Temporospatial hierarchy and allele-specific expression of zygotic genome activation revealed by distant interspecific urochordate hybrids.

Zygotic genome activation (ZGA) is a universal process in early embryogenesis of metazoan, when the quiescent zygotic nucleus initiates global transcription. However, the mechanisms related to massive genome activation and allele-specific expression (ASE) remain not well understood. Here, we develop hybrids from two deeply diverged (120 Mya) ascidian species to symmetrically document the dynamics of ZGA. We identify two coordinated ZGA waves represent early developmental and housekeeping gene reactivation, respectively. Single-cell RNA sequencing reveals that the major expression wave exhibits spatial heterogeneity and significantly correlates with cell fate. Moreover, allele-specific expression occurs in a species- rather than parent-related manner, demonstrating the divergence of cis-regulatory elements between the two species. These findings provide insights into ZGA in chordates.

Spatially resolved single-cell atlas of ascidian endostyle provides insight into the origin of vertebrate pharyngeal organs.

The pharyngeal endoderm, an innovation of deuterostome ancestors, contributes to pharyngeal development by influencing the patterning and differentiation of pharyngeal structures in vertebrates; however, the evolutionary origin of the pharyngeal organs in vertebrates is largely unknown. The endostyle, a distinct pharyngeal organ exclusively present in basal chordates, represents a good model for understanding pharyngeal organ origins. Using Stereo-seq and single-cell RNA sequencing, we constructed aspatially resolved single-cell atlas for the endostyle of the ascidian Styela clava. We determined the cell composition of the hemolymphoid region, which illuminates a mixed ancestral structure for the blood and lymphoid system. In addition, we discovered a cluster of hair cell-like cells in zone 3, which has transcriptomic similarity with the hair cells of the vertebrate acoustico-lateralis system. These findings reshape our understanding of the pharynx of the basal chordate and provide insights into the evolutionary origin of multiplexed pharyngeal organs.

Evaluation of the possible association of PDCD-1 and LAG3 gene polymorphisms with hepatocellular carcinoma risk.

PURPOSE: Programmed death-1 (PDCD-1) and lymphocyte activating 3 (LAG3), two important immunosuppressive molecules, play crucial roles in immune escape of tumor cells. This study evaluated the effects of PDCD-1 (rs10204525 and rs36084323), and LAG3 (rs870849 and rs1882545) gene polymorphisms on hepatocellular carcinoma (HCC) risk. METHODS: 341 patients with HCC and 350 cancer-free controls in the South Chinese population were included in a population-based case-control study. DNAs were extracted from peripheral blood samples. Genotypes were analyzed using multiplex PCR and sequencing. SNPs were analyzed using multiple inheritance models (co-dominant, dominant, recessive, and over-dominant). RESULTS: The allele and genotype frequencies of neither of the four polymorphisms, adjusted for age and gender, differed between HCC patients and controls. The differences were also not significant after stratifying by gender and age. According to our results, HCC patients with rs10204525 TC genotype had significantly lower AFP levels than HCC patients with rs10204525 TT genotype (P = 0.004). Moreover, the frequency of PDCD-1 rs36084323 CT genotype reduced the risk of TNM grade (CT vs. C/C-T/T: OR = 0.57, 95%CI = 0.37-0.87, P = 0.049). CONCLUSION: Our results demonstrated that the PDCD-1 (rs10204525 and rs36084323), and LAG3 (rs870849 and rs1882545) polymorphism did not influence the risk of HCC, PDCD-1 rs10204525 TC genotype was associated with the lower AFP levels and rs36084323 CT genotypes were related to HCC tumor grades in the South Chinese samples.

Transcriptional Analysis of the Endostyle Reveals Pharyngeal Organ Functions in Ascidian.

