The astroglial and stem cell functions of adult rat folliculostellate cells

Abstract

The mammalian pituitary gland is a complex organ consisting of hormone-producing cells, anterior lobe folliculostellate cells (FSCs), posterior lobe pituicytes, vascular pericytes and endothelial cells, and Sox2-expressing stem cells. We present single-cell RNA sequencing and immunohistofluorescence analyses of pituitary cells of adult female rats with a focus on the transcriptomic profiles of nonhormonal cell types. Samples obtained from whole pituitaries and separated anterior and posterior lobe cells contained all expected pituitary resident cell types and lobe-specific vascular cell subpopulations. FSCs and pituicytes expressed S100B, ALDOC, EAAT1, ALDH1A1, and VIM genes and proteins, as well as other astroglial marker genes, some common and some cell type-specific. We also found that the SOX2 gene and protein were expressed in ~15% of pituitary cells, including FSCs, pituicytes, and a fraction of hormone-producing cells, arguing against its stem cell specificity. FSCs comprised two Sox2-expressing subclusters; FS1 contained more cells but lower genetic diversity, while FS2 contained proliferative cells, shared genes with hormone-producing cells, and expressed genes consistent with stem cell niche formation, regulation of cell proliferation and stem cell pluripotency, including the Hippo and Wnt pathways. FS1 cells were randomly distributed in the anterior and intermediate lobes, while FS2 cells were localized exclusively in the marginal zone between the anterior and intermediate lobes. These data indicate the identity of the FSCs as anterior pituitary-specific astroglia, with FS1 cells representing differentiated cells equipped for classical FSC roles and FS2 cells exhibiting additional stem cell-like features.

Keywords: Sox2; astrocyte marker genes; endothelial cells; folliculostellate cells; hormone-producing cells; pericytes; pituicytes; pituitary gland; pituitary stem/progenitor cells; stem cell marker genes.

FIGURE 1.

Identification of pituitary resident cell types. (a) Heatmap showing selected marker genes characteristic of identified cell types for a random subsample of up to 150 cells per type. We identified six hormone producing cells – melanotrophs (M), corticotrophs (C), gonadotrophs (G), thyrotrophs (T), somatotrophs (S), and lactotrophs (L); four nonhormonal cell types – folliculostellate cells (FSC), pituicytes (Pc), vascular endothelial cells (EC) and pericyte (Pe); and blood cells – immune cells/leukocytes (Le). Red blood cells were excluded, and in further analysis, we excluded Le. (b) UMAP embedding showing identified resident pituitary cell types. Cells encircled with a dotted line expressed cell cycle marker genes. (c-e) UMAP embeddings showing the expression of (c) non-endocrine cell expression of Fstl1, (d) FSC and pituicyte expression of Aldoc, and (e) FSC-localized expression of Slc15a2.

FIGURE 2.

Transcriptomic profiles of nonhormonal FSC, pituicytes, and vascular cells. (a) UMAP plot showing nonhormonal cells. Anterior lobe- and posterior lobe-specific clusters of pericytes and ECs are indicated with subscripts a and p, respectively. (b) Venn diagrams showing the number of genes in the intersections of pituicyte- and FSC-dominant genes relative to all other cell types (top), and for pituicyte, FSC, pericyte, and EC dominant genes relative to HPC cell types (bottom). Pericyte and EC dominant genes were identified separately but pooled together for visualization (for full results see Supplemental Data 1 and 2). (c) Dot plot showing dominant genes that were highly specific to pituicytes (top group), FSCs (second group), common to pituicytes and FSCs (third group), and common to combinations of non-endocrine cells (bottom group). (d) UMAP plots showing single cell expression levels for selected genes from panel c.

FIGURE 3.

Astrocyte marker gene signatures of FSCs and pituicytes. (a) UMAP plots showing selected astrocyte markers with common expression between FSCs and pituicytes (Slc1a3, Gpr37l, Fam107a), pituicyte-specific expression (Acsbg1), FSC-specific (Pla2g7), and FSC-dominant expression (Aldh1a1). (b) Dot plot showing the expression of 28 of 36 astrocyte marker genes that were identified in at least four of seven published sources and were FSC or pituicyte-dominant expression compared to all other cell types (see also Supplemental Data 3). (c) Gene scores for a focused list of 36 FSC or Pc dominant astrocyte markers appearing in at least four of the seven reference astrocyte marker gene lists. (d) Correlation of mean expression from scRNAseq with qRT-PCR expression for 19 astrocyte marker genes in whole pituitary or separated anterior and posterior lobe samples. Whole pituitary scRNAseq was computed from all cells, while anterior and posterior expression was computed from cells assigned to each lobe via k-nearest neighbor classification (Methods, Figure S7).

FIGURE 4.

