination of evolutionary potential., (© 2023 The Authors. Molecular Ecology published by John Wiley & Sons Ltd.)

Multinucleated cells, or syncytia, are found in diverse taxa. Their biological function is often associated with the compartmentalization of biochemical or cellular activities within the syncytium. How such compartments are generated and maintained is poorly understood. The sea urchin embryonic skeleton is secreted by a syncytium, and local patterns of skeletal growth are associated with distinct sub-domains of gene expression within the syncytium. For such molecular compartments to be maintained and to control local patterns of skeletal growth: (1) the mobility of TFs must be restricted to produce stable differences in the transcriptional states of nuclei within the syncytium; and (2) the mobility of biomineralization proteins must also be restricted to produce regional differences in skeletal growth. To test these predictions, we expressed fluorescently tagged forms of transcription factors and biomineralization proteins in sub-domains of the skeletogenic syncytium. We found that both classes of proteins have restricted mobility within the syncytium and identified motifs that limit their mobility. Our findings have general implications for understanding the functional and molecular compartmentalization of syncytia., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

Nanostructured cellulose sponges (CNS) have been developed as eco-friendly and sustainable engineered materials for marine environmental remediation. Despite their functionality, sensitivity, efficiency and specificity have been proved, CNS application is still limited since their environmental safety (eco-safety) has not been completely assessed. In this study, CNS were allowed to leach in natural seawater simulating the remediation process condition and the eco-safety of CNS leachate on sea urchin reproduction has been assessed by carrying out a multi-response integrated approach, combining standardized ecotoxicity tests, innovative bioassays and gamete quality assessment. Overall, the ecotoxicity data indicate that CNS leachate affects gamete quality, gamete fertilisation competence, and embryo development probably associated with the release of chemical additives used during the synthesis process. However, in the framework of the eco-design approach, consecutive leaching treatments and conditioning of CNS in seawater open the route for a new safety protocol successfully solving the ecotoxicity while maintaining CNS sorbent properties. A safe environmental application of the resulting conditioned CNS for seawater pollution remediation is envisaged., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Alessandra Gallo reports a relationship with Zoological Station Anton Dohrn that includes: employment., (Copyright © 2023. Published by Elsevier Ltd.)

CCCTC-binding factor (CTCF), an insulator protein with 11 zinc fingers, is enriched at the boundaries of topologically associated domains (TADs) in eukaryotic genomes. In this study, we isolated and analyzed the cDNAs encoding HpCTCF, the CTCF homolog in the sea urchin Hemicentrotus pulcherrimus, to investigate its expression patterns and functions during the early development of sea urchin. HpCTCF contains nine zinc fingers corresponding to fingers 2-10 of the vertebrate CTCF. Expression pattern analysis revealed that HpCTCF mRNA was detected at all developmental stages and in the entire embryo. Upon expressing the HpCTCF-GFP fusion protein in early embryos, we observed its uniform distribution within interphase nuclei. However, during mitosis, it disappeared from the chromosomes and subsequently reassembled on the chromosome during telophase. Moreover, the morpholino-mediated knockdown of HpCTCF resulted in mitotic arrest during the morula to blastula stage. Most of the arrested chromosomes were not phospholylated at serine 10 of histone H3, indicating that mitosis was arrested at the telophase by HpCTCF depletion. Furthermore, impaired sister chromatid segregation was observed using time-lapse imaging of HpCTCF-knockdown embryos. Thus, HpCTCF is essential for mitotic progression during the early development of sea urchins, especially during the telophase-to-interphase transition. However, the normal development of pluteus larvae in CRISPR-mediated HpCTCF-knockout embryos suggests that disruption of zygotic HpCTCF expression has little effect on embryonic and larval development., (© 2023 Japanese Society of Developmental Biologists.)

One presenilin gene (PSEN) is expressed in the sea urchin embryo, in the vegetal pole of the gastrula and then mainly in cilia cells located around the digestive system of the pluteus, as we recently have reported. PSEN expression must be accurately regulated for correct execution of these two steps of development. While investigating PSEN expression changes in embryos after expansion of endoderm with LiCl or of ectoderm with Zn2+ by whole-mount in situ hybridization (WISH) and quantitative PCR (qPCR), we detected natural antisense transcription of PSEN. We then found that Endo16 and Wnt5, markers of endo-mesoderm, and of Hnf6 and Gsc, markers of ectoderm, are also sense and antisense transcribed. We discuss that general gene expression could depend on both sense and antisense transcription. This mechanism, together with the PSEN gene, should be included in gene regulatory networks (GRNs) that theorize diverse processes in this species. We suggest that it would also be relevant to investigate natural antisense transcription of PSEN in the field of Alzheimer's disease (AD) where the role of human PSEN1 and PSEN2 is well known., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

