An RNA interference approach for functional studies in the sea urchin and its use in analysis of nodal signaling gradients.

Dicer substrate interfering RNAs (DsiRNAs) destroy targeted transcripts using the RNA-Induced Silencing Complex (RISC) through a process called RNA interference (RNAi). This process is ubiquitous among eukaryotes. Here we report the utility of DsiRNA in embryos of the sea urchin Lytechinus variegatus (Lv). Specific knockdowns phenocopy known morpholino and inhibitor knockdowns, and DsiRNA offers a useful alternative to morpholinos. Methods are described for the design of specific DsiRNAs that lead to destruction of targeted mRNA. DsiRNAs directed against pks1, an enzyme necessary for pigment production, show how successful DsiRNA perturbations are monitored by RNA in situ analysis and by qPCR to determine relative destruction of targeted mRNA. DsiRNA-based knockdowns phenocopy morpholino- and drug-based inhibition of nodal and lefty. Other knockdowns demonstrate that the RISC operates early in development as well as on genes that are first transcribed hours after gastrulation is completed. Thus, DsiRNAs effectively mediate destruction of targeted mRNA in the sea urchin embryo. The approach offers significant advantages over other widely used methods in the urchin in terms of cost, and ease of procurement, and offers sizeable experimental advantages in terms of ease of handling, injection, and knockdown validation.

Stable germline transgenesis using the Minos Tc1/mariner element in the sea urchin Lytechinus pictus.

Stable transgenesis is a transformative tool in model organism biology. Although the sea urchin is one of the oldest animal models in cell and developmental biology, studies in this animal have largely relied on transient manipulation of wild animals, without a strategy for stable transgenesis. Here, we build on recent progress to develop a more genetically tractable sea urchin species, Lytechinus pictus, and establish a robust transgene integration method. Three commonly used transposons (Minos, Tol2 and piggyBac) were tested for non-autonomous transposition, using plasmids containing a polyubiquitin promoter upstream of a H2B-mCerulean nuclear marker. Minos was the only transposable element that resulted in significant expression beyond metamorphosis. F0 animals were raised to sexual maturity, and spawned to determine germline integration and transgene inheritance frequency, and to characterize expression patterns of the transgene in F1 progeny. The results demonstrate transgene transmission through the germline, the first example of a germline transgenic sea urchin and, indeed, of any echinoderm. This milestone paves the way for the generation of diverse transgenic resources that will dramatically enhance the utility, reproducibility and efficiency of sea urchin research.

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.

Generation of a homozygous mutant drug transporter (ABCB1) knockout line in the sea urchin Lytechinus pictus.

Sea urchins are premier model organisms for the study of early development. However, the lengthy generation times of commonly used species have precluded application of stable genetic approaches. Here, we use the painted sea urchin Lytechinus pictus to address this limitation and to generate a homozygous mutant sea urchin line. L. pictus has one of the shortest generation times of any currently used sea urchin. We leveraged this advantage to generate a knockout mutant of the sea urchin homolog of the drug transporter ABCB1, a major player in xenobiotic disposition for all animals. Using CRISPR/Cas9, we generated large fragment deletions of ABCB1 and used these readily detected deletions to rapidly genotype and breed mutant animals to homozygosity in the F2 generation. The knockout larvae are produced according to expected Mendelian distribution, exhibit reduced xenobiotic efflux activity and can be grown to maturity. This study represents a major step towards more sophisticated genetic manipulation of the sea urchin and the establishment of reproducible sea urchin animal resources.

Developmental single-cell transcriptomics in the Lytechinus variegatus sea urchin embryo.

Using scRNA-seq coupled with computational approaches, we studied transcriptional changes in cell states of sea urchin embryos during development to the larval stage. Eighteen closely spaced time points were taken during the first 24 h of development of Lytechinus variegatus (Lv). Developmental trajectories were constructed using Waddington-OT, a computational approach to 'stitch' together developmental time points. Skeletogenic and primordial germ cell trajectories diverged early in cleavage. Ectodermal progenitors were distinct from other lineages by the 6th cleavage, although a small percentage of ectoderm cells briefly co-expressed endoderm markers that indicated an early ecto-endoderm cell state, likely in cells originating from the equatorial region of the egg. Endomesoderm cells also originated at the 6th cleavage and this state persisted for more than two cleavages, then diverged into distinct endoderm and mesoderm fates asynchronously, with some cells retaining an intermediate specification status until gastrulation. Seventy-nine out of 80 genes (99%) examined, and included in published developmental gene regulatory networks (dGRNs), are present in the Lv-scRNA-seq dataset and are expressed in the correct lineages in which the dGRN circuits operate.

