Establishment Of The Zebrafish Tac3a^-/-Tac3b^-/-Gnrh3^-/- Mutant Lines And Phenotype Assessment

Neurokinin B?NKB?and Gonadotropin-releasing hormone?GnRH?play pivotal roles in the regulation of reproduction in vertebrates.NKB,a member of the tachykin-in-peptide family,is encoded by tac2/3 gene.Previous study in humans has demonstr-ated that NKB/NK3R null mutations result in isolated hypogonatropic hypogonadism?IHH?,and similar phenotypes were also found in mice when genetical knockout of tac2/3 gene.GnRH is the major regulator of hypotahlamic-pituitary-gonadal?HPG?axis in vertebrate,and is known to be the critical regulator in stimulating the release and synthesis of follicle-stimulating hormone?FSH?and luteinizing hormone?LH?.In order to reveal their authentic functions in fish,in this study,two genes encoding NKB and the gnrh3 gene were functional mutated in zebrafish?Danio rerio?using the Transcription Activator-like Effector Nucleases?TALEN?technology.We established tac3a^-/-tac3b^-/-,tac3a^-/-gnrh3^-/-and tac3a^-/-tac3b^-/-gnrh3^-/-zebrafish lines,and investigated their reproductive performance and ontogeny.The main topics of this study were summarized as follows:1.Through breeding and screening by sequencing,we had successfully established the homozygous tac3a,tac3b and gnrh3 gene knockout lines from the mosaic P0 generation fish,respectively.The mutations had induced new stop codons just in the coding sequence for the mature peptides.The tac3a^-/-tac3b^-/-,tac3a^-/-gnrh3^-/-and tac3a^-/-tac3b^-/-gnrh3^-/-zebrafish lines were obtained from homozygous tac3a,tac3b and gnrh3 gene knockout lines.Furthermore,RT-PCR analysis indicated that tac3a,tac3b and gnrh3 genes were successfully knocked out in the corresponding mutant zebrafish lines.2.In tac3a^-/-tac3b^-/-zebrafish lines,the development of growth and reproduction were not affected.The spermatogenesis and folliculogenesis were not impaired in mature individuals.Gene expression studies found that the compensation mechanism was found between tac3a and tac3b genes.In tac3a^-/-gnrh3^-/-zebrafish lines,the development of growth was slower than the wildtype,but spermatogenesis and folliculogenesis were also not impaired in mature individuals.3.The growth of tac3a^-/-tac3b^-/-gnrh3^-/-zebrafish were slower than the wildtype at the stage of sexual differentiation,puberty onset and sexual maturation.Further studies found that tac3a^-/-tac3b^-/-gnrh3^-/-zebrafish line leaded to all-male-offspring because it failed to accomplish ovarian differentiation.However,NKB and LH releasing hormone?LHRH?peptides treatment could partically rescue this phenotype.Our results indicate that the absence of both NKB and GnRH3 signalling can impair the sexual differentiation in zebrafish.For the first time,the tac3a;tac3b;gnrh3deficient model has been established in vertebrates.It provides an effective model for the study of NKB and GnRH3,which helps to reveal new mechanisms for the regulation of sexual differentiation in animals.