In-vitro analysis of Pitx3 in mesodiencephalic dopaminergic neuron maturation.
The transcription factor Pitx3 is expressed exclusively by mesodiencephalic dopaminergic neurons; however, ablation of Pitx3 results in selective degeneration of primarily dopaminergic neurons of the substantia nigra pars compacta, the neuronal population that is most vulnerable in Parkinson's disease. Although the exact molecular mechanisms of the action of Pitx3 are unclear, roles in both terminal maturation and/or survival of substantia nigra dopaminergic neurons have been suggested. To investigate the connection between Pitx3 and selective neurodegeneration, we generated embryonic stem cells from a Pitx3-deficient mouse (aphakia) for in-vitro differentiation to dopaminergic neurons. This 'loss of function'in-vitro system allowed us to examine characteristic features in dopaminergic neuron development and to assess the role that Pitx3 plays in the differentiation/maturation process. We found that aphakia embryonic stem cells generated 50% fewer tyrosine hydroxylase-positive/microtubule-associated protein (Map)2-positive mature neurons compared with control cultures. The expression of dopamine transport regulators and vesicle release proteins was reduced and dopamine release was unregulated in the Pitx3-deficient tyrosine hydroxylase-positive neurons generated. Treatment of aphakia embryonic stem cell cultures with retinoic acid resulted in a significant increase in mesodiencephalic tyrosine hydroxylase-positive neurons, providing further support for the role of Pitx3 in dopaminergic neuron specification through the retinoic acid pathway. Our study, using Pitx3-deficient embryonic stem cells in an in-vitro differentiation culture system, allowed us to assess the role of Pitx3 in the specification and final maturation of dopaminergic neurons.
Papanikolaou
Theodora
T
Department of Physiology and Neurobiology, University of Connecticut, Storrs, CT 06269-4243, USA.
Amano
Tomokazu
T
Lennington
Jessica
J
Sink
Kelly
K
Farrar
Andrew M
AM
Salamone
John
J
Yang
Xiangzhong
X
Conover
Joanne C
JC
D000818:Animals; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000235:genetics*; D002478:Cells, Cultured; D004027:Diencephalon / Q000166:cytology / Q000196:embryology / Q000378:metabolism; D004298:Dopamine / Q000378:metabolism*; D050483:Dopamine Plasma Membrane Transport Proteins / Q000378:metabolism; D053595:Embryonic Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D018398:Homeodomain Proteins / Q000235:genetics*; D008636:Mesencephalon / Q000166:cytology / Q000196:embryology / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D055495:Neurogenesis / Q000235:genetics*; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics*; D014212:Tretinoin / Q000378:metabolism / Q000494:pharmacology; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism
D050483:Dopamine Plasma Membrane Transport Proteins; D018398:Homeodomain Proteins; D008869:Microtubule-Associated Proteins; D014157:Transcription Factors; C433629:homeobox protein PITX3; D014212:Tretinoin; D014446:Tyrosine 3-Monooxygenase; D004298:Dopamine