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，在脊椎动物物种的繁殖中起重要作用。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切割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，这使我们能够检查从大脑中收获的单个神经元中新型和GnRH mRNA的存在和数量成熟和未成熟雄性罗非鱼。在这里，我们提出的证据表明视前GnRH 1对性腺成熟很重要，而GnRH 2和GnRH 3可能在生殖行为或非生殖功能中发挥补充作用。此外，我们首次在非哺乳动物脊椎动物中分离并克隆了GPR 54。GPR 54是一种新的G蛋白偶联受体，被认为是性发育所必需的。罗非鱼GPR 54 cDNA全长1131 bp，编码377个氨基酸，与人GPR 54的同源性为56%。在单细胞水平上，只有成熟雄性大鼠GnRH 1 mRNA水平与GPR 54 mRNA水平呈负相关（P<0.002）。GPR 54在成熟的GnRH 1、GnRH 2和GnRH 3神经元中表达的比例很高（45.0-60.0%），在迁移到脑内最终位置附近的未成熟的GnRH 3神经元中也有表达;相反，只有5.0%的未成熟的GnRH 1和GnRH 2神经元有GPR 54转录本（P<0.001）。因此，使用一种新的创新的单细胞基因分析技术，我们提供的证据表明，GPR 54的表达是一个“停止信号”的GnRH 1，GnRH 2和GnRH 3神经元迁移，导致细胞生长的抑制和GnRH分泌的调制，这是重要的正常性发育。

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.