A tyrosine hydroxylase-yellow fluorescent protein knock-in reporter system labeling dopaminergic neurons reveals potential regulatory role for the first intron of the rodent tyrosine hydroxylase gene

Abstract

Degeneration of the dopaminergic neurons of the substantia nigra is a hallmark of Parkinson's disease. To facilitate the study of the differentiation and maintenance of this population of dopaminergic neurons both in vivo and in vitro, we generated a knock-in reporter line in which the yellow fluorescent protein (YFP) replaced the first exon and the first intron of the tyrosine hydroxylase (TH) gene in one allele by homologous recombination. Expression of YFP under the direct control of the entire endogenous 5' upstream region of the TH gene was predicted to closely match expression of TH from the wild type allele, thus marking functional dopaminergic neurons. We found that YFP was expressed in dopaminergic neurons differentiated in vitro from the knock-in mouse embryonic stem cell line and in dopaminergic brain regions in knock-in mice. Surprisingly, however, YFP expression did not overlap completely with TH expression, and the degree of overlap varied in different TH-expressing brain regions. Thus, the reporter gene did not identify functional TH-expressing cells with complete accuracy. A DNaseI hypersensitivity assay revealed a cluster of hypersensitivity sites in the first intron of the TH gene, which was deleted by insertion of the reporter gene, suggesting that this region may contain cis-acting regulatory sequences. Our results suggest that the first intron of the rodent TH gene may be important for accurate expression of TH.

Fig. 1

Generation of TH^YFP/+ knock-in mES cells and mice. (A) Schematic of targeting strategy used to insert the gene for YFP into the TH gene locus by homologous recombination. Southern blot digestion sites and PCR primers for genotyping are indicated. (B) Southern blot analysis of genomic DNA after digestion with SpeI confirmed homologous recombination and in vitro Neo excision. (C) PCR of mouse tail DNA confirmed heterozygous TH/YFP genotype and excision of Neo.

Fig. 2

YFP reporter expression in differentiated TH^YFP/+ knock-in mES cells and adult TH^YFP/+ knock-in mouse midbrain. (A, B) TH^YFP/+ mES cells were differentiated in vitro for 9 days on PA6 cells into dopaminergic neurons and co-stained with antibodies to TH (red) and YFP (green). (C) TH^YFP/+ mES cells were differentiated for 9 days and co-stained for TH (red), YFP (green) and the nuclear neuronal marker NeuN (blue) or for TH (red), YFP (green) and the neuronal marker TuJ1 (blue). Triple overlap in expression appears purple. Cryosections of TH^YFP/+ heterozygous adult brain were co-stained with antibodies to TH (red) and YFP (green) in the ventral tegmental area (D) and substantia nigra (E). Examples of cells with overlapping expression are indicated by arrowheads. A higher magnification confocal stack image of neurons in the substantia nigra is seen in (F). (G) Quantification of differentiated TH and YFP expressing cells in vitro. Of the total cells (n=1508), 54% expressed TH alone (n=804), 17% expressed YFP alone (n=262), and 29% expressed both TH and YFP (n=442). (H) Quantification of TH and YFP expressing cells in the mouse midbrain. Of the total cells (n=426), 90% expressed TH alone (n=382), 5% expressed YFP alone (n=21), and 5% expressed both TH and YFP (n=23).

Fig. 3

YFP and TH expression in other dopaminergic and non-dopaminergic TH-expressing cell groups. Cryosections of TH^YFP/+ heterozygous adult brains were co-stained with antibodies to TH (red) and YFP (green) in the olfactory bulb (A) and hypothalamus (B). Examples of cells with overlapping expression are indicated by arrowheads. (C) Quantification of TH and YFP expressing cells in the olfactory bulb. Of the total cells (n=174), 37% expressed TH alone (n=65), 41% expressed YFP alone (n=70), and 22% expressed both TH and YFP (n=39). (D) Quantification of TH and YFP expressing cells in the dopaminergic cell groups in the hypothalamus. Of the total cells (n=274), 35% expressed TH alone (n=97), 36% expressed YFP alone (n=98), and 29% expressed both TH and YFP (n=79). Cryosections of TH^YFP/+ heterozygous adult brain were co-stained with antibodies to TH (red) and YFP (green) in the locus ceruleus (E) and C1 cell group (F). Examples of cells with overlapping expression are indicated by arrowheads.

Fig. 4

DNaseI hypersensitivity assay of the TH gene in C6 glioma cells and PC12 cells. (A) Southern blot of chromosomal DNA from TH-negative C6 glioma cells and TH-positive PC12 cells treated with increasing concentrations of DNaseI. DNA was digested with EcoRI and probed using an EcoRI-PstI rat cDNA fragment that spans from nucleotides 117–1137 bp (exons 2–11) within the TH coding region. (B) Schematic of hypersensitivity sites identified in the TH gene. Sites HS3-6 fall within the region deleted in the reporter system. (C) Schematic map of the first exon, first intron and second exon of the mouse, rat and human TH genes.