Bone morphogenetic protein 9 induces the transcriptome of basal forebrain cholinergic neurons

Abstract

Basal forebrain cholinergic neurons (BFCN) participate in processes of learning, memory, and attention. Little is known about the genes expressed by BFCN and the extracellular signals that control their expression. Previous studies showed that bone morphogenetic protein (BMP) 9 induces and maintains the cholinergic phenotype of embryonic BFCN. We measured gene expression patterns in septal cultures of embryonic day 14 mice and rats grown in the presence or absence of BMP9 by using species-specific microarrays and validated the RNA expression data of selected genes by immunoblot and immunocytochemistry analysis of their protein products. BMP9 enhanced the expression of multiple genes in a time-dependent and, in most cases, reversible manner. The set of BMP9-responsive genes was concordant between mouse and rat and included genes encoding cell-cycle/growth control proteins, transcription factors, signal transduction molecules, extracellular matrix, and adhesion molecules, enzymes, transporters, and chaperonins. BMP9 induced the p75 neurotrophin receptor (NGFR), a marker of BFCN, and Cntf and Serpinf1, two trophic factors for cholinergic neurons, suggesting that BMP9 creates a trophic environment for BFCN. To determine whether the genes induced by BMP9 in culture were constituents of the BFCN transcriptome, we purified BFCN from embryonic day 18 mouse septum by using fluorescence-activated cell sorting of NGFR(+) cells and profiled mRNA expression of these and NGFR(-) cells. Approximately 30% of genes induced by BMP9 in vitro were overexpressed in purified BFCN, indicating that they belong to the BFCN transcriptome in situ and suggesting that BMP signaling contributes to maturation of BFCN in vivo.

Fig. 1.

BMP9 induction of gene expression in cultured cells from E14 mouse septum. Cells were grown for varying periods of time in the presence or absence of BMP9 (10 ng/ml). Microarray analysis was performed on the RNA purified from the cultures. (A) The abundance of particular mRNA species, in parts per million (ppm) based on internal standard curve calibrations, in BMP9-treated cells versus their abundance in control cells, depicting the trends at the beginning and the end of the time course (i.e., at 6 and 144 h, respectively). Genes whose expression is up-regulated at least 3-fold by BMP9 are indicated by black circles. Time course (Bottom) of the total number of affected genes (black circles), including those whose expression remained elevated 72 h after BMP9 was removed from the cultures (blue circles). (B–H) Graphs represent those genes whose expression was up-regulated at least 3-fold by BMP9 (black circle) over controls (red circles) at any two of the six time points examined. Some cells were treated for 3 days with BMP9, washed on the third day, and were incubated for an additional 3 days in the absence of BMP9 (blue circles). The data are expressed in ppm on the ordinates and time (hours) on the abscissas. (B) Cell cycle-associated genes. (C) Transcription factors. (D) Signaling molecules. (E) Extracellular matrix and adhesion molecules. (F) Enzymes and enzyme inhibitors. (G) Transporters. (H) Chaperonins.

Fig. 2.

Analysis of FACS-purified BFCN. (A) Visualization of NGFR positive and negative septal cells after FACS. After immunostaining of dissociated cells from E18 mouse septa with anti-NGFR polyclonal antibody and Alexa-fluor conjugated secondary antibody, the cells were sorted and purified by FACS (see Materials and Methods), and aliquots of the positive and negative fractions were analyzed by phase and fluorescence microscopy. (B) RT-PCR of BFCN markers for Vacht (814 bp), Cht1 (840 bp), Chat (267 bp), Ngfr (657 bp), and Gapd (983 bp; as control) of RNAs obtained from NGFR positive and negative cell fractions.

Fig. 3.

Analysis of BMP9-induced proteins. (Upper) Western blots of selected proteins from control and BMP9-treated neuronal cultures. Septal cultures from E14 mice were treated for 3 days with BMP9 (10 ng/ml) or vehicle. Cells were harvested and processed for SDS/PAGE as described in Materials and Methods. (Lower) Immunocytochemistry (A–J) of cultures are treated as in Upper.(A–D) Double immunofluorescence staining with anti-Na⁺/K⁺ ATPase α-1 and anti-VACHT antibodies, done in parallel with negative and positive controls. (E and F) Immunofluorescence staining with anti-NGFR antibody. Phase-contrast (G and H) and immunofluorescence (I and J) staining with anti-Noggin antibody of the same field. All pictures were obtained with a ×20 objective.