Marine larvae have factored heavily in pursuits to understand the origin and evolution of animal life cycles. Recent comparisons of gene expression and chromatin state in different species of sea urchin and annelid show how evolutionary changes in embryonic gene regulation can lead to markedly different larval forms., Competing Interests: Declaration of interests The authors declare no conflicts of interest., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Echinometra lucunter, the rock-boring sea urchin, is a widely distributed echinoid and a model for ecological studies of reproduction, responses to climate change, and speciation. We present a near chromosome-level genome assembly of E. lucunter, including 21 scaffolds larger than 10 Mb predicted to represent each of the chromosomes of the species. The 760.4 Mb assembly includes a scaffold N50 of 30.0 Mb and BUSCO (benchmarking universal single-copy orthologue) single copy and a duplicated score of 95.8% and 1.4%, respectively. Ab-initio gene model prediction and annotation with transcriptomic data constructed 33,989 gene models composing 50.4% of the assembly, including 37,036 transcripts. Repetitive elements make up approximately 39.6% of the assembly, and unresolved gap sequences are estimated to be 0.65%. Whole genome alignment with Echinometra sp. EZ revealed high synteny and conservation between the two species, further bolstering Echinometra as an emerging genus for comparative genomics studies. This genome assembly represents a high-quality genomic resource for future evolutionary and developmental studies of this species and more broadly of echinoderms., (Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution 2023.)

Defining pattern formation mechanisms during embryonic development is important for understanding the etiology of birth defects and to inform tissue engineering approaches. In this study, we used tricaine, a voltage-gated sodium channel (VGSC) inhibitor, to show that VGSC activity is required for normal skeletal patterning in Lytechinus variegatus sea urchin larvae. We demonstrate that tricaine-mediated patterning defects are rescued by an anesthetic-insensitive version of the VGSC LvScn5a. Expression of this channel is enriched in the ventrolateral ectoderm, where it spatially overlaps with posterolaterally expressed Wnt5. We show that VGSC activity is required to spatially restrict Wnt5 expression to this ectodermal region that is adjacent and instructive to clusters of primary mesenchymal cells that initiate secretion of the larval skeleton as triradiates. Tricaine-mediated Wnt5 spatial expansion correlates with the formation of ectopic PMC clusters and triradiates. These defects are rescued by Wnt5 knockdown, indicating that the spatial expansion of Wnt5 is responsible for the patterning defects induced by VGSC inhibition. These results demonstrate a previously unreported connection between bioelectrical status and the spatial control of patterning cue expression during embryonic pattern formation., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

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., (© 2023 Wiley Periodicals LLC.)

Echinometra is the most widespread genus of sea urchin and has been the focus of a wide range of studies in ecology, speciation, and reproduction. However, available genetic data for this genus are generally limited to a few select loci. Here, we present a chromosome-level genome assembly based on 10x Genomics, PacBio, and Hi-C sequencing for Echinometra sp. EZ from the Persian/Arabian Gulf. The genome is assembled into 210 scaffolds totaling 817.8 Mb with an N50 of 39.5 Mb. From this assembly, we determined that the E. sp. EZ genome consists of 2n = 42 chromosomes. BUSCO analysis showed that 95.3% of BUSCO genes were complete. Ab initio and transcript-informed gene modeling and annotation identified 29,405 genes, including a conserved Hox cluster. E. sp. EZ can be found in high-temperature and high-salinity environments, and we therefore compared E. sp. EZ gene families and transcription factors associated with environmental stress response ("defensome") with other echinoid species with similar high-quality genomic resources. While the number of defensome genes was broadly similar for all species, we identified strong signatures of positive selection in E. sp. EZ noncoding elements near genes involved in environmental response pathways as well as losses of transcription factors important for environmental response. These data provide key insights into the biology of E. sp. EZ as well as the diversification of Echinometra more widely and will serve as a useful tool for the community to explore questions in this taxonomic group and beyond., (© The Author(s) 2022. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

