Application of RNAi Technology to the Inhibition of Zebrafish GtHα, FSHβ, and LHβ Expression and to Functional Analyses

Huang, Wei‐Tung; Hsieh, Jung-Chen; Chiou, Ming-Jyun; Chen, Jyh-Yih; Wu, Jen‐Leih; Kuo, Ching-Ming

doi:10.2108/zsj.25.614

Cited by 17 publications

(8 citation statements)

References 34 publications

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“…The abnormal phenotypes were likely caused by the lack of Fsh production, because there was a strong correlation between the expression of fshb and the development of these phenotypes in fshb-MO fish. In agreement with our results, a recent study in the zebrafish showed that knockdown of cga (the common a subunit of Fsh, Lh, and Tsh) expression by RNA interference (RNAi) or morpholino also caused severe developmental defects in the embryos, including truncated tail and enlarged heart [41].…”

Section: Temporal Expression Of Gonadotropins In Zebrafishsupporting

confidence: 93%

Ontogenic Expression Profiles of Gonadotropins (fshb and lhb) and Growth Hormone (gh) During Sexual Differentiation and Puberty Onset in Female Zebrafish1

Chen

2012

Biology of Reproduction

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In the zebrafish model, the ontogenic expression profiles of all pituitary hormones have been reported except gonadotropins, partly because they are not supposed to be expressed in the embryonic stage. The spatiotemporal expression patterns of gonadotropins, namely follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh), in this species therefore remain largely unknown. As the master hormones controlling reproduction, the information on this issue would be valuable for understanding the roles of gonadotropins in early sexual development. Using double-colored fluorescent in situ hybridization (FISH) and real-time quantitative PCR (qPCR), this study was undertaken to analyze the ontogenic expression patterns of FSHbeta (fshb) and LHbeta (lhb) subunits in the zebrafish pituitary, with particular emphasis on the stage of sexual differentiation (∼25-30 dpf [days postfertilization]) and puberty onset (∼45 dpf). As a control, growth hormone (gh) was also examined throughout the study. The zebrafish were collected at different time points of early development, including 4, 5, 6, 8, 10, 13, 16, 19, 22, 25, 28, 38, 48, and 53 dpf. The head of each fish, including the brain and pituitary, was sampled for double-colored FISH analysis, whereas the body was fixed for histological examination of sex and gonadal developmental stage. Our results showed that the expression of fshb started much earlier than that of lhb, with its mRNA signal detectable (∼2-3 cells per pituitary) shortly after hatching (4 dpf). In contrast, lhb expression became detectable much later, at the time of sex differentiation (∼25 dpf). In female zebrafish, the first morphological sign for puberty is the first wave of follicle transition from the primary growth to previtellogenic stage, which occurs around 45 dpf and is marked by the appearance of cortical alveoli in the oocytes. Interestingly, the number of lhb-expressing cells was very low (∼5-6 cells per pituitary) before this transition but increased dramatically during and after the transition. In contrast, the expression of fshb was abundant before puberty, with only a slight increase in cell number during puberty onset. The increased expression of fshb and lhb at puberty was also supported by real-time qPCR analysis at the single pituitary level. Interestingly, the fshb-expressing cells changed their spatial distribution significantly during puberty, from a predominantly peripheral to a central location. As the control, the expression of gh was abundant throughout prepubertal and pubertal periods. Our results strongly suggest an important role for Lh at the puberty onset of female zebrafish, similar to the situation in mammals, and its expression could be a sign for puberty at the pituitary level. However, the significance of the location change of Fsh cells during this period will be interesting to investigate.

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Section: Temporal Expression Of Gonadotropins In Zebrafishsupporting

confidence: 93%

Ontogenic Expression Profiles of Gonadotropins (fshb and lhb) and Growth Hormone (gh) During Sexual Differentiation and Puberty Onset in Female Zebrafish1

Chen

2012

Biology of Reproduction

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“…The expression of GtH mRNA was more efficiently suppressed by GtHα RNAi expression compared with the other two subunits. They suggested that GtHα RNAi may be an essential factor in the further development of sterility technology of transgenic fish for biosafety purposes [76]. However, we discovered a new alternative splicing transcript of the GtH alpha subunit from the pituitary glands and ovaries of common carp.…”

Section: Reproductive Containment Strategies Of Transgenic Fishmentioning

confidence: 81%

“…Huang et al [76] reported using RNA interference (RNAi) to knockdown GtHα, FSHβ and LHβ genes. The RNA or protein expression levels of the GtH genes were monitored by RT-PCR, Southern blotting, and GFP analyses.…”

Section: Reproductive Containment Strategies Of Transgenic Fishmentioning

confidence: 99%

Integration mechanisms of transgenes and population fitness of GH transgenic fish

Zhang

2010

Sci. China Life Sci.