New techniques for creating parthenogenetic larvae of the sea urchin Lytechinus pictus for gene expression studies.

BACKGROUND: Sea urchins are model organisms for studying the spatial-temporal control of gene activity during development. The Southern California species, Lytechinus pictus, has a sequenced genome and can be raised in the laboratory from egg to egg in 4 to 5 months. RESULTS: Here, we present new techniques for generating parthenogenetic larvae of this species and include a gallery of photomicrographs of morphologically abnormal larvae that could be used for transcriptomic analysis. CONCLUSIONS: Comparison of gene expression in parthenogenotes to larvae produced by fertilization could provide novel insights into gene expression controls contributed by sperm in this important model organism. Knowledge gained from transcriptomics of sea urchin parthenogenotes could contribute to parthenogenetic studies of mammalian embryos.

Chromosomal-Level Genome Assembly of the Painted Sea Urchin Lytechinus pictus: A Genetically Enabled Model System for Cell Biology and Embryonic Development.

The painted urchin Lytechinus pictus is a sea urchin in the family Toxopneustidae and one of several sea urchin species that are routinely used as an experimental research organism. Recently, L. pictus has emerged as a tractable model system for establishing transgenic sea urchin lines due to its amenability to long term laboratory culture. We present the first published genome of L. pictus. This chromosomal-level assembly was generated using Illumina sequencing in conjunction with Oxford Nanopore Technologies long read sequencing and HiC chromatin conformation capture sequencing. The 998.9-Mb assembly exhibits high contiguity and has a scaffold length N50 of 46.0 Mb with 97% of the sequence assembled into 19 chromosomal-length scaffolds. These 19 scaffolds exhibit a high degree of synteny compared with the 19 chromosomes of a related species Lytechinus variegatus. Ab initio and transcript evidence gene modeling, combined with sequence homology, identified 28,631 gene models that capture 92% of BUSCO orthologs. This annotation strategy was validated by manual curation of gene models for the ABC transporter superfamily, which confirmed the completeness and accuracy of the annotations. Thus, this genome assembly, in conjunction with recent high contiguity assemblies of related species, positions L. pictus as an exceptional model system for comparative functional genomics and it will be a key resource for the developmental, toxicological, and ecological biology scientific communities.

Toposome: Source of antimicrobial molecules in the gonads of the sea urchin Lytechinus variegatus (Lamarck, 1816).

Sea urchins live in a challenging environment that requires rapid and efficient responses against pathogens and invaders. This response may be also important in reproductive processes once males and females release their gametes into water. In addition, the gonads are organs with dual function: reproductive organ and nutrient reserve, therefore it needs efficient protective mechanisms to preserve the nutrients as well as the reproductive cells. The aim of this study was to evaluate the presence and characterize antimicrobial molecules in the male and female gonads of the sea urchin Lytechinus variegatus. Through HPLC purification, antimicrobial activity test and mass spectrometry several antimicrobial molecules were found in the gonads of both gender. Computational in silico analyses showed that they are fragments of a glycoprotein called toposome, also known as major yolk protein (MYP) which is one of the major proteins found in the gonads. Although different functions have been reported for this protein, this is the first description of a direct antimicrobial activity in Lytechinus variegatus. The results indicate that when undergoing proteolysis the toposome generates different fragments with antimicrobial activity which may indicate the importance of a rapid defense response strategy against invading microorganisms in the gonads used by both males and females sea urchins.

Genomic insights of body plan transitions from bilateral to pentameral symmetry in Echinoderms.

Echinoderms are an exceptional group of bilaterians that develop pentameral adult symmetry from a bilaterally symmetric larva. However, the genetic basis in evolution and development of this unique transformation remains to be clarified. Here we report newly sequenced genomes, developmental transcriptomes, and proteomes of diverse echinoderms including the green sea urchin (L. variegatus), a sea cucumber (A. japonicus), and with particular emphasis on a sister group of the earliest-diverged echinoderms, the feather star (A. japonica). We learned that the last common ancestor of echinoderms retained a well-organized Hox cluster reminiscent of the hemichordate, and had gene sets involved in endoskeleton development. Further, unlike in other animal groups, the most conserved developmental stages were not at the body plan establishing phase, and genes normally involved in bilaterality appear to function in pentameric axis development. These results enhance our understanding of the divergence of protostomes and deuterostomes almost 500 Mya.