With rising ocean temperatures, extreme weather events such as marine heatwaves (MHWs) are increasing in frequency and duration, pushing marine life beyond their physiological limits. The potential to respond to extreme conditions through physiological acclimatization, and pass on resistance to the next generation, fundamentally depends on the capacity of an organism to cope within their thermal tolerance limits. To elucidate whether heat conditioning of parents could benefit offspring development, we exposed adult sea urchins (Heliocidaris erythrogramma) to ambient summer (23°C), moderate (25°C) or strong (26°C) MHW conditions for 10 days. Offspring were then reared at constant temperature along a thermal gradient (22-28°C) and development was tracked to the 14-day juvenile stage. Progeny from the MHW-conditioned adults developed through to metamorphosis faster than those of ambient conditioned parents, with most individuals from the moderate and strong heatwaves developing to the larval stage across all temperatures. In contrast, the majority of offspring from the control summer temperature died before metamorphosis at temperatures above 25°C (moderate MHW). Juveniles produced from the strong MHW-conditioned adults were also larger across all temperatures, with the largest juveniles in the 26°C treatment. In contrast, the smallest juveniles were from control (current-day summer) parents (and reared at 22 and 25°C). Surprisingly, initial survival was higher in the progeny of MHW exposed parents, even at temperatures hotter than predicted MHWs (28°C). Importantly, however, there was substantial mortality of juveniles from the strong MHW parents by day 14. Therefore, while carryover effects of parental conditioning to MHWs resulted in faster growing, larger progeny, this benefit will only persist beyond the more sensitive juvenile stage and enhance survival if conditions return promptly to normal seasonal temperatures within current thermal tolerance limits., (© 2022 The Authors. Global Change Biology published by John Wiley & Sons Ltd.)

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., Competing Interests: Declaration of competing interest Synthego, IDT, and Aldevron each provided free samples of Cas9 and sgRNAs to initiate this project. All products thereafter were purchased at market value., (Copyright © 2022 Elsevier Inc. All rights reserved.)

In this work, we used Nanostring N-counter technology, to evaluate the mRNA expression level of more than 330 regulatory genes over 34 time points covering the first three days of development of the sea urchin larvae. The hierarchical clustering of the mRNAs expression levels has identified groups corresponding to the major developmental landmarks (e.g. maternal to zygotic transition and gastrulation). Furthermore, comparison with previous experiments indicates high reproducibility of mRNA level temporal dynamics across batches. Finally, we generated an online tool to visualise gene expression during sea urchin larval development. The site can be accessed at and https://www621.lamp.le.ac.uk/nanostring_app/nanostring/., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

Background: Understanding how gene regulatory networks (GRNs) control developmental progression is a key to the mechanistic understanding of morphogenesis. The sea urchin larval skeletogenesis provides an excellent platform to tackle this question. In the early stages of sea urchin skeletogenesis, skeletogenic genes are uniformly expressed in the skeletogenic lineage. Yet, during skeletal elongation, skeletogenic genes are expressed in distinct spatial sub-domains. The regulation of differential gene expression during late skeletogenesis is not well understood., Results: Here we reveal the dynamic expression of the skeletogenic regulatory genes that define a specific regulatory state for each pair of skeletal rods, in the sea urchin Paracentrotus lividus. The vascular endothelial growth factor (VEGF) signaling, essential for skeleton formation, specifically controls the migration of cells that form the postoral and distal anterolateral skeletogenic rods. VEGF signaling also controls the expression of regulatory genes in cells at the tips of the postoral rods, including the transcription factors Pitx1 and MyoD1. Pitx1 activity is required for normal skeletal elongation and for the expression of some of VEGF target genes., Conclusions: Our study illuminates the fine-tuning of the regulatory system during the transition from early to late skeletogenesis that gives rise to rod-specific regulatory states., (© 2022 The Authors. Developmental Dynamics published by Wiley Periodicals LLC on behalf of American Association for Anatomy.)

