Single-cell stabilization method identifies gonadotrope transcriptional dynamics and pituitary cell type heterogeneity.

Frederique Ruf-Zamojski,Marlon Stoeckius,Harold Swerdlow,William Stephenson,Venugopalan Nair,Chirine Toufaily,Boris M Hartmann,Jessica Tome-Garcia,Miguel Fribourg,Yongchao Ge,Nadejda M Tsankova,Stuart C Sealfon,Gregory R Smith,Hanna Pincas,Michel Zamojski,Daniel J Bernard,Judith L Turgeon,Peter Smibert

doi:10.1093/nar/gky991

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.

Highlights

Immediate-early response genes (IEGs) are rapidly upregulated in response to various external stimuli such as growth factors, hormones, or stress [1,2]
We compared RNA isolated from L␤T2 gonadotrope cells preserved for 10 days at 4◦C with RNA-Best, to that of cells stored at −80◦C with other single cells (SCs) preservation methods [19,20,21], and to RNA isolated from fresh cells
The high RNA integrity numbers (RINs) seen in fresh cells were only found with the RNA-Best and cryopreservation protocols (Figure 1B, Supplementary Figure S1)

Summary

Introduction

Immediate-early response genes (IEGs) are rapidly upregulated in response to various external stimuli such as growth factors, hormones, or stress [1,2]. IEGs respond to external stimuli within minutes, without requiring de novo protein synthesis. Most IEGs encode transcription factors, which regulate genes involved in various cellular functions [3]. IEG induction by hypothalamic gonadotropin-releasing hormone (GnRH) is involved in the regulation of gonadotropin subunit gene (Lhb and Fshb) expression in pituitary gonadotropes and in gonadotrope cell lines [5,6,7,8,9]. The pattern of GnRH signal is pulsatile, which influences gonadotropin gene regulation and is critical for reproductive function [10,11]

Methods

Results

Conclusion