Identification of novel genes expressed in migrating and nonmigrating GnRH neurons

促性腺激素释放激素（GnRH）是一种高度保守的分子，在脊椎动物的生殖中起着重要作用。本研究旨在鉴定控制GnRH神经元分化、迁移和基因表达的分子。为了解决这个问题，我们成功地将免疫荧光定义的激光显微解剖的解剖特异性与实时定量RT-PCR （RT-Q-RT-PCR）的灵敏度结合起来，这使我们能够检测从成熟和未成熟罗非鱼（Oreochromis niloticus）雄性大脑中采集的单个神经元中新颖mrna和GnRH mrna的存在和数量。在这里，我们提供的证据表明，视前GnRH1对性腺成熟很重要，而GnRH2和GnRH3可能在生殖行为或非生殖功能中起补充作用。此外，我们还首次在非哺乳动物脊椎动物中分离并克隆了GPR54。GPR54是一种新的G蛋白偶联受体，据推测对性发育至关重要。罗非鱼GPR54 cDNA全长1131 bp，编码377个氨基酸，与人类GPR54具有56%的同源性。在单细胞水平上，仅在成熟雄性中，GnRH1 mRNA水平与GPR54 mRNA水平呈负相关（P<0.002）。GPR54在成熟GnRH1、GnRH2和GnRH3神经元以及迁移到其最终脑内位置附近的未成熟GnRH3神经元中表达的比例显著高（45.0-60.0%），而在未成熟GnRH1和GnRH2神经元中，只有5.0%的未成熟GnRH1和GnRH2神经元有GPR54转录本（P<0.001）。因此，利用一种新颖的单细胞基因分析技术，我们提供了证据，证明GPR54的表达是GnRH1、GnRH2和GnRH3神经元迁移的“停止信号”，导致细胞生长受到抑制，GnRH分泌受到调节，这对正常的性发育至关重要。

Gonadotropin-releasing hormone(GnRH) is a highly conserved molecule, which is important for reproduction in vertebrate species. The present study was undertaken to identify molecules that control GnRH neuronal differentiation, migration and gene expression. To address this issue, we successfully coupled the anatomic specificity of immunofluorescently defined laser microdissection with the sensitivity of real-time quantitative RT-PCR (RT-Q-RT-PCR), which enabled us to examine the presence and quantity of novel and GnRH mRNAs in individual neurons harvested from the brain of mature and immature males of tilapia, Oreochromis niloticus. Here, we present evidence that shows preoptic GnRH1 is important for gonadal maturation, whereas GnRH2 and GnRH3 might have supplementary roles in reproductive behaviors or nonreproductive functions. Further, we isolated and cloned GPR54 for the first time in a non-mammalian vertebrate. GPR54, a novel G protein-coupled receptor, is speculated to be essential for sexual development. The tilapia GPR54 cDNA contains an open reading frame of 1131 bp encoding 377 amino acids and exhibits 56% identity to human GPR54. At the single-cell level, only in mature males, GnRH1 mRNA levels were inversely related to GPR54 mRNA (P<0.002). GPR54 was expressed in a significantly high percentage (45.0-60.0%) of mature GnRH1,GnRH2,and GnRH3 neurons and in immature GnRH3 neurons, which had migrated to the vicinity of their final locations in the brain : on the contrary, only 5.0% of immature GnRH1 and GnRH2 neurons had GPR54 transcripts (P<0.001). Thus, using a novel innovative single-cell gene profiling technique, we provide evidence that the expression of GPR54 is a "stop signal" for GnRH1,GnRH2,and GnRH3 neuronal migration, leading to suppression of cell growth and modulation of GnRH secretion, which is important for normal sexual development.