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., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Sea urchin (Mseocentrotus nudus) is an economically important mariculture species in several Asian countries. The growth rate and immunocompetence differ by sex in this species. However, the mechanisms of sex determination in M. nudus have remain unclear. In the present study, we focus on the dmrt1 gene of M. nudus (Mndmrt1) to investigate its dynamic expression pattern during different developmental stages. Real-time quantitative PCR (RT-qPCR) revealed that Mndmrt1 exhibits testis-specific expression and undetectable during the whole embryogenesis. With the development of ontogenetic, Mndmrt1 transcripts are first detected at 9 months post-fertilization (mpf). In addition, both the transcripts and protein of Mndmrt1 gene were specifically expressed in spermatogonia and spermatocytes, indicating that it might be a male germ cells marker in sea urchin. Significantly, the 1441 bp promoter sequence of Mndmrt1 gene was obtained by DNA walking, and one positive regulatory region at -1197/ -968 in the promoter, as well as one negative regulatory region at -1441/ -1198 have been identified by promoter activity analysis. Moreover, two regulatory regions contain multiple putative binding sites for transcription factors, including Sp1, Egr1, Sox5, CEBP, GATA and SRY. These findings suggest that Mndmrt1 may be related to testis differentiation and spermatogenesis in sea urchin and will provide an insight into understanding the regulatory mechanism of the dmrt1 gene., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The micromeres of the sea urchin embryo are distinct from other blastomeres. After they arise through an asymmetric cell division at the 8- to 16-cell stage, micromeres immediately function as organizers. They also commit themselves to specific cell fates such as larval skeletogenic cells and primordial germ cells, while other blastomeres remain plastic and uncommitted at the 16-cell stage. In the phylum Echinodermata, only the sea urchin (class Echinoidea) embryo forms micromeres that serve as apparent organizers during early embryogenesis. Therefore, it is considered that micromeres are the derived features and that modification(s) of the developmental system allowed evolutionary introduction of this unique cell lineage. In this chapter, we summarize the both historic and recent observations that demonstrate unique properties of micromeres and discuss how this lineage of micromeres may have arisen during echinoderm evolution., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Echinoderms, including sea urchins and starfish, have played important roles in cell, developmental and evolutionary biology research for more than a century. However, since most of them take a long time to mature sexually and their breeding seasons are limited, it has been difficult to obtain subsequent generations in the laboratory, resulting in them not being recognized as model organisms in recent genetics research. To overcome this issue, we maintained and obtained gametes from several nonmodel sea urchins in Japan and finally identified Temnopleurus reevesii as a suitable model for sea urchin genetics. Genomic and transcriptomic information was obtained for this model, and the DNA database TrBase was made publicly available. In this review, we describe how we found this species useful for biological research and show an example of CRISPR/Cas9 based knockout sea urchin., (© 2021 Japanese Society of Developmental Biologists.)

This review reports recent findings on the specification and patterning of neurons that establish the larval nervous system of the sea urchin embryo. Neurons originate in three regions of the embryo. Perturbation analyses enabled construction of gene regulatory networks controlling the several neural cell types. Many of the mechanisms described reflect shared features of all metazoans and others are conserved among deuterostomes. This nervous system with a very small number of neurons supports the feeding and swimming behaviors of the larva until metamorphosis when an adult nervous system replaces that system., (Copyright © 2022 Elsevier Inc. All rights reserved.)

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., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Early in animal development many cells are conditionally specified based on observations that those cells can be directed toward alternate fates. The endomesoderm is so named because early specification produces cells that often have been observed to simultaneously express both early endoderm and mesoderm transcription factors. Experiments with these cells demonstrate that their progeny can directed entirely toward endoderm or mesoderm, whereas normally they establish both germ layers. This review examines the mechanisms that initiate the conditional endomesoderm state, its metastability, and the mechanisms that resolve that state into definitive endoderm and mesoderm., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs) represent the most common pollutants in the marine sediments. Previous investigations demonstrated short-term sublethal effects of sediments polluted with both contaminants on the sea urchin Paracentrotus lividus after 2 months of exposure in mesocosms. In particular, morphological malformations observed in P. lividus embryos deriving from adults exposed to PAHs and PCBs were explained at molecular levels by de novo transcriptome assembly and real-time qPCR, leading to the identification of several differentially expressed genes involved in key physiological processes. Here, we extensively explored the genes involved in the response of the sea urchin P. lividus to PAHs and PCBs. Firstly, 25 new genes were identified and interactomic analysis revealed that they were functionally connected among them and to several genes previously defined as molecular targets of response to the two pollutants under analysis. The expression levels of these 25 genes were followed by Real Time qPCR, showing that almost all genes analyzed were affected by PAHs and PCBs. These findings represent an important further step in defining the impacts of slight concentrations of such contaminants on sea urchins and, more in general, on marine biota, increasing our knowledge of molecular targets involved in responses to environmental stressors.

An ultimate benefit of marine protected areas (MPAs) is to reverse trophic cascades caused by human-driven collapse of critical ecological interactions. Here we demonstrate that, despite a small scale (0.28 km 2 ) and not being fully protected, an MPA with strict fishing management and habitat enhancement by artificial reefs (ARs) in southwest Japan can lead to well-established macroalgal communities on widespread sea urchin barrens through cascading effects of predator recovery. Areas with low urchin densities occurred in and around daytime lobster (Panulirus japonicus) shelters primarily formed by quarry-rock ARs inside the MPA. We confirmed in the laboratory that lobsters preyed on two dominant sea urchins (Echinometra sp. A and Heliocidaris crassispina), with size- and species-dependent predation. The area with few urchins extended farther (˜65 m) from an AR with numerous lobsters than from a natural shelter (patch reef) with far fewer lobsters. Causation of this pattern was confirmed by a tethering experiment showing that predation on urchins was similarly high at and near lobster shelters but decreased at ˜100 m from the AR to a similar level as at an unprotected site. Time-lapse photography revealed that predation on tethered urchins was due mostly to the largest size class of lobsters (>100 mm carapace length), which comprised only 7% of the population, highlighting the importance of large-sized lobsters in controlling urchin abundance in localized areas adjacent to urchin-dominated barrens. Despite an ongoing once-a-year fishing event permitted within the MPA, lobster populations were persistent, demonstrating that the cascading effect of the lobsters on urchins and ultimately macroalgae was robust to temporary reductions in predator population size. Erect macroalgal cover was not simply accounted for by snapshot urchin density or biomass, suggesting a hysteresis effect of the phase shifts between macroalgal dominance and urchin barren states., (© 2021 by the Ecological Society of America.)

