RNA-seq of Isolated Chromaffin Cells Highlights the Role of Sex-Linked and Imprinted Genes in Adrenal Medulla Development

Wing Hei Chan,Masayuki Komada,Matthew J Wakefield,Colin R Anderson,Toshiaki Fukushima,E Michelle Southard-Smith

doi:10.1038/s41598-019-40501-0

Abstract

Adrenal chromaffin cells and sympathetic neurons synthesize and release catecholamines, and both cell types are derived from neural crest precursors. However, they have different developmental histories, with sympathetic neurons derived directly from neural crest precursors while adrenal chromaffin cells arise from neural crest-derived cells that express Schwann cell markers. We have sought to identify the genes, including imprinted genes, which regulate the development of the two cell types in mice. We developed a method of separating the two cell types as early as E12.5, using differences in expression of enhanced yellow fluorescent protein driven from the tyrosine hydroxylase gene, and then used RNA sequencing to confirm the characteristic molecular signatures of the two cell types. We identified genes differentially expressed by adrenal chromaffin cells and sympathetic neurons. Deletion of a gene highly expressed by adrenal chromaffin cells, NIK-related kinase, a gene on the X-chromosome, results in reduced expression of adrenaline-synthesizing enzyme, phenyl-N-methyl transferase, by adrenal chromaffin cells and changes in cell cycle dynamics. Finally, many imprinted genes are up-regulated in chromaffin cells and may play key roles in their development.

Highlights

One gene previously noted to be upregulated in developing adrenal chromaffin cells is Delta-like 1 homolog (Dlk1)[7,8,9,10], an imprinted gene
We describe a technique for separating adrenal chromaffin and sympathetic neuron precursors in embryonic mice before they segregate anatomically, which allowed us to generate a resource of RNA sequencing data on genes differentially expressed by the two cell types, identify a role for Nrk in adrenal chromaffin cell development and produce evidence that many imprinted genes are upregulated during adrenal chromaffin cell development
The separation of developing adrenal chromaffin cells from sympathetic neuroblasts in TH-Cre::R26R-enhanced yellow fluorescent protein (EYFP) mice by fluorescence-activated cell sorting (FACS) depended on the differing intensity of the EYFP reporter in the two cell types

Summary

Introduction

One gene previously noted to be upregulated in developing adrenal chromaffin cells is Delta-like 1 homolog (Dlk1)[7,8,9,10], an imprinted gene. Separation of embryonic adrenal chromaffin cells from sympathetic neuroblasts is technically challenging due to www.nature.com/scientificreports/. In the present study we used TH-Cre activation of enhanced yellow fluorescent protein (EYFP) expression in transgenic mice coupled with fluorescence-activated cell sorting (FACS) to isolate and collect sufficient number of sympathetic neuroblasts and adrenal chromaffin cells at E12.5 for transcriptomic analysis by RNA sequencing. This allowed the assessment of all differentially expressed genes, and the identification of potentially important transcription and cell signaling genes. Subsequent studies tested the leading candidate gene for a role in chromaffin cell development along with assessing the expression of imprinted genes

Methods

Results

Conclusion