The Effect of Synthetic Polyamine BPA-C8 on the Fertilization Process of Intact and Denuded Sea Urchin Eggs.

Sea urchin eggs are covered with layers of extracellular matrix, namely, the vitelline layer (VL) and jelly coat (JC). It has been shown that sea urchin eggs' JC components serve as chemoattractants or ligands for the receptor on the fertilizing sperm to promote the acrosome reaction. Moreover, the egg's VL provides receptors for conspecific sperm to bind, and, to date, at least two sperm receptors have been identified on the surface of sea urchin eggs. Interestingly, however, according to our previous work, denuded sea urchin eggs devoid of the JC and VL do not fail to become fertilized by sperm. Instead, they are bound and penetratedby multiple sperm, raising the possibility that an alternative pathway independent of the VL-residing sperm receptor may be at work. In this research, we studied the roles of the JC and VL using intact and denuded eggs and the synthetic polyamine BPA-C8. BPA-C8 is known to bind to the negatively charged macromolecular complexes in the cells, such as the JC, VL, and the plasma membrane of echinoderm eggs, as well as to the actin filaments in fibroblasts. Our results showed that, when added to seawater, BPA-C8 significantly repressed the Ca2+ wave in the intact P. lividus eggs at fertilization. In eggs deprived of the VL and JC, BPA-C8 binds to the plasma membrane and increases fibrous structures connecting microvilli, thereby allowing the denuded eggs to revert towards monospermy at fertilization. However, the reduced Ca2+ signal in denuded eggs was nullified compared to the intact eggs because removing the JC and VL already decreased the Ca2+ wave. BPA-C8 does not cross the VL and the cell membrane of unfertilized sea urchin eggs to diffuse into the cytoplasm at variance with the fibroblasts. Indeed, the jasplakinolide-induced polymerization of subplasmalemmal actin filaments was inhibited in the eggs microinjected with BPA-C8, but not in the ones bath-incubated with the same dose of BPA-C8.

miR-31-mediated local translation at the mitotic spindle is important for early development.

miR-31 is a highly conserved microRNA that plays crucial roles in cell proliferation, migration and differentiation. We discovered that miR-31 and some of its validated targets are enriched on the mitotic spindle of the dividing sea urchin embryo and mammalian cells. Using the sea urchin embryo, we found that miR-31 inhibition led to developmental delay correlated with increased cytoskeletal and chromosomal defects. We identified miR-31 to directly suppress several actin remodeling transcripts, including ß-actin, Gelsolin, Rab35 and Fascin. De novo translation of Fascin occurs at the mitotic spindle of sea urchin embryos and mammalian cells. Importantly, miR-31 inhibition leads to a significant a increase of newly translated Fascin at the spindle of dividing sea urchin embryos. Forced ectopic localization of Fascin transcripts to the cell membrane and translation led to significant developmental and chromosomal segregation defects, highlighting the importance of the regulation of local translation by miR-31 at the mitotic spindle to ensure proper cell division. Furthermore, miR-31-mediated post-transcriptional regulation at the mitotic spindle may be an evolutionarily conserved regulatory paradigm of mitosis.

Visualizing metabolic regulation using metabolic biosensors during sea urchin embryogenesis.

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 H2O2, 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.

Combinatorial regulatory states define cell fate diversity during embryogenesis.

Cell fate specification occurs along invariant species-specific trajectories that define the animal body plan. This process is controlled by gene regulatory networks that regulate the expression of the limited set of transcription factors encoded in animal genomes. Here we globally assess the spatial expression of ~90% of expressed transcription factors during sea urchin development from embryo to larva to determine the activity of gene regulatory networks and their regulatory states during cell fate specification. We show that >200 embryonically expressed transcription factors together define >70 cell fates that recapitulate the morphological and functional organization of this organism. Most cell fate-specific regulatory states consist of ~15-40 transcription factors with similarity particularly among functionally related cell types regardless of developmental origin. Temporally, regulatory states change continuously during development, indicating that progressive changes in regulatory circuit activity determine cell fate specification. We conclude that the combinatorial expression of transcription factors provides molecular definitions that suffice for the unique specification of cell states in time and space during embryogenesis.

Diazirine-AIOC-NAADP, a Clickable-Photoactive NAADP Analog for Sea Urchin NAADP Binding Proteins.