Chromosomal-Level Genome Assembly of the Sea Urchin Lytechinus variegatus Substantially Improves Functional Genomic Analyses.

Lytechinus variegatus is a camarodont sea urchin found widely throughout the western Atlantic Ocean in a variety of shallow-water marine habitats. Its distribution, abundance, and amenability to developmental perturbation make it a popular model for ecologists and developmental biologists. Here, we present a chromosomal-level genome assembly of L. variegatus generated from a combination of PacBio long reads, 10× Genomics sequencing, and HiC chromatin interaction sequencing. We show L. variegatus has 19 chromosomes with an assembly size of 870.4 Mb. The contiguity and completeness of this assembly are reflected by a scaffold length N50 of 45.5 Mb and BUSCO completeness score of 95.5%. Ab initio and transcript-informed gene modeling and annotation identified 27,232 genes with an average gene length of 12.6 kb, comprising an estimated 39.5% of the genome. Repetitive regions, on the other hand, make up 45.4% of the genome. Physical mapping of well-studied developmental genes onto each chromosome reveals nonrandom spatial distribution of distinct genes and gene families, which provides insight into how certain gene families may have evolved and are transcriptionally regulated in this species. Lastly, aligning RNA-seq and ATAC-seq data onto this assembly demonstrates the value of highly contiguous, complete genome assemblies for functional genomics analyses that is unattainable with fragmented, incomplete assemblies. This genome will be of great value to the scientific community as a resource for genome evolution, developmental, and ecological studies of this species and the Echinodermata.

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

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

The first mitochondrial genome of the model echinoid Lytechinus variegatus and insights into Odontophoran phylogenetics.

Assembly of publically available next-generation sequence data facilitated the generation of three camarodont echinoid mitogenomes: two for the Green Urchin (Lytechinus variegatus) and one for the Red Urchin (Mesocentrotus franciscanus). The data generated are exploited in a phylogenomic analysis of the superfamily Odontophora, originally proposed for echinoids with tooth supports on the epiphyses of the jaw. The analysis highly supports this taxon and its current subdivision into three families: the Echinometridae, Toxopneustidae, and Strongylocentrotidae. The analysis furthermore implies that historical taxonomic issues between two members of the genus Strongylocentrotus (S. pallidus and S. droebachiensis) may have a genetic basis. The novel mitogenomes for the model species L. variegatus complements the draft genome available for this taxon, one of only three genome-enabled echinoid species. The assembly method applied herein, follows a divide-and-conquer approach that provides for reduced computational requirements and facilitates resolving assembly problems when processing ultra-high coverage next-generation sequence data.

Neurogenesis in the sea urchin embryo is initiated uniquely in three domains.

Many marine larvae begin feeding within a day of fertilization, thus requiring rapid development of a nervous system to coordinate feeding activities. Here, we examine the patterning and specification of early neurogenesis in sea urchin embryos. Lineage analysis indicates that neurons arise locally in three regions of the embryo. Perturbation analyses showed that when patterning is disrupted, neurogenesis in the three regions is differentially affected, indicating distinct patterning requirements for each neural domain. Six transcription factors that function during proneural specification were identified and studied in detail. Perturbations of these proneural transcription factors showed that specification occurs differently in each neural domain prior to the Delta-Notch restriction signal. Though gene regulatory network state changes beyond the proneural restriction are largely unresolved, the data here show that the three neural regions already differ from each other significantly early in specification. Future studies that define the larval nervous system in the sea urchin must therefore separately characterize the three populations of neurons that enable the larva to feed, to navigate, and to move food particles through the gut.

The Role of Male Variation in Fertilization Success in Determining the Costs and Benefits of Polyandry in the Broadcast Spawning Urchin Lytechinus variegatus.

