Lmx1a and Msx1 Are Expressed in Midbrain Dopamine Progenitor Cells
To identify HD proteins expressed in DA progenitor cells in the vMB, embryonic day (E) 10.5 mouse embryo vMB cDNA was used as a template to screen for HD-encoding transcripts by PCR. This approach, combined with a large-scale in situ hybridization screen, identified Lmx1a and Msx1 as two transcription factors with relevant expression patterns. Whole-mount in situ hybridization at E11.5 showed that Lmx1a and Msx1 were both expressed in the vMB and at the caudalmost part of the diencephalon (Figures 1A and 1B). We applied immunohistochemistry to further examine the expression of Lmx1a and Msx1 relative to defined markers of postmitotic DA neurons, including Nurr1, Lmx1b, Pitx3, and tyrosine hydroxylase (TH), the rate-limiting enzyme in DA synthesis (Figures 1F–1K and 1V; data not shown). At E12.5, a stage when DA neurons are actively generated, Lmx1a and Msx1 were coexpressed in DA progenitors located immediately above differentiating DA neurons (Figures 1C–1F). Expression of Lmx1a was maintained in postmitotic Nurr1+/TH+ DA neurons (Figures 1C and 1D), while Msx1 expression was exclusively confined to mitotic DA progenitors that had not yet initiated expression of Nurr1 and TH (Figures 1C, 1E, and 1F).
Lmx1a was expressed prior to Msx1 and was first detected in ventral midline cells at E9 (Figure 1G). At this stage, Lmx1a was coexpressed with Nkx6.1, a HD protein that is broadly expressed in ventral progenitor cells (Vallstedt et al., 2001), including motor neuron progenitors located lateral to DA progenitors (Figure 1K). From E9.5 and onward, the expression of Nkx6.1 was progressively extinguished in presumptive DA progenitor cells (Figures 1K–1R). The downregulation of Nkx6.1 expression correlated precisely with the initiation of Msx1 expression at E9.5 (Figures 1O–1R). Msx2 was also expressed in a pattern similar to Msx1 but at lower levels (data not shown).Lmx1b is structurally related to Lmx1a and has previously been implicated in the maturation of DA neurons (Smidt et al., 2000). While expressed in vMB progenitors, Lmx1b was not restricted to DA progenitors at early developmental stages (Figures 1S–1V). Also, expression of Lmx1b was downregulated in DA progenitors at E11.5, approximately 2 days before the cessation of midbrain DA neuron generation, while the expression of Lmx1a and Msx1 was maintained throughout this period (Figure 1V; data not shown). Thus, the expression patterns of Lmx1a and Msx1, but not that of Lmx1b, correlate with DA neuron specification. Taken together, a Lmx1a+/Msx1+/Nkx6.1− expression profile defines DA progenitor cells in the vMB.
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