Fus在斑马鱼生长发育及两性生长异形中的功能（英文）
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摘要
为研究肉瘤融合基因(fus)在鱼类中的功能,在斑马鱼中利用CRISPR/Cas9基因突变技术使fus基因产生移码突变。fus~(-/-)纯合突变体斑马鱼发育正常并且完全可育,但体长和体重在幼鱼和成年阶段都小于野生型斑马鱼。此外,相较于野生型斑马鱼, fus~(-/-)纯合突变体斑马鱼不存在雌鱼身体比例大于雄鱼的性别异形现象。在fus~(-/-)纯合突变体斑马鱼早期幼鱼发育阶段,一些生长相关基因包括gh1、ghra、igf1、igf2a、stat5.1和socs6的表达水平显著低于野生型斑马鱼但其运动能力并未受到影响。因此,不同于fus基因在哺乳动物中的功能,它在斑马鱼中不参与运动神经元的发育,但在调节鱼类躯体生长发育与两性生长异形方面有着重要的功能。
The Fused in sarcoma gene(FUS) is a RNA-binding protein implicated in the regulation of transcription and pre-mRNA splicing. Mutations in FUS lead to neurodegenerative diseases including ALS(amyotrophic lateral sclerosis) in mammals. However, little is known about fus function in fish species. Here,we generated frame-shift alleles in the zebrafish fus~(-/-) gene using CRISPR/Cas9 mutagenesis. Homozygous fuszebrafish developed normally and were fully fertile, except that their body size and body weight were smaller than that of wild-type zebrafish at both the larvae and adult stages. In addition, the female-biased sexual size dimorphism in adult zebrafish was also eliminated when fus was mutated. The expression levels of several growth-related genes including gh1, ghra, igf1, igf2 a, stat5.1 and socs6 were significantly reduced in fus~(-/-) zebrafish larvae compared with the wild-type. However, there was no effect on locomotor activity in fus~(-/-) zebrafish. Therefore, different from its functions in mammals, fus is not associated with motoneuron development and has a divergent function in regulating somatic growth and sexual size dimorphism in fish species.
The Fused in sarcoma gene(FUS) is a RNA-binding protein implicated in the regulation of transcription and pre-mRNA splicing. Mutations in FUS lead to neurodegenerative diseases including ALS(amyotrophic lateral sclerosis) in mammals. However, little is known about fus function in fish species. Here,we generated frame-shift alleles in the zebrafish fus~(-/-) gene using CRISPR/Cas9 mutagenesis. Homozygous fuszebrafish developed normally and were fully fertile, except that their body size and body weight were smaller than that of wild-type zebrafish at both the larvae and adult stages. In addition, the female-biased sexual size dimorphism in adult zebrafish was also eliminated when fus was mutated. The expression levels of several growth-related genes including gh1, ghra, igf1, igf2 a, stat5.1 and socs6 were significantly reduced in fus~(-/-) zebrafish larvae compared with the wild-type. However, there was no effect on locomotor activity in fus~(-/-) zebrafish. Therefore, different from its functions in mammals, fus is not associated with motoneuron development and has a divergent function in regulating somatic growth and sexual size dimorphism in fish species.
