The growing variety of RNA classes,such as mRNAs,lncRNAs,and circRNAs,plays pivotal roles in both developmental processes and various pathophysiological conditions.Nonetheless,our comprehension of RNA functions in liv...The growing variety of RNA classes,such as mRNAs,lncRNAs,and circRNAs,plays pivotal roles in both developmental processes and various pathophysiological conditions.Nonetheless,our comprehension of RNA functions in live organisms remains limited due to the absence of durable and effective strategies for directly influencing RNA levels.In this study,we combined the CRISPR-RfxCas13d system with spermlike stem cell-mediated semi-cloning techniques,which enabled the suppressed expression of different RNA species.This approach was employed to interfere with the expression of three types of RNA molecules:Sfmbt2 mRNA,Fendrr lncRNA,and circMan1a2(2,3,4,5,6).The results confirmed the critical roles of these RNAs in embryonic development,as their loss led to observable phenotypes,including embryonic lethality,delayed embryonic development,and embryo resorption.In summary,our methodology offers a potent toolkit for silencing specific RNA targets in living organisms without introducing genetic alterations.展开更多
Paternal imprinted genes(H19 and Gtl2)are pivotal for prenatal embryonic development in mice.Nongrowing oocytes and sperm-or oocyte-originated haploid embryonic stem cells(ha ESCs)carrying both H19-DMR(differentially ...Paternal imprinted genes(H19 and Gtl2)are pivotal for prenatal embryonic development in mice.Nongrowing oocytes and sperm-or oocyte-originated haploid embryonic stem cells(ha ESCs)carrying both H19-DMR(differentially DNA-methylated region)and IG(intergenic)-DMR deletions that partially mimic paternal imprinting of H19-Igf2 and Dlk1-Dio3 can be employed as sperm replacement to efficiently support full-term embryonic development.However,how H19-DMR and IG-DMR act together to regulate embryonic development is still largely unknown.Here,using androgenetic ha ESC(AG-ha ESC)-mediated semi-cloned(SC)technology,we showed that paternal H19-DMR and IG-DMR are not essential for pre-implantation development of SC embryos generated through injection of AG-ha ESCs into oocytes.H19-DMR plays critical roles before 12.5 days of gestation while IG-DMR is essential for late-gestation of SC embryos.Interestingly,we found that combined deletions of H19 and H19-DMR can further improve the efficiency of normal development of SC embryos at mid-gestation compared to DKO SC embryos.Transcriptome and histology analyses revealed that H19 and H19-DMR combined deletions rescue the placental defects.Furthermore,we showed that H19,H19-DMR and IG-DMR deletions(TKO)give rise to better prenatal and postnatal embryonic development of SC embryos compared to DKO.Together,our results indicate the temporal regulation of paternal imprinted loci during embryonic development.展开更多
The use of two inhibitors of Mek1/2 and Gsk3β(2i)promotes the generation of mouse diploid and haploid embryonic stem cells(ESCs)from the inner cell mass of biparental and uniparental blastocysts,respectively.However,...The use of two inhibitors of Mek1/2 and Gsk3β(2i)promotes the generation of mouse diploid and haploid embryonic stem cells(ESCs)from the inner cell mass of biparental and uniparental blastocysts,respectively.However,a system enabling long-term maintenance of imprints in ESCs has proven challenging.Here,we report that the use of a two-step a2i(alternative two inhibitors of Src and Gsk3β,TSa2i)derivation/culture protocol results in the establishment of androgenetic haploid ESCs(AG-haESCs)with stable DNA methylation at paternal DMRs(differentially DNA methylated regions)up to passage 60 that can efficiently support generating mice upon oocyte injection.We also show coexistence of H3K9me3 marks and ZFP57 bindings with intact DMR methylations.Furthermore,we demonstrate that TSa2itreated AG-haESCs are a heterogeneous cell population regarding paternal DMR methylation.Strikingly,AGhaESCs with late passages display increased paternal-DMR methylations and improved developmental potential compared to early-passage cells,in part through the enhanced proliferation of H19-DMR hypermethylated cells.Together,we establish AG-haESCs that can longterm maintain paternal imprints.展开更多
Fish, like many other animals, have two major cell lineages, namely the germline and soma. The germ-soma separation is one of the earliest events of embryonic development. Germ cells can be specifically labeled and is...Fish, like many other animals, have two major cell lineages, namely the germline and soma. The germ-soma separation is one of the earliest events of embryonic development. Germ cells can be specifically labeled and isolated for culture and transplan-tation, providing tools for reproduction of endangered species in close relatives, such as surrogate production of trout in salmon. Haploid cell cultures, such as medaka haploid embryonic stem cells have recently been obtained, which are capable of mimicking sperm to produce fertile offspring, upon nuclear being directly transferred into normal eggs. Such fish originated from a mosaic oocyte that had a haploid meiotic nucleus and a transplanted haploid mitotic cell culture nucleus. The first semi-cloned fish is Holly. Here we review the current status and future directions of understanding and manipulating fish germ cells in basic research and reproductive technology.展开更多