Although the benefits to males mating with multiple females have been well documented, the benefits to females mating with multiple males (polyandry) are less studied, particularly the mechanism that might drive these potential benefits. Benefits of polyandry might stem from increasing the chance of mating with a high-quality or compatible male or stem from the ability of multiple males to fertilize more eggs than any single male. We examine the fertilization consequences of polyandry in the sea urchin Lytechinus variegatus. This species has variation in spine color, and we conducted matings between individual and pooled sperm from two males that matched or mismatched in color. The results indicate that (1) males with white spines achieved higher fertilization and were more likely to cause polyspermy than males with purple spines, and there was no effect of female spine color on fertilization; (2) when comparing the average success of individual matings with pooled-sperm matings, there was a net benefit to polyandry when purple-spine males were pooled, a net cost when white-spine males were pooled, and no difference when mismatched spine color males were pooled; and (3) the success under pooled-sperm trials, with any of the spine color combinations, never exceeded the success of the more successful male in the individual-male trials. Together these results suggest that the consequences of polyandry depend on the relation between sperm availability and the sensitivity of eggs to sperm limitation and polyspermy with respect to the specific set of available males. The potential fertilization consequences of a female spawning with multiple males might be associated primarily with increasing the amount of sperm available to fertilize her eggs and secondarily with increasing the chances of mating with a higher-quality or more compatible male, as opposed to a diversity of males.

Acetylation Mimics Within a Single Nucleosome Alter Local DNA Accessibility In Compacted Nucleosome Arrays.

The activation of a silent gene locus is thought to involve pioneering transcription factors that initiate changes in the local chromatin structure to increase promoter accessibility and binding of downstream effectors. To better understand the molecular requirements for the first steps of locus activation, we investigated whether acetylation of a single nucleosome is sufficient to alter DNA accessibility within a condensed 25-nucleosome array. We found that acetylation mimics within the histone H4 tail domain increased accessibility of the surrounding linker DNA, with the increased accessibility localized to the immediate vicinity of the modified nucleosome. In contrast, acetylation mimics within the H3 tail had little effect, but were able to synergize with H4 tail acetylation mimics to further increase accessibility. Moreover, replacement of the central nucleosome with a nucleosome free region also resulted in increased local, but not global DNA accessibility. Our results indicate that modification or disruption of only a single target nucleosome results in significant changes in local chromatin architecture and suggest that very localized chromatin modifications imparted by pioneer transcription factors are sufficient to initiate a cascade of events leading to promoter activation.

Genomes of Strongylocentrotus franciscanus and Lytechinus variegatus: are there any genomic explanations for the two order of magnitude difference in the lifespan of sea urchins?

Sea urchins are marine invertebrates of extreme diversity of life span. Red sea urchin S. franciscanus is among the longest living creatures of the Ocean. Its lifetime is estimated to exceed a century, while the green sea urchin L. variegatus hardly survives more than four years. We sequenced and compared the genomes of these animals aiming at determination of the genetic basis of their longevity difference. List of genes related to the longevity of other animal species was created and used for homology search among the genomic data obtained in this study. Amino acid sequences of longevity related proteins of S. fransciscanus and L. variegatus as well as from a set of model species, were aligned and grouped on the basis of the species lifespan. Amino acid residues specific for a longevity group were identified. Proteins containing amino acids whose identity correlated with the lifespan were clustered on the basis of their function.

Induction of innate immune gene expression following methyl methanesulfonate-induced DNA damage in sea urchins.

Sea urchins are noted for the absence of neoplastic disease and represent a novel model to investigate cellular and systemic cancer protection mechanisms. Following intracoelomic injection of the DNA alkylating agent methyl methanesulfonate, DNA damage was detected in sea urchin cells and tissues (coelomocytes, muscle, oesophagus, ampullae and gonad) by the alkaline unwinding, fast micromethod. Gene expression analyses of the coelomocytes indicated upregulation of innate immune markers, including genes involved in NF-κB signalling. Results suggest that activation of the innate immune system following DNA damage may contribute to the naturally occurring resistance to neoplastic disease observed in sea urchins.

Evolutionary origins of retinoid active short-chain dehydrogenases/reductases of SDR16C family.

