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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 ​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.

Nanos is a translational regulator required for the survival and maintenance of primordial germ cells during embryogenesis. Three nanos homologs are present in the genome of the sea urchin Strongylocentrotus purpuratus (Sp), and each nanos mRNA accumulates specifically in the small micromere (sMic) lineage. We found that a highly conserved element in the 3′ UTR of nanos2 is sufficient for reporter expression selectively in the sMic lineage: microinjection into a Sp fertilized egg of an RNA that contains the GFP open reading frame followed by Sp nanos2 3′UTR leads to selective reporter enrichment in the small micromeres in blastulae. The same result was seen with nanos2 from the sea urchin Hemicentrotus pulcherrimus (Hp). In both species, the 5′UTR alone is not sufficient for the sMic localization but it always increased the sMic reporter enrichment when present with the 3′UTR. We defined an element conserved between Hp and Sp in the nanos2 3′UTR which is necessary and sufficient for protein enrichment in the sMic, and refer to it as GNARLE (Global Nanos Associated RNA Lability Element). We also found that the nanos2 3′UTR is essential for the selective RNA retention in the small micromeres; GNARLE is required but not sufficient for this process. These results show that a combination of selective RNA retention and translational control mechanisms instills nanos accumulation uniquely in the sMic lineage.

Two distinct types of orthodenticle-related proteins (HpOtxE/L) have been implicated as transcription activators of the aboral ectoderm-specific arylsulfatase (Ars) gene. Here, we describe the structure of HpOtx gene and present evidence that mRNAs ofHpOtxE/Lare transcribed from a single HpOtx gene by altering the transcription start site and by alternative splicing. By transactivation experiments, we have also demonstrated that HpOtxLactivates theArspromoter in the gastrula-stage embryo.

The sea urchin (Hemicentrotus pulcherrimus) arylsulfatase (HpArs) gene, expressed specifically in aboral ectoderm, contains a 229-bp enhancer in its first intron that is required for the activation ofHpArsgene expression. Deletion analysis shows that a tandem repeat of orthodenticle-related protein (Otx) binding sites are responsible for the activity of this enhancer. Gel mobility shift analysis reveals that three types of Otx-proteins, which show different mobilities in gel shift assays, form complexes with the enhancer. Band I appears before hatching and gradually decreases by the gastrula stage. Band III appears at the blastula stage and Band II appears at the mesenchyme blastula stage; the levels of Band II and III remain constant until the gastrula stage. Two distinct types of HpOtx cDNA clones have been isolated from cDNA libraries of unfertilized eggs and gastrulae. Nucleotide sequences of the homeobox and downstream regions are well conserved in the two types of HpOtx cDNAs, while the region upstream from the homeobox has different nucleotide sequences. By genomic Southern blot analysis, only a single copy ofHpOtxgene is detectable in the Hp genome, making it likely that two HpOtx isoforms are generated from the same gene. Results from Northern blot analysis confirm the presence of two types of HpOtx transcripts. Transcriptional regulation of theHpArsgene may, in part, be carried out through switching of Otx isoforms.

The possible role of the egg nucleus in regulating the behavior of sperm nuclei and the cycling of maturation-promoting factor (MPF) was investigated in the physiologically polyspermic eggs of the newt Cynops pyrrhogaster. Many sperm entered all areas of the egg, but only one sperm pronucleus, the principal sperm pronucleus, moved to the center of the animal hemisphere to form a zygote nucleus with the egg pronucleus. All sperm and egg pronuclei synthesized DNA, but the zygote nucleus completed the synthesis of DNA 0.5-1 hr earlier than the accessory sperm nuclei. Entrance into M phase by the accessory sperm nuclei was delayed to a greater and greater extent with increasing distance of these nuclei from the zygote nucleus. When DNA in the egg nucleus was damaged by uv irradiation, not only were both prophase and M phase in the zygote nucleus prolonged, but also the MPF cycle was delayed. Some accessory sperm nuclei in the animal hemisphere escaped degeneration to form additional bipolar spindles, so that delayed multipolar cleavage occurred. The MPF activity in the vegetal hemisphere was less than 25% of that found in the animal hemisphere at M phase in normally fertilized eggs. The levels of immunologically detectable proteins that contained the sequence PSTAIR in vegetal hemispheres were less than 25% of those in animal hemispheres. These results indicate that the egg nucleus of the Cynops egg is involved in the control of the activation of MPF and that the accessory sperm nuclei in the vegetal hemisphere degenerate as a result of the lack of components that are indispensable for entry into M phase.

We showed that engineered zinc-finger nucleases (ZFNs), which consist of a zinc-finger DNA-binding array and a nuclease domain of the restriction enzyme FokI, can introduce mutations at a specific genomic site in the sea urchin embryo. Using bacterial one-hybrid screening with zinc-finger randomized libraries and a single-strand annealing assay in cultured cells, ZFNs targeting the sea urchin Hemicentrotus pulcherrimus homologue of HesC (HpHesC) were efficiently selected. Consistent with the phenotype observed in embryos injected with an antisense morpholino oligonucleotide against HpHesC, an increase in the primary mesenchyme cell population was observed in embryos injected with a pair of HpHesC ZFN mRNAs. In addition, sequence analysis of the mutations showed that deletions and insertions occurred at the HpHesC target site in the embryos injected with the HpHesC ZFN mRNAs. These results suggest that targeted gene disruption using ZFNs is feasible for the sea urchin embryo.