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Volume 25, Issue 5, Pages 1194-1205.e4 (May 2017)
Deciphering Pancreatic Islet β Cell and α Cell Maturation Pathways and Characteristic Features at the Single-Cell Level Wei-Lin Qiu, Yu-Wei Zhang, Ye Feng, Lin-Chen Li, Liu Yang, Cheng-Ran Xu Cell Metabolism Volume 25, Issue 5, Pages e4 (May 2017) DOI: /j.cmet Copyright © 2017 Elsevier Inc. Terms and Conditions
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Cell Metabolism 2017 25, 1194-1205.e4DOI: (10.1016/j.cmet.2017.04.003)
Copyright © 2017 Elsevier Inc. Terms and Conditions
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Figure 1 Overview of the β and α Cell Transcriptomes
(A) Three-dimensional principal component analysis (PCA) of 538 β cells and 328 α cells. Cells are color-coded by developmental stage and are shape-coded by cell type. (B) Identification of genes highly expressed in β lineage and α lineage (adjusted p < 0.05, fold change > 4); each column represents a single cell. (C) The expression patterns of selected marker genes for β cells and α cells. (D) Two-dimensional PCA of β cells and α cells. The arrows indicate the directions of cell maturation (M) and proliferation (P). (E) Hierarchical clustering of differentially expressed genes selected from the first two principal components (PCs) (p < 10−13 in β cells and p < 10−8 in α cells; transcripts per million [TPM] < 1 in at least 10% cells). Each column represents a single β or α cell in pseudotime ordering. (F and G) Selected GO terms of clusters 1–3 in β cells (F) and clusters 1′ and 2′ in α cells (G). See also Figure S1 and Tables S1, S2, and S3. Cell Metabolism , e4DOI: ( /j.cmet ) Copyright © 2017 Elsevier Inc. Terms and Conditions
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Figure 2 Characterization of Proliferative β and α Cells
(A) Hierarchical clustering of cell-cycle-related genes from cluster 1 in β or cluster 1′ in α cells (Figure 1E). For both β and α cells, cluster G1/S and G2/M genes are listed in the boxes. Proliferative cells are further divided into three groups (G1/S, S/G2/M, and G2/M). (B) Distributions of β and α cells at different cell-cycle phases on the PCA map. Cells are color-coded by cell-cycle phase. (C) Stage-specific expressions of p57 and p18 in β and α cells. Dot size represents the expression level (TPM). (D) Boxplots show the relative expression of p57 and p18 in β and α cells at E17.5, P3 (β cell only), P9, and P60. Each dot represents the relative expression level in a single cell measured by real-time PCR with Gapdh normalization. n, number of cells. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, Wilcoxon rank-sum test. (E) Immunofluorescence staining of p18 and DAPI on sorted P3 and P60 β cells. The arrows point to the p18+ cells. (F) Statistics for the percentage of p18+ cells in all β cells at P3 or P60. Data show the mean + SEM (error bar); n, number of independent biological replicates. Cell #, the number of counted cells. ∗∗∗p < 0.001, t test. See also Figure S2 and Table S3. Cell Metabolism , e4DOI: ( /j.cmet ) Copyright © 2017 Elsevier Inc. Terms and Conditions
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Figure 3 Proliferative Cells Mature Synchronously with Quiescent Cells
(A) Hierarchical clustering of differently expressed genes from clusters 2–3 in Figure 1E in β cells or 2′–3′ in α cells. Each column represents a single cell at P0, P3, or P9 (β cells), and E17.5, P0, or P9 (α cells) in pseudotime ordering. Individual cells are marked in yellow (proliferative) or black (quiescent) according to cell-cycle states. (B) Empty circles and solid circles represent proliferative cells and quiescent cells, respectively. The x axis represents the pseudotime ordering in the maturation process. (C) The model demonstrates the coordination between proliferation and maturation during β cell development. See also Figure S3. Cell Metabolism , e4DOI: ( /j.cmet ) Copyright © 2017 Elsevier Inc. Terms and Conditions
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Figure 4 β and α Cell Heterogeneity during Development
(A and C) Hierarchical clustering of quiescent β (A) and α cells (C) at different developmental stages (E17.5–P18) with their corresponding PCAs on the right. Differentially expressed genes are selected from the first PC (p < 0.005). Points on the PCA plot are color-coded in the same manner as the group colors. (B and D) Maturation states of β cell (B) and α cell (D) subpopulations. Each point represents one cell and is consistently colored with the PCA plot. The green points in (B) represent Ngn3+hormone− cells. (E and F) Hierarchical clustering of quiescent P60 β (E) and P60 α cells (F) with their corresponding PCAs on the right. Differentially expressed genes are selected from the first two PCs (p < 0.005). See also Figure S4 and Table S4. Cell Metabolism , e4DOI: ( /j.cmet ) Copyright © 2017 Elsevier Inc. Terms and Conditions
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Figure 5 Bulk-Cell RNA-Seq to Analyze β Cell Maturation
(A) Hierarchical clustering of 397 cell-cycle-related genes in bulk β cells of seven developmental stages. (B) Hierarchical clustering of 1,716 maturation-associated genes after cell-cycle-related genes were excluded. The arrow indicates the transition of immature β cells to mature β cells. TFs that have been reported to regulate β cell maturation are listed on the right. (C and D) Selected GO items of clusters “immature” genes (C) and “mature” genes (D) in (B). See also Figure S5 and Tables S1 and S5. Cell Metabolism , e4DOI: ( /j.cmet ) Copyright © 2017 Elsevier Inc. Terms and Conditions
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