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It has been more than 20 years since the first batch of transgenic fish was produced. Five stable germ-line transmitted growth hormone (GH) transgenic fish lines have been generated. This paper reviews the mechanisms of integration and gene targeting of the transgene, as well as the viability, reproduction and transgenic approaches for the reproductive containment of GH-transgenic fish. Further, we propose that it should be necessary to do the following studies, in particularly, of the breeding of transgenic fish: to assess the fitness of transgenic fish in an aqueous environment with a large space and a complex structure; and to develop a controllable on-off strategy of reproduction in transgenic fish. transgene, integration mechanism, GH transgenic fish, population fitness Citation:Hu [6,7]. In cooperation with Hunan Normal University, we produced a transgenic triploid fish with a growth rate that is 15% faster than that of the control carp, with a forage utilization rate increased by 11.1%. The transgenic triploid fish is completely sterile [8]. Such completely sterile transgenic triploid carp such as the all-fish GH transgenic carp may be suitable for industrialized conditions. The US Food and Drug Administration (FDA) is evaluating the ecological effects of current and potential uses of the field release of GH transgenic Atlantic salmon, which would promote the commercial farming of transgenic fish. No transgenic animals have been released into a natural environment for commercialized cultivation as food. Transgenic fish would be the breakthrough model as the first commercial farming transgenic animal. We review the mechanisms of integration and controllable regulatory expression of transgenes and the population fitness assessments of GH transgenic fish. We will conclude by providing suggestions for the thorough study of genetic engineering in breeding fish for potential commercial uses.

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“…Data obtained in this study would also be a useful basis for future application of living fluorescence color reporters to genomic and transgenic researches with this species. Also the transgenic O. dancena lines to stably express the fluorescent reporter in whole body throughout entire lifespan might serve as a fundamental platform to facilitate the set-up and/or designation of the developmental stage-or tissue-specific RNA interference experiments (see Xie et al 2005;Huang et al 2008). …”

Section: Regulation Of Rfp Transgene At Transcription Level In Transgmentioning

confidence: 99%

Functional ability of cytoskeletal β-actin regulator to drive constitutive and ubiquitous expression of a fluorescent reporter throughout the life cycle of transgenic marine medaka Oryzias dancena

et al. 2011

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Marine medaka Oryzias dancena, a candidate model organism, represents many attractive merits as a material for experimental transgenesis and/or heterologous expression assay particularly in the field of ecotoxicology and developmental biology. In this study, cytoskeletal β-actin gene was characterized from O. dancena and the functional capability of its promoter to drive constitutive expression of foreign reporter protein was evaluated. The O. dancena β-actin gene possessed a conserved genomic organization of vertebrate major cytoplasmic actin genes and the bioinformatic analysis of its 5'-upstream regulatory region predicted various transcription factor binding motifs. Heterologous expression assay using a red fluorescent protein (RFP) reporter construct driven by the O. dancena β-actin regulator resulted in stunningly bright expression of red fluorescence signals in not only microinjected embryos but also grown-up transgenic adults. Although founder transgenics exhibited mosaic patterns of RFP expression, transgenic offspring in subsequent generations displayed a vivid and uniform expression of RFP continually from embryos to adults. Based on the blot hybridization assays, two transgenic lines established in this study were proven to possess high copy numbers of transgene integrants (approximately 240 and 34 copies, respectively), and the transgenic genotype in both lines could successfully be passed stably up to three generations, although the rate of transgene transmission in one of the two transgenic lines was significantly lower than expected Mendelian ratio. Significant red fluorescence color could be ubiquitously observable in all the tissues or organs of the transgenics. Quantitative real-time RT-PCR represented that the expression pattern of transgene under the regulation of β-actin promoter would resemble, in overall, the regulation of endogenous β-actin gene in adult tissues, although putative mechanism for competitive or independent regulation between transgene and endogenous gene could also be found in several tissues. Results from this study undoubtedly indicate that the O. dancena β-actin promoter would be powerful enough to fluorescently visualize most cell types in vivo throughout its whole lifespan. This study could be a useful start point for a variety of transgenic experiments with this species concerning the constitutive expression of living fluorescent color reporters and other foreign proteins.

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Application of RNAi Technology to the Inhibition of Zebrafish GtHα, FSHβ, and LHβ Expression and to Functional Analyses

Cited by 17 publications

References 34 publications

Ontogenic Expression Profiles of Gonadotropins (fshb and lhb) and Growth Hormone (gh) During Sexual Differentiation and Puberty Onset in Female Zebrafish1

Ontogenic Expression Profiles of Gonadotropins (fshb and lhb) and Growth Hormone (gh) During Sexual Differentiation and Puberty Onset in Female Zebrafish1

Integration mechanisms of transgenes and population fitness of GH transgenic fish

Functional ability of cytoskeletal β-actin regulator to drive constitutive and ubiquitous expression of a fluorescent reporter throughout the life cycle of transgenic marine medaka Oryzias dancena

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