Vertebrate enzymes that belong to the 16C family of short-chain dehydrogenases/reductases (SDR16C) were shown to play an essential role in the control of retinoic acid (RA) levels during development. To trace the evolution of enzymatic function of SDR16C family, and to examine the origins of the pathway for RA biosynthesis from vitamin A, we identified putative SDR16C enzymes through the extensive search of available genome sequencing data in a subset of species representing major metazoan phyla. The phylogenetic analysis revealed that enzymes from protostome, non-chordate deuterostome and invertebrate chordate species are found in three clades of SDR16C family containing retinoid active enzymes, which are retinol dehydrogenase 10 (RDH10), retinol dehydrogenases E2 (RDHE2) and RDHE2-similar, and dehydrogenase reductase (SDR family) member 3 (DHRS3). For the initial functional analysis, we cloned RDH10- and RDHE2-related enzymes from the early developmental stages of a non-chordate deuterostome, green sea urchin Lytechinus variegatus, and an invertebrate chordate, sea squirt Ciona intestinalis. In situ hybridization revealed that these proteins are expressed in a pattern relevant to development, while assays performed on proteins expressed in mammalian cell culture showed that they possess retinol-oxidizing activity as their vertebrate homologs. The existence of invertebrate homologs of DHRS3 was inferred from the analysis of phylogeny and cofactor-binding residues characteristic of preference for NADP(H). The presence of invertebrate homologs in the DHRS3 group of SDR16C is interesting in light of the complex mutually activating interaction, which we have recently described for human RDH10 and DHRS3 enzymes. Further functional analysis of these homologs will establish whether this interaction evolved to control retinoid homeostasis only in vertebrates, or is also conserved in pre-vertebrates.

Sub-circuits of a gene regulatory network control a developmental epithelial-mesenchymal transition.

Epithelial-mesenchymal transition (EMT) is a fundamental cell state change that transforms epithelial to mesenchymal cells during embryonic development, adult tissue repair and cancer metastasis. EMT includes a complex series of intermediate cell state changes including remodeling of the basement membrane, apical constriction, epithelial de-adhesion, directed motility, loss of apical-basal polarity, and acquisition of mesenchymal adhesion and polarity. Transcriptional regulatory state changes must ultimately coordinate the timing and execution of these cell biological processes. A well-characterized gene regulatory network (GRN) in the sea urchin embryo was used to identify the transcription factors that control five distinct cell changes during EMT. Single transcription factors were perturbed and the consequences followed with in vivo time-lapse imaging or immunostaining assays. The data show that five different sub-circuits of the GRN control five distinct cell biological activities, each part of the complex EMT process. Thirteen transcription factors (TFs) expressed specifically in pre-EMT cells were required for EMT. Three TFs highest in the GRN specified and activated EMT (alx1, ets1, tbr) and the 10 TFs downstream of those (tel, erg, hex, tgif, snail, twist, foxn2/3, dri, foxb, foxo) were also required for EMT. No single TF functioned in all five sub-circuits, indicating that there is no EMT master regulator. Instead, the resulting sub-circuit topologies suggest EMT requires multiple simultaneous regulatory mechanisms: forward cascades, parallel inputs and positive-feedback lock downs. The interconnected and overlapping nature of the sub-circuits provides one explanation for the seamless orchestration by the embryo of cell state changes leading to successful EMT.

Nucleosome disruption by DNA ligase III-XRCC1 promotes efficient base excision repair.

Each day, approximately 20,000 oxidative lesions form in the DNA of every nucleated human cell. The base excision repair (BER) enzymes that repair these lesions must function in a chromatin milieu. We have determined that the DNA glycosylase hNTH1, apurinic endonuclease (APE), and DNA polymerase ß (Pol ß), which catalyze the first three steps in BER, are able to process their substrates in both 601- and 5S ribosomal DNA (rDNA)-based nucleosomes. hNTH1 formed a discrete ternary complex that was displaced by the addition of APE, suggesting an orderly handoff of substrates from one enzyme to the next. In contrast, DNA ligase IIIα-XRCC1, which completes BER, was appreciably active only at concentrations that led to nucleosome disruption. Ligase IIIα-XRCC1 was also able to bind and disrupt nucleosomes containing a single base gap and, because of this property, enhanced both its own activity and that of Pol ß on nucleosome substrates. Collectively, these findings provide insights into rate-limiting steps that govern BER in chromatin and reveal a unique role for ligase IIIα-XRCC1 in enhancing the efficiency of the final two steps in the BER of lesions in nucleosomes.