Single-cell molecular and cellular architecture of the mouse neurohypophysis

Abstract

AcknowledgementsWe thank Yael Kuperman for her help in neurohypophysis dissection; Hagit Dafni for providing C57BL6 mice for dissociation protocol optimization; Stefan Jung for providing he Cx3cr1:GFP mice and Shalev Itzkovitz for the smFISH probes; Amrutha Swaminathan and Ludmila Gordon for their valuable comments on the text and figures and Hanjie Li for his advices on cluster annotation. Abstract:The neurohypophysis (NH), located at the posterior lobe of the pituitary, is a major neuroendocrine tissue, which mediates osmotic balance, blood pressure, reproduction, and lactation by means of releasing the neurohormones oxytocin and arginine-vasopressin from the brain into the peripheral blood circulation. The major cellular components of the NH are hypothalamic axonal termini, fenestrated endothelia and pituicytes, the resident astroglia. However, despite the physiological importance of the NH, the exact molecular signature defining neurohypophyseal cell types and in particular the pituicytes, remains unclear. Using single cell RNA sequencing, we captured seven distinct cell types in the NH and intermediate lobe (IL) of adult male mouse. We revealed novel pituicyte markers showing higher specificity than previously reported. Single molecule in situ hybridization revealed spatial organization of the major cell types implying intercellular communications. We present a comprehensive molecular and cellular characterization of neurohypophyseal cell-types serving as a valuable resource for further functional research. Significance StatementThe neurohypophysis (NH) is a major neuroendocrine interface, which allows the brain to regulate the function of peripheral organs in response to specific physiological demands. Despite its importance, a comprehensive molecular description of cell identities in the NH is still lacking. Utilizing single cell RNA sequencing technology, we identified the transcriptomes of five major neurohypophyseal cell types in the adult male mice and mapped the spatial distribution of selected cell types in situ. We revealed an unexpected cellular heterogeneity of the neurohypophysis and provide novel molecular markers for neurohypophyseal cell types with higher specificity than previously reported.

Neural plate progenitors give rise to both anterior and posterior pituitary cells

“…( D ) Histogram showing over-representation analyses (ORA) comparing zebrafish pituicyte to scRNA-Seq gene sets of adult mouse neurointermediate pituitary lobe (Chen Q et al, 2020) or human fetus pituitary (Zhang et al, 2020). The bar lengths indicate -log 10 (FDR), and the color-coded scale bars depict the number of overlapping genes.…”

“…We have recently reported that commonly used marker genes, which have been so far considered as ‘pituicyte-specific’ display promiscuous cell-type specificity (Chen Q et al, 2020). This highlighted the need for a better molecular and cellular characterization, as well as an improved markers definition of pituitary cells and in particular the pituicytes, ideally leading to the generation of new transgenic tools in rodents and zebrafish.…”

“…Antibody staining for eGFP (GFP Rabbit Polyclonal #A11122 or GFP Chicken Polyclonal #A10262, Invitrogen, Thermo Fisher, USA) and/or mCherry (Living Colors DsRed Polyclonal Antibody #632496, Takara, Japan) after in situ hybridization were carried out as described in (Chen Q et al, 2020). The samples were then mounted in 75% glycerol, and imaged using a Zeiss LSM800 confocal microscope with 40X oil lens.…”

“…Notably, although the top differentially-enriched markers defining pituicytes of mouse, human and zebrafish were not identical, the evolutionary conservation between the three species was evident at the transcriptome level. This was demonstrated by an unbiased bioinformatic ORA analysis (Boyle et al, 2004) of the entire transcriptome of zebrafish pituicytes compared to that of mouse (Chen Q et al, 2020) and human (Zhang et al, 2020).…”

Renewing an old interest: Pituitary folliculostellate cells

“…Recent reports analysing single cells RNA sequencing (scR-NAseq) of rodent anterior pituitary cells utilising unbiased clustering methods have variably described FS and Sox2+ve cells as single or multiple cell types. [54][55][56][57] It is important to note that differences in pituitary dissection, with the inclusion of the intermediate and posterior pituitary that contains cell types such as pituicytes and epithelial-like cells expressing markers associated with FS cells, 58 may affect the identification of potential subpopulations. Indeed, this recent description of the expression in pituicytes and epitheliallike cells of markers used in previous analyses of FS cell function may require the reinterpretation of some older studies, in particular those using dispersed pituitary cells.…”