Figure 1.
Sim1 mRNA expression by RT-PCR and cellular localization of Sim1 in mouse tissues by immunohistochemistry.
A: Sim1 transcript was detected in mouse E12 ventral hindbrain, ventral midbrain, and isthmus region using sim1 primers corresponding to nucleotides (nt) 736-756 and nt:1052-1032 of the mouse Sim1 gene (Genbank accession number: NM011376). B: double immunolabeling on mouse fixed paraffin sections for TH (blue) and Sim1 (brown) showed Sim1 localization in the cell nucleus and cytoplasmic staining pattern for TH in midbrain substantia nigra pars compacta (SNc) and ventral tegmental area (VTA). Inset at higher magnification shown in C: co-localization of TH and Sim1 is apparent. Arrows point to cells that are immunopositive for both TH and Sim1. Asterisks indicate Sim1 positive, but TH negative cells. D, E: double immunofluorescence for 5-HT (green) and Sim1 (red) in mouse hindbrain revealed co-localization of the proteins.
Figure 2.
Phenotype analysis for midbrain dopaminergic and rostral serotonergic neurons of Sim1-/- mutants.
Counting of TH positive cells after immunostaining in midbrain tissue sections revealed no differences in the number of midbrain dopaminergic neurons between wild type (wt) and Sim1-/- mutants at embryonic day (E)14.5 (A) and newborn (P0; B). ns: not significant. C-D: 5-HT immunofluorescence in frontal sections of wt (C) and Sim1-/- embryos (D) at E14.5 (DRN: dorsal raphe nucleus; Aq: aqueduct). E: Quantification of cell counts: numbers of 5-HT immunopositive cells were comparable between wt and Sim1-/-. F-J: Whereas quantification of cell counts revealed no statistical differences (ns) in the total number of rostral 5-HT neurons, median raphe, and paramedian raphe 5-HT neurons between Sim1-/- (H) and their wt littermates (F), the number of 5-HT immunoreactive cells in the dorsal raphe nucleus (DRN) was found significantly decreased in Sim1-/-, compared to the wild type. 5-HT immunolabeling in mouse rostral raphe nuclei at P0 in wt (G) and Sim1 mutants (I). Arrow points to dorsal raphe nucleus (DRN). Aq: aqueduct.
Figure 3.
MN9D cells, a suitable in vitro model system.
A: Expression of determinants of the serotonergic lineage in the MN9D cell line by RT-PCR. Gata2, Pet1, and Tph2 could be amplified from MN9D cells cDNA. The Sim1 primers used correspond to nucleotides (nt) 37-66 and nt:152-126 of the mouse Sim1. In addition, expression of the neural stem cell marker nestin was also detected. B: RT-PCR analysis of Sim1 expression in MN9D cells cDNA, 24 hours, 48 hours, and 72 hours after transfection of the cells with either pcDNA3::Sim1 or pcDNA3 expression vectors (ctl) and GAPDH as housekeeping gene. Sim1 expression was clearly increased in pcDNA3::Sim1-transfected MN9D cells, compared to the pcDNA3-transfected cells. C: Sim1 protein abundance by immunoblotting in homogenates of MN9D cells transfected either with pcDNA3::Sim1 or pcDNA3 expression vectors (ctl) 24 hours and 48 hours after transfection. The immunoblots were probed either with monoclonal antibody against GAPDH or with rabbit polyclonal antibody against Sim1. Sim1 protein 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 Sim1: GAPDH and Student's t-test, n = 3). Arrow points to the band of the expected size. The blots are representative for three different experiments. 30 µg protein was loaded per lane.
Figure 4.
Impact of Sim1 on protein abundance of early and late markers of the dopaminergic (A) and serotonergic (B) lineage.
Immunoblotting in homogenates of MN9D cells transfected either with pcDNA3::Sim1 or pcDNA3 expression vectors (ctl) 24 hours after transfection. The immunoblots were probed either with an antibody against GAPDH or with Pitx3, TH, Pet1, or TPH2 antibody. Pitx3 and TH protein expression was comparable between controls and pcDNA3::Sim1-transfected MN9D cells. In contrast, Pet1 and TPH2 protein abundance was significantly upregulated in pcDNA3::Sim1-transfected MN9D cells, compared to the pcDNA3-transfected cells. (*P<0.05 after densitometric analysis of the signal ratio Pet1:GAPDH or TPH2:GAPDH and Student's t-test; n = 3). The blots are representative for three different experiments. 30 µg protein was loaded per lane.
Figure 5.
Differential regulation of serotonergic lineage determinants by Sim1.
Quantitative real-time PCR analysis of Gata2 (A), Mash1 (B), Pet1 (C) and Tph2 (D) mRNA levels in pcDNA3::Sim1-transfected MN9D cells and in pcDNA3-transfected cells. Expression of individual gene is shown as 2−ΔΔCt±s. Gata2 and Mash1 expression remained unchanged in pcDNA3::Sim1-transfected MN9D cells, compared to pcDNA3-transfected cells 24 hours, 48 hours, and 72 hours after transfection. In contrast, Pet1 and Tph2 expression was significantly up-regulated in pcDNA3::Sim1-transfected MN9D cells, compared to the controls 48 hours and 72 hours after transfection (*p<0.05; **p<0.01; and ***p<0.0001 using the Student's t-test, n = 3).
Figure 6.
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.
Figure 7.
Sim1 loss-of-function in vitro and in vivo.
A: Quantitative real-time PCR analysis for candidate genes in MN9D cells transfected either with control siRNA (ctl) or with specific Sim1 siRNA. Gene expression is shown as 2−ΔΔCt±s. Pet1, Gata2, Brn3.2, and Tph2, but not RGS4 expression was significantly down-regulated in Sim1 siRNA-transfected cells, compared to the controls, 24 hours after transfection (*p<0.05 and **p<0.01, using the Student's t-test, n = 3). B-M: In situ hybridization for Pet1 (B-E), RGS4 (F-I) and Brn3.2 (J-M) in wild type (wt) and Sim1-/- mutants at P0. Pet1 expression was observed in the area of rostral 5-HT neurons (B) including the area of dorsal raphe in wt (C: magnification of the inset in B), but was considerably decreased in Sim1-/- (D). E: magnification of the inset illustrating the area of the dorsal raphe. RGS4 (F, and magnification of the inset in G) was also expressed in the dorsal raphe in wt, and was considerably decreased in Sim1 mutants (H and magnification of the inset in I). In contrast, although Brn3.2 expression in wt was present (J, asterisks) the area of rostral 5-HT neurons was devoid of Brn3.2 expression (J, arrow and magnification of the inset in K). In Sim1-/- mutants Brn3.2 expression was absent (L, and magnification of the inset in M). N: immunoblot analysis for Pet1, RGS4, and Brn3.2 in hindbrain of wt and Sim1-/- mutants. Pet1, RGS4, and Brn3.2 protein abundance was significantly downregulated in Sim1-/- hindbrain, compared to wt. (*p<0.05 after densitometric analysis of the signal ratio Pet1:GAPDH, RGS4:GAPDH or Brn3.2:GAPDH and Student's t-test; n = 4). The blots are representative for four different experiments. 30 µg protein was loaded per lane.
Table 1.
Primer sequences used for RT-PCR gene expression analysis.