Mathematical Modeling of Neurons and Endocrine Cells

与Stojilkovic实验室和NICHD的基因组学核心一起，我们在这些研究中开创了一个新的方向，即单细胞RNAseq分析垂体前叶的基因表达。该腺体由5种激素分泌细胞、神经胶质样滤泡星形细胞、内皮细胞和血细胞组成，构成了垂体窦毛细血管网络。垂体生理学的研究重点包括三维组织、组织结构和细胞间通讯、垂体细胞的发育和再生以及滤泡星状细胞的异质性和功能。人们为这些目标付出了巨大的努力，但许多分子基础仍然未知。 我们进行了第一个单细胞转录组研究的前垂体细胞。我们分析了来自青春期后雄性和雌性大鼠的6700多个新鲜分散的细胞。除了确认已知的标记外，我们的转录组分析还突出了许多新的遗传标记，这些标记有助于垂体细胞类型识别和性别二型性。我们首次提供了毛囊星状细胞百分比的估计，并确定了这种异质细胞群体的至少两种主要亚型。我们的数据支持这样的假设，即滤泡星状细胞为激素产生细胞提供结构支持，因为它们表达许多编码解毒酶的基因。我们对发育、干细胞/祖细胞和神经内分泌标记物的表达分析表明，卵泡星状细胞和激素产生细胞是共同起源的姐妹细胞。我们还提供了编码细胞外基质、细胞黏附和内源性配体蛋白的基因的细胞类型相关表达的详细视图，这些基因对于垂体前叶的三维组织及其组成细胞之间的相互作用至关重要。 我们期望所鉴定的垂体腺细胞类型的标记基因将为进一步研究垂体腺的结构、功能和病理生理学，以及为研究出生后垂体腺的发育、再生和重组奠定坚实的基础。结果发表在参考文献中。#1. 持续给予下丘脑促性腺激素释放激素(GnRH)，而不是脉冲式给药，会导致垂体促性腺激素黄体生成素和卵泡刺激素的分泌显著减少，生殖功能受损。在此，我们研究了促性腺激素亚单位和GnRH受体基因在体外和体内的表达谱，以阐明它们在不存在和持续存在GnRH的情况下的表达谱。在无GnRH的条件下培养的垂体细胞下调FSHB、CGA和GnRHr的表达，而GnRH激动剂持续处理后CgA的表达逐渐上调，GnRHr和FSHB的表达短暂上调，同时FSHB的表达被长期阻断，但GnRHr的表达不被抑制。相反，LHB的表达对内源性GnRH的丧失和GnRH的持续治疗相对不敏感，可能反映了Egr1和Nr5a1的表达状况。体内注射促性腺激素释放激素激动剂后也观察到类似的反应模式。然而，持续使用促性腺激素释放激素在体外和体内刺激黄体生成素的分泌，导致黄体生成素细胞含量下降，尽管基础LHb表达水平较高。这些数据表明，在持续GnRH治疗过程中，FSHB表达受阻和促黄体生成素分泌池枯竭是导致促性腺激素分泌功能减弱的两个主要因素。研究结果发表在《参考文献》上。#2.

Together with the Stojilkovic lab and the Genomics core of NICHD, we have initiated a new direction in these studies, single-cell RNAseq analysis of gene expression in the anterior pituitary. This gland is composed of five hormone producing cell types, glia-like folliculostellate cells, and endothelial and blood cells comprising the pituitary sinusoidal capillary network. Some key lines of inquiry in pituitary physiology include three-dimensional organization tissue organization and intercellular communication among cells, development and regeneration of pituitary cells, and the heterogeneity and function of folliculostellate cells. Great efforts have been dedicated towards these aims, but many of the molecular underpinnings remain unknown. We have carried out the first single-cell transcriptome study of anterior pituitary cells. We profiled over 6700 freshly dispersed cells from postpubertal male and female rats. In addition to confirming known markers, our transcriptome analysis highlights many novel genetic markers contributing to pituitary cell type identity and sexual dimorphism. We provide for the first time an estimate of the percentage of folliculostellate cells, and we identify at least two main subtypes of this heterogeneous cell population. Our data support the hypothesis that folliculostellate cells provide structural support for the hormone producing cells, as they express many genes encoding detoxification enzymes. Our analysis of expression of developmental, stem cell/progenitor, and neuroendocrine markers suggests that folliculostellate and hormone producing cells are sister cells with a common origin. We also provide a detailed view of cell type-dependent expression of genes encoding extracellular matrix, cell adhesion, and endogenous ligand proteins, critical for the tridimensional organization of the anterior pituitary and the cross-talk between its constituent cells. We expect the marker genes identified for pituitary cell types will serve as a solid base for future investigations into pituitary structure, function, and pathophysiology, as well as for the study of postnatal pituitary development, regeneration, and reorganization. The results were reported in ref. #1. Continuous, as opposed to pulsatile, delivery of hypothalamic gonadotropin-releasing hormone (GnRH) leads to a marked decrease in secretion of pituitary gonadotropins LH and FSH and impairment of reproductive function. Here we studied the expression profile of gonadotropin subunit and GnRH receptor genes in rat pituitary in vitro and in vivo to clarify their expression profiles in the absence and continuous presence of GnRH. Culturing of pituitary cells in GnRH-free conditions downregulated Fshb, Cga, and Gnrhr expression, whereas continuous treatment with GnRH agonists upregulated Cga expression progressively and Gnrhr and Fshb expression transiently, accompanied by a prolonged blockade of Fshb but not Gnrhr expression. In contrast, Lhb expression was relatively insensitive to loss of endogenous GnRH and continuous treatment with GnRH, probably reflecting the status of Egr1 and Nr5a1 expression. Similar patterns of responses were observed in vivo after administration of a GnRH agonist. However, continuous treatment with GnRH stimulated LH secretion in vitro and in vivo, leading to decrease in LH cell content despite high basal Lhb expression. These data suggest that blockade of Fshb expression and depletion of the LH secretory pool are two major factors accounting for weakening of the gonadotroph secretory function during continuous GnRH treatment. The results were published in ref. #2.