Cytochrome P450 (Cyp)17A1 has both 17α-hydroxylase and 17,20-lyase activities, which are involved in the steroidogenic pathway that produces androgens and estrogens. Previously, a phenotype of all-male cyp17a1-deficient zebrafish generated by transcription activatorlike effector nuclease has been reported. In the current study, the mechanisms relating to Cyp17a1 that are involved in the development of sexual traits, especially gonadal differentiation and testicular development, were characterized. We found that the cyp17a1-deficient fish at 3 months postfertilization (mpf) were all fertile males with normal testis and spermatogenesis but compromised male-typical mating behaviors and secondary sex characters (SSCs), including breeding tubercles, body pigmentation, and anal fin coloration. These results demonstrate that spermatogenesis and testicular development are not as susceptible to androgen deficiency compared with the formation of male-typical SSCs and mating behaviors in zebrafish. The differentiation of the juvenile ovary into the mature ovary failed during the critical sexual differentiation stage. This all-male phenotype of the cyp17a1-deficient fish could be restored with testosterone or estradiol treatment. For testicular development in cyp17a1-deficient fish, a gradually increasing number of spermatozoa and testis hypertrophy from 3 to 6 mpf were observed, accompanied by constitutively upregulated pituitary gonadotropin FSH subunit β (fshβ). The hypertrophic testis and enhanced spermatogenesis in the cyp17a1-deficient fish at 6 mpf could be effectively rescued by fshβ depletion. These results confirm that adequate estrogen is essential for maintaining ovarian differentiation, and they provide new insight into the role of FSHβ in male testicular development and spermatogenesis.

Myostatin (MSTN) is a negative regulator of myogenesis in vertebrates. Depletion of mstn resulted in elevated muscle growth in several animal species. However, the report on the complete ablation of mstn in teleost fish has not yet become available. In this study, two independent mstnb-deficient mutant lines in zebrafish were generated with the TALENs technique. In the mstnb-deficient zebrafish, enhanced muscle growth with muscle fibre hyperplasia was achieved. Beginning at the adult stage (80 dpf), the mstnb-deficient zebrafish exhibited increased circumferences and body weights compared to the wild-type sibling control fish. Although the overall total lipid/body weight ratios remained similar between the mstnb-deficient zebrafish and the control fish, the distribution of lipids was altered. The size of the visceral adipose tissues (VAT) became smaller while more lipids accumulated in skeletal muscle in the mstnb-deficient zebrafish than in the wild-type control fish. Based on the transcriptional expression profiles, our results revealed that lipid metabolism, including lipolysis and lipogenesis processes, were highly activated in the mstnb-deficient zebrafish, which indicated the transition of energy metabolism from protein-dependent to lipid-dependent in mstnb-deficient zebrafish. Our mstnb-deficient model could be valuable in understanding not only the growth trait regulation in teleosts but also the mechanisms of teleost energy metabolism.

1α,25(OH)2D3 (vitamin D3) is crucial for mineral homeostasis in mammals, but the precise effects of 1α,25(OH)2D3 in adipose tissue remain to be clarified in vivo. The initial 25-hydroxylation is catalyzed by liver microsomal cytochrome P450 2R1 (CYP2R1), which is conserved in vertebrates. To probe the physiological function(s) of 1α,25(OH)2D3 in teleosts, we generated two independent cyp2r1-deficient zebrafish lines. These mutants exhibit retarded growth and increased obesity, especially in the visceral adipose tissue (VAT). These defects could be rescued with 25(OH)D3 treatments. ChIP-PCR analyses demonstrated that pgc1a is the target of the vitamin D receptor in the liver and VAT of zebrafish. Significantly decreased protein levels of Pgc1a, impaired mitochondrial biogenesis, and free fatty acid oxidation are also observed in the cyp2r1 mutant VAT. Our results demonstrate that regulation of 1α,25(OH)2D3 during lipid metabolism occurs through the regulation of Pgc1a for mitochondrial biogenesis and oxidative metabolism within zebrafish VAT.

Specification of arteries and veins is a key process for establishing functional vasculature during embryogenesis and involves distinctly different signaling mechanisms. Vascular endothelial growth factor-A (VEGFA) is required for differentiation of arteries; however, the upstream angiogenic factor for vein specification is unknown. Klippel–Trenaunay syndrome (KTS) is a congenital vascular disease associated with capillary and venous malformations (VMs), but not with arterial defects. We have previously reported that upregulation of angiogenic factor AGGF1 is associated with KTS, but the molecular mechanism is not clear. Here, we show that AGGF1 is involved in establishing venous identity in zebrafish embryos. Overexpression of AGGF1 led to increased angiogenesis and increased lumen diameter of veins, whereas knockdown of AGGF1 expression resulted in defective vasculogenesis and angiogenesis. Overexpression of AGGF1 increased expression of venous markers (e.g. flt4), but had little effect on arterial markers (e.g. notch5). Knockdown of AGGF1 expression resulted in a loss of venous identity (loss of expression of flt4, ephb4 and dab2), but had no effect on the expression of arterial development. We further show that AGGF1 activates AKT, and that decreased AGGF1 expression inhibits AKT activation. Overexpression of constitutively active AKT rescues the loss of venous identity caused by AGGF1 downregulation. Our study establishes AGGF1 as an angiogenic factor with an important role in the specification of vein identity and suggests that AGGF1-mediated AKT signaling is responsible for establishing venous cell fate. We propose that increased AGGF1 expression leads to increased vein differentiation by inducing activation of AKT signaling, resulting in VMs s in KTS patients.

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.

Prolactin (PRL) is an anterior pituitary hormone with a broad range of functions. Its ability to stimulate lactogenesis, maternal behavior, growth and development, osmoregulation and epithelial ion transport has been reported in many vertebrates. In our present study, we have targeted the zebrafish prl locus via transcription activator-like effector nucleases (TALENs). Two independent targeted mutant lines with premature termination of the putative sequence of PRL peptides were generated. All prl-deficient zebrafish progeny died at 6–16 days post-fertilization stage (dpf) in egg water. However, the prl-deficient larvae thrived and survived through adulthood in brackish water (5175 mg/L ocean salts), without obvious defects in somatic growth or reproduction. When raised in egg water, the expression levels of certain key Na⁺/Cl⁻ cotransporters in the gills and Na⁺/K⁺-ATPase subunits, Na⁺/H⁺ exchangers and Na⁺/Cl⁻ transporters in the pronephros of prl-deficient larvae were down-regulated at 5 dpf, which caused Na⁺/K⁺/Cl⁻ uptake defects in the mutant fish at 6 dpf. Our present results demonstrate that the primary function of zebrafish prl is osmoregulation via governing the uptake and homeostasis of Na⁺, K⁺ and Cl⁻. Our study provides valuable evidence to understand the mechanisms of PRL function better through both phylogenetic and physiological perspectives.