Calcium ions (Ca2+) play a vital role as intracellular messengers, regulating essential cellular processes. Nicotinic acid adenine dinucleotide phosphate (NAADP) serves as a potent second messenger, responsible for releasing Ca2+ in both mammals and echinoderms. Despite identification of two human NAADP receptor proteins, their counterparts in sea urchins remain elusive. Sea urchin NAADP binding proteins are important due to their unique identities and NAADP binding properties which may illuminate new signaling modalities in other species. Consequently, the development of new photoactive and clickable NAADP analogs with specificity for binding targets in sea urchin egg homogenates is a priority. We designed and synthesized diazirine-AIOC-NAADP, a photoactive and "clickable" NAADP analog, to specifically label and identify sea urchin NAADP receptors. This analog, synthesized using a chemo-enzymatic approach, induced Ca2+ release from sea urchin egg homogenates at low-micromolar concentrations. The ability of diazirine-AIOC-NAADP to mobilize Ca2+ in cultured human cells was investigated by microinjection of the probe into U2OS cells. Microinjected NAADP elicited a robust Ca2+ release, but even 6000-fold higher concentrations of diazirine-AIOC-NAADP were unable to release Ca2+. Our results indicate that our new probe is specifically recognized at low concentration by sea urchin egg NAADP receptors but not by the NAADP receptors in a human cultured cell line.

Tropomyosin induces the synthesis of magnesian calcite in sea urchin spines.

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.

Edible Sea urchins Echinus esculentus from Norwegian waters- Effect of season on nutritional quality and chemical contaminants.

This study aimed to characterize Echinus esculentus gonads in terms of biometric parameters and nutritional quality at two sites in Mid-Norway at four different seasons. The chemical contamination of the gonads was also investigated for the first time through the evaluation of 28 macro- and trace elements and 32 components from the emerging and persistent group per- and polyfluoroalkyl substances (PFAS). The spawning period was determined in summer, given that the gonad index was the lowest in this season for both sites. Protein concentrations were constant (8%-10%). However, lipid contents (1%-3%) were noticed to be higher in gonads during autumn and winter. The gonads had high contents of PUFA mainly EPA and DHA, followed by SFA, and MUFA year around for both locations. E. esculentus gonads constitute a good source of fatty acids, macro, and trace elements. This species could also be a bioindicator for the monitoring of marine environments.

microRNA-1 regulates sea urchin skeletogenesis by directly targeting skeletogenic genes and modulating components of signaling pathways.

microRNAs are evolutionarily conserved non-coding RNAs that direct post-transcriptional regulation of target transcripts. In vertebrates, microRNA-1 (miR-1) is expressed in muscle and has been found to play critical regulatory roles in vertebrate angiogenesis, a process that has been proposed to be analogous to sea urchin skeletogenesis. Results indicate that both miR-1 inhibitor and miR-1 mimic-injected larvae have significantly less F-actin enriched circumpharyngeal muscle fibers and fewer gut contractions. In addition, miR-1 regulates the positioning of skeletogenic primary mesenchyme cells (PMCs) and skeletogenesis of the sea urchin embryo. Interestingly, the gain-of-function of miR-1 leads to more severe PMC patterning and skeletal branching defects than its loss-of-function. The results suggest that miR-1 directly suppresses Ets1/2, Tbr, and VegfR7 of the skeletogenic gene regulatory network, and Nodal, and Wnt1 signaling components. This study identifies potential targets of miR-1 that impacts skeletogenesis and muscle formation and contributes to a deeper understanding of miR-1's function during development.

Application of Raman spectroscopy to the evaluation of F-actin changes in sea urchin eggs at fertilization.

The actin filaments on the surface of echinoderm oocytes and eggs readily undergo massive reorganization during meiotic maturation and fertilization. In sea urchin eggs, the actin cytoskeletal response to the fertilizing sperm is fast enough to accompany Ca2+ signals and to guide sperm's entry into the egg. Although recent work using live cell imaging technology confirmed changes in the actin polymerization status in fertilized eggs, as was previously shown using light and electron microscopy, it failed to provide experimental evidence of F-actin depolymerization a few seconds after insemination, which is concurrent with the sperm-induced Ca2+ release. In the present study, we applied Raman microspectroscopy to tackle this issue by examining the spectral profiles of the egg's subplasmalemmal regions before and after treating the eggs with actin drugs or fertilizing sperm. At both early (15 s) and late (15 min) time points after fertilization, specific peak shifts in the Raman spectra revealed change in the actin structure, and Raman imaging detected the cytoskeletal changes corresponding to the F-actin reorganization visualized with LifeAct-GFP in confocal microscopy. Our observation suggests that the application of Raman spectroscopy, which does not require microinjection of fluorescent probes and exogenous gene expression, may serve as an alternative or even advantageous method in disclosing rapid subtle changes in the subplasmalemmal actin cytoskeleton that are difficult to resolve.

