Mathematical Modeling of Neurons and Endocrine Cells

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

We are continuing our work with the Stojilkovic lab and the Genomics core of NICHD, providing bioinformatic support for their to single-cell RNAseq analyses 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 the multiple cell types in the pituitary. The primary function of endocrine pituitary cells is to secrete hormones via regulated exocytosis, for which the triggering role of intracellular calcium is well established. However, this process relies on many calcium independent cellular components. Phosphoinositides, low-abundance membrane lipids, have critical roles in exocytosis due to their interactions with vesicle priming and fusion proteins, but their role in pituitary hormone secretion has received little attention. Seven distinct phosphoinositides can be generated via phosphorylation of phosphatidylinositol by distinct families of kinases. This study found that blockade of phosphoinositide production with Wortmannin, at doses blocking PI3 and PI4 kinases, abolished prolactin secretion from lactotrophs downstream of calcium signaling. The genes coding for all major families of these kinases were detected in all hormone-producing pituitary cell types by scRNAseq and quantitative RT-PCR, so a systematic pharmacological approach was used to identify PI4KA as the essential kinase underlying the prolactin secretion blockade. This establishes PI4KA as a key enzyme regulating prolactin secretion. The work has been published (ref. #1). The pineal gland produces a daily rhythm in melatonin in all vertebrates, peaking at night to provide a signal of darkness. The generation of this time signal reflects coordinated circadian transcriptomic changes, driven by neural input, that control melatonin synthesis in pinealocytes, the dominant cells in the mature gland. While a large body of work has focused on the mature pineal gland, relatively little is known about the transcriptional programs underlying its development. Here we applied single-cell RNA sequencing (scRNAseq) to the developing rat pineal gland, using tissues obtained from late-embryonic to adult time points. RNAScope in situ hybridization was used as independent validation of scRNAseq results. All mature pineal cell types were identified, including pinealocytes, astrocytes, vascular cells, and microglia, as well as immature pinealocytes and proliferative progenitor cells. The latter, identified predominantly in embryonic samples, declined sharply in number by postnatal day 5. Mature pinealocytes and astrocytes showed a developmental connection to the same population of embryonic progenitors, suggesting a common origin of these cells. The transcriptomic profile of progenitor cells suggests novel progenitor marker genes and supports a role for Notch signaling in the developing rat pineal gland. Trajectory analysis further revealed transcriptional changes associated with post-mitotic pinealocyte differentiation, as well as a marked similarity between adult astrocytes and pineal progenitors. These scRNAseq data offer a resource and a solid basis for future studies of the mammalian pineal gland development. A manuscript is in preparation. The somatostatin (SST) receptor family controls pituitary hormone secretion, but the distribution and specific roles of these receptors on the excitability and voltage-gated calcium signaling of hormone producing pituitary cells have not been fully characterized. Here we show that the rat pituitary gland expressed Sstr1, Sstr2, Sstr3, and Sstr5 receptor genes in a cell type-specific manner: Sstr2 in thyrotrophs, Sstr3 in gonadotrophs and lactotrophs, all genes in somatotrophs, and none in corticotrophs and melanotrophs. Most gonadotrophs and thyrotrophs spontaneously fired high-amplitude single action potentials, which were silenced by SST without affecting intracellular calcium concentrations. In contrast, lactotrophs and somatotrophs spontaneously fired low-amplitude plateau-bursting action potentials in conjunction with calcium transients, both of which were silenced by SST. Moreover, SST inhibited GPCR-induced voltage-gated calcium signaling and hormone secretion in all cell types expressing SST receptors, but the inhibition was more pronounced in somatotrophs. The pattern of inhibition of electrical activity and calcium signaling was consistent with both direct and indirect inhibition of voltage-gated calcium channels, the latter being driven by cell type-specific hyperpolarization. These results indicate that the action of SST in somatotrophs is enhanced by the expression of several types of SST receptors and their slow desensitization, that SST may play a role in the electrical resynchronization of gonadotrophs, thyrotrophs, and lactotrophs, and that lack of SST receptors in corticotrophs and melanotrophs keeps them excitable and ready to respond to stress. See Ref. #1.