Mechanisms of development of the pituitary progenitors with pit-1 expression into growth hormone and prolactin cells

生长激素(GH)和催乳素(PRL)被认为来源于一个共同的祖先基因，而在哺乳动物中，这两种激素是由不同类型的脑垂体腺分泌的。人们也普遍认为GH和PRL细胞不同于共同的前体细胞，因为这些细胞类型通常表达垂体特异性转录因子PIT-1。然而，导致这两种细胞类型分化的机制仍然不清楚。在本研究中，我试图检测这两种细胞类型是独立于前体细胞分化，还是其中一种细胞首先分化成为另一种细胞类型的母细胞。通过聚合酶链式反应(PCR)扩增报告基因特异性表达所需的生长激素(GH)、催乳素(PRL)和PIT-1的5‘上游区域，并在这些启动子的指导下构建表达荧光蛋白CFP的报告载体。将这些质粒导入成年雄性大鼠分离的垂体细胞，成功地用CFP标记了GH或PRL细胞。然而，将CFP导入胎儿脑垂体后，其表达水平极低，可能是由于胎儿脑下垂体细胞启动子活性较弱所致。这些结果表明，将该方法应用于胎儿脑垂体腺细胞类型鉴定的研究中，还需要进一步提高检测系统的灵敏度。结果显示，近50%的生长激素细胞也表达PRL mRNA，这表明意外地存在大量表达多个mRNAs的细胞。在单个细胞中建立GH和PRL基因表达的调控机制具有重要意义。

It is believed that growth hormone(GH) and prolactin(PRL) are derived from a common ancestral gene, whereas each hormone is secreted from distinct cell type in the anterior pituitary gland in mammals. It is also widely accepted that GH and PRL cells are differentiate from the common precursor cells since these cell types commonly express pituitary specific transcription factor, pit-1. The mechanisms responsible for the differentiation of these two cell types are, however, still obscure. In this study, I tried to examine whether these two cell types differentiate from precursors independently or one of them differentiate first and become the mother cells of another cell type.The 5'-upstream region of GH, PRL, and pit-1 that required for the cell type specific expression of the reporter gene were PCR amplified and the reporter plasmids were constructed that express the fluolescent protein, CFP, under the direction of these promoters. These plasmids successfully labeled GH or PRL cells with CFP when introduced into isolated pituitary cells from adult male rat. However, the expression of CFP was extremely low when introduced into fetal pituitary gland, probably due to the weak promoter activity in the fetal pituitary cells. These results indicate that further enhancement of the sensitivity of the detection system is required for the application of this method to the study of cell type determination that occurs in the fetal pituitary gland.I also established the PCR-technology for the detection of the hormone mRNA expression in the single pituitary cells. As a consequence, it is revealed that nearly 50% of GH cells also express PRL mRNA, suggesting that there are an unexpectedly large number of cells that express multiple mRNAs. It is considered to be of importance to establish the regulatory mechanisms for the expression of GH and PRL gene in the single cells.