Tracing blastomere fate choices of early embryos in single cell culture

Blastomeres of early vertebrate embryos undergo numerous fate choices for division, motility, pluripotency maintenance and restriction culminating in various cell lineages. Tracing blastomere fate choices at the single cell level in vitro has not been possible because of the inability to isolate and cultivate early blastomeres as single cells. Here we report the establishment of single cell culture system in the fish medaka, enabling the isolation and cultivation of individual blastomeres from 16- to 64-cell embryos for fate tracing at the single cell level in vitro. Interestingly, these blastomeres immediately upon isolation exhibit motility, lose synchronous divisions and even stop dividing in ≥50% cases, suggesting that the widely accepted nucleocytoplasmic ratio controlling synchronous divisions in entire embryos does not operate on individual blastomeres. We even observed abortive division, endomitosis and cell fusion. Strikingly, ~5% of blastomeres in single cell culture generated extraembryonic yolk syncytial cells, embryonic stem cells and neural crest-derived pigment cells with timings mimicking their appearance in embryos. We revealed the maternal inheritance of key lineage regulators and their differential expression in cleavage embryos. Therefore, medaka blastomeres possess the accessibility for single cell culture, previously unidentified heterogeneity in motility, division, gene expression and intrinsic ability to generate major extraembryonic and embryonic lineages without positioning cues. Our data demonstrate the fidelity and potential of the single cell culture system for tracking blastomere fate decisions under defined conditions in vitro

Medaka Cleavage Embryos Are Capable of Generating ES-Like Cell Cultures

Mammalian embryos at the blastocyst stage have three major lineages, which in culture can give rise to embryonic stem (ES) cells from the inner cell mass or epiblast, trophoblast stem cells from the trophectoderm, and primitive endoderm stem cells. None of these stem cells is totipotent, because they show gene expression profiles characteristic of their sources and usually contribute only to the lineages of their origins in chimeric embryos. It is unknown whether embryos prior to the blastocyst stage can be cultivated towards totipotent stem cell cultures. Medaka is an excellent model for stem cell research. This laboratory fish has generated diploid and even haploid ES cells from the midblastula embryo with ~2000 cells. Here we report in medaka that dispersed cells from earlier embryos can survive, proliferate and attach in culture. We show that even 32-cells embryos can be dissociated into individual cells capable of producing continuously growing ES-like cultures. Our data point to the possibility to derive stable cell culture from cleavage embryos in this organism

Two Novel lncRNAs Regulate Primordial Germ Cell Development in Zebrafish

Long noncoding RNAs (lncRNAs) are regulatory transcripts in various biological processes. However, the role of lncRNAs in germline development remains poorly understood, especially for fish primordial germ cell (PGC) development. In this study, the lncRNA profile of zebrafish PGC was revealed by single cell RNA-sequencing and bioinformatic prediction. We established the regulation network of lncRNA-mRNA associated with PGC development, from which we identified three novel lncRNAs—lnc172, lnc196, and lnc304—highly expressing in PGCs and gonads. Fluorescent in situ hybridization indicated germline-specific localization of lnc196 and lnc304 in the cytoplasm and nucleus of spermatogonia, spermatocyte, and occyte, and they were co-localized with vasa in the cytoplasm of the spermatogonia. By contrast, lnc172 was localized in the cytoplasm of male germline, myoid cells and ovarian somatic cells. Loss- and gain-of-function experiments demonstrated that knockdown and PGC-specific overexpression of lnc304 as well as universal overexpression of lnc172 significantly disrupted PGC development. In summary, the present study revealed the lncRNA profile of zebrafish PGC and identified two novel lncRNAs associated with PGC development, providing new insights for understanding the regulatory mechanism of PGC development

MiR-202-5p Inhibits RIG-I-Dependent Innate Immune Responses to RGNNV Infection by Targeting TRIM25 to Mediate RIG-I Ubiquitination

The RIG-I-like receptors (RLRs) signaling pathway is essential for inducing type I interferon (IFN) responses to viral infections. Meanwhile, it is also tightly regulated to prevent uncontrolled immune responses. Numerous studies have shown that microRNAs (miRNAs) are essential for the regulation of immune processes, however, the detailed molecular mechanism of miRNA regulating the RLRs signaling pathway remains to be elucidated. Here, our results showed that miR-202-5p was induced by red spotted grouper nervous necrosis virus (RGNNV) infection in zebrafish. Overexpression of miR-202-5p led to reduced expression of IFN 1 and its downstream antiviral genes, thus facilitating viral replication in vitro. In comparison, significantly enhanced levels of IFN 1 and antiviral genes and significantly low viral burden were observed in the miR-202-5p-/- zebrafish compared to wild type zebrafish. Subsequently, zebrafish tripartite motif-containing protein 25 (zbTRIM25) was identified as a target of miR-202-5p in both zebrafish and humans. Ectopic expression of miR-202-5p suppressed zbTRIM25-mediated RLRs signaling pathway. Furthermore, we showed that miR-202-5p inhibited zbTRIM25-mediated zbRIG-I ubiquitination and activation of IFN production. In conclusion, we demonstrate that RGNNV-inducible miR-202-5p acts as a negative regulator of zbRIG-I-triggered antiviral innate response by targeting zbTRIM25. Our study reveals a novel mechanism for the evasion of the innate immune response controlled by RGNNV

Marine medaka heat shock protein 90ab1 is a receptor for red-spotted grouper nervous necrosis virus and promotes virus internalization through clathrin-mediated endocytosis.

