Transcriptional Responses of Cultured Rat Sympathetic Neurons during BMP-7-Induced Dendritic Growth

Abstract

BackgroundDendrites are the primary site of synapse formation in the vertebrate nervous system; however, relatively little is known about the molecular mechanisms that regulate the initial formation of primary dendrites. Embryonic rat sympathetic neurons cultured under defined conditions extend a single functional axon, but fail to form dendrites. Addition of bone morphogenetic proteins (BMPs) triggers these neurons to extend multiple dendrites without altering axonal growth or cell survival. We used this culture system to examine differential gene expression patterns in naïve vs. BMP-treated sympathetic neurons in order to identify candidate genes involved in regulation of primary dendritogenesis.Methodology/Principal FindingsTo determine the critical transcriptional window during BMP-induced dendritic growth, morphometric analysis of microtubule-associated protein (MAP-2)-immunopositive processes was used to quantify dendritic growth in cultures exposed to the transcription inhibitor actinomycin-D added at varying times after addition of BMP-7. BMP-7-induced dendritic growth was blocked when transcription was inhibited within the first 24 hr after adding exogenous BMP-7. Thus, total RNA was isolated from sympathetic neurons exposed to three different experimental conditions: (1) no BMP-7 treatment; (2) treatment with BMP-7 for 6 hr; and (3) treatment with BMP-7 for 24 hr. Affymetrix oligonucleotide microarrays were used to identify differential gene expression under these three culture conditions. BMP-7 significantly regulated 56 unique genes at 6 hr and 185 unique genes at 24 hr. Bioinformatic analyses implicate both established and novel genes and signaling pathways in primary dendritogenesis.Conclusions/SignificanceThis study provides a unique dataset that will be useful in generating testable hypotheses regarding transcriptional control of the initial stages of dendritic growth. Since BMPs selectively promote dendritic growth in central neurons as well, these findings may be generally applicable to dendritic growth in other neuronal cell types.

Reactive oxygen species are involved in BMP-induced dendritic growth in cultured rat sympathetic neurons

“…Previous studies of podocytes demonstrated that BMP-2 increased NAPDH oxidase-dependent ROS production via upregulation of Id-1, a negative regulator of bHLH transcription factors (Pache et al, 2006). Interestingly, it has been shown that Id-1 is upregulated in cultured sympathetic neurons following BMP-7 treatment (Garred et al, 2011). However, the effects of Id-1 on BMP-7 induced dendritic growth and on NOX2 expression in sympathetic neurons have not yet been explored.…”

Regulatory mechanisms underlying the differential growth of dendrites and axons

“…Consistent with this model, Garred and colleagues found that Actinomycin-D, a transcriptional inhibitor, blocks BMP7-induced dendritic growth in cultured sympathetic neurons. Microarray analysis of cultured sympathetic neurons treated with BMP7 for six hours showed changes in the transcript level of a number of transcriptional repressors belonging to the inhibitor of DNA binding (Id) family [25] , an effect which may subsequently lead to the regulation of other transcriptional programs. BMP7 might also promote dendritic growth by enhancing the expression of the microtubule-associated protein MAP2 [26] .…”

Regulation of Dendritogenesis in Sympathetic Neurons

“…A comprehensive analyses of Smad-dependent dendritic growth in sympathetic neurons provided evidence for early transcriptional regulation of dendritic growth downstream of BMP-7 [52]. In neuronal cell cultures from embryonic rat SCG, BMP-7-induced dendritic growth could be blocked by pharmacologic inhibition of transcription with actinomycin-D when the inhibitor was added within the first 24 hours of BMP-7 exposure but not when it was added after 48 hours of BMP-7 exposure.…”