引文
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[2]Armstrong G A,Drapeau P.Loss and gain of FUS function impair neuromuscular synaptic transmission in a genetic model of ALS[J].Human Molecular Genetics,2013,22(21):4282-4292
[3]Yamazaki T,Chen S,Yu Y,et al.FUS-SMN protein interactions link the motor neuron diseases ALS and SMA[J].Cell Reports,2012,2(4):799-806
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[7]Trautmann M,Menzel J,Bertling C,et al.FUS-DDIT3 fusion protein driven IGF-IR signaling is a therapeutic target in myxoid liposarcoma[J].Clinical Cancer Research,2017,23(20):6277-6238
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[10]Kabashi E,Bercier V,Lissouba A,et al.FUS and TARDBPbut not SOD1 interact in genetic models of amyotrophic lateral sclerosis[J].PLoS Genetics,2011,7(8):e1002214
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[13]Xiong S,Wu J,Jing J,et al.Loss of stat3 function leads to spine malformation and immune disorder in zebrafish[J].Science Bulletin,2017,62(3):185-196
[14]Jing J,Xiong S,Li Z,et al.A feedback regulatory loop involving p53/miR-200 and growth hormone endocrine axis controls embryo size of zebrafish[J].Scientific Reports,2015,5:15906
[15]Dranow D B,Tucker R P,Draper B W.Germ cells are required to maintain a stable sexual phenotype in adult zebrafish[J].Developmental Biology,2013,376(1):43-50
[16]Mei J,Yue H M,Li Z,et al.C1q-like Factor,a target of mir-430,regulates primordial germ cell development in early embryos of Carassius auratus[J].International Journal of Biological Sciences,2014,10(1):15-24
[17]Xiong S,Mei J,Huang P,et al.Essential roles of stat5.1/stat5b in controlling fish somatic growth[J].Journal of Genetics and Genomics,2017,44(12):577-585
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[19]Mei J,Gui J F.Genetic basis and biotechnological manipulation of sexual dimorphism and sex determination in fish[J].Science China Life Sciences,2015,58(2):124-136
[20]Leroith D,Nissley P.Knock your SOCS off![J].Journal of Clinical Investigation,2005,115(2):233-236
[21]Shinsuke I,Akio M,Yusuke F,et al.Position-dependent FUS-RNA interactions regulate alternative splicing events and transcriptions[J].Scientific Reports,2012,2(30):529
[22]Nakaya T,Alexiou P,Maragkakis M,et al.FUS regulates genes coding for RNA-binding proteins in neurons by binding to their highly conserved introns[J].RNA-a Publication of the RNA Society,2013,19(4):498-509
[23]Masuda A,Takeda J,Okuno T,et al.Position-specific binding of FUS to nascent RNA regulates mRNA length[J].Genes&Development,2015,29(10):1045-1057
[24]Baechtold H,Kuroda M,Sok J,et al.Human 75-kDa DNA-pairing protein is identical to the pro-oncoprotein TLS/FUSand is able to promote D-loop formation[J].Journal of Biological Chemistry,1999,274(48):34337-34342
[25]Kwiatkowski TJ Jr,Bosco D A,Leclerc A L,et al.Mutations in the FUS/TLS gene on chromosome 16 cause familial amyotrophic lateral sclerosis[J].Science,2009,323(5918):1205-1208
[26]Kino Y,Washizu C,Kurosawa M,et al.FUS/TLS deficiency causes behavioral and pathological abnormalities distinct from amyotrophic lateral sclerosis[J].Acta Neuropathologica Communications,2015,3(1):24
[27]Hicks G G,Singh N,Nashabi A,et al.Fus deficiency in mice results in defective B-lymphocyte development and activation,high levels of chromosomal instability and perinatal death[J].Nature Genetics,2000,24(2):175-179
[28]Rossi A,Kontarakis Z,Gerri C,et al.Genetic compensation induced by deleterious mutations but not gene knockdowns[J].Nature,2015,524(7564):230-233
[29]Li-Villarreal N,Forbes M M,Loza A J,et al.Dachsous1b cadherin regulates actin and microtubule cytoskeleton during early zebrafish embryogenesis[J].Development,2015,142(15):2704-2718
