Benzo[a]pyrene(BaP),a ubiquitous pollutant,raises environmental health concerns due to induction of bone toxicity in the unexposed offspring.Exposure of F0 ancestor medaka(Oryzias latipes)to 1μg/L BaP for 21 days cau...Benzo[a]pyrene(BaP),a ubiquitous pollutant,raises environmental health concerns due to induction of bone toxicity in the unexposed offspring.Exposure of F0 ancestor medaka(Oryzias latipes)to 1μg/L BaP for 21 days causes reduced vertebral bone thickness in the unexposed F3 male offspring.To reveal the inherited modifications,osteoblast(OB)abundance and molecular signaling pathways of transgenerational BaP-induced bone thinning were assessed.Histomorphometric analysis showed a reduction in OB abundance.Analyses of the miRNA and mRNA transcriptomes revealed the dysregulation of Wnt signaling(frzb/ola-miR-1–3p,sfrp5/ola-miR-96–5p/miR-455–5p)and bone morphogenetic protein(Bmp)signaling(bmp3/ola-miR-96–5p/miR-181b-5p/miR-199a-5p/miR-205–5p/miR-455–5p).Both pathways are major indicators of impaired bone formation,while the altered Rank signaling in osteoclasts(c-fos/miR-205–5p)suggests a potentially augmented bone resorption.Interestingly,a typical BaP-responsive pathway,the Nrf2-mediated oxidative stress response(gst/ola-miR-181b-5p/miR-199a-5p/miR-205),was also affected.Moreover,mRNA levels of epigenetic modification enzymes(e.g.,hdac6,hdac7,kdm5b)were found dysregulated.The findings indicated that epigenetic factors(e.g.,miRNAs,histone modifications)may directly regulate the expression of genes associated with transgenerational BaP bone toxicity and warrants further studies.The identified candidate genes and miRNAs may serve as potential biomarkers for BaP-induced bone disease and as indicators of historic exposures in wild fish for conservation purposes.展开更多
In mammals,lipoprotein lipase(LPL)has been found to play an important role in lipid mentalismand deposition.LPL deficiency in humans(Homo sapiens)and mice(Mus musculus)tends to cause hypertriglyceridemia.The lpl gene ...In mammals,lipoprotein lipase(LPL)has been found to play an important role in lipid mentalismand deposition.LPL deficiency in humans(Homo sapiens)and mice(Mus musculus)tends to cause hypertriglyceridemia.The lpl gene is not expressed in adult mammalian liver,but is in adult fish liver.The functions provided by the lpl gene are diverse in vertebrates.Here,we knocked out the lpl gene in Japanese medaka(Oryzias latipes)with the CRISPR/Cas9 system.The lpl-knockout(KO)homozygous individuals showed severe developmental defects with an extremely emaciated and deformedbody and onlyaccounted for about5%of the F2fish.This is consistentwiththefindings inmicebut disaccords with the results for zebrafish(Danio rerio).Compared with wild-type(WT)madaka,the mRNA level of lpl in lpl-KO heterozygous mutant was significantly higher in the muscle,showed no significant difference in the liver,and was significantly lower in the heart.Under lpl heterozygous deficiency,the relative area of Oil RedO and triglycerides(TG)level in the liver,heart and muscle tissue covaried with levels of lpl mRNA in medaka.The lpl heterozygous deficiency did not affect the levelsofTG,low-density lipoprotein cholesterol(LDL-C),high-density lipoprotein cholesterol(HDL-C)and total cholesterol(TC)in the plasma of medaka,which is inconsistent with the findings in mammals.In general,the lpl gene plays an important role in the growth and development and is closely related to lipid deposition of medaka.展开更多