The endostyle is a pharyngeal organ with an opening groove and cilia in invertebrate chordates (amphioxus and ascidian) and cyclostomate (lamprey), serving as a filter-feeding tract and thyroid-secreting location. Emerging evidence implies its complex cellular composition and potentially versatile functions. Multiple cell types in the endostyle have been thought to be progenitors of complex organs in advanced vertebrates. To describe the expression profile and the potential functions, bulk RNA sequencing on the endostyle in ascidian Styela clava was conducted and distinct markers were selected by multileveled comparative analysis. Transcriptional data assay and qRT-PCR-verified results showed the regional expression patterns of Hox genes in the longitudinal axis. Organ-specific markers of the endostyle was proposed by comparing expression with the main organs of the ascidian. A cross-species transcriptional profile projection between the endostyle and organs from Danio rerio and Homo sapiens indicates a robust homogenous relationship to the thyroid and digestive system of the endostyle. The high similarity between the endostyle and the head kidney in zebrafish/the bone marrow in human implies uniquely profound functions of the pharyngeal organ in proto-vertebrates. Our result revealed that the transcriptional profile of the human parathyroid gland was similar to the ascidian endostyle, indicating the evolutionary origin of vertebrate hormone secretion organs.

EDomics: a comprehensive and comparative multi-omics database for animal evo-devo.

Evolutionary developmental biology (evo-devo) has been among the most fascinating interdisciplinary fields for decades, which aims to elucidate the origin and evolution of diverse developmental processes. The rapid accumulation of omics data provides unprecedented opportunities to answer many interesting but unresolved evo-devo questions. However, the access and utilization of these resources are hindered by challenges particularly in non-model animals. Here, we establish a comparative multi-omics database for animal evo-devo (EDomics, http://edomics.qnlm.ac) containing comprehensive genomes, bulk transcriptomes, and single-cell data across 40 representative species, many of which are generally used as model organisms for animal evo-devo study. EDomics provides a systematic view of genomic/transcriptomic information from various aspects, including genome assembly statistics, gene features and families, transcription factors, transposable elements, and gene expressional profiles/networks. It also exhibits spatiotemporal gene expression profiles at a single-cell level, such as cell atlas, cell markers, and spatial-map information. Moreover, EDomics provides highly valuable, customized datasets/resources for evo-devo research, including gene family expansion/contraction, inferred core gene repertoires, macrosynteny analysis for karyotype evolution, and cell type evolution analysis. EDomics presents a comprehensive and comparative multi-omics platform for animal evo-devo community to decipher the whole history of developmental evolution across the tree of life.

Single-Cell RNA Sequencing of the Testis of Ciona intestinalis Reveals the Dynamic Transcriptional Profile of Spermatogenesis in Protochordates.

Spermatogenesis is a complex and continuous process of germ-cell differentiation. This complex process is regulated by many factors, of which gene regulation in spermatogenic cells plays a decisive role. Spermatogenesis has been widely studied in vertebrates, but little is known about spermatogenesis in protochordates. Here, for the first time, we performed single-cell RNA sequencing (scRNA-seq) on 6832 germ cells from the testis of adult Ciona intestinalis. We identified six germ cell populations and revealed dynamic gene expression as well as transcriptional regulation during spermatogenesis. In particular, we identified four spermatocyte subtypes and key genes involved in meiosis in C. intestinalis. There were remarkable similarities and differences in gene expression during spermatogenesis between C. intestinalis and two other vertebrates (Chinese tongue sole and human). We identified many spermatogenic-cell-specific genes with functions that need to be verified. These findings will help to further improve research on spermatogenesis in chordates.

Nuclear Factor of Activated T Cells-5 Regulates Notochord Lumenogenesis in Chordate Larval Development.

Osmoregulation is essential for organisms to adapt to the exterior environment and plays an important role in embryonic organogenesis. Tubular organ formation usually involves a hyperosmotic lumen environment. The mechanisms of how the cells respond and regulate lumen formation remain largely unknown. Here, we reported that the nuclear factor of activated T cells-5 (NFAT5), the only transcription factor in the NFAT family involved in the cellular responses to hypertonic stress, regulated notochord lumen formation in chordate Ciona. Ciona NFAT5 (Ci-NFAT5) was expressed in notochord, and its expression level increased during notochord lumen formation and expansion. Knockout and expression of the dominant negative of NFAT5 in Ciona embryos resulted in the failure of notochord lumen expansion. We further demonstrated that the Ci-NFAT5 transferred from the cytoplasm into nuclei in HeLa cells under the hyperosmotic medium, indicating Ci-NFAT5 can respond the hypertonicity. To reveal the underly mechanisms, we predicted potential downstream genes of Ci-NFAT5 and further validated Ci-NFAT5-interacted genes by the luciferase assay. The results showed that Ci-NFAT5 promoted SLC26A6 expression. Furthermore, expression of a transport inactivity mutant of SLC26A6 (L421P) in notochord led to the failure of lumen expansion, phenocopying that of Ci-NFAT5 knockout. These results suggest that Ci-NFAT5 regulates notochord lumen expansion via the SLC26A6 axis. Taken together, our results reveal that the chordate NFAT5 responds to hypertonic stress and regulates lumen osmotic pressure via an ion channel pathway on luminal organ formation.