[30]Campbell P D,Heim A E,Smith M Z,et al.Kinesin-1 interacts with Bucky ball to form germ cells and is required to pattern the zebrafish body axis[J].Development,2015,142(17):2996-3008
[31]Ma W,Wu J,Zhang J,et al.Sex differences in the expression of GH/IGF axis genes underlie sexual size dimorphism in the yellow catfish(Pelteobagrus fulvidraco)[J].Science China,2016,59(4):431
[32]Zhang J,Ma W,He Y,et al.Sex biased expression of ghrelin and GHSR associated with sexual size dimorphism in yellow catfish[J].Gene,2016,578(2):169-176
[33]Zhang J,Ma W,He Y,et al.Potential contributions of miR-200a/-200b and their target gene-leptin to the sexual size dimorphism in yellow catfish[J].Frontiers in Physiology,2017,8:970
[34]Degani G,Tzchori I,Yom-Din S,et al.Growth differences and growth hormone expression in male and female European eels[Anguilla anguilla(L.)][J].General&Comparative Endocrinology,2003,134(1):88
[35]Reindl K M,Kittilson J D,Bergan H E,et al.Growth hormone-stimulated insulin-like growth factor-1 expression in rainbow trout(Oncorhynchus mykiss)hepatocytes is mediated by ERK,PI3K-AKT,and JAK-STAT[J].American Journal of Physiology Regulatory Integrative&Comparative Physiology,2011,301(1):R236
[36]Trautmann M,Menzel J,Bertling C,et al.FUS-DDIT3 fusion protein-driven IGF-IR signaling is a therapeutic target in myxoid liposarcoma[J].Clinical Cancer Research,2017,23(20):6227-6238
[2]Armstrong G A,Drapeau P.Loss and gain of FUS function impair neuromuscular synaptic transmission in a genetic model of ALS[J].Human Molecular Genetics,2013,22(21):4282-4292
[3]Yamazaki T,Chen S,Yu Y,et al.FUS-SMN protein interactions link the motor neuron diseases ALS and SMA[J].Cell Reports,2012,2(4):799-806
[4]Takata T.A neuropathological report of familial amyotrophic lateral sclerosis with an R521S FUS/TLS mutation[J].Journal of the Neurological Sciences,2017,381:714
[5]Kuroda M,Sok J,Webb L,et al.Male sterility and enhanced radiation sensitivity in TLS-/-mice[J].Embo Journal,2014,19(3):453-462
[6]Dichmann D S,Harland R M.Fus/TLS orchestrates splicing of developmental regulators during gastrulation[J].Genes&Development,2012,26(12):1351-1363
[7]Trautmann M,Menzel J,Bertling C,et al.FUS-DDIT3 fusion protein driven IGF-IR signaling is a therapeutic target in myxoid liposarcoma[J].Clinical Cancer Research,2017,23(20):6277-6238
[8]Luisa C,NicolòR,Paolo P,et al.IGF1 is a common target gene of ewing’s sarcoma fusion proteins in mesenchymal progenitor cells[J].PLoS One,2008,3(7):e2634
[9]Ward C L,Boggio K J,Johnson B N,et al.A loss of FUS/TLS function leads to impaired cellular proliferation[J].Cell Death&Disease,2014,5(12):e1572
[10]Kabashi E,Bercier V,Lissouba A,et al.FUS and TARDBPbut not SOD1 interact in genetic models of amyotrophic lateral sclerosis[J].PLoS Genetics,2011,7(8):e1002214
[11]Lebedeva S,de Jesus Domingues A M,Butter F,et al.Characterization of genetic loss-of-function of Fus in zebrafish [J].RNA Biology,2017,14(1):29-35
[12]Kimmel C B,Ballard W W,Kimmel S R,et al.Stages of embryonic development of the zebrafish[J].Developmental Dynamics,1995,203(3):253-310
[13]Xiong S,Wu J,Jing J,et al.Loss of stat3 function leads to spine malformation and immune disorder in zebrafish[J].Science Bulletin,2017,62(3):185-196
[14]Jing J,Xiong S,Li Z,et al.A feedback regulatory loop involving p53/miR-200 and growth hormone endocrine axis controls embryo size of zebrafish[J].Scientific Reports,2015,5:15906
[15]Dranow D B,Tucker R P,Draper B W.Germ cells are required to maintain a stable sexual phenotype in adult zebrafish[J].Developmental Biology,2013,376(1):43-50
[16]Mei J,Yue H M,Li Z,et al.C1q-like Factor,a target of mir-430,regulates primordial germ cell development in early embryos of Carassius auratus[J].International Journal of Biological Sciences,2014,10(1):15-24