Species-specific sperm-egg interactions are essential for sexual reproduction. Broadcast spawning of marine organisms is under particularly stringent conditions, since eggs released into the water column can be exposed to multiple different sperm. Bindin isolated from the sperm acrosome results in insoluble particles that cause homospecific eggs to aggregate, whereas no aggregation occurs with heterospecific eggs. Therefore, Bindin is concluded to play a critical role in fertilization, yet its function has never been tested. Here we report that Cas9-mediated inactivation of the bindin gene in a sea urchin results in perfectly normal-looking embryos, larvae, adults, and gametes in both males and females. What differed between the genotypes was that the bindin -/- sperm never fertilized an egg, functionally validating Bindin as an essential gamete interaction protein at the level of sperm-egg cell surface binding., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Background: Doublecortin-like kinase1 and 2 (DCLKs) are protein Ser/Thr kinases important for neuronal development. More recently, they are also reported to regulate plasticity such as cell proliferation and differentiation of stem cells and cancer cells, but the details of their functions in this biological context are still unclear. With an attempt to reveal the functions of DCLKs in plasticity regulation, we here used the sea urchin embryo that undergoes highly regulative development as an experimental model., Results: We found that both the transcripts and the proteins of DCLKs are uniformly present during early embryogenesis and with some enrichment in mesenchymal cells after gastrula stage. Knockdown of DCLKs induced general developmental delay and defects at day 2. Further, the damage on the embryo/larva induced ectopic expression of DCLKs in the ectoderm where the damage was most severe. Under a tumor-prone or -suppressive condition, DCLKs expression was upregulated or downregulated, respectively, after damage. In both cases, the embryos showed severe developmental defects., Conclusions: Taken together, a transient upregulation of DCLKs appears to be involved in a damage response both during normal and abnormal development, and which could result in different phenotypes in a context dependent manner., (© 2021 American Association of Anatomists.)

Otopetrins comprise a family of proton-selective channels that are critically important for the mineralization of otoliths and statoconia in vertebrates but whose underlying cellular mechanisms remain largely unknown. Here, we demonstrate that otopetrins are critically involved in the calcification process by providing an exit route for protons liberated by the formation of CaCO 3 Using the sea urchin larva, we examined the otopetrin ortholog otop2l , which is exclusively expressed in the calcifying primary mesenchymal cells (PMCs) that generate the calcitic larval skeleton. otop2l expression is stimulated during skeletogenesis, and knockdown of otop2l impairs spicule formation. Intracellular pH measurements demonstrated Zn 2+ -sensitive H + fluxes in PMCs that regulate intracellular pH in a Na + /HCO 3 - -independent manner, while Otop2l knockdown reduced membrane proton permeability. Furthermore, Otop2l displays unique features, including strong activation by high extracellular pH (>8.0) and check-valve-like outwardly rectifying H + flux properties, making it into a cellular proton extrusion machine adapted to oceanic living conditions. Our results provide evidence that otopetrin family proton channels are a central component of the cellular pH regulatory machinery in biomineralizing cells. Their ubiquitous occurrence in calcifying systems across the animal kingdom suggest a conserved physiological function by mediating pH at the site of mineralization. This important role of otopetrin family proton channels has strong implications for our view on the cellular mechanisms of biomineralization and their response to changes in oceanic pH., Competing Interests: The authors declare no competing interest.

Presenilins (PSENs) are widely expressed across eukaryotes. Two PSENs are expressed in humans, where they play a crucial role in Alzheimer's disease (AD). Each PSEN can be part of the γ-secretase complex, which has multiple substrates, including Notch and amyloid-β precursor protein (AβPP) - the source of amyloid-β (Aβ) peptides that compose the senile plaques during AD. PSENs also interact with various proteins independently of their γ-secretase activity. They can then be involved in numerous cellular functions, which makes their role in a given cell and/or organism complex to decipher. We have established the Paracentrotus lividus sea urchin embryo as a new model to study the role of PSEN. In the sea urchin embryo, the PSEN gene is present in unduplicated form and encodes a protein highly similar to human PSENs. Our results suggest that PSEN expression must be precisely tuned to control the course of the first mitotic cycles and the associated intracellular Ca2+ transients, the execution of gastrulation and, probably in association with ciliated cells, the establishment of the pluteus. We suggest that it would be relevant to study the role of PSEN within the gene regulatory network deciphered in the sea urchin., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