Cultivation of gut microorganisms of the marine ascidian Halocynthia roretzi reveals their potential roles in the environmental adaptation of their host.

It has long been known that abundant symbiotic bacteria exist in the tunic and gut of marine ascidians, and that these play crucial roles in host development, physiological metabolism, and environmental adaptation. However, the identity, roles and functions of these symbiotic bacteria are known for only a few strains. In this study, we isolated and cultivated 263 strains of microorganisms from the intestine of the marine ascidian Halocynthia roretzi through a combination of aerobic and anaerobic culture approaches. Most cultivated species, both aerobic and anaerobic, from ascidian stool samples belonged to the genus Bacillus based on 16S rDNA sequencing identification and phylogenetic assays. The distribution of cultured bacteria varied with seasonal changes in environmental conditions. To explore the functions of cultured bacteria, we screened out a strain of Serratia sp. whose extracts showed high antibacterial activity against aquatic pathogens. These findings revealed the potential roles of gut microorganisms in ascidian defense and environmental adaptation, thus providing insights into the interaction and co-evolution between gut bacteria and their hosts. Supplementary Information: The online version contains supplementary material available at 10.1007/s42995-022-00131-4.

Genome-Wide Identification, Comparison, and Expression Analysis of Transcription Factors in Ascidian Styela clava.

Tunicates include diverse species, as they are model animals for evolutionary developmental biology study. The embryonic development of tunicates is known to be extensively regulated by transcription factors (TFs). Styela clava, the globally distributed invasive tunicate, exhibits a strong capacity for environmental adaptation. However, the TFs were not systematically identified and analyzed. In this study, we reported 553 TFs categorized into 60 families from S. clava, based on the whole genome data. Comparison of TFs analysis among the tunicate species revealed that the gene number in the zinc finger superfamily displayed the most significant discrepancy, indicating this family was under the highly evolutionary selection and might be related to species differentiation and environmental adaptation. The greatest number of TFs was discovered in the Cys2His2-type zinc finger protein (zf-C2H2) family in S. clava. From the point of temporal view, more than half the TFs were expressed at the early embryonic stage. The expression correlation analysis revealed the existence of a transition for TFs expression from early embryogenesis to the later larval development in S. clava. Eight Hox genes were identified to be located on one chromosome, exhibiting different arrangement and expression patterns, compared to Ciona robusta (C. intestinalis type A). In addition, a total of 23 forkhead box (fox) genes were identified in S. clava, and their expression profiles referred to their potential roles in neurodevelopment and sensory organ development. Our data, thus, provides crucial clues to the potential functions of TFs in development and environmental adaptation in the leathery sea squirt.

Anti-virus effects of interferon regulatory factors (IRFs) identified in ascidian Ciona savignyi.

