Neurokinin Bs and neurokinin B receptors in zebrafish-potential role in controlling fish reproduction.

The endocrine regulation of vertebrate reproduction is achieved by the coordinated actions of several peptide neurohormones, tachykinin among them. To study the evolutionary conservation and physiological functions of neurokinin B (NKB), we identified tachykinin (tac) and tac receptor (NKBR) genes from many fish species, and cloned two cDNA forms from zebrafish. Phylogenetic analysis showed that piscine Tac3s and mammalian neurokinin genes arise from one lineage. High identity was found among different fish species in the region encoding the NKB; all shared the common C-terminal sequence. Although the piscine Tac3 gene encodes for two putative tachykinin peptides, the mammalian ortholog encodes for only one. The second fish putative peptide, referred to as neurokinin F (NKF), is unique and found to be conserved among the fish species when tested in silico. tac3a was expressed asymmetrically in the habenula of embryos, whereas in adults zebrafish tac3a-expressing neurons were localized in specific brain nuclei that are known to be involved in reproduction. Zebrafish tac3a mRNA levels gradually increased during the first few weeks of life and peaked at pubescence. Estrogen treatment of prepubertal fish elicited increases in tac3a, kiss1, kiss2, and kiss1ra expression. The synthetic zebrafish peptides (NKBa, NKBb, and NKF) activated Tac3 receptors via both PKC/Ca(2+) and PKA/cAMP signal-transduction pathways in vitro. Moreover, a single intraperitoneal injection of NKBa and NKF significantly increased leuteinizing hormone levels in mature female zebrafish. These results suggest that the NKB/NKBR system may participate in neuroendocrine control of fish reproduction.

Targeted gonadotropin-releasing hormone-3 neuron ablation in zebrafish: effects on neurogenesis, neuronal migration, and reproduction.

Hypophysiotropic GnRH neurons are located in the preoptic area and ventral hypothalamus of sexually mature vertebrates. In several species, the embryonic origin of hypophysiotropic GnRH neurons remains unclear. Using the Tg(GnRH3:EGFP) zebrafish line, in which GnRH3 neurons express EGFP, GnRH3 neurons in the olfactory region were specifically and individually ablated during early development using laser pulses. After ablation, the olfactory region maintained the capacity to regenerate GnRH3 neurons. However, this capacity was time-limited. When ablation of GnRH3 cells was conducted at 2 d after fertilization, high regeneration rates were observed, but regeneration capacity significantly decreased when ablation was performed at 4 or 6 d after fertilization. Unilateral GnRH3 neuron ablation results in unilateral soma presence. These unilateral somata are capable of projecting fiber extensions bilaterally. Successful bilateral GnRH3 soma ablation during development resulted in complete lack of olfactory, terminal nerve, preoptic area, and hypothalamic GnRH3 neurons and fibers in 12-wk-old animals. Mature animals lacking GnRH3 neurons exhibited arrested oocyte development and reduced average oocyte diameter. Animals in which GnRH3 neurons were partially ablated exhibited normal oocyte development; however, their fecundity was significantly reduced. These findings demonstrate that the hypophysiotropic GnRH3 populations in zebrafish consist of neurons that originate in the olfactory region during early development. The presence of GnRH3 neurons of olfactory region origin in reproductively mature zebrafish is a prerequisite for normal oocyte development and reproduction.

Cxcl12a-Cxcr4b signaling is important for proper development of the forebrain GnRH system in zebrafish.

Hypothalamic gonadotropin-releasing hormone (GnRH) neurons control pituitary gonadotropin secretion and gametogenesis. In the course of development, these neurons migrate from the olfactory placode to the hypothalamus. The precise molecular mechanism of this neuronal migration is unclear. Here, we investigated whether the chemokine receptor, Cxcr4b, and its cognate ligand, Cxcl12a, are required for proper migration of GnRH3 neurons in zebrafish. Deviated GnRH3 axonal projections and neuronal migration were detected in larvae that carry a homozygote cxcr4b mutation. Similarly, knockdown of Cxcr4b or Cxcl12a led to the appearance of abnormal GnRH3 axonal projections and cell migration, including absence of the characteristic lateral crossing of GnRH3 axons at the anterior commissure and optic chiasm. Double-labeling analysis has shown that cxcr4b and cxcl12a are expressed along the GnRH3 migration pathway (i.e. olfactory placode, terminal nerve and the optic chiasm). The results of this study suggest that the Cxcl12a-Cxcr4b ligand-receptor pair are involved in the migration of GnRH3 neurons in zebrafish, and are therefore crucial for the development of this system.