Generation of mutants with clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)is commonly carried out in fish species by co-injecting a mixture of Cas9 messenger RNA(mRN...Generation of mutants with clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)is commonly carried out in fish species by co-injecting a mixture of Cas9 messenger RNA(mRNA)or protein and transcribed guide RNA(gRNA).However,the appropriate expression system to produce functional gRNAs in fish embryos and cells is rarely present.In this study,we employed a poly-transfer RNA(tRNA)-gRNA(PTG)system driven by cytomegalovirus(CMV)promoter to target the medaka(Oryzias latipes)endogenous gene tyrosinase(tyr)or paired box 6.1(pax6.1)and illustrated its function in a medaka cell line and embryos.The PTG system was combined with the CRISPR/Cas9 system under high levels of promoter to successfully induce gene editing in medaka.This is a valuable step forward in potential application of the CRISPR/Cas9 system in medaka and other teleosts.展开更多
Balbiani body （BB） is a large distinctive organelle aggregate uniquely present in developing oocytes of diverse animal species. BB is thought as a stage-specific structure that resembles germ plasm, the cytoplasmic ...Balbiani body （BB） is a large distinctive organelle aggregate uniquely present in developing oocytes of diverse animal species. BB is thought as a stage-specific structure that resembles germ plasm, the cytoplasmic organelle of germ cells. The role and function of BB have remained speculative because of a highly dynamic structure and a lack of genetic and molecular data. BB has been found to contain proteins and RNAs, none of them -- except the zebrafish foxH1 RNA, is or encodes a transcription factor. Here we report in the fish medaka （Oryzias latipes） that RNAs encoding microphthalmia-associated transcription factor （Mitf） are prominent components of the BB. By fluorescence in situ hybridization on ovarian section, we revealed that the transcripts of both mitfl and mitf2 genes concentrated in the BB, in which they co-localized with the dazl RNA, a definitive BB marker highly conserved in vertebrates. Therefore, the mitfproduct may play dual roles in germ gene transcription and BB formation and/or function in this organism. Our data provide the second evidence that the RNA of a transcription factor can be a prominent component of the BB in a vertebrate.展开更多
基金supported by the Southern Marine Science and Engineering Guangdong Laboratory(Guangzhou)(No.SMSEGL20SC02)the National Natural Science Foundation of China(No.41977371)+1 种基金a Project grant from the Shenzhen and Technology Innovation Commission(No.JCYJ20170818094137791)supported by the National Institute of Environmental Health Sciences of the National Institutes of Health under award number 1R15ES032936-01.
文摘Benzo[a]pyrene(BaP),a ubiquitous pollutant,raises environmental health concerns due to induction of bone toxicity in the unexposed offspring.Exposure of F0 ancestor medaka(Oryzias latipes)to 1μg/L BaP for 21 days causes reduced vertebral bone thickness in the unexposed F3 male offspring.To reveal the inherited modifications,osteoblast(OB)abundance and molecular signaling pathways of transgenerational BaP-induced bone thinning were assessed.Histomorphometric analysis showed a reduction in OB abundance.Analyses of the miRNA and mRNA transcriptomes revealed the dysregulation of Wnt signaling(frzb/ola-miR-1–3p,sfrp5/ola-miR-96–5p/miR-455–5p)and bone morphogenetic protein(Bmp)signaling(bmp3/ola-miR-96–5p/miR-181b-5p/miR-199a-5p/miR-205–5p/miR-455–5p).Both pathways are major indicators of impaired bone formation,while the altered Rank signaling in osteoclasts(c-fos/miR-205–5p)suggests a potentially augmented bone resorption.Interestingly,a typical BaP-responsive pathway,the Nrf2-mediated oxidative stress response(gst/ola-miR-181b-5p/miR-199a-5p/miR-205),was also affected.Moreover,mRNA levels of epigenetic modification enzymes(e.g.,hdac6,hdac7,kdm5b)were found dysregulated.The findings indicated that epigenetic factors(e.g.,miRNAs,histone modifications)may directly regulate the expression of genes associated with transgenerational BaP bone toxicity and warrants further studies.The identified candidate genes and miRNAs may serve as potential biomarkers for BaP-induced bone disease and as indicators of historic exposures in wild fish for conservation purposes.