[17]Xiong S,Mei J,Huang P,et al.Essential roles of stat5.1/stat5b in controlling fish somatic growth[J].Journal of Genetics and Genomics,2017,44(12):577-585
[18]Gui J F,Zhu Z Y.Molecular basis and genetic improvement of economically important traits in aquaculture animals[J].Chinese Science Bulletin,2012,57(15):1751-1760
[19]Mei J,Gui J F.Genetic basis and biotechnological manipulation of sexual dimorphism and sex determination in fish[J].Science China Life Sciences,2015,58(2):124-136
[20]Leroith D,Nissley P.Knock your SOCS off![J].Journal of Clinical Investigation,2005,115(2):233-236
[21]Shinsuke I,Akio M,Yusuke F,et al.Position-dependent FUS-RNA interactions regulate alternative splicing events and transcriptions[J].Scientific Reports,2012,2(30):529
[22]Nakaya T,Alexiou P,Maragkakis M,et al.FUS regulates genes coding for RNA-binding proteins in neurons by binding to their highly conserved introns[J].RNA-a Publication of the RNA Society,2013,19(4):498-509
[23]Masuda A,Takeda J,Okuno T,et al.Position-specific binding of FUS to nascent RNA regulates mRNA length[J].Genes&Development,2015,29(10):1045-1057
[24]Baechtold H,Kuroda M,Sok J,et al.Human 75-kDa DNA-pairing protein is identical to the pro-oncoprotein TLS/FUSand is able to promote D-loop formation[J].Journal of Biological Chemistry,1999,274(48):34337-34342
[25]Kwiatkowski TJ Jr,Bosco D A,Leclerc A L,et al.Mutations in the FUS/TLS gene on chromosome 16 cause familial amyotrophic lateral sclerosis[J].Science,2009,323(5918):1205-1208
[26]Kino Y,Washizu C,Kurosawa M,et al.FUS/TLS deficiency causes behavioral and pathological abnormalities distinct from amyotrophic lateral sclerosis[J].Acta Neuropathologica Communications,2015,3(1):24
[27]Hicks G G,Singh N,Nashabi A,et al.Fus deficiency in mice results in defective B-lymphocyte development and activation,high levels of chromosomal instability and perinatal death[J].Nature Genetics,2000,24(2):175-179
[28]Rossi A,Kontarakis Z,Gerri C,et al.Genetic compensation induced by deleterious mutations but not gene knockdowns[J].Nature,2015,524(7564):230-233
[29]Li-Villarreal N,Forbes M M,Loza A J,et al.Dachsous1b cadherin regulates actin and microtubule cytoskeleton during early zebrafish embryogenesis[J].Development,2015,142(15):2704-2718
[30]Campbell P D,Heim A E,Smith M Z,et al.Kinesin-1 interacts with Bucky ball to form germ cells and is required to pattern the zebrafish body axis[J].Development,2015,142(17):2996-3008
[31]Ma W,Wu J,Zhang J,et al.Sex differences in the expression of GH/IGF axis genes underlie sexual size dimorphism in the yellow catfish(Pelteobagrus fulvidraco)[J].Science China,2016,59(4):431
[32]Zhang J,Ma W,He Y,et al.Sex biased expression of ghrelin and GHSR associated with sexual size dimorphism in yellow catfish[J].Gene,2016,578(2):169-176
[33]Zhang J,Ma W,He Y,et al.Potential contributions of miR-200a/-200b and their target gene-leptin to the sexual size dimorphism in yellow catfish[J].Frontiers in Physiology,2017,8:970
[34]Degani G,Tzchori I,Yom-Din S,et al.Growth differences and growth hormone expression in male and female European eels[Anguilla anguilla(L.)][J].General&Comparative Endocrinology,2003,134(1):88
[35]Reindl K M,Kittilson J D,Bergan H E,et al.Growth hormone-stimulated insulin-like growth factor-1 expression in rainbow trout(Oncorhynchus mykiss)hepatocytes is mediated by ERK,PI3K-AKT,and JAK-STAT[J].American Journal of Physiology Regulatory Integrative&Comparative Physiology,2011,301(1):R236
[36]Trautmann M,Menzel J,Bertling C,et al.FUS-DDIT3 fusion protein-driven IGF-IR signaling is a therapeutic target in myxoid liposarcoma[J].Clinical Cancer Research,2017,23(20):6227-6238