The role of the homeodomain protein Pitx3 in the development and survival of midbrain dopaminergic neurons.

Abstract

There is much interest in the study of midbrain dopaminergic (mDA) neurons as their functions include the regulation of motor function, emotion and reward pathways. Furthermore the dysfunction of these neurons is implicated in a number of human disorders such as Parkinson’s disease (PD), addiction and schizophrenia. PD is characterised by the degeneration of mDA neurons of the substantia nigra pars compacta (SNc), therefore, research into the specification and development of mDA neurons is of particular interest in relation to this disease. An understanding of the development of mDA neurons may lead to new methods of preventing their degeneration or potentially a human ES cell derived source of mDA neurons that could be used for transplantation in PD patients. Pitx3 is a bicoid-related homeodomain protein with an expression pattern restricted to the mDA neurons of the SNc and ventral tegmental area (VTA), within the central nervous system. To directly investigate a role for Pitx3 in mDA neuron development, I have analysed a line of transgenic mice with a green fluorescent protein (GFP) reporter under the control of the endogenous Pitx3 promoter. Use of the targeted GFP reporter as a midbrain dopaminergic lineage marker in the phenotypically normal heterozygous mice identified previously unrecognised ontogenetically distinct subpopulations of dopaminergic cells within the ventral midbrain. These subpopulations were detectable at E12.5 based on their temporal and topographical expression of Pitx3 and TH. Analysis of the Pitx3 null mice revealed that Pitx3 is required for the survival of a subset of nascent mDA neurons at the beginning of their terminal differentiation. The loss of mDA neurons via apoptosis continued throughout development resulting in a complete absence of SNc neurons whilst the VTA remained relatively intact in adult Pitx3 null mice. In addition, during embryonic development Pitx3 deficiency caused a loss of tyrosine hydroxylase (TH) expression specifically in the SNc dopaminergic neurons. Analysis of chimeric mice made with Pitx3 null and Pitx3 heterozygous ES cells revealed that Pitx3 acts in a cell autonomous manner. These findings point to two roles for Pitx3 in SNc mDA neurons, one in their survival and the other in regulation of TH expression. Taken together, these studies suggest that the ontogenetically distinct subpopulations may provide the molecular basis for the specific dependence of substantia nigra DA neurons on Pitx3. In addition, to establish whether the subpopulations identified at E12.5 do form the SNc and VTA, respectively, a strategy to track the fate of the earliest Pitx3- expressing cells has been initiated. In order to achieve this I have created transgenic mice in which a tamoxifen inducible form of Cre recombinase is under the control of the endogenous Pitx3 promoter. These mice can be crossed with existing mice which contain a ubiquitously expressed Cre-inducible reporter, such as LacZ or GFP, to give a temporally and spatially restricted reporter expression.