Identification of candidate genes involved in the development of serotonergic neurons.
A: Quantitative real-time PCR analysis showed that expression of the transcription factors Brn3.2 and Lhx8, and of a regulator of G protein signalling and modulator of 5-HT1A-mdiated neurotransmitter release, RGS4, are significantly upregulated in mouse ventral hindbrain tissue compared to ventral midbrain primary tissue at embryonic day 11.5. B-C: Regulation of expression of candidate genes by Sim1, assessed by quantitative real-time PCR. Rgs4 (B) and Brn3.2 (C) mRNA levels in pcDNA3::Sim1-transfected MN9D cells and in pcDNA3-transfected cells were analyzed. Expression of individual gene is shown as 2−ΔΔCt±s. Rgs4 and Brn3.2 expression was significantly up-regulated 24 hours and 48 hours, respectively, after transfection of MN9D cells with pcDNA3::Sim1 expression vector, compared with the controls (**P<0.01, using the Student's t-test, n = 3). D: Immunoblotting for RGS4 and Brn3.2 protein abundance in homogenates of MN9D cells transfected either with pcDNA3::Sim1 or pcDNA3 expression vectors (ctl) 48 hours after transfection. The immunoblots were probed either with monoclonal antibody against GAPDH or with a goat polyclonal antibody against RGS4 or Brn3.2. RGS4 protein expression was comparable between controls and pcDNA3::Sim1-transfected MN9D cells. In contrast, Brn3.2 protein abundance was significantly upregulated in pcDNA3::Sim1-transfected MN9D cells, compared to the pcDNA3-transfected cells. (**p<0.01 after densitometric analysis of the signal ratio Brn3.2: GAPDH and Student's t-test; n = 3). The blots are representative for three different experiments. 30 µg protein was loaded per lane. E, G: In situ hybridization for expression of candidate genes in mouse at E14.5 using antisense probes. Brn3.2 expression was present in hindbrain, but not in the area of rostral serotonergic neurons (E), whereas RGS4 expression was detectable in rostral serotonergic neurons (G). F: immunofluorescence for 5-HT at E14.5. H, J: In situ hybridization for expression of candidate genes in mouse at E18.5 using antisense probes. Brn3.2 expression was present in hindbrain, but not in the area of rostral serotonergic neurons (H), whereas RGS4 expression was detectable in rostral serotonergic neurons (J). I: immunofluorescence for 5-HT at E18.5. K, M: In situ hybridization at E18.5 for expression of Brn3.2 (K) and RGS4 (M) using sense probes revealed no detectable staining. L: schematic presentation of a sagittal section of mouse brain at E18.5 with a line drawn to indicate the approximate level of brain sections used for H-M (r: rostral, c: caudal). Aq: aqueduct.