Interferon regulatory factors (IRFs) are key transcription factors that function in the immune system via the interferon (IFN) pathway. In the current study, we identified and characterized three IRFs (CsIRFL1, CsIRFL2, and CsIRFL3) from ascidian Ciona savignyi. Phylogenetic analysis showed that CsIRFL1 was clustered with two IRFs from Ciona robusta and shrimp IRF apart from the vertebrate IRFs, whereas CsIRFL2 and CsIRFL3 were grouped with an unnamed protein from Oikopleura dioica into a sub-branch highly identifying with the vertebrate IRF4, IRF8, and IRF9. Gene expression analysis revealed that CsIRFL1 and CsIRFL2 expressed in all the examined adult tissues (stomach, intestines, eggs, hemocytes, gonad, heart, and pharynx) and predominantly in hemocytes. However, the expression of CsIRFL3 was undetectable in the tested adult tissues. Furthermore, in situ hybridization showed that CsIRFL1 and CsIRFL2 mainly expressed in immunocytes within hemolymph, including phagocytes, macrophage-like cells, morula cells, and amoebocytes, suggesting CsIRFL1 and CsIRFL2 were involved in ascidian immune responses. We then performed LPS and poly(I:C) challenge assay and found that CsIRFL1 highly expressed in the cultured hemocytes following LPS infection for 24 h. After viral analogue poly(I:C) stimulation, the expression of CsIRFL2 was dramatically upregulated from 12 to 24 h. Meanwhile, two critical components of the IFN signaling pathways, STAT and TBK1, showed the increased expression as well after poly(I:C) induction, indicating that CsIRFL2 and IFN pathways genes were activated under the infection of viral analogue. Thus, our findings suggested that CsIRFL2 was a potential transcriptional regulatory factor that participated in regulating the ascidian anti-virus immune response.

Genomic basis of environmental adaptation in the leathery sea squirt (Styela clava).

Tunicates occupy the evolutionary position at the boundary of invertebrates and vertebrates. It exhibits adaptation to broad environmental conditions and is distributed globally. Despite hundreds of years of embryogenesis studies, the genetic basis of the invasive habits of ascidians remains largely unknown. The leathery sea squirt, Styela clava, is an important invasive species. We used the chromosomal-level genome and transcriptome of S. clava to explore its genomic- and molecular-network-based mechanisms of adaptation to environments. Compared with Ciona intestinalis type A (C. robusta), the size of the S. clava genome was expanded by 2-fold, although the gene number was comparable. An increase in transposon number and variation in dominant types were identified as potential expansion mechanisms. In the S. clava genome, the number of genes encoding the heat-shock protein 70 family and members of the complement system was expanded significantly, and cold-shock protein genes were transferred horizontally into the S. clava genome from bacteria. The expanded gene families potentially play roles in the adaptation of S. clava to its environments. The loss of key genes in the galactan synthesis pathway might explain the distinct tunic structure and hardness compared with the ascidian Ciona species. We demonstrated further that the integrated thyroid hormone pathway participated in the regulation of larval metamorphosis that provides S. clava with two opportunities for adapting to their environment. Thus, our report of the chromosomal-level leathery sea squirt genome provides a comprehensive genomic basis for the understanding of environmental adaptation in tunicates.

Publisher Correction: Evolutionary transcriptomics of metazoan biphasic life cycle supports a single intercalation origin of metazoan larvae.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Evolutionary transcriptomics of metazoan biphasic life cycle supports a single intercalation origin of metazoan larvae.

The transient larva-bearing biphasic life cycle is the hallmark of many metazoan phyla, but how metazoan larvae originated remains a major enigma in animal evolution. There are two hypotheses for larval origin. The 'larva-first' hypothesis suggests that the first metazoans were similar to extant larvae, with later evolution of the adult-added biphasic life cycle; the 'adult-first' hypothesis suggests that the first metazoans were adult forms, with the biphasic life cycle arising later via larval intercalation. Here, we investigate the evolutionary origin of primary larvae by conducting ontogenetic transcriptome profiling for Mollusca-the largest marine phylum characterized by a trochophore larval stage and highly variable adult forms. We reveal that trochophore larvae exhibit rapid transcriptome evolution with extraordinary incorporation of novel genes (potentially contributing to adult shell evolution), and that cell signalling/communication genes (for example, caveolin and innexin) are probably crucial for larval evolution. Transcriptome age analysis of eight metazoan species reveals the wide presence of young larval transcriptomes in both trochozoans and other major metazoan lineages, therefore arguing against the prevailing larva-first hypothesis. Our findings support an adult-first evolutionary scenario with a single metazoan larval intercalation, and suggest that the first appearance of proto-larva probably occurred after the divergence of direct-developing Ctenophora from a metazoan ancestor.

Ascidian caveolin induces membrane curvature and protects tissue integrity and morphology during embryogenesis.

