PhD Student UCSF Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, United States
Abstract: Exposure to nicotine (NIC) during pregnancy is associated with a reduction in fetal cortical grey matter volume, driven by vulnerable cell types and molecular pathways that are not clearly understood. There is evidence of acetylcholine signaling to fetal cortical germinal centers from cholinergic tracts, but the effect of activation of nicotinic acetylcholine receptors (nAChRs) in progenitor cells and radial glia (RG) of the developing human cortex remains unclear. We find two nACHR subunits, CHRNA7 and the human-specific subunit CHRFAM7A, expressed in SOX2+ progenitors, with CHRFAM7A highly enriched along RG apical endfeet. To explore potential functions of cholinergic signaling, we exposed dissociated primary cortical cultures to nAChR agonists, including NIC, or performed nAChR knockdown followed by bulk and single-cell (sc) RNA-sequencing. ScRNAseq revealed that downstream effects of NIC exposure included upregulation of semaphorin-plexin axon guidance associated genes in ventricular RG, outer Radial Glia (oRG) and excitatory neurons (ENs) at GW15-16. We also observed an upregulation of genes associated with chromatin silencing, DNA methylation, and DNA alkylation changes in RG at GW 19-22. Many genes critical for cortex development including DCHS1, SHANK3, LAMA5, NLGN2, LRP1, KIF1A, were downregulated in ENs following NIC exposure. While some DEGs are downstream of both CHRNA7 and CHRFAM7A, a large fraction of DEGs were unique to each nAChR subunit, suggesting a diversification of the regulatory networks controlled by CHRFAM7A during human cortical evolution. Using immunostaining, we observed that activation of nAChRs through agonists increased the number of SOX2+, HOPX+, and KI67+ RG and decreased NEUN+, deep layer CTIP2+ and upper layer SATB2+ neurons. ShRNA-mediated nAChR knockdown produced inverse phenotypic changes, with a reduction in RG cells and an increase in neuron number. Our results suggest that prenatal NIC exposure is able to change RG cell fate, which we hypothesize may be causing some of the clinical phenotypes observed in the offspring of smokers.
Funding Source: Boehringer Ingelheim Fonds PhD Fellowship R35NS097305-07 Swiss National Science Foundation
