Expression signatures of dopaminergic neurons of ventral SNc (Pitx3-dependent), dorsal SNc (Pitx3-independent) and VTA

Pitx3 is a transcription factor that is expressed in all midbrain dopaminergic (mDA) neurons during early development, but later becomes restricted in dopaminergic subsets of substantia nigra compacta (SNc) and of the ventral tegmental are (VTA) that are vulnerable to neurodegenerative stress (MPTP, 6-OHDA, rotenone, Parkinson's disease). Overall, in mice, Pitx3 is required for developmental survival of ventral SNc neurons and for postnatal survival of VTA neurons (after postnatal day 40). With the aim of determining the gene networks that distinguish Pitx3-vulnerable (Pitx3-positive) from Pitx3-resistant (Pitx3-negative) subsets of SNc and VTA, we performed a comparison at the transcriptome level between FAC-sorted mDA neurons of SNc and VTA that were obtained from wild-type and Pitx3-/- newborn mice. The latter mice have already lost the majority of their TH+Calb1- mDA neurons of ventral SNc (Pitx3-dependent), but their TH+Calb1+ neurons of dorsal SNc (Pitx3-independent), including all of VTA neurons (50% are Pitx3-dependent and 50% Pitx3-independent), are unaffected by Pitx3 deletion. At postnatal day 40, Pitx3-/- mice display a marked loss of dopaminergic subsets of VTA that normally co-express Pitx3 and Calb1 (Pitx3-dependent neurons of VTA). Ventral midbrains were dissected from Pitx3+/+;TH-EGFP+/- and Pitx3-/-;TH-EGFP+/- mouse pups (P1-P4). SNc and VTA were separated for both mouse genotypes (4 sample types: VTA WT, VTA KO, SNc WT and SNc KO) and then tissues were digested in papain solution. Propidium-iodide (PI) was then added to the single cell suspensions and EGFP+/PI- mDA neurons were sorted by flow cytometry into RNAlater solution (3000 cells per 30uL). Batches of EGFP+ cells in RNAlater were stored at -80°C until the necessary number of cells had been accumulated for enough total RNA (100ng) per sample type, as recommended by Affymetrix and Nugen companies. Prior to purification of total RNA, corresponding batches of cells were thawed from -80°C and pooled together in order to achieve the required amount of total RNA for each sample type (VTA WT, VTA KO, SNc WT and SNc KO) in duplicate (8 samples in total).