Supplementary Figure 3: Expression analysis of mDA neuron markers and catecholamines in Lmx1a-, Lmx1b- and Lmx1a/b-ablated mice.

(a) Tyrosine hydroxylase (TH) immunostaining in ventral midbrain sections of 2 months (2m) old cLmx1a/b^Ctrl (cCtrl) and cLmx1a/b^DatCre (cDatCre) mice. Scale bar, 100 μm. (b) Unbiased stereological counting of TH-immunopositive cells (TH-positive) in the substantia nigra (SN) and the ventral tegmental area (VTA) of 2m old cCtrl and cDatCre mice. The sections were counterstained with Nissl staining and the Nissl-positive neuronal cells that were negative for TH immunostaining (Nissl) were counted. The total number of TH-positive and TH-negative neurons in the SN and VTA was also estimated (Total). Numbers correspond to the estimated cell numbers in one hemisphere. n=6 animals per genotype. (c) Tyrosine hydroxylase (TH) immunostaining in striatal sections of 2m old cLmx1a^DatCre, cLmx1b^DatCre mice and their corresponding control littermates (cLmx1a^Ctrl and cLmx1b^Ctrl). Scale bar, 100 μm. (d) TH immunostaining in ventral midbrain sections of 2m old cLmx1a^DatCre, cLmx1b^DatCre mice and their corresponding control littermates (cLmx1a^Ctrl and cLmx1b^Ctrl). Scale bar, 100 μm. (e) Optical densitometry from TH and Dopamine transporter (DAT) immunostained terminals from the dorsal striatum (Dorsal) and the ventral striatum (Ventral) areas of young (2m) cLmx1a^DatCre, cLmx1b^DatCre mice and their corresponding control littermates (cLmx1a^Ctrl and cLmx1b^Ctrl), n=3 animals per genotype. Data is shown in Arbitrary Units (AU). (f) TH immunostaining in striatal sections of 2m old cLmx1a^DatCre, cLmx1b^DatCre mice and their corresponding control littermates (cLmx1a^Ctrl and cLmx1b^Ctrl), showing nerve terminals and abnormally large profiles in cLmx1b^DatCre mice. Scale bar, 50 μm. (g) HPLC measurements of Dopamine and the dopamine metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and Homovanillic acid (HVA) of 16 to 20 months old cLmx1a^Ctrl and cLmx1a^DatCre mice. Separate analyses were performed on tissue extracts from the Striatum and the Nucleus accumbens (NAcc). n=10 cCtrl and 10 cDatCre animals per genotype. (h) HPLC measurements of Dopamine, DOPAC and HVA of 20 months old cLmx1b^Ctrl and cLmx1b^DatCre mice. Separate analyses were performed on tissue extracts from the Striatum and the NAcc. n=10 cCtrl and 10 cDatCre animals per genotype. (i) HPLC measurements of Dopamine, DOPAC and HVA of 9 months old cLmx1a/b^Ctrl (cCtrl) and cLmx1a/b^DatCre (cDatCre) mice. Separate analyses were performed on tissue extracts from the different brain areas indicated (Substantia nigra, SN; Ventral tegmental area, VTA; Cerebellum, CB). n=10 cCtrl and 13 cDatCre animals per genotype. All data is represented as mean±sem; Mann Whitney test; *p<0.05, **p<0.05, #p=0.06.