The Roles of follistatin 1 in Regulation of Zebrafish Fecundity and Sexual Differentiation

Follistatin 1 (Fst1) is a binding protein of activin and some other members of the transforming growth factor beta superfamily. It plays a key role in the regulation of gonadal function in vertebrates. An oocyte-specific promoter, derived from the zona pellucida 3 (zp3) gene, was used to create transgenic fst1 zebrafish (Danio rerio). Three independent oocyte-specific overexpression fst1 transgenic zebrafish lines were generated. Decreased levels of phosphorylated Smad3 were observed in ovarian tissues in fst1 transgenic fish compared with those from their control female siblings. Analyses on the numbers of mature eggs also indicated the attenuated oocyte maturation in the fst1 transgenic fish and in the females administered recombinant human Fst protein. Remarkably, when raised in the same tank with their control siblings, a significantly larger proportion of the fst1 transgenic population developed as males compared to the controls. Moreover, assessing the levels of active caspase 3 in gonadal tissues at 30 days postfertilization, we observed increased levels of apoptosis in the transitioning gonads of the transgenic fish compared to nontransgenic control siblings. Our results demonstrate that zebrafish Fst1 not only acts as an inhibitory binding protein of activin in the regulation of oocyte maturation in adult females but also plays a potential role in the masculinization of juveniles. Overall, the present study contributes to our understanding of the paracrine roles of fst1 as well as normal oocyte maturation and gonadal differentiation.Follistatin 1 (Fst1) is a binding protein of activin and some other members of the transforming growth factor beta superfamily. It plays a key role in the regulation of gonadal function in vertebrates. An oocyte-specific promoter, derived from the zona pellucida 3 (zp3) gene, was used to create transgenic fst1 zebrafish (Danio rerio). Three independent oocyte-specific overexpression fst1 transgenic zebrafish lines were generated. Decreased levels of phosphorylated Smad3 were observed in ovarian tissues in fst1 transgenic fish compared with those from their control female siblings. Analyses on the numbers of mature eggs also indicated the attenuated oocyte maturation in the fst1 transgenic fish and in the females administered recombinant human Fst protein. Remarkably, when raised in the same tank with their control siblings, a significantly larger proportion of the fst1 transgenic population developed as males compared to the controls. Moreover, assessing the levels of active caspase 3 in gonadal tissues at 30 days postfertilization, we observed increased levels of apoptosis in the transitioning gonads of the transgenic fish compared to nontransgenic control siblings. Our results demonstrate that zebrafish Fst1 not only acts as an inhibitory binding protein of activin in the regulation of oocyte maturation in adult females but also plays a potential role in the masculinization of juveniles. Overall, the present study contributes to our understanding of the paracrine roles of fst1 as well as normal oocyte maturation and gonadal differentiation

Neuroendocrine regulation of somatic growth in fishes

Growth is a polygenic trait that is under the influence of multiple physiological pathways regulating energy metabolism and muscle growth. Among the possible growth-regulating pathways in vertebrates, components of the somatotropic axis are thought to have the greatest influence. There is growing body of literature focusing on the somatotropic axis and its role regulating growth in fish. This includes research into growth hormone, upstream hypothalamic hormones, insulin-like growth factors, and downstream signaling molecules. Many of these signals have both somatic effects stimulating the growth of tissues and metabolic effects that play a role in nutrient metabolism. Signals of other endocrine axes exhibit profound effects on the function of the somatotropic axis in vivo. In this review we highlight recent advances in our understanding of the teleost fish endocrine somatotropic axis, including emerging research using genetic modified models. These studies have revealed new aspects and challenges associated with regulation of the important steps of somatic growth

Sufficient Numbers of Early Germ Cells Are Essential for Female Sex Development in Zebrafish