Echinoderms are important experimental models for analyzing embryonic development, but a lack of spatial and temporal control over gene perturbations has hindered developmental studies using these animals. Morpholino antisense oligonucleotides (MOs) have been used successfully by the echinoderm research community for almost two decades, and MOs remain the most widely used tool for acute gene knockdowns in these organisms. Echinoderm embryos develop externally and are optically transparent, making them ideally-suited to many light-based approaches for analyzing and manipulating development. Studies using zebrafish embryos have demonstrated the effectiveness of photoactivatable (caged) MOs for conditional gene knockdowns. Here we show that caged MOs, synthesized using nucleobase-caged monomers, provide light-regulated control over gene expression in sea urchin embryos. Our work provides the first robust approach for conditional gene silencing in this prominent model system., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Objectives: Echinometra mathaei was known to have potential antioxidant activities because it contains of  polyhydroxy-naphthoquinone ( echinochrome and spinochromes ). The antioxidant properties contributed to the hepatoprotective effect by binding to free radicals compound that causes oxidative stress and necrosis in the hepatocytes. The research aimed to determine the hepatorepair effects of the E. mathaei ethanol extract on high-dose paracetamol-induced hepatic damage in Wistar rats., Methods: This research used a true experimental method. Thirty white male rats were divided into sixth groups, i.e., normal control group, group II-VI was induced paracetamol 2,000 mg/kg BW for three days. After paracetamol-induced, group III-VI was treated with curcumin 800 mg/kg BW, E. mathaei extract 400, 800, and 1,200 mg/kg BW for seven days. The hepatorepair parameter was obtained from AST/ALT, MDA tissue levels, and the number of hepatocyte necrosis cells. The data results were analyzed using the ANOVA test, followed by the LSD test to determine the difference between each treatment., Results: The results showed that E. mathaei significantly (p<0.05) decreased the AST levels, MDA levels and the number of hepatocyte necrosis cells at a dose of 800 mg/kg BW per orally treatment., Conclusions: The E. mathaei ethanol extract repaired the hepatic damage induced by paracetamol., (© 2021 Walter de Gruyter GmbH, Berlin/Boston.)

Adaptive landscapes are a common way of conceptualizing the phenotypic evolution of lineages across deep time. Although multiple approaches exist to implement this concept into operational models of trait evolution, inferring adaptive landscapes from comparative datasets remains challenging. Here, I explore the macroevolutionary dynamics of echinoid body size using data from over 5000 specimens and a phylogenetic framework incorporating a dense fossil sampling and spanning approximately 270 million years. Furthermore, I implement a novel approach of exploring alternative parameterizations of adaptive landscapes that succeeds in finding simpler, yet better-fitting models. Echinoid body size has been constrained to evolve within a single adaptive optimum for much of the clade's history. However, most of the morphological disparity of echinoids was generated by multiple regime shifts that drove the repeated evolution of miniaturized and gigantic forms. Events of body size innovation occurred predominantly in the Late Cretaceous and were followed by a drastic slowdown following the Cretaceous-Paleogene mass extinction. The discovery of these patterns is contingent upon directly sampling fossil taxa. The macroevolution of echinoid body size is therefore characterized by a late increase in disparity (likely linked to an expansion of ecospace), generated by active processes driving lineages toward extreme morphologies., (© 2021 The Authors. Evolution © 2021 The Society for the Study of Evolution.)

Background : Echinochrome A (EchA) is a pigment from sea urchins. EchA is a polyhydroxylated 1,4-naphthoquinone that contains several hydroxyl groups appropriate for free-radical scavenging and preventing redox imbalance. EchA is the most studied molecule of this family and is an active principle approved to be used in humans, usually for cardiopathies and glaucoma. EchA is used as a pharmaceutical drug. Methods : A comprehensive literature and patent search review was undertaken using PubMed, as well as Google Scholar and Espacenet search engines to review these areas. Conclusions : In the bloodstream, EchA can mediate cellular responses, act as a radical scavenger, and activate the glutathione pathway. It decreases ROS imbalance, prevents and limits lipid peroxidation, and enhances mitochondrial functions. Most importantly, EchA contributes to the modulation of the immune system. EchA can regulate the generation of regulatory T cells, inhibit pro-inflammatory IL-1β and IL-6 cytokine production, while slightly reducing IL-8, TNF-α, INF-α, and NKT, thus correcting immune imbalance. These characteristics suggest that EchA is a candidate drug to alleviate the cytokine storm syndrome (CSS).

