Illumina HiSeq 2000 sequencing; GSM2664661: E12.5_A1 [single cell RNA-seq]; Mus musculus; RNA-Seq

E12.5_A1 [single cell RNA-seq]

Cells producing adrenalin are largely derived from nerve-associated Schwann cell precursors via an intermediate progenitor “bridge” cell. We demonstrate that large numbers of chromaffin cells arise from peripheral glial stem cells, termed Schwann cell precursors (SCPs) Overall design: SCPs migrate along the visceral motor nerve to the vicinity of the forming adrenal gland where they detach from the nerve and form post-synaptic neuroendocrine chromaffin cells. An intricate molecular logic drives two sequential phases of gene expression, one unique for a distinct transient cellular state and another for cell-type specification. Subsequently, these programs downregulate SCP- and upregulate chromaffin-cell-gene networks. The adrenal medulla forms through limited cell expansion and requires the recruitment of numerous SCPs. Thus, peripheral nerves serve as a stem cell niche for neuroendocrine system development.

Multipotent Peripheral Glial Cells Generate Neuroendocrine Cells of the Adrenal Medulla

Submitted by Gene Expression Omnibus on 11-JUL-2017

Timed-pregnant mice were sacrificed by deep anesthesia followed by cervical dislocation. Dissection of embryos and adrenal anlages was carried out using a stereotactic microscope equipped with a fluorescent light source. Biological materials were transferred into DMEM/F12 medium with 0.05% Trypsin/ 0.02% EDTA (Sigma) and incubated at 37⁰ C for 5 min. Single cells were isolated by gentle trituration using P1000 tips and then added cold 10% FCS to stop the enzymatic reaction. After a double wash procedure using PBS with RNAse inhibitor, cells were filtered through a 40 uM nylon cell strainer and collected into a FACS tube on ice. Samples remained on ice throughout the process and total processing time from adrenal anlages isolation to sorting was limited to 1 hour. Dissection of Wnt1-Cre/Tomato+ sympatho-adrenal anlages at E12.5 and E13.5 stages was carried out using a stereomicroscope equipped with a fluorescent light source. The dissected fragments were transferred into DMEM/F12 medium with 0.05% Trypsin/ 0.02% EDTA (Sigma) and incubated at 37⁰ C for 5 min. Single cells were released by gentle trituration using P1000 tips, and then ice-cold 10% FCS was added to stop the enzymatic reaction. After a double wash procedure using PBS with RNAse inhibitor, cells were filtered through a 40 uM nylon cell strainer and collected into a FACS tube on ice. After that the samples remained on ice or were kept at +4C throughout the rest of the processing that included single cell FACS sorting of Tomato+ cells into 384-well plates with pre-filled lysis buffer.The sequencing libraries were generated using the published protocol for Smartseq2 sequencing (Picelli et al., Nature Protocols 2014). Resulting libraries were sequenced on the Illumina® HiSeqTM 2000 sequencing platform. The extended analysis is published in Furlan, Dyachuk and Kastriti et al., Science 2016".

Multipotent Peripheral Glial Cells Generate Neuroendocrine Cells of the Adrenal Medulla

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