Nervous necrosis virus (NNV) can infect many species of fish and causes serious acute or persistent infection. However, its pathogenic mechanism is still far from clear. Specific cellular surface receptors are crucial determinants of the species tropism of a virus and its pathogenesis. Here, the heat shock protein 90ab1 of marine model fish species marine medaka (MmHSP90ab1) was identified as a novel receptor of red-spotted grouper NNV (RGNNV). MmHSP90ab1 interacted directly with RGNNV capsid protein (CP). Specifically, MmHSP90ab1 bound to the linker region (LR) of CP through its NM domain. Inhibition of MmHSP90ab1 by HSP90-specific inhibitors or MmHSP90ab1 siRNA caused significant inhibition of viral binding and entry, whereas its overexpression led to the opposite effect. The binding of RGNNV to cultured marine medaka hMMES1 cells was inhibited by blocking cell surface-localized MmHSP90ab1 with anti-HSP90β antibodies or pretreating virus with recombinant MmHSP90ab1 or MmHSP90ab1-NM protein, indicating MmHSP90ab1 was an attachment receptor for RGNNV. Furthermore, we found that MmHSP90ab1 formed a complex with CP and marine medaka heat shock cognate 70, a known NNV receptor. Exogenous expression of MmHSP90ab1 independently facilitated the internalization of RGNNV into RGNNV impenetrable cells (HEK293T), which was blocked by chlorpromazine, an inhibitor of clathrin-dependent endocytosis. Further study revealed that MmHSP90ab1 interacted with the marine medaka clathrin heavy chain. Collectively, these data suggest that MmHSP90ab1 is a functional part of the RGNNV receptor complex and involved in the internalization of RGNNV via the clathrin endocytosis pathway

The CXC Chemokine Receptors in Four-Eyed Sleeper (Bostrychus sinensis) and Their Involvement in Responding to Skin Injury

CXC Chemokine signaling plays an important role in wound healing. The four-eyed sleeper (Bostrychus sinensis) is a commercially important marine fish, which is prone to suffer skin ulceration at high temperature seasons, leading to mass mortality of fish in aquaculture farms. The genetic background related to skin ulceration and wound healing has remained unknown in this fish. Herein, we identified 10 differentially expressed Bostrychus sinensis CXC chemokine receptors (BsCXCRs) in skin ulcerated fish by de novo transcriptome sequencing. The transcripts of these BsCXCRs were classified in seven types, including BsCXCR1a/1b, BsCXCR2, BsCXCR3a1/3a2, BsCXCR4a/4b, and BsCXCR5-7, and BsCXCR6 was the first CXCR6 homologue experimentally identified in teleost fish. These BsCXCRs were further characterized in gene and protein structures, as well as phylogenetics, and the results revealed that BsCXCRs have expanded to divergent homologues. Our results showed that, in healthy fish, the BsCXCR transcripts was mainly distributed in the muscle and immune related organs, and that BsCXCR1a/1b proteins located in the cytomembrane, BsCXCR4a/4b/5/6 in the cytomembrane and perinuclear region, and BsCXCR3a1/3a2/7 in the cytomembrane, perinuclear region, and nuclear membrane, respectively. In skin injured fish, the transcripts of all BsCXCRs were transiently increased within one hour after injury, suggesting the involvement of BsCXCRs into the early inflammatory response to skin injury in the four-eyed sleeper. These results are valuable for understanding the evolutionary events of fish CXCR genes and provide insights into the roles of CXCR family in fish skin injury

Medaka Cleavage Embryos Are Capable of Generating ES-Like Cell Cultures

Mammalian embryos at the blastocyst stage have three major lineages, which in culture can give rise to embryonic stem (ES) cells from the inner cell mass or epiblast, trophoblast stem cells from the trophectoderm, and primitive endoderm stem cells. None of these stem cells is totipotent, because they show gene expression profiles characteristic of their sources and usually contribute only to the lineages of their origins in chimeric embryos. It is unknown whether embryos prior to the blastocyst stage can be cultivated towards totipotent stem cell cultures. Medaka is an excellent model for stem cell research. This laboratory fish has generated diploid and even haploid ES cells from the midblastula embryo with &#126;2000 cells. Here we report in medaka that dispersed cells from earlier embryos can survive, proliferate and attach in culture. We show that even 32-cells embryos can be dissociated into individual cells capable of producing continuously growing ES-like cultures. Our data point to the possibility to derive stable cell culture from cleavage embryos in this organism.</p