Organisms can uptake minerals, shape them in different forms and generate teeth, skeletons or shells that support and protect them. Mineral uptake, trafficking and nucleation are tightly regulated by the biomineralizing cells through networks of specialized proteins. Specifically, matrix metalloproteases (MMPs) digest various extracellular substrates and allow for mineralization in the vertebrates' teeth and bones, but little is known about their role in invertebrates' systems. The sea urchin embryo provides an excellent invertebrate model for genetic and molecular studies of biomineralization. MMP inhibition prevents the growth of the calcite spicules of the sea urchin larval skeleton, however, the molecular mechanisms and genes that underlie this response are not well understood. Here we study the spatial expression and regulation of two membrane type MMPs that were found to be occluded in the sea urchin spicules, Pl-MmpL7 and Pl-MmpL5, and investigate the function of Pl-MmpL7 in skeletogenesis. The inhibition of MMPs does not change the volume of the calcium vesicles in the skeletogenic cells. The expression of Pl-MmpL7 and Pl-MmpL5 is regulated by the Vascular Endothelial Growth Factor (VEGF) signaling, from the time of skeleton initiation and on. The expression of these genes is localized to the subsets of skeletogenic cells where active spicule growth occurs throughout skeletogenesis. Downregulation of Pl-MmpL7 expression delays the growth of the skeletal rods and in some cases, strongly perturbs skeletal shape. The localized expression of Pl-MmpL7 and Pl-MmpL5 to the active growth zone and the effect of Pl-MmpL7 perturbations on skeletal growth, suggest that these genes are essential for normal spicule elongation in the sea urchin embryo., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: Light is essential for various biological activities. In particular, visual information through eyes or eyespots is very important for most of animals, and thus, the functions and developmental mechanisms of visual systems have been well studied to date. In addition, light-dependent non-visual systems expressing photoreceptor Opsins have been used to study the effects of light on diverse animal behaviors. However, it remains unclear how light-dependent systems were acquired and diversified during deuterostome evolution due to an almost complete lack of knowledge on the light-response signaling pathway in Ambulacraria, one of the major groups of deuterostomes and a sister group of chordates., Results: Here, we show that sea urchin larvae utilize light for digestive tract activity. We found that photoirradiation of larvae induces pyloric opening even without addition of food stimuli. Micro-surgical and knockdown experiments revealed that this stimulating light is received and mediated by Go(/RGR)-Opsin (Opsin3.2 in sea urchin genomes) cells around the anterior neuroectoderm. Furthermore, we found that the anterior neuroectodermal serotoninergic neurons near Go-Opsin-expressing cells are essential for mediating light stimuli-induced nitric oxide (NO) release at the pylorus. Our results demonstrate that the light>Go-Opsin>serotonin>NO pathway functions in pyloric opening during larval stages., Conclusions: The results shown here will lead us to understand how light-dependent systems of pyloric opening functioning via neurotransmitters were acquired and established during animal evolution. Based on the similarity of nervous system patterns and the gut proportions among Ambulacraria, we suggest the light>pyloric opening pathway may be conserved in the clade, although the light signaling pathway has so far not been reported in other members of the group. In light of brain-gut interactions previously found in vertebrates, we speculate that one primitive function of anterior neuroectodermal neurons (brain neurons) may have been to regulate the function of the digestive tract in the common ancestor of deuterostomes. Given that food consumption and nutrient absorption are essential for animals, the acquirement and development of brain-based sophisticated gut regulatory system might have been important for deuterostome evolution.

The ABC transporter ABCB1 plays an important role in the disposition of xenobiotics. Embryos of most species express high levels of this transporter in early development as a protective mechanism, but its native substrates are not known. Here, we used larvae of the sea urchin Strongylocentrotus purpuratus to characterize the early life expression and role of Sp-ABCB1a, a homolog of ABCB1. The results indicate that while Sp-ABCB1a is initially expressed ubiquitously, it becomes enriched in the developing gut. Using optimized CRISPR/Cas9 gene editing methods to achieve high editing efficiency in the F0 generation, we generated ABCB1a crispant embryos with significantly reduced transporter efflux activity. When infected with the opportunistic pathogen Vibrio diazotrophicus, Sp-ABCB1a crispant larvae demonstrated significantly stronger gut inflammation, immunocyte migration and cytokine Sp-IL-17 induction, as compared with infected control larvae. The results suggest an ancestral function of ABCB1 in host-microbial interactions, with implications for the survival of invertebrate larvae in the marine microbial environment., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