Functional mimicry of sea urchin biomineralization proteins with CaCO3-binding peptides selected by phage display.

The intricate process of biomineralization, e.g. in sea urchins, involves the precise interplay of highly regulated mineralization proteins and the spatiotemporal coordination achieved through compartmentalization. However, the investigation of biomineralization effector molecules, e.g. proteins, is challenging, due to their very low abundance. Therefore, we investigate the functional mimicry in the bioinspired precipitation of calcium carbonate (CaCO3) with artificial peptides selected from a peptide library by phage display based on peptide-binding to calcite and aragonite, respectively. The structure-directing effects of the identified peptides were compared to those of natural protein mixes isolated from skeletal (test) structures of two sea urchin species (Arbacia lixula and Paracentrotus lividus). The calcium carbonate samples deposited in the absence or presence of peptides were analyzed with a set of complementary techniques with regard to morphology, polymorph, and nanostructural motifs. Remarkably, some of the CaCO3-binding peptides induced morphological features in calcite that appeared similar to those obtained in the presence of the natural protein mixes. Many of the peptides identified as most effective in exerting a structure-directing effect on calcium carbonate crystallization were rich in basic amino acid residues. Hence, our in vitro mineralization study further highlights the important, but often neglected, role of positively charged soluble organic matrices associated with biological and bioinspired CaCO3 deposition.

Structure and electromechanical coupling of a voltage-gated Na+/H+ exchanger.

Voltage-sensing domains control the activation of voltage-gated ion channels, with a few exceptions1. One such exception is the sperm-specific Na+/H+ exchanger SLC9C1, which is the only known transporter to be regulated by voltage-sensing domains2-5. After hyperpolarization of sperm flagella, SLC9C1 becomes active, causing pH alkalinization and CatSper Ca2+ channel activation, which drives chemotaxis2,6. SLC9C1 activation is further regulated by cAMP2,7, which is produced by soluble adenyl cyclase (sAC). SLC9C1 is therefore an essential component of the pH-sAC-cAMP signalling pathway in metazoa8,9, required for sperm motility and fertilization4. Despite its importance, the molecular basis of SLC9C1 voltage activation is unclear. Here we report cryo-electron microscopy (cryo-EM) structures of sea urchin SLC9C1 in detergent and nanodiscs. We show that the voltage-sensing domains are positioned in an unusual configuration, sandwiching each side of the SLC9C1 homodimer. The S4 segment is very long, 90 Å in length, and connects the voltage-sensing domains to the cytoplasmic cyclic-nucleotide-binding domains. The S4 segment is in the up configuration-the inactive state of SLC9C1. Consistently, although a negatively charged cavity is accessible for Na+ to bind to the ion-transporting domains of SLC9C1, an intracellular helix connected to S4 restricts their movement. On the basis of the differences in the cryo-EM structure of SLC9C1 in the presence of cAMP, we propose that, upon hyperpolarization, the S4 segment moves down, removing this constriction and enabling Na+/H+ exchange.

Structures of a sperm-specific solute carrier gated by voltage and cAMP.

The newly characterized sperm-specific Na+/H+ exchanger stands out by its unique tripartite domain composition1,2. It unites a classical solute carrier unit with regulatory domains usually found in ion channels, namely, a voltage-sensing domain and a cyclic-nucleotide binding domain1,3, which makes it a mechanistic chimera and a secondary-active transporter activated strictly by membrane voltage. Our structures of the sea urchin SpSLC9C1 in the absence and presence of ligands reveal the overall domain arrangement and new structural coupling elements. They allow us to propose a gating model, where movements in the voltage sensor indirectly cause the release of the exchanging unit from a locked state through long-distance allosteric effects transmitted by the newly characterized coupling helices. We further propose that modulation by its ligand cyclic AMP occurs by means of disruption of the cytosolic dimer interface, which lowers the energy barrier for S4 movements in the voltage-sensing domain. As SLC9C1 members have been shown to be essential for male fertility, including in mammals2,4,5, our structure represents a potential new platform for the development of new on-demand contraceptives.