The sex determination for zebrafish is controlled by a combination of genetic and environmental factors. The determination of sex in zebrafish has been suggested to rely on a mechanism that is affected by germ cell-derived signals. To begin our current study, a simplified and efficient germ cell-specific promoter of the dead end (dnd) gene was identified. Utilizing the metrodinazole (MTZ)/bacterial nitroreductase (NTR) system for inducible germ cell ablation, several stable Tg (dnd:NTR-EGFP(-3'UTR)) and Tg (dnd:NTR-EGFP(+3'UTR)) zebrafish lines were then generated with the identified promoter. A thorough comparison of the expression patterns and tissue distributions of endogenous dnd and ntr-egfp transcripts in vivo revealed that the identified 2032-bp zebrafish dnd promoter can recapitulate dnd expression faithfully in stable transgenic zebrafish. The correlation between the levels of the germ cell-derived signals and requirement for maintaining the female fate has been also explored with different durations of the MTZ treatments. Our results revealed the decreasing ratios of female presented in the treated transgenic group are fairly associated with the reducing levels of the early germ cell-derived signals. After the juvenile transgenic fish treated with 5 mM MTZ for 20 days, all MTZ-treated transgenic fish exclusively developed into males with subfertilities. Taken together, our results identified here a simplified and efficient dnd promoter, and provide clear evidence indicating that it was not the presence but the sufficiency of signals derived from germ cells that is essential for female sex development in zebrafish. Our model also provides a unique system for sex control in zebrafish studies

Generation and characterization of gsu alpha:EGFP transgenic zebrafish for evaluating endocrine-disrupting effects

The glycoprotein subunit alpha(gsu alpha) gene encodes the shared a subunit of the three pituitary heterodimeric glycoprotein hormones: follicle-stimulating hormone beta (Fsh beta), luteinizing hormone beta (Lh beta) and thyroid stimulating hormone beta (Tsh beta). In our current study, we identified and characterized the promoter region of zebrafish gsu alpha and generated a stable gsu alpha:EGFP transgenic line, which recapitulated the endogenous gsu alpha expression in the early developing pituitary gland. A relatively conserved regulatory element set is presented in the promoter regions of zebrafish and three other known mammalian gsu alpha promoters. Our results also demonstrated that the expression patterns of the gsu alpha:EGFP transgene were all identical to those expression patterns of the endogenous gsu alpha expression in the pituitary tissue when our transgenic fish were treated with various endocrine chemicals, including forskolin (FSK), SP600125, trichostatin A (TSA), KCIO4, dexamethasone (Dex),beta-estradiol and progesterone. Thus, this gsu alpha:EGFP transgenic fish reporter line provides another valuable tool for investigating the lineage development of gsu alpha-expressing gonadotrophins and the coordinated regulation of various glycoprotein hormone subunit genes. These reporter fish can serve as a novel platform to perform screenings of endocrine-disrupting chemicals (EDCs) in vivo as well. (C) 2014 Elsevier Inc. All rights reserved.The glycoprotein subunit alpha(gsu alpha) gene encodes the shared a subunit of the three pituitary heterodimeric glycoprotein hormones: follicle-stimulating hormone beta (Fsh beta), luteinizing hormone beta (Lh beta) and thyroid stimulating hormone beta (Tsh beta). In our current study, we identified and characterized the promoter region of zebrafish gsu alpha and generated a stable gsu alpha:EGFP transgenic line, which recapitulated the endogenous gsu alpha expression in the early developing pituitary gland. A relatively conserved regulatory element set is presented in the promoter regions of zebrafish and three other known mammalian gsu alpha promoters. Our results also demonstrated that the expression patterns of the gsu alpha:EGFP transgene were all identical to those expression patterns of the endogenous gsu alpha expression in the pituitary tissue when our transgenic fish were treated with various endocrine chemicals, including forskolin (FSK), SP600125, trichostatin A (TSA), KCIO4, dexamethasone (Dex),beta-estradiol and progesterone. Thus, this gsu alpha:EGFP transgenic fish reporter line provides another valuable tool for investigating the lineage development of gsu alpha-expressing gonadotrophins and the coordinated regulation of various glycoprotein hormone subunit genes. These reporter fish can serve as a novel platform to perform screenings of endocrine-disrupting chemicals (EDCs) in vivo as well. (C) 2014 Elsevier Inc. All rights reserved

Sept6 Is Required for Ciliogenesis in Kupffer's Vesicle, the Pronephros, and the Neural Tube during Early Embryonic Development

