The development of sympathoadrenal progenitor cells into sympathetic neurons and adrenal chromaffin cells
AuthorChan, Wing Hei
AffiliationAnatomy and Neuroscience
Document TypePhD thesis
Access StatusOpen Access
© 2017 Dr. Wing Hei Chan
Sympathetic neurons and adrenal chromaffin cells originate from a common precursor, the neural crest-derived sympathoadrenal (SA) progenitor cell. Specification of trunk neural crest cells into SA progenitor cells is under the influence of bone morphogenetic proteins (BMPs) produced from the wall of the dorsal aorta. SA progenitor cells then further differentiate into either sympathetic neurons of the sympathetic ganglia or chromaffin cells in the adrenal medulla. However, the mechanism underlying the segregation of chromaffin and neuronal cell fates remain unknown and the time course of SA segregation and regulation of cell fate is poorly understood. To discover novel regulators, and to have a broader view of the transcriptional networks involved in the segregation events, large scale gene screening is required. Isolation of sympathetic neuroblasts and adrenal chromaffin precursor cells for molecular studies, however, is challenging, as they are hard to discriminate during early development. Therefore, the aims of this study were to 1), identify effective marker(s) for early discrimination of neuroblasts and chromaffin precursor cells in order to 2), investigate their patterns of proliferation and differentiation and 3), develop an isolation method by fluorescence-activated cell sorting (FACS) for the two cell types, and finally 4), perform a comparative transcriptomic analysis by using RNA sequencing (RNA-seq) to identify candidate regulator genes that control fate determination of developing adrenal chromaffin cells and sympathetic neuroblasts. The temporal and spatial expression patterns of tyrosine hydroxylase (TH) and cocaine and amphetamine regulated transcript (CART) were first analysed by quadruple-label immunofluorescence. The proliferative behaviour of SA cells and their derivatives were also analysed. Our data showed that sympathetic neuroblasts and chromaffin precursor cells could be discriminated as early as E12.5 by differences in the intensity of TH immunostaining, expression of CART and by proliferative activity. Quantitative data showed higher TH immunostaining in chromaffin cells compared with most ganglion cells, while CART expression was widespread in embryonic sympathetic neuroblasts but absent from adrenal chromaffin cells. Thus CART expression and the levels of TH expression are identified as markers for distinguishing between sympathetic neurons and chromaffin cells as they differentiate. The possibility of isolating sympathetic neuroblasts and adrenal chromaffin precursor cells by FACS was then examined. Isolation based on the differential levels of TH expression by FACS was first investigated in transgenic animals that express fluorescent marker proteins driven from the TH promoter. In cells from transgenic mice where TH-Cre drove enhanced yellow fluorescent protein (EYFP) expression, the intensity of EYFP expression was found to separate the two cell types for isolation. A FACS-based isolation strategy developed in this study permitted routine isolation of purified sympathetic neuroblasts and adrenal chromaffin cells in E12.5 mice. The cell type-specific transcription profiles of the isolated sympathetic neuroblasts and adrenal chromaffin precursor cells during the key segregation event at E12.5 mice were examined by RNA-seq. Comparative transcriptome analysis revealed more than 4,000 differentially expressed genes between adrenal chromaffin cells and sympathetic neuroblasts. Among these, 10 genes including Elf3, Elf4, Nrk, Msx2, Dlx1, Dlx2, Dll4, Dlk1, Foxq1 and Fzd10, were identified as potential regulators based on their differential expression level and potential relevancy to development. The temporal gene expression patterns of these 10 genes during SA development in E11.5 to E14.5 mice were further examined by droplet digital PCR (ddPCR) and all of 10 genes were shown to be expressed transiently at E12.5 and E13.5, suggesting a potential role during the key stage of lineages segregation. Among these, the differential protein expression patterns of DLK1 and FOXQ1 in the adrenal medulla and sympathetic ganglia, revealed by immunohistochemistry, were also found to be highly consistent with the gene expression patterns revealed by both RNA-seq and ddPCR. For Nrk, loss-of-function analysis in E18.75 mutant mice showed a defect in adrenergic phenotype acquisition in adrenal chromaffin cells and cell proliferation, suggesting a role in adrenergic chromaffin cell fate acquisition. The RNA-seq data also raised the possibility of epigenetic regulation in SA lineage cell fate acquisition through genomic imprinting and X-chromosome inactivation.
Keywordssympathetic neuron; chromaffin cell; neuroblastoma; RNA-seq; sympathoadrenal
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