Lineage and stage specific requirement for Dicer1 in sympathetic ganglia and adrenal medulla formation and maintenance

Abstract

The development of sympathetic neurons and chromaffin cells is differentially controlled at distinct stages by various extrinsic and intrinsic signals. Here we use conditional deletion of Dicer1 in neural crest cells and noradrenergic neuroblasts to identify stage specific functions in sympathoadrenal lineages. Conditional Dicer1 knockout in neural crest cells of Dicer1(Wnt1Cre) mice results in a rapid reduction in the size of developing sympathetic ganglia and adrenal medulla. In contrast, Dicer1 elimination in noradrenergic neuroblasts of Dicer1(DbhiCre) animals affects sympathetic neuron survival starting at late embryonic stages and chromaffin cells persist at least until postnatal week 1. A differential function of Dicer1 signaling for the development of embryonic noradrenergic and cholinergic sympathetic neurons is demonstrated by the selective increase in the expression of Tlx3 and the cholinergic marker genes VAChT and ChAT at E16.5. The number of Dbh, Th and TrkA expressing noradrenergic neurons is strongly decreased in Dicer1-deficient sympathetic ganglia at birth, whereas Tlx3(+)/ Ret(+) cholinergic neurons cells are spared from cell death. The postnatal death of chromaffin cells is preceded by the loss of Ascl1, mir-375 and Pnmt and an increase in the markers Ret and NF-M, which suggests that Dicer1 is required for the maintenance of chromaffin cell differentiation and survival. Taken together, these findings demonstrate distinct stage and lineage specific functions of Dicer1 signaling in differentiation and survival of sympathetic neurons and adrenal chromaffin cells.

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

“…Known markers of sympathetic neurons expressed at between 2.5 and 4-fold difference in sympathetic neuroblasts (Fig. 4D) included Nefl ( neurofilament, light) 9,19,23 , Nefm (neurofilament, medium) 19,24 , Vmat2 or Slc18a2 (vesicle monoamine transporter 2 or solute carrier family 18 member A2) 10,25–27 , Tuj1 or Tubb3 ( Tubulin Beta 3 Class III), 20,28,29 , Ret ( Receptor tyrosine kinase) 30,31 and Isl1 (Insulin gene enhancer protein) 10,22,27 . Of particular significance was Cartpt mRNA 17 , which was more than 8-fold enriched in sympathetic neuroblasts over adrenal chromaffin cells.…”

“…Loss of Meg3 and the microRNAs it controls leads to reduced neural differentiation in human embryonic stem cells 43 . Recently, microRNAs have been implicated in adrenal chromaffin cell fate determination 24 , as deletion of the RNAse, Dicer, which processes miRNAs, leads to a reversion of developing chromaffin cells to a more neuronal phenotype 24 .…”

From proliferation to target innervation: signaling molecules that direct sympathetic nervous system development

“…Knock-out of Dicer1 therefore disturbs siRNA and miRNA production. In mice in which there is loss of Dicer1 from TH-expressing cells, sympathetic neurons show disturbed induction of neuronal markers and increased cell death in late embryonic ages (Stubbusch, et al, 2015), indicating that siRNA and/or miRNA are important. (Stubbusch, et al, 2013).…”

“…Dicer1, via the siRNA and miRNA it generates (see above) may also play a role in the segregation of neuronal and chromaffin cell lineages. Conditional knockout of Dicer1 leads to a maintenance of the neuronal markers that are normally rapidly down-regulated in developing chromaffin cells (Stubbusch, et al, 2013) and leads to increased postnatal death of the cells (Stubbusch, et al, 2015).…”

Overview of Methods to Differentiate Sympathetic Neurons from Human Pluripotent Stem Cells

“…Differentiation of NC cells into sympathetic neuroblasts is supported by BMPs (Saito et al., ). Also, loss of Dicer1 from TH‐expressing cells was shown to prevent symN differentiation (Stubbusch et al., ). SymNs are marked at the early stage by ASCL1 (also called MASH1), PHOX2A, PHOX2B, INSM1, HAND2, GATA3, SOX4, SOX11, ISL1, and TFAP2A/B (Chan, Anderson, & Gonsalvez, ).…”