Figure 1.
Strategy for isolation of FACS-purified Pitx3-dependent (red) and Pitx3-independent (white) mDA neurons for expression profiling analysis.
Dissected SN and VTA from newborn TH-EGFP transgenic Pitx3+/+ and Pitx3−/− mice were used for FACS purification of catecholaminergic neurons. The EGFP+ cells consists in various proportions (approximate % shown) of Pitx3+ (red), Pitx3− (white) and Pitx3del (yellow) mDA neurons, depending on the region dissected and mDA neuronal loss resulting from Pitx3 inactivation. RNA from four cell preparations (SNc WT, VTA WT, SNc KO, VTA KO) were analyzed by hybridization in duplicates to Affymetrix Mouse Gene 1.0ST microarrays.
Figure 2.
Subset-specific expression signatures of mDA neurons.
(A) Differentially expressed probesets were clustered in an unbiased manner into three-dimensional Cartesian-type coordinates defined according to the relative enrichment attributes for each probeset in the comparisons between SNc WT/VTA WT, SNc KO/SNc WT and VTA KO/VTA WT. Each cluster was named according to the observed preferential expression pattern (label and shown in red in diagrams). The heatmaps show relative enrichments for 20 genes that are highly enriched and/or that have documented functions (the complete gene lists for each subset is provided in Table S1). The total number of genes (not probesets) in each cluster is indicated together with the number of those that are either activated or repressed by Pitx3 based on the VTA KO/WT comparison. (B) qRT-PCR quantification of mRNAs with preferential expression in either SN or VTA assessed in FACS-sorted cells from WT or Pitx3−/− (KO) tissues. mRNA levels are normalized relative to Gapdh mRNA. Data are shown as means ± S.D.
Figure 3.
Restricted expression of Rgs6 in Pitx3-positive (Pitx3+) dopaminergic neurons of ventral SNc.
(A) Rgs6 expression revealed by immunohistofluorescence (red) together with TH (green) in mDA neurons of SNc but not VTA. Scale bar 410 µm. (B) Co-immunofluorescence analysis of TH (green) and Rgs6 or Pitx3 (red, as indicated) in SNc and VTA of adult mice. Arrowheads point to Pitx3-negative or Rgs6-negative mDA neurons in dorsal SNc. Scale bar 100 µm. (C) Triple immunofluorescence staining for TH (green), Pitx3 (red) and Calb1 (blue) on tissue sections of adult mice indicates that the majority of TH+Pitx3− cells of dSNc (arrowheads) are positive for Calb1, while TH+Pitx3+ cells of vSNc are negative for Calb1, consistent with depiction in A. Scale bar 100 µm.
Figure 4.
Unilateral loss of Pitx3-positive dopaminergic neurons in ventral SNc of Rgs6−/− mice.
(A) Immunoperoxidase staining for TH on representative coronal midbrain sections showing less SNc TH+ neurons on one side of Rgs6−/− mice at 1 year of age compared to sib control. Sections are identified with Bregma position. Scale bar 400 µm. (B) Number of TH+ cells in SNc and VTA of TH-stained coronal sections across midbrain (every 30 µm). Cell counts are represented as means +/− S.D. (***p<0.005). (C) Nissl staining of vSNc sections contiguous to A shows fewer cell bodies and abnormal elongated neurons in Rgs6−/− mice compared to control. Scale bar 100 µm. (D) Double immunofluorescence staining for TH (green) and Pitx3 (red) on sections contiguous to A showing a marked loss of TH+Pitx3+ cells in Rgs6−/− mice. Scale bar 100 µm.
Figure 5.
Unilateral degeneration of vSNc neurons in a subset of Rgs6−/− mice.
