Co-profiling of single-cell gene expression and chromatin landscapes in stickleback pituitary

Abstract

The pituitary gland is a key endocrine gland with various physiological functions including metabolism, growth, and reproduction. It comprises several distinct cell populations that release multiple polypeptide hormones. Although the major endocrine cell types are conserved across taxa, the regulatory mechanisms of gene expression and chromatin organization in specific cell types remain poorly understood. Here, we performed simultaneous profiling of the transcriptome and chromatin landscapes in the pituitary cells of the three-spined stickleback (Gasterosteus aculeatus), which represents a good model for investigating the genetic mechanisms underlying adaptive evolution. We obtained pairwise gene expression and chromatin profiles for 5184 cells under short- and long-day conditions. Using three independent clustering analyses, we identified 16 distinct cell clusters and validated their consistency. These results advance our understanding of the regulatory dynamics in the pituitary gland and provide a reference for future research on comparative physiology and evolutionary biology.

Fig. 1

Experimental and bioinformatics workflow of single-cell multiome ATAC + Gene Expression (GEX). (a) Experimental workflow. (b) Bioinformatics workflow. The acronym L/D refers to the light/dark cycle used during the experiment. Figure 1a was created with icons from biorender.com.

Fig. 2

Quality assessment of ATAC and cDNA libraries. (a,b) Electropherogram profiles of ATAC and cDNA library quality, respectively. The integrity and size distribution were evaluated using a Bioanalyzer. SD: Short-day; LD: Long-day.

Fig. 3

GEX quality control metrics. Percent Mitochondrial: Percent of mitochondrial reads detected in each cell. RNA Counts: Count of GEX reads detected in each cell. RNA Features: Number of genes/features detected in each cell.

Fig. 4

ATAC quality control metrics. (a) Transcription Start Site (TSS) enrichment. (b) Nucleosome Signals. nCount_ATAC: ATAC reads detected in each cell. The red vertical and horizontal lines indicate the 5th, 10th, 90th, and 95th quantiles of the data distribution (left to right/bottom to top).

Fig. 5

Clustering and annotation of gene expression and chromatin accessibility of the stickleback pituitary under short- (left) and long-day conditions (right). Independent analyses based on (a) gene expression profiles, (b) chromatin accessibility, and (c) Weighted Nearest Neighbor (WNN) analysis. Cells are visualized by Uniform Manifold Approximation and Projection for Dimension Reduction (UMAP). Cell annotation and their corresponding GEX_, ATAC_ and WNN_cluster ID are listed in Table 4.

Fig. 6

Gene expression and chromatin accessibility of representative cell markers. Uniform Manifold Approximation and Projection for Dimension Reduction (UMAP) visualizations represent the expression of the marker genes. The adjacent genomic track plots display chromatin accessibility near the marker genes. Cluster numbers, corresponding to the gene expression-based analysis, are noted to the left of the genomic tracks.

Fig. 7

Differentially expressed genes (DEGs) and differentially accessible regions (DARs) in each cell cluster. (a) Expression patterns of the top 20 DEGs in each cluster. (b) Accessibility patterns for top 20 DARs in each cluster.

Fig. 8

Motif enrichment across cell clusters and those under different photoperiodic conditions. (a) Top four enriched motifs in representative cell clusters: red blood cells, progenitor cells, and major hormone producing cells. (b) Binding motifs for transcription factors showing changes in their enrichment in DARs in response to photoperiodic shifts to long-day conditions. The corresponding transcription factors (TFs) are shown at the top of each subplot. The cluster numbers correspond to the cell cluster identities determined by gene expression-based clustering (Fig. 5a).