Directed intercellular movement of diverse small molecules, including metabolites, signal molecules and xenobiotics, is a key feature of multicellularity. Networks of small molecule transporters (SMTs), including several ATP Binding Cassette (ABC) transporters, are central to this process. While small molecule transporters are well described in differentiated organs, little is known about their patterns of expression in early embryogenesis. Here we report the pattern of ABC-type SMT expression and activity during the early development of sea urchins. Of the six major ABCs in this embryo (ABCB1, -B4, -C1, -C4, -C5 and -G2), three expression patterns were observed: 1) ABCB1 and ABCC1 are first expressed ubiquitously, and then become enriched in endoderm and ectoderm-derived structures. 2) ABCC4 and ABCC5 are restricted to a ring of mesoderm in the blastula and ABCC4 is later expressed in the coelomic pouches, the embryonic niche of the primordial germ cells. 3) ABCB4 and ABCG2 are expressed exclusively in endoderm-fated cells. Assays with fluorescent substrates and inhibitors of transporters revealed a ring of ABCC4 efflux activity emanating from ABCC4 + mesodermal cells. Similarly, ABCB1 and ABCB4 efflux activity was observed in the developing gut, prior to the onset of feeding. This study reveals the early establishment of unique territories of small molecule transport during embryogenesis. A pattern of ABCC4/C5 expression is consistent with signaling functions during gut invagination and germ line development, while a later pattern of ABCB1/B4 and ABCG2 is consistent with roles in the embryonic gut. This work provides a conceptual framework with which to examine the function and evolution of SMT networks and to define the specific developmental pathways that drive the expression of these genes., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2021. Published by Elsevier Inc.)

Activation of Wnt/β-catenin (cWnt) signaling at the future posterior end of early bilaterian embryos is a highly conserved mechanism for establishing the anterior-posterior (AP) axis. Moreover, inhibition of cWnt at the anterior end is required for development of anterior structures in many deuterostome taxa. This phenomenon, which occurs around the time of gastrulation, has been fairly well characterized, but the significance of intracellular inhibition of cWnt signaling in cleavage-stage deuterostome embryos for normal AP patterning is less well understood. To investigate this process in an invertebrate deuterostome, we defined Axin function in early sea urchin embryos. Axin is ubiquitously expressed at relatively high levels in early embryos and functional analysis revealed that Axin suppresses posterior cell fates in anterior blastomeres by blocking ectopic cWnt activation in these cells. Structure-function analysis of sea urchin Axin demonstrated that only its GSK-3β-binding domain is required for cWnt inhibition. These observations and results in other deuterostomes suggest that Axin plays a crucial conserved role in embryonic AP patterning by preventing cWnt activation in multipotent early blastomeres, thus protecting them from assuming ectopic cell fates., Competing Interests: Competing interestsThe authors declare no competing or financial interests. Author contributionsConceptualization: H.S., C.J.P., A.H.W.; Methodology: H.S., C.J.P., H.F., A.H.W.; Validation: H.S., C.J.P., L.W., A.H.W.; Formal analysis: H.S., C.J.P., L.W., H.F., A.H.W.; Investigation: H.S., C.J.P., L.W., A.H.W.; Resources: A.H.W.; Data curation: H.S., C.J.P., L.W., H.F., A.H.W.; Writing - original draft: H.S., C.J.P., A.H.W.; Writing - review & editing: H.S., A.H.W.; Visualization: H.S.; Supervision: A.H.W.; Project administration: A.H.W.; Funding acquisition: A.H.W., (© 2021. Published by The Company of Biologists Ltd.)

We found that the extract of the body wall of the sea urchin, Pseudocentrotus depressus, agglutinate Escherichia coli and is inhibited by mannose. A mannose-binding protein of 22 kDa was purified via affinity chromatography using mannose-agarose. Amino acid sequences obtained by Edman degradation and liquid chromatography quadrupole time-of-flight mass spectrometry followed by de novo sequencing suggested that the protein is a C-type lectin. Products of PCR with a degenerate primer pair and of RACE PCR for the cDNA of the 22 kDa protein were sequenced and produced two full-length cDNA sequences encoding C-type lectins. These two lectins, named P. depressus mannose-binding C-type lectin (PdMBCL) 1 and 2 are composed of 187 and 189 amino acid residues, including signal peptides, respectively, and share 86% identity in their mature form. PdMBCLs agglutinated Lactococcus garvieae, a Gram-positive fish pathogen. Reverse transcription PCR showed that both the genes for the PdMBCLs were expressed in the body wall and in other tissues. Furthermore, the lectins were detected from a rinse of the body surface. Taken together, the present study showed that PdMBCLs function as anti-microbial agents on the body surface of P. depressus., (Copyright © 2020 Elsevier Ltd. All rights reserved.)