Abstract
The transcription factor engrailed 1 (En1) is essential in mdDA neuron maintenance, both during embryonic development and adulthood. The overall aims of this thesis were to unravel the function of En1, at a molecular level, in mdDA neuron development and to determine its relationship to canonical Wnt signalling during the
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specification of these neurons. We have disclosed the downstream cascades of En1 both in a dopaminergic cell line and in vivo (in the midbrain). The analysis of these data provides evidence that En1 is crucial for the induction of dopaminergic identity in mdDA neurons, being involved in the regulation of key mdDA genes, like Th, Aadc and Ahd2. Furthermore, we have unravelled the mechanism behind this regulation: En1 might be part of the Nurr1 transcriptional complex. We describe the interaction between En1 and the Nurr1 co-repressor Psf. Moreover, we show that En1 uses the same transcriptional mechanism in vivo as Nurr1 does on the Th promoter – i.e., En1 requires the release of HDAC-mediated repression for Th transcription to occur. These data suggest that En1 might be part of the Nurr1 transcriptional complex driving mdDA neuron specification. Wnt signaling is critical for proper brain development, including the mdDA system. However, it was not clear whether its activity overlaped with mdDA differentiation in later stages of midbrain development. We address this problem and demonstrate that canonical Wnt signaling is active in the midbrain mainly in the progenitor- and Nurr1-expression domains, while its activity is substantially reduced as development progresses. In addition, considering the known interaction between En1 and Wnt signaling in early development, we investigated whether En1 and canonical Wnt signalling might interact during mdDA neuron specification. We show that, around this developmental stage, the En1 mutant midbrain presents reduced ?-catenin activity, implicating En1 as a positive regulator of Wnt signaling. Furthermore, En1 and Nurr1 both interact with ?-catenin in vivo, leading to the hypothesis that ?-catenin, Nurr1 and En1 might be in a complex which could play a relevant role in early mdDA differentiation. Overall, our data contribute considerably to a deeper understanding of the mdDA system development, especially on how terminal differentiation in mdDA neurons is induced. Hopefully this knowledge will contribute to future improvement of cell replacement therapy in PD, whereby the specific differentiation of a stem cell into a DA neuronal phenotype is accomplished.
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