Stem cells are present in developing embryos and adult tissues of multicellular organisms. Owing to their unique features, stem cells provide excellent opportunities for experimental analyses of basic developmental pr...Stem cells are present in developing embryos and adult tissues of multicellular organisms. Owing to their unique features, stem cells provide excellent opportunities for experimental analyses of basic developmental processes such as pluripotency control and cell fate decision and for regenerative medicine by stem cell-based therapy. Stem cell cultures have been best studied in 3 vertebrate organisms. These are the mouse, human and a small laboratory fish called medaka. Specifically, medaka has given rise to the first embryonic stem (ES) cells besides the mouse, the first adult testis-derived male stem cells spermatogonia capable of test-tube sperm production, and most recently, even haploid ES cells capable of producing Holly, a semi-cloned fertile female medaka from a mosaic oocyte created by microinjecting a haploid ES cell nucleus directly into a normal oocyte. These breakthroughs make medaka a favoring vertebrate model for stem cell research, the topic of this review.展开更多
基金supported by the Strategic Priority Research Program of the Chinese Academy of Science(XDB0570000)the CAS Project for Young Scientists in Basic Research(YSBR-009)+3 种基金the National Key Research and Development Program of China(2021YFA1100203,2020YFA0509000)the National Natural Science Foundation of China(31821004,32030029,32293230)Shanghai Municipal Science and Technology Major Project(23HC1401000,22YS1400900)support from the Xplorer Prize and New Cornerstone Science Foundation(NCI202232).
文摘The growing variety of RNA classes,such as mRNAs,lncRNAs,and circRNAs,plays pivotal roles in both developmental processes and various pathophysiological conditions.Nonetheless,our comprehension of RNA functions in live organisms remains limited due to the absence of durable and effective strategies for directly influencing RNA levels.In this study,we combined the CRISPR-RfxCas13d system with spermlike stem cell-mediated semi-cloning techniques,which enabled the suppressed expression of different RNA species.This approach was employed to interfere with the expression of three types of RNA molecules:Sfmbt2 mRNA,Fendrr lncRNA,and circMan1a2(2,3,4,5,6).The results confirmed the critical roles of these RNAs in embryonic development,as their loss led to observable phenotypes,including embryonic lethality,delayed embryonic development,and embryo resorption.In summary,our methodology offers a potent toolkit for silencing specific RNA targets in living organisms without introducing genetic alterations.
基金partly supported by the Genome Tagging Project,Fountain-Valley Life Sciences Fund of University of Chinese Academy of Sciences Education Foundation and grants from the Chinese Academy of Sciences(XDB19010204,OYZDJ-SSW-SMC023 and Facility-based Open Research Program)the National Natural Science Foundation of China(31530048,81672117,31730062,31821004,and31601163)+1 种基金the Ministry of Science and Technology of China(2019YFA0109900)Shanghai Municipal Commission for Science and Technology(16JC1420500,17JC1420102,17JC1400900,and17411954900)
文摘Paternal imprinted genes(H19 and Gtl2)are pivotal for prenatal embryonic development in mice.Nongrowing oocytes and sperm-or oocyte-originated haploid embryonic stem cells(ha ESCs)carrying both H19-DMR(differentially DNA-methylated region)and IG(intergenic)-DMR deletions that partially mimic paternal imprinting of H19-Igf2 and Dlk1-Dio3 can be employed as sperm replacement to efficiently support full-term embryonic development.However,how H19-DMR and IG-DMR act together to regulate embryonic development is still largely unknown.Here,using androgenetic ha ESC(AG-ha ESC)-mediated semi-cloned(SC)technology,we showed that paternal H19-DMR and IG-DMR are not essential for pre-implantation development of SC embryos generated through injection of AG-ha ESCs into oocytes.H19-DMR plays critical roles before 12.5 days of gestation while IG-DMR is essential for late-gestation of SC embryos.Interestingly,we found that combined deletions of H19 and H19-DMR can further improve the efficiency of normal development of SC embryos at mid-gestation compared to DKO SC embryos.Transcriptome and histology analyses revealed that H19 and H19-DMR combined deletions rescue the placental defects.Furthermore,we showed that H19,H19-DMR and IG-DMR deletions(TKO)give rise to better prenatal and postnatal embryonic development of SC embryos compared to DKO.Together,our results indicate the temporal regulation of paternal imprinted loci during embryonic development.
