Mathematical Modeling of Neurons and Endocrine Cells

National Institute Of Diabetes And Digestive And Kidney Diseases

Investigators

Linked publications & trials

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). A main focus this year has been to apply our single-cell RNA sequencing (scRNAseq) expertise to a non-pituitary tissue, the pineal gland, which 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 controls 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 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.