Cell morphology and tissue integrity are essential for embryogenesis. Caveolins are membrane proteins that induce the formation of surface pits called caveolae that serve as membrane reservoirs for cell and tissue protection during development. In vertebrates, caveolin 1 (Cav1) and caveolin 3 (Cav3) are required for caveola formation. However, the formation of caveola and the function of caveolins in invertebrates are largely unknown. In this study, three caveolins, Cav-a, Cav-b, and CavY, are identified in the genome of the invertebrate chordate Ciona spp. Based on phylogenetic analysis, Cav-a is found to be closely related to the vertebrate Cav1 and Cav3. In situ hybridization shows that Cav-a is expressed in Ciona embryonic notochord and muscle. Cell-free experiments, model cell culture systems, and in vivo experiments demonstrate that Ciona Cav-a has the ability to induce membrane curvature at the plasma membrane. Knockdown of Cav-a in Ciona embryos causes loss of invaginations in the plasma membrane and results in the failure of notochord elongation and lumenogenesis. Expression of a dominant-negative Cav-a point mutation causes cells to change shape and become displaced from the muscle and notochord to disrupt tissue integrity. Furthermore, we demonstrate that Cav-a vesicles show polarized trafficking and localize at the luminal membrane during notochord lumenogenesis. Taken together, these results show that the invertebrate chordate caveolin from Ciona plays crucial roles in tissue integrity and morphology by inducing membrane curvature and intracellular vesicle trafficking during embryogenesis.

Seasonal dynamics and starvation impact on the gut microbiome of urochordate ascidian Halocynthia roretzi.

BACKGROUND: Gut microbiota plays important roles in host animal metabolism, homeostasis and environmental adaptation. However, the interplay between the gut microbiome and urochordate ascidian, the most closet relative of vertebrate, remains less explored. In this study, we characterized the gut microbial communities of urochordate ascidian (Halocynthia roretzi) across the changes of season and starvation stress using a comprehensive set of omic approaches including 16S rRNA gene amplicon sequencing, shotgun metagenomics, metabolomic profiling, and transcriptome sequencing. RESULTS: The 16S rRNA gene amplicon profiling revealed that ascidians harbor indigenous gut microbiota distinctly different to the marine microbial community and significant variations in composition and abundance of gut bacteria, with predominant bacterial orders representing each season. Depressed alpha-diversities of gut microbiota were observed across starvation stress when compared to the communities in aquafarm condition. Synechococcales involving photosynthesis and its related biosynthesis was reduced in abundance while the enrichments of Xanthomonadales and Legionellales may facilitate bile acid biosynthesis during starvation. Metabolomics analysis found that long chain fatty acids, linolenic acid, cyanoamino acid, and pigments derived from gut bacteria were upregulated, suggesting a beneficial contribution of the gut microbiome to the ascidian under starvation stress. CONCLUSIONS: Our findings revealed seasonal variation of ascidian gut microbiota. Defense and energy-associated metabolites derived from gut microbiome may provide an adaptive interplay between gut microbiome and ascidian host that maintains a beneficial metabolic system across season and starvation stress. The diversity-generating metabolisms from both microbiota and host might lead to the co-evolution and environmental adaptation.

Identification and functional characterization of solute carrier family 6 genes in Ciona savignyi.

The solute carrier 6 (SLC6) gene family, functioning as neurotransmitter transporters, plays the crucial roles in neurotransmission, cellular and organismal homeostasis. In this study, we found an expansion of SLC6 family gene in the genome of chordate invertebrate Ciona savignyi. A total of 40 candidate genes including 29 complete and 11 putative genes were identified as SLC6 family gene homologs. Phylogenetic analysis revealed that most of these Ciona SLC6 genes were highly conserved with the vertebrate ones, although gene duplication and gene losses did exist. Four genes were selected from SLC6 subfamilies to be further investigated for their functional characteristics on cell growth and migration through overexpression approach in cultured cell lines. The results showed both SLC6A7 and SLC6A17 from amino acid transporters AA1 and AA2 sub-families, respectively, significantly suppressed the cell proliferation and migration. While SLC6A1 and SLC6A4, which were classified into GABA and monoamine transporters, respectively, did not affect the cell proliferation and migration in HEK293T, HeLa, and MCF7 cells. The whole set of C. savignyi SLC6 genes identified in this study provides an important genomic resource for future biochemical, physiological, and phylogenetic studies on SLC6 gene family. Our experimental data demonstrated that Ciona amino acid transporters, such as SLC6A7 and SLC6A17, were essential for cell physiology and behaviors, indicating their crucially potential roles in the control of cell proliferation and migration during ascidian embryogenesis.