基金This study was supported by Genome Tagging Project and grants from the Chinese Academy of Sciences,the National Key Research and Development Program of Chinathe National Natural Science Foundation of China(2019YFA0109900,2020YFA0509000,XDB19010204,QYZDJ-SSW-SMC023,Facility-based Open Research Program,31821004,32030029,and 31730062).
文摘The use of two inhibitors of Mek1/2 and Gsk3β(2i)promotes the generation of mouse diploid and haploid embryonic stem cells(ESCs)from the inner cell mass of biparental and uniparental blastocysts,respectively.However,a system enabling long-term maintenance of imprints in ESCs has proven challenging.Here,we report that the use of a two-step a2i(alternative two inhibitors of Src and Gsk3β,TSa2i)derivation/culture protocol results in the establishment of androgenetic haploid ESCs(AG-haESCs)with stable DNA methylation at paternal DMRs(differentially DNA methylated regions)up to passage 60 that can efficiently support generating mice upon oocyte injection.We also show coexistence of H3K9me3 marks and ZFP57 bindings with intact DMR methylations.Furthermore,we demonstrate that TSa2itreated AG-haESCs are a heterogeneous cell population regarding paternal DMR methylation.Strikingly,AGhaESCs with late passages display increased paternal-DMR methylations and improved developmental potential compared to early-passage cells,in part through the enhanced proliferation of H19-DMR hypermethylated cells.Together,we establish AG-haESCs that can longterm maintain paternal imprints.
基金supported by the Biomedical Research Council of Singapore (Grant Nos. R-05-1-21-19-404, R-08-1-21-19-585 and SBIC-SSCC C-002-2007)the Ministry of Education of Singapore (Grant No. R-154-000-285-112)+1 种基金the National University of Singapore (Grant No. R-154-000-153-720)the National Key Basic Research Program of China (Grant Nos. 2004CB117406 and 2010CB126301)
文摘Fish, like many other animals, have two major cell lineages, namely the germline and soma. The germ-soma separation is one of the earliest events of embryonic development. Germ cells can be specifically labeled and isolated for culture and transplan-tation, providing tools for reproduction of endangered species in close relatives, such as surrogate production of trout in salmon. Haploid cell cultures, such as medaka haploid embryonic stem cells have recently been obtained, which are capable of mimicking sperm to produce fertile offspring, upon nuclear being directly transferred into normal eggs. Such fish originated from a mosaic oocyte that had a haploid meiotic nucleus and a transplanted haploid mitotic cell culture nucleus. The first semi-cloned fish is Holly. Here we review the current status and future directions of understanding and manipulating fish germ cells in basic research and reproductive technology.
基金supported by the Biomedical Research Council of Singapore (Grant Nos. R-05-1-21-19-404, R-08-1-21-19-585 and SBIC-SSCC-002-2007)the Ministry of Education of Singapore (Grant No. R-154-000-285-112)the National University of Singapore (Grant No. R-154-000-153-720)
文摘Stem cells are present in developing embryos and adult tissues of multicellular organisms. Owing to their unique features, stem cells provide excellent opportunities for experimental analyses of basic developmental processes such as pluripotency control and cell fate decision and for regenerative medicine by stem cell-based therapy. Stem cell cultures have been best studied in 3 vertebrate organisms. These are the mouse, human and a small laboratory fish called medaka. Specifically, medaka has given rise to the first embryonic stem (ES) cells besides the mouse, the first adult testis-derived male stem cells spermatogonia capable of test-tube sperm production, and most recently, even haploid ES cells capable of producing Holly, a semi-cloned fertile female medaka from a mosaic oocyte created by microinjecting a haploid ES cell nucleus directly into a normal oocyte. These breakthroughs make medaka a favoring vertebrate model for stem cell research, the topic of this review.