基金the National Natural Science Foundation of China(31972809)China Agriculture Research System(CARS-46)the National Key R&D Program of China(2019YFD0900500).
文摘In mammals,lipoprotein lipase(LPL)has been found to play an important role in lipid mentalismand deposition.LPL deficiency in humans(Homo sapiens)and mice(Mus musculus)tends to cause hypertriglyceridemia.The lpl gene is not expressed in adult mammalian liver,but is in adult fish liver.The functions provided by the lpl gene are diverse in vertebrates.Here,we knocked out the lpl gene in Japanese medaka(Oryzias latipes)with the CRISPR/Cas9 system.The lpl-knockout(KO)homozygous individuals showed severe developmental defects with an extremely emaciated and deformedbody and onlyaccounted for about5%of the F2fish.This is consistentwiththefindings inmicebut disaccords with the results for zebrafish(Danio rerio).Compared with wild-type(WT)madaka,the mRNA level of lpl in lpl-KO heterozygous mutant was significantly higher in the muscle,showed no significant difference in the liver,and was significantly lower in the heart.Under lpl heterozygous deficiency,the relative area of Oil RedO and triglycerides(TG)level in the liver,heart and muscle tissue covaried with levels of lpl mRNA in medaka.The lpl heterozygous deficiency did not affect the levelsofTG,low-density lipoprotein cholesterol(LDL-C),high-density lipoprotein cholesterol(HDL-C)and total cholesterol(TC)in the plasma of medaka,which is inconsistent with the findings in mammals.In general,the lpl gene plays an important role in the growth and development and is closely related to lipid deposition of medaka.
基金This study was supported by the National Natural Science Foundation of China(Nos.31771648 and 31672653)the Scientific Research Foundation of Jimei University(No.ZQ2020003)the National Key Basic Research Program of China(No.2013CB967700).
文摘Generation of mutants with clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)is commonly carried out in fish species by co-injecting a mixture of Cas9 messenger RNA(mRNA)or protein and transcribed guide RNA(gRNA).However,the appropriate expression system to produce functional gRNAs in fish embryos and cells is rarely present.In this study,we employed a poly-transfer RNA(tRNA)-gRNA(PTG)system driven by cytomegalovirus(CMV)promoter to target the medaka(Oryzias latipes)endogenous gene tyrosinase(tyr)or paired box 6.1(pax6.1)and illustrated its function in a medaka cell line and embryos.The PTG system was combined with the CRISPR/Cas9 system under high levels of promoter to successfully induce gene editing in medaka.This is a valuable step forward in potential application of the CRISPR/Cas9 system in medaka and other teleosts.
基金supported by the National Research Foundation Singapore(R-154-000-529-281)Biomedical Research Council of Singapore(R154-000-427-305)
文摘Balbiani body （BB） is a large distinctive organelle aggregate uniquely present in developing oocytes of diverse animal species. BB is thought as a stage-specific structure that resembles germ plasm, the cytoplasmic organelle of germ cells. The role and function of BB have remained speculative because of a highly dynamic structure and a lack of genetic and molecular data. BB has been found to contain proteins and RNAs, none of them -- except the zebrafish foxH1 RNA, is or encodes a transcription factor. Here we report in the fish medaka （Oryzias latipes） that RNAs encoding microphthalmia-associated transcription factor （Mitf） are prominent components of the BB. By fluorescence in situ hybridization on ovarian section, we revealed that the transcripts of both mitfl and mitf2 genes concentrated in the BB, in which they co-localized with the dazl RNA, a definitive BB marker highly conserved in vertebrates. Therefore, the mitfproduct may play dual roles in germ gene transcription and BB formation and/or function in this organism. Our data provide the second evidence that the RNA of a transcription factor can be a prominent component of the BB in a vertebrate.