S100 gene expression in the pituitary gland. (a) UMAP scatter plots showing expression of S100b, common to FSC and Pc, S100a6, expressed in FSC and Pe, and S100g, expressed in Pc and a subset of S and L. (b) Dot plot indicating the mean expression of S100 genes in each cell type, with EC and Pe divided by lobe of origin (Methods, Figure S7). (c) qRT-PCR measurements of S100 gene expression in dispersed cells and pituitary tissue from female rats. Solid line – linear fit; dotted lines – 95% confidence intervals. (d and e) Immunohistofluorescence analysis of S100B and S100A protein expression in the anterior (d) and posterior (e) lobes of female rat pituitary gland. Expression of S100B (green, left) and S100A (red, middle) proteins and their overlay (right). Cell nuclei are stained with DAPI (blue). Scale bar (applies to all images), 10 μm.

FIGURE 5.

Immunohistofluorescence analysis of S100A and S100B protein expression. Expression of S100B (green, left) and EAAT1/SLC1A3 (red, middle) proteins and their overlay expression (right) in the anterior (upper panels) and posterior (lower panels) lobes of the female rat pituitary gland. Cell nuclei are stained with DAPI (blue). Scale bar (applies to all images), 10 μm.

FIGURE 6.

Immunohistofluorescence analysis of astrocyte marker protein expression in the anterior lobe of female rat pituitary gland. Upper panels display expression of S100B (green, left) and ALDOC (red, middle) proteins and their colocalization (right). Lower panels show appearance of S100B (green, left) and ALDH1A1 (red, middle) and their overlay expression (right). Cell nuclei are labeled with DAPI (blue). Scale bar (applies to all images), 10 μm.

FIGURE 7.

Identification of two FSC subtypes. (a) UMAP embedding showing unsupervised clustering of FSCs into two subtypes. A small portion of FS2 cells (circled) express cell cycle markers. (b) Violin plot of the distributions of number of genes per cell in FS1 and FS2, with median values of 2099 and 3137, respectively. (c) Venn diagram showing the number of genes identified as FS1-dominant, FS2-dominant, and common to both, relative to other cell types except pituicytes (full results in Supplemental Data 4). (d) Dot plot showing selected top FSC-expressed genes: dominant in both subtypes (first group), FS1-dominant (second group), FS2-dominant (third group), and co-expressed in FS2 and HPC (fourth group). (e) UMAP scatter plots showing the expression patterns of selected genes from panel d.

FIGURE 8.

Gene Ontology and Pathway enrichment analysis for FS1-, FS2-, and Pc-dominant genes. (a) UMAP plot of GO and pathway enriched terms for FSCs and Pc (Methods). The Euler diagram indicates the number of terms enriched for each combination of cell subtypes, with the colors of intersections corresponding to the colors of the points in the UMAP plot. (b) UMAP plot showing cluster identities of terms. (c) Dot plot of enrichment analysis results for selected terms per cluster, indicating the fold-enrichment relative to chance (color) and p-value (size). The left y-axis represents the type of term, with colors corresponding to the clusters in panel b. Full results for 315 enriched terms are given in Supplemental Data 5. BP, Biological Process; CC, Cellular Component; MF, Molecular Function; KEGG, Kyoto Encyclopedia of Genes and Genomes; REAC, Reactome; ECM, extracellular matrix.

FIGURE 9.

Development and differentiation signature genes. (a) Anterior lobe expression of key genes from the KEGG Hippo signaling pathway term. Pe_a and EC_a – anterior lobe clusters of pericytes and endothelial cells. (b) FSC-targeted ligand-receptor (L-R) interactions based on CellChat analysis (Methods). Rows represent the L-R interactions, and columns labels indicate source-target directionality. Point color represents potential interaction strength and size indicates the percentage of target cells expressing the receptor (or the minimum % expression for receptor complex genes when the receptor is a complex). All points shown have bootstrap p-value < 1e-3, and both source and target cell types express the ligand or receptor gene in at least 10% of cells. Several ligands could interact via multiple receptor subtypes (See Supplemental Data 9); the most highly expressed one is shown. (c) UMAP embeddings showing the expression per cell for selected marker genes. (d) Dot plot of the mean expression and percentage of cells expressing selected genes associated with developmental and differentiation processes in the pituitary, grouped by cell type expression pattern.

FIGURE 10.

Immunohistofluorescence survey of S100B and SOX2 patterns of expression in female rat pituitary gland. S100B (green) and SOX2 (red) proteins were expressed in all three lobes of pituitary gland as well as in the marginal zone. Arrows indicate SOX2 expression in melanotrophs in intermediate lobe and unidentified cells other than FSCs in the anterior lobe. Cell nuclei are stained with DAPI (blue). Scale bar, 10 μm.

FIGURE 11.

Expression of Cytokeratin 17 (KRT17) in the female rat pituitary gland. Immunohistofluorescence analysis revealed expression of KRT17 (red), a marker protein of FSC2, limited to the marginal zone, where it is coexpressed with S100B (green), marker of all FSCs. Cell nuclei are stained with DAPI (blue). Scale bar, 10 μm.