The zebrafish as a model system for forebrain GnRH neuronal development.

Development and function of the forebrain gonadotropin-releasing hormone (GnRH) neuronal system has long been the focus of study in various vertebrate species. This system is crucial for reproduction and an important model for studying tangential neuronal migration. In addition, the finding that multiple forms of GnRH exist in the CNS as well as in non-CNS tissues, coupled with the fact that GnRH fibers project to many CNS regions, implies that GnRH has a variety of functions in addition to its classic reproductive role. The study of the GnRH system and its functions is, however, limited by available model systems and methodologies. The transgenic (Tg) GnRH3:EGFP zebrafish line, in which GnRH3 neurons express EGFP, allows in vivo study of the GnRH3 system in the context of the entire animal. Coupling the use of this line with the attributes and molecular tools available in zebrafish has expanded our ability to study the forebrain GnRH system. Herein, we discuss the use of the Tg(GnRH3:EGFP) zebrafish line as a model for studying forebrain GnRH neurons, both in developing larvae and in sexually mature animals. We also discuss the potential use of this line to study regulation of GnRH3 system development.

Nasal embryonic LHRH factor plays a role in the developmental migration and projection of gonadotropin-releasing hormone 3 neurons in zebrafish.

The initiation of puberty and the functioning of the reproductive system depend on proper development of the hypophysiotropic gonadotropin-releasing hormone (GnRH) system. One critical step in this process is the embryonic migration of GnRH neurons from the olfactory area to the hypothalamus. Using a transgenic zebrafish model, Tg(gnrh3:EGFP), in which GnRH3 neurons and axons are fluorescently labeled, we investigated whether zebrafish NELF is essential for the development of GnRH3 neurons. The zebrafish nelf cDNA was cloned and characterized. During embryonic development, nelf is expressed in GnRH3 neurons and in target sites of GnRH3 projections and perikarya, before the initiation of their migration. Nelf knockdown resulted in a disruption of the GnRH3 system which included absence or misguiding of GnRH3 axonal outgrowth and incorrect or arrested migration of GnRH3 perikarya. These results suggest that Nelf is an important factor in the developmental migration and projection of GnRH3 neurons in zebrafish.

Ontogeny of the GnRH systems in zebrafish brain: in situ hybridization and promoter-reporter expression analyses in intact animals.

The ontogeny of two gonadotropin-releasing-hormone (GnRH) systems, salmon GnRH (sGnRH) and chicken GnRH-II (cGnRH-II), was investigated in zebrafish (Danio rerio). In situ hybridization (ISH) first detected sGnRH mRNA-expressing cells at 1 day post-fertilization (pf) anterior to the developing olfactory organs. Subsequently, cells were seen along the ventral olfactory organs and the olfactory bulbs, reaching the terminal nerve (TN) ganglion at 5-6 days pf. Some cells were detected passing posteriorly through the ventral telencephalon (10-25 days pf), and by 25-30 days pf, sGnRH cells were found in the hypothalamic/preoptic area. Continuous documentation in live zebrafish was achieved by a promoter-reporter expression system. The expression of enhanced green fluorescent protein (EGFP) driven by the sGnRH promoter allowed the earlier detection of cells and projections and the migration of sGnRH neurons. This expression system revealed that long leading processes, presumably axons, preceded the migration of the sGnRH neuron somata. cGnRH-II mRNA expressing cells were initially detected (1 day pf) by ISH analysis at lateral aspects of the midbrain and later on (starting at 5 days pf) at the midline of the midbrain tegmentum. Detection of red fluorescent protein (DsRed) driven by the cGnRH-II promoter confirmed the midbrain expression domain and identified specific hindbrain and forebrain cGnRH-II-cells that were not identified by ISH. The forebrain DsRed-expressing cells seemed to emerge from the same site as the sGnRH-EGFP-expressing cells, as revealed by co-injection of both constructs. These studies indicate that zebrafish TN and hypothalamic sGnRH cell populations share a common embryonic origin and migratory path, and that midbrain cGnRH-II cells originate within the midbrain.