SUMMARYDevelopment of meso-diencephalic dopamine (mdDA) neurons requires the combined actions of the orphan nuclear receptor Nurr1 and the paired-like homeobox transcription factor Pitx3. Whereas all mdDA neurons require Nurr1 for expression of Th and survival, dependence on Pitx3 is displayed only by the mdDA subpopulation that will form the substantia nigra (SNc). Previously, we have demonstrated that Pitx3 -/-embryos lack the expression of the retinoic acid (RA)-generating enzyme Ahd2, which is normally selectively expressed in the Pitx3-dependent DA neurons of the SNc. Restoring RA signaling in Pitx3 -/-embryos revealed a selective dependence of SNc neurons on the presence of RA for differentiation into Th-positive neurons and maintenance throughout embryonic development. Whereas these data are suggestive of an important developmental role for RA in neurons of the SNc, it remained unclear whether other Nurr1 and Pitx3 target genes depend on RA signaling in a manner similar to Th. In the search for genes that were affected in Pitx3-deficient mdDA neurons and restored upon embryonic RA treatment, we provide evidence that Delta-like 1, D2R (Drd2) and Th are regulated by Pitx3 and RA signaling, which influences the mdDA terminal differentiated phenotype. Furthermore, we show that regulation of Ahd2-mediated RA signaling represents only one aspect of the Pitx3 downstream cascade, as Vmat2, Dat, Ahd2 (Aldh1a1), En1, En2 and Cck were unaffected by RA treatment and are (subset) specifically modulated by Pitx3. In conclusion, our data reveal several RA-dependent and -independent aspects of the Pitx3-regulated gene cascade, suggesting that Pitx3 acts on multiple levels in the molecular subset-specification of mdDA neurons.

Normal rat kidney (NRK) fibroblasts exhibit growth-dependent changes in electrophysiological properties and intracellular calcium dynamics. The transition from a quiescent state to a density-arrested state results in altered calcium entry characteristics. This coincides with modulation of the expression of the genes encoding the calcium channels Trpc1, Trpc6 and Orai1, and of the intracellular calcium sensor Stim1. In the present study we have used gene selective short hairpin (sh) RNAs against these various genes to investigate their role in (a) capacitative store-operated calcium entry (SOCE); (b) non-capacitative OAG-induced receptor-operated calcium entry (ROCE); and (c) prostaglandin F(2α) (PGF(2α))-induced Ca(2+)-oscillations in NRK fibroblasts. Intracellular calcium measurements revealed that knockdown of the genes encoding Trpc1, Orai1 and Stim1 each caused a significant reduction of SOCE in NRK cells, whereas knockdown of the gene encoding Trpc6 reduced only the OAG-induced ROCE. Furthermore, our data show that knockdown of the genes encoding Trpc1, Orai1 and Stim1, but not Trpc6, substantially reduced the frequency (up to 60%) of PGF(2α)-induced Ca(2+) oscillations in NRK cells. These results indicate that in NRK cells distinct calcium channels control the processes of SOCE, ROCE and PGF(2α)-induced Ca(2+) oscillations.

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