Widespread priming of transcriptional regulatory elements by incipient accessibility or RNA polymerase II pause in early embryos of the sea urchin Strongylocentrotus purpuratus.

Transcriptional regulatory elements (TREs) are the primary nodes that control developmental gene regulatory networks. In embryo stages, larvae, and adult differentiated red spherule cells of the sea urchin Strongylocentrotus purpuratus, transcriptionally engaged TREs are detected by Precision Run-On Sequencing (PRO-seq), which maps genome-wide at base pair resolution the location of paused or elongating RNA polymerase II (Pol II). In parallel, TRE accessibility is estimated by the Assay for Transposase-Accessible Chromatin using Sequencing (ATAC-seq). Our analysis identifies surprisingly early and widespread TRE accessibility in 4-cell cleavage embryos that is not necessarily followed by concurrent or subsequent transcription. TRE transcriptional differences identified by PRO-seq provide more contrast among embryonic stages than ATAC-seq accessibility differences, in agreement with the apparent excess of accessible but inactive TREs during embryogenesis. Global TRE accessibility reaches a maximum around the 20-hour late blastula stage, which coincides with the consolidation of major embryo regionalizations and peak histone variant H2A.Z expression. A transcriptional potency model based on labile nucleosome TRE occupancy driven by DNA sequences and the prevalence of histone variants is proposed in order to explain the basal accessibility of transcriptionally inactive TREs during embryogenesis. However, our results would not reconcile well with labile nucleosome models based on simple A/T sequence enrichment. In addition, a large number of distal TREs become transcriptionally disengaged during developmental progression, in support of an early Pol II paused model for developmental gene regulation that eventually resolves in transcriptional activation or silencing. Thus, developmental potency in early embryos may be facilitated by incipient accessibility and transcriptional pause at TREs.

Possible role of translation in unfertilized sea urchin eggs.

Unfertilized eggs of animals contain maternal messenger RNAs (mRNAs) and proteins, which are required for the maintenance of metabolism and regulation of development during the initial stages of embryogenesis. Unfertilized eggs are transcriptionally and translationally quiescent. After fertilization, activated translation of maternal mRNAs is one of the major forces that direct the early stages of embryogenesis before activation of the zygotic genome. However, a low rate and level of protein synthesis have been detected in unfertilized sea urchin eggs indicating that translation is not completely inhibited. Analysis of translatomes of unfertilized eggs and early embryos detected three sets of maternal mRNAs translated either before or after fertilization, or both before and after fertilization. Proteins encoded by maternal mRNAs, which are translated in unfertilized eggs, perform many different functions required for homeostasis, fertilization, egg activation, and early development. This suggests that translation in unfertilized sea urchin eggs may be required to renew the pool of proteins involved in these processes. Thus, translation may be necessary to maintain the fertility and developmental potential of sea urchin eggs during the long-term storage of eggs in ovaries until spawning begins.

In Vitro Anti-Inflammatory and Vasculoprotective Effects of Red Cell Extract from the Black Sea Urchin Arbacia lixula.

Sea urchins have emerged as an important source of bioactive compounds with anti-inflammatory and antioxidant properties relevant to human health. Since inflammation is a crucial pathogenic process in the development and progression of atherosclerosis, we here assessed the potential anti-inflammatory and vasculoprotective effects of coelomic red-cell methanolic extract of the black sea urchin Arbacia lixula in an in vitro model of endothelial cell dysfunction. Human microvascular endothelial cells (HMEC-1) were pretreated with A. lixula red-cell extract (10 and 100 µg/mL) before exposure to the pro-inflammatory cytokine tumor necrosis factor (TNF)-α. The extract was non-toxic after 24 h cell treatment and was characterized by antioxidant power and phenol content. The TNF-α-stimulated expression of adhesion molecules (VCAM-1, ICAM-1) and cytokines/chemokines (MCP-1, CCL-5, IL-6, IL-8, M-CSF) was significantly attenuated by A. lixula red-cell extract. This was functionally accompanied by a reduction in monocyte adhesion and chemotaxis towards activated endothelial cells. At the molecular level, the tested extract significantly counteracted the TNF-α-stimulated activation of the pro-inflammatory transcription factor NF-κB. These results provide evidence of potential anti-atherosclerotic properties of A. lixula red-cell extract, and open avenues in the discovery and development of dietary supplements and/or drugs for the prevention or treatment of cardiovascular diseases.