Septins are conserved filament-forming GTP-binding proteins that act as cellular scaffolds or diffusion barriers in a number of cellular processes. However, the role of septins in vertebrate development remains relatively obscure. Here, we show that zebrafish septin 6 (sept6) is first expressed in the notochord and then in nearly all of the ciliary organs, including Kupffer's vesicle (KV), the pronephros, eye, olfactory bulb, and neural tube. Knockdown of sept6 in zebrafish embryos results in reduced numbers and length of cilia in KV. Consequently, cilium-related functions, such as the left-right patterning of internal organs and nodal/spaw signaling, are compromised. Knockdown of sept6 also results in aberrant cilium formation in the pronephros and neural tube, leading to cilium-related defects in pronephros development and Sonic hedgehog (Shh) signaling. We further demonstrate that SEPT6 associates with acetylated alpha-tubulin in vivo and localizes along the axoneme in the cilia of zebrafish pro-nephric duct cells as well as cultured ZF4 cells. Our study reveals a novel role of sept6 in ciliogenesis during early embryonic development in zebrafish.Septins are conserved filament-forming GTP-binding proteins that act as cellular scaffolds or diffusion barriers in a number of cellular processes. However, the role of septins in vertebrate development remains relatively obscure. Here, we show that zebrafish septin 6 (sept6) is first expressed in the notochord and then in nearly all of the ciliary organs, including Kupffer's vesicle (KV), the pronephros, eye, olfactory bulb, and neural tube. Knockdown of sept6 in zebrafish embryos results in reduced numbers and length of cilia in KV. Consequently, cilium-related functions, such as the left-right patterning of internal organs and nodal/spaw signaling, are compromised. Knockdown of sept6 also results in aberrant cilium formation in the pronephros and neural tube, leading to cilium-related defects in pronephros development and Sonic hedgehog (Shh) signaling. We further demonstrate that SEPT6 associates with acetylated alpha-tubulin in vivo and localizes along the axoneme in the cilia of zebrafish pro-nephric duct cells as well as cultured ZF4 cells. Our study reveals a novel role of sept6 in ciliogenesis during early embryonic development in zebrafish

Zebrafish pituitary gene expression before and after sexual maturation

Sexual maturation and somatic growth cessation are associated with adolescent development, which is precisely controlled by interconnected neuroendocrine regulatory pathways in the endogenous endocrine system. The pituitary gland is one of the key regulators of the endocrine system. By analyzing the RNA sequencing (RNA-seq) transcriptome before and after sexual maturation, in this study, we characterized the global gene expression patterns in zebrafish pituitaries at 45 and 90 days post-fertilization (dpf). A total of 15 043 annotated genes were expressed in the pituitary tissue, 3072 of which were differentially expressed with a greater than or equal to twofold change between pituitaries at 45 and 90 dpf. In the pituitary transcriptome, the most abundant transcript was gh. The expression levels of gh remained high even after sexual maturation at 90 dpf. Among the eight major pituitary hormone genes, lhb was the only gene that exhibited a significant change in its expression levels between 45 and 90 dpf. Significant changes in the pituitary transcripts included genes involved in the regulation of immune responses, bone metabolism, and hormone secretion processes during the juvenile-sexual maturity transition. Real-time quantitative PCR analysis was carried out to verify the RNA-seq transcriptome results and demonstrated that the expression patterns of the eight major pituitary hormone genes did not exhibit a significant gender difference at 90 dpf. For the first time, we report the quantitative global gene expression patterns at the juvenile and sexual maturity stages. These expression patterns may account for the dynamic neuroendocrine regulation observed in body metabolism.Sexual maturation and somatic growth cessation are associated with adolescent development, which is precisely controlled by interconnected neuroendocrine regulatory pathways in the endogenous endocrine system. The pituitary gland is one of the key regulators of the endocrine system. By analyzing the RNA sequencing (RNA-seq) transcriptome before and after sexual maturation, in this study, we characterized the global gene expression patterns in zebrafish pituitaries at 45 and 90 days post-fertilization (dpf). A total of 15 043 annotated genes were expressed in the pituitary tissue, 3072 of which were differentially expressed with a greater than or equal to twofold change between pituitaries at 45 and 90 dpf. In the pituitary transcriptome, the most abundant transcript was gh. The expression levels of gh remained high even after sexual maturation at 90 dpf. Among the eight major pituitary hormone genes, lhb was the only gene that exhibited a significant change in its expression levels between 45 and 90 dpf. Significant changes in the pituitary transcripts included genes involved in the regulation of immune responses, bone metabolism, and hormone secretion processes during the juvenile-sexual maturity transition. Real-time quantitative PCR analysis was carried out to verify the RNA-seq transcriptome results and demonstrated that the expression patterns of the eight major pituitary hormone genes did not exhibit a significant gender difference at 90 dpf. For the first time, we report the quantitative global gene expression patterns at the juvenile and sexual maturity stages. These expression patterns may account for the dynamic neuroendocrine regulation observed in body metabolism