Penaeid shrimp brachyury: sequence analysis and expression during gastrulation.

Gastrulation occurs by a variety of morphogenetic movements, often correlated with diverse expression of the T-box transcription factor Brachyury (Bra). Bra may be expressed in ectoderm, mesoderm, or endoderm, but its role in cell fate specification or regulation of gastrulation movements has not been studied in the development of crustaceans. Penaeid shrimp (Decapoda: Dendrobranchiata: Penaeidae) develop by complete cleavage and gastrulation by invagination to a free-swimming nauplius larva. Penaeid gastrulation diverges from other decapods and from insects, occurring early at a low cell number with the formation of a radial invagination. Toward a better understanding of gastrulation movements in penaeid shrimp, bra was identified from newly available penaeid shrimp genomes and transcriptomes of Litopenaeus vannamei, Marsupenaeus japonicus, and Penaeus monodon. Additional bra homologs were obtained from the outgroups Sicyonia ingentis (Decapoda: Dendrobranchiata: Sicyoniidae) and the caridean shrimp Caridina multidentata (Decapoda: Pleocymata). The genes encoded penaeid shrimp Bra proteins of 551-552 amino acids, containing the highly conserved T-box DNA-binding region. The N-terminal Smad1-binding domain, conserved in most animals, was absent in shrimp Bra. The R1 repressor domain was the best conserved of the C-terminal regulatory domains, which were widely divergent compared to other species. The penaeid shrimp bra gene consisted of six exons, with splice sites conserved with other phyla across the animal kingdom. Real-time qPCR and FPKM analysis showed that shrimp bra mRNA was strongly expressed during gastrulation. These findings begin to address the evolution of gastrulation in shrimp at the molecular level.

Identification and expression analysis of long noncoding RNAs in embryogenesis and larval metamorphosis of Ciona savignyi.

Long noncoding RNAs (lncRNAs) play important roles in diverse developmental and pathological processes through chromatin reprogramming, cis regulation and posttranscriptional modification. They have been extensively studied in both vertebrates and invertebrates. However, the information of lncRNAs in urochordate is still lacking. In this study, we used the RNA-Seq data from three developmental stages (18, 21 and 42 hours post fertilization, hpf) of embryos and larvae in Ciona savignyi to identify candidate lncRNAs and analyze their expression profiles. A total of 29,944 unigenes were predicted as lncRNAs, five of which had hits with lncRNAs in NONCODE database. The acquired lncRNAs had an average length of 466 nt. The peaks of length, GC content and minimum free energy of the lncRNAs were significantly lower than that of the message RNAs (mRNAs). The average expression levels of lncRNAs were also lower than those of mRNAs. Among the three developmental stages, highly expressed lncRNAs concentrated in 18 hpf embryos. While, for those lncRNAs specifically up-regulated in 21 hpf embryos, their co-expressed mRNAs were enriched in GO terms of membrane, indicating these lncRNAs are involved in the regulation of luminal membrane biogenesis, and extracellular matrix secretion through membrane localized proteins during Ciona notochord tubulogenesis. The lncRNAs in 42 hpf larvae were distinct from those in 18 and 21 hpf embryos. This result is associated with the fact that swimming larvae are transiting into metamorphic juveniles at this stage, indicating lncRNAs are involved in the regulation of larval metamorphosis. Overall, our study identified a large number of lncRNAs in C. savignyi and revealed their expression characteristics and dynamics during Ciona embryogenesis and larval metamorphosis. The results will help to further understand the function of lncRNAs in chordate development and the evolution of lncRNAs.

Identification and characterization of microRNAs involved in ascidian larval metamorphosis.