(A) Immunoperoxidase staining for TH on representative coronal midbrain sections showing dysmorphic TH+ neurons (low TH staining, THlow, inset) in ventral SNc of Rgs6−/− mice. The dSNc and VTA mDA neurons are unaffected (strong/normal TH staining, inset). Scale bar 200 µm. (B) Triple staining for TH (red), Dapi (blue) and Fluoro-Jade C (FJC, green) showing presence of degenerating THlow cells in vSNc of 1 y-old Rgs6−/− mice (middle panels) and not in control vSNc (upper panels) or in dorsal SNc (lower panels). Scale bar 20 µm. (C) Bilateral cell counts of FJC+ mDA neurons in SNc and VTA of coronal sections from control and 1 y-old Rgs6−/− mice. (D) Co-immunostaining for TH (green) and LC3B (red) in vSNc of WT and Rgs6−/− mice. Scale bar 20 µm. Arrowheads indicate unaffected neurons while arrows point to THlow cells. (E) Co-immunostaining for TH (green) and phosphorylated p27Kip1 (phospho-p27, red) in vSNc of WT and Rgs6−/− mice. Scale bar 20 µm.
Figure 6.
Expression of familial PD genes is altered in degenerating neurons of vSNc.
Double immunofluorescence staining against TH (green) and (A) DJ-1 (red) or (B) Pink1 (red) or (C) Lrrk2 (red) in SNc of control and 1 y-old Rgs6−/− mice that display dysmorphic THlow mDA neurons. Arrowheads indicate unaffected neurons while arrows point to THlow cells. Scale bar 20 µm.
Figure 7.
Reduced expression of Pitx3 and its target genes in degenerating neurons.
(A) Double immunofluorescence staining against TH (green) and Pitx3 (red) in SNc of control and 1 y-old Rgs6−/− mice that display dysmorphic mDA neurons. Arrowheads indicate Pitx3-negative neurons of dSNc. Scale bar 20 µm. (B) Co-immunofluorescence staining against TH (green), nuclear Dapi (blue) and Vmat2 (red) in SNc of WT and Rgs6−/− mice. Scale bar 20 µm. (C) Co-immunofluorescence staining against TH (green), nuclear Dapi (blue) and Bdnf (red) in SNc and VTA of WT and Rgs6−/− mice. Scale bar 20 µm. (D) Double immunofluorescence staining against TH (green) and Pitx3, Aldh1a1, Fgf10 (red) in control and 1 y-old Rgs6−/− mice that display dysmorphic mDA neurons. Arrowheads indicate unaffected dSNc neurons and arrows point to affected vSNc neurons. Scale bar 100 µm.
Figure 8.
Drd2-related changes of gene expression in degenerating neurons of vSNc.
(A) Immunohistofluorescence staining for TH and Drd2 in coronal sections of 1 y-old Rgs6−/− mice and WT controls. mDA neurons of vSNc express higher levels of glycosylated dopamine receptor D2 (Drd2) than those of dSNc (arrowheads). Scale bar 50 µm. (B) Phospho-Erk1/2 (red) staining is only present in degenerating vSNc THlow (green) neurons of Rgs6−/− mice and not in WT controls. Scale bar 20 µm. Arrowheads indicate unaffected neurons. (C) DAT (red) staining is stronger in degenerating vSNc THlow (green) neurons of Rgs6−/− mice than in WT controls. Scale bar 20 µm.
Figure 9.
Rgs6−/− mice as a model for Parkinsonian neurodegeneration.
(A) Rgs6-dependent signaling pathways in vSNc mDA neurons control Pitx3 expression, which itself controls expression of Aldh1a1, TH, Fgf10, Vmat2 and Bdnf. Together, Rgs6 and Pitx3 define a survival pathway in these neurons. The dopamine receptor Drd2 may be a target of Rgs6 action and consistent with this hypothesis, expression of the dopamine transporter DAT is up-regulated in Rgs6−/− vSNc. (B) Schematic representation of major subsets of SNc mDA neurons present at different ages in midbrains of Rgs6−/− mice showing progressive degeneration (small pale green) followed by loss of vSNc mDA neurons.