Physicochemical characterization and phase II metabolic profiling of echinochrome A, a bioactive constituent from sea urchin, and its physiologically based pharmacokinetic modeling in rats and humans.

Echinochrome A, a natural naphthoquinone pigment found in sea urchins, is increasingly being investigated for its nutritional and therapeutic value associated with antioxidant, anticancer, antiviral, antidiabetic, and cardioprotective activities. Although several studies have demonstrated the biological effects and therapeutic potential of echinochrome A, little is known regarding its biopharmaceutical behaviors. Here, we aimed to investigate the physicochemical properties and metabolic profiles of echinochrome A and establish a physiologically-based pharmacokinetic (PBPK) model as a useful tool to support its clinical applications. We found that the lipophilicity, color variability, ultraviolet/visible spectrometry, and stability of echinochrome A were markedly affected by pH conditions. Moreover, metabolic and pharmacokinetic profiling studies demonstrated that echinochrome A is eliminated primarily by hepatic metabolism and that four possible metabolites, i.e., two glucuronidated and two methylated conjugates, are formed in rat and human liver preparations. A whole-body PBPK model incorporating the newly identified hepatic phase II metabolic process was constructed and optimized with respect to chemical-specific parameters. Furthermore, model simulations suggested that echinochrome A could exhibit linear disposition profiles without systemic and local tissue accumulation in clinical settings. Our proposed PBPK model of echinochrome A could be a valuable tool for predicting drug interactions in previously unexplored scenarios and for optimizing dosage regimens and drug formulations.

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

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

Soluble adenylyl cyclase coordinates intracellular pH homeostasis and biomineralization in calcifying cells of a marine animal.

Biomineralizing cells concentrate dissolved inorganic carbon (DIC) and remove protons from the site of mineral precipitation. However, the molecular regulatory mechanisms that orchestrate pH homeostasis and biomineralization of calcifying cells are poorly understood. Here, we report that the acid-base sensing enzyme soluble adenylyl cyclase (sAC) coordinates intracellular pH (pHi) regulation in the calcifying primary mesenchyme cells (PMCs) of sea urchin larvae. Single-cell transcriptomics, in situ hybridization, and immunocytochemistry elucidated the spatiotemporal expression of sAC during skeletogenesis. Live pHi imaging of PMCs revealed that the downregulation of sAC activity with two structurally unrelated small molecules inhibited pHi regulation of PMCs, an effect that was rescued by the addition of cell-permeable cAMP. Pharmacological sAC inhibition also significantly reduced normal spicule growth and spicule regeneration, establishing a link between PMC pHi regulation and biomineralization. Finally, increased expression of sAC mRNA was detected during skeleton remineralization and exposure to CO2-induced acidification. These findings suggest that transcriptional regulation of sAC is required to promote remineralization and to compensate for acidic stress. This work highlights the central role of sAC in coordinating acid-base regulation and biomineralization in calcifying cells of a marine animal.

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

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

microRNA-124 regulates Notch and NeuroD1 to mediate transition states of neuronal development.

MicroRNAs regulate gene expression by destabilizing target mRNA and/or inhibiting translation in animal cells. The ability to mechanistically dissect miR-124's function during specification, differentiation, and maturation of neurons during development within a single system has not been accomplished. Using the sea urchin embryo, we take advantage of the manipulability of the embryo and its well-documented gene regulatory networks (GRNs). We incorporated NeuroD1 as part of the sea urchin neuronal GRN and determined that miR-124 inhibition resulted in aberrant gut contractions, swimming velocity, and neuronal development. Inhibition of miR-124 resulted in an increased number of cells expressing transcription factors (TFs) associated with progenitor neurons and a concurrent decrease of mature and functional neurons. Results revealed that in the early blastula/gastrula stages, miR-124 regulates undefined factors during neuronal specification and differentiation. In the late gastrula/larval stages, miR-124 regulates Notch and NeuroD1 during the transition between neuronal differentiation and maturation. Overall, we have improved the neuronal GRN and identified miR-124 to play a prolific role in regulating various transitions of neuronal development.