BACKGROUND: Metamorphosis takes place within the life cycle of most marine invertebrates. The marine ascidian is a classical model to study complex cellular processes and underlying molecular mechanisms involved in its larval metamorphosis. The detailed molecular signaling pathways remain elusive, though extracellular signal-regulated kinases (ERKs) and c-Jun N-terminal kinase (JNK) have been revealed to regulate cell migration, differentiation, and apoptosis in ascidian larval organ regression and juvenile organ development. MicroRNAs (miRNAs) are small non-coding RNAs that modulate gene expression at the post-transcriptional level. Large numbers of miRNAs have been demonstrated to be involved in many developmental and metamorphic processes. However, the identification of miRNAs in ascidian larval metamorphosis has not yet been investigated. RESULTS: Totally, 106 known and 59 novel miRNAs were screened out through RNA-sequencing of three small RNA libraries from 18 to 21-h post-fertilization (hpf) tailbud embryos as well as from 42 hpf larvae (after tail regression) in Ciona savignyi. Expression profiling of miRNAs was confirmed by quantitative real-time PCR, showing that the expression levels of csa-miR-4040, csa-miR-4086, csa-miR-4055, csa-miR-4060, csa-miR-216a, csa-miR-216b, csa-miR-217, csa-miR-183, and csa-miR-92c were significantly higher in 42 hpf larvae, whereas those of csa-miR-4018a, csa-miR-4018b, and csa-miR-4000f were higher in 18 and 21 hpf embryos; then, their expression in 42 hpf larvae became significantly low. For these 12 miRNAs, whose expression levels significantly changed, we predicted their target genes through the combination of miRanda and TargetScan. This prediction analysis revealed 332 miRNA-target gene pairs that were associated with the ERK, JNK, and transforming growth factor beta signaling pathways, suggesting that the identified miRNAs are involved in the regulation of C. savignyi larval metamorphosis via controlling the expression of their target genes. Furthermore, we validated the expression of five selected miRNAs by northern blotting. Among the selected miRNAs, the expression patterns of csa-miR-4018a, csa-miR-4018b, and csa-miR-4000f were further examined by whole-mount in situ hybridization. The results showed that all three miRNAs were specifically expressed in a cell population resembling mesenchymal cells at the head and trunk part in swimming larvae but not in metamorphic larvae. Utilizing the luciferase assay, we also confirmed that miR-4000f targeted Mapk1, suggesting that the csa-miR-4018a/csa-miR-4018b/csa-miR-4000f cluster regulates larval metamorphosis through the Mapk1-mediated signaling pathway. CONCLUSIONS: Totally, 165 miRNAs, including 59 novel ones, were identified from the embryos and larvae of C. savignyi. Twelve of them showed significant changes in expression before and during metamorphosis. In situ hybridization and northern blotting results revealed that three miRNAs are potentially involved in the signaling regulatory network for the migration and differentiation of mesenchymal cells in larval metamorphosis. Furthermore, the luciferase reporter assay revealed that Mapk1 is a target of csa-miR-4000f. Our results not only present a list and profile of miRNAs involved in Ciona metamorphosis but also provide informative cues to further understand their function in ascidian larval metamorphosis.

Architectural delineation and molecular identification of extracellular matrix in ascidian embryos and larvae.

The extracellular matrix (ECM) not only provides essential physical scaffolding for cellular constituents but also initiates crucial biochemical and biomechanical cues that are required for tissue morphogenesis. In this study, we utilized wheat germ agglutinin (WGA) staining to characterize the ECM architecture in ascidian embryos and larvae. The results showed three distinct populations of ECM presenting in Ciona embryogenesis: the outer layer localized at the surface of embryo, an inner layer of notochord sheath and the apical ECM secreted by the notochord. To further elucidate the precise structure of Ciona embryonic ECM, we employed scanning and transmission electron microscopy, and found that the outer membrane was relatively thick with short fibres, whereas the ECM layer in notochord sheath was not as thick as the outer membrane but more regular arranged; the lumen between notochord cells was hydrostatic and sticky. Then, we used the RNA sequencing data from the embryos and larvae of Ciona savignyi to identify ECM genes and acquire their expression patterns. We identified 115 unigenes as 67 ECM genes, and 77 unigenes showed dynamic expression changes between different stages. Our results reveal the architecture, molecular composition and dynamic expression profile of ECM in ascidian embryogenesis, and may increase understanding of the function of the ECM in chordate development.