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, 46 (21), 11370-11380

Single-cell Stabilization Method Identifies Gonadotrope Transcriptional Dynamics and Pituitary Cell Type Heterogeneity

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Single-cell Stabilization Method Identifies Gonadotrope Transcriptional Dynamics and Pituitary Cell Type Heterogeneity

Frederique Ruf-Zamojski et al. Nucleic Acids Res.

Abstract

Immediate-early response genes (IEGs) are rapidly and transiently induced following an extracellular signal. Elucidating the IEG response patterns in single cells (SCs) requires assaying large numbers of timed samples at high accuracy while minimizing handling effects. To achieve this, we developed and validated RNA stabilization Buffer for Examination of Single-cell Transcriptomes (RNA-Best), a versatile single-step cell and tissue preservation protocol that stabilizes RNA in intact SCs without perturbing transcription patterns. We characterize for the first time SC heterogeneity in IEG responses to pulsatile gonadotropin-releasing hormone (GnRH) stimuli in pituitary gonadotrope cells. Our study identifies a gene-specific hierarchical pattern of all-or-none transcript induction elicited by increasing concentrations of GnRH. This quantal pattern of gene activation raises the possibility that IEG activation, when accurately resolved at the SC level, may be mediated by gene bits that behave as pure binary switches.

Figures

Figure 1.
Figure 1.
RNA-Best preservation of cultured gonadotropes maintains IEG transcript levels under basal and stimulated conditions. (A, B) RNA yield (A) and quality (B) from fresh cells, RNA-Best-preserved cells, or cells preserved using cryopreservation, methanol fixation, or FRISCR. *P < 0.05 and **P < 0.01; bars show mean ± s.e.m. (C) qPCR analysis of Fos and Egr1 basal expression and GnRH induction (fold change); 2 nM GnRH treatment for 35 min; *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001. Note that Egr1 fold change using FRISCR equals 1, due to no difference between vehicle and GnRH treatment. (D) Time course of GnRH induction of Fos and Egr1 in fresh vs. RNA-Best-preserved cells (n = 6 biological replicates per time point and protocol). Data in A–D are from one of four representative experiments.
Figure 2.
Figure 2.
RNA-Best preserves transcriptome profiles from bulk gonadotrope cells. (A) Pairwise correlations between bulk RNA-seq data from three fresh and three RNA-Best samples. Expression counts were converted to log2 (counts per million +1). Pearson correlations are indicated. (B) Heat map of selected GnRH-regulated genes and commonly used house-keeping (HK) genes in the same samples as in A.
Figure 3.
Figure 3.
RNA-Best preserves cell cycle stage assignments in single gonadotrope cells exposed to GnRH. (A, B) Cell cycle and GnRH-cell cycle interaction effects on SC transcriptome in fresh (1992 vehicle- and 1889 GnRH-treated) vs. RNA-Best-preserved cells (3579 vehicle- and 2753 GnRH-treated) using GEM Drop-seq. (A) Each cell on the x-axis is aligned by cell cycle progression. Color coding indicates score assignment to the cell cycle phase. The five cell cycle phases are indicated. (B) Summary of the percentage of cells assigned to different cycles. (C) Pairwise correlations between SC gene expression measurements averaged across vehicle- vs. GnRH-treated cells that were either directly lysed (fresh cells) or preserved in RNA-Best. Expression counts were converted to log2 (counts per million +1). Pearson correlations are indicated.
Figure 4.
Figure 4.
IEGs show bimodal SC responses to GnRH pulse stimulation. (A) Schematic of the time course experiment design. Cells were exposed to four pulses of GnRH (blue arrows) and collected at short time intervals around the 4th pulse (yellow arrows). (B, C) Graphs showing the probability for a SC to express a house-keeping gene (either Eef1a, H2fz, Rps11, or Rps25; (B)) or a regulated gene (as indicated; (C)) following either vehicle or GnRH treatment. Error bars are based on the binomial standard deviation on the number of gene-expressing cells. In C, Right panel, the +25 min and +35 min data points are statistically different from −1 min.
Figure 5.
Figure 5.
SC probability of IEG induction is concentration-dependent and varies for each IEG. (A–D) Cells were exposed to increasing concentrations of GnRH and collected 35 min after the fourth pulse. (A) Vertical scatter plots of Egr2, Fosb, and Rps25 expression in SCs. In parentheses is indicated the number of gene-expressing (top) and non-gene expressing cells (bottom); each square represents a cell; the green dotted line signifies the average expression in all analyzed cells at a given GnRH concentration. (B) Plots of the percentages of cells expressing a regulated gene, as indicated. Error bars are based on the binomial standard deviation on the number of gene-expressing cells. (C) Bar graphs of average gene expression in gene expressing (i.e. induced) cells. Error bars represent standard deviation. ANOVA shows no significant differences. (D) Venn diagrams illustrating the overlap of Fos, Egr1, Egr2, and Fosb expression in all analyzed cells at +35 min.

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