الفهرس 
Acknowledgement
Early embryogenesis and CNS developmentOnly 14 pages are availabe for public view
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Abstract
Dopaminergic neuron of the midbrain is one of the most intensively studied neuronal groups due to its involvement in several mental and neurological diseases. The midbrain dopaminergic neurons have a prominent function in voluntary movements, reward behaviors and emotions. The degeneration of dopaminergic neurons in the substantia nigra and the subsequent loss of striatal target innervation underlie the motor symptoms of the Parkinson disease (PD), which is recognized as the second most common neurodegenerative disorder. A better understanding of the molecular mechanisms that controlling the development of the midbrain dopaminergic neurons might provide valuable information for the generation of new and better therapeutic alternatives for these ailments, as well as give further insights for the regenerative medicine field. Although much research have been performed related to the development and function of the mDA system, the molecular mechanisms underlying , induction, specification and survival of mDA neurons are still not fully elucidated.
The aim of this study was to develop a better understanding for the molecular mechanisms underlying the development of the mesencephalic dopaminergic neurons with focus on the signalling molecules sonic hedgehog (Shh) and transforming growth factor beta (TGF-β).
Shh is a morphogen secreted by the notochord and the floor plate. It is well known that the embryonic expression of Shh is strongly associated with the dorsoventral patterning of neural tube and that Shh is apparently required for patterning and phenotype specification in vivo and consequently in induction of midbrain dopaminergic neurons. The signaling pathways and the molecular determinants downstream of Shh have been well characterized, however, recently, new accessory receptors for Shh binding and signaling, namely Gas1 and Boc, have been identified. In the present study, we have addressed the question whether the Shh novel components Boc and Gas1 are involved in the differentiation and survival of midbrain dopaminergic neurons in vivo. In the current study, I examined for the first time the phenotypic characterization and the molecular expression for Boc and Gas1 in the midbrain dopaminergic area during the differentiation and survival of these neurons in C57BL/6 mice at E14 and E18 (as determined by immunohistochemical staining). The results revealed that Shh accessory receptors were not expressed in the midbrain dopaminergic area during the differentiation and survival of these neurons, their expression was restricted in the ventricular zone of the aqueduct. Afterwards, we have presented a detailed analysis for the gene expression of the Shh signalling component Gli1, Gli2, Gli3 and Ptch1. To address their expression pattern,in situ hybridization method was applied using brain cryo-sections at E12, E14 and E18.Ourdetailed analysis revealed that the Shh signalling components were not expressed in the mDA area while their expression was restricted around the ventricular zone of the aqueduct. These observations clearly demonstrate that Shh signalling components are not directly involved in the development of mDA neurons during their differentiation and survival. To ensure the specificity of our results, we investigated the expression of Boc, Gas1 and other Shh signalling components in the developing cerebellum, a tissue which was used as a positive control.These results were very surprising and at the same time intriguing and have raised many questions related to the biological significance of Shh signaling during midbrain development.
To address the question of the mode of action of Shh in the development of mDA neurons, the experimental design has been extended to in vitro analyses as well, using the MN9D cell line, an established model for studying dopaminergic neurons. The results have demonstrated that Ptch1 and Smoothened receptors were expressed at the protein level in MN9D cells (as determined with immunocytochemistry). To examine the gene expression of the Shh signalling components in this cell line, a reverse transcription polymerase chain reaction (RT-PCR) was performed using specific primers against Shh, Ptch1, Gli1 and Gli2. Our results demonstrated that the in vitro expression of Ptch1 and Smoothened receptors, Gli1 and Gli2 transcription factors in the MN9D cell line does not correspond to our in vivo data. I observed that this cell line has mixed cell populations;either progenitor cells which found in clusters or cells adopted more mature neuronal morphology with extended neurites. We conclude that the MN9D cell line may not be a suitable line to study the differentiation and survival of dopaminergic neurons.
Since Shh is known to co-operate with other signalling molecules such as TGF-βduring development of midbrain dopaminergic neurons. So wefinally addressed the question of the role of TGF-β signaling in the development of midbrain dopaminergic neurons. Therefore, we generated a conditional knock-out mouse model in which the TGF-β receptor 2 is knocked-out (deleted) in the midbrain cells expressing Engrailed-1. The phenotypic characterization of the TGFβR2 flox/flox: En1Cre/+ (cKO) at E18 revealed a significant decrease in the midbrain TH-immunoreactive neurons in cKO compared to wild-type littermates. We demonstrated that the intensity of aldehyde dehydrogenase and cholecystokinin expression in the substania nigra and ventral tegmental area respectively, was reduced in the cKO mice compared to wild-type littermates. Together, these results strengthened our hypothesis that TGF-β signalling is necessary for the differentiation and survival of the subpopulations (SN and VTA) of mDA neurons. To address whether these loss was due to neuronal loss or neurotransmitter loss, I investigated that the decrease in the number of mDA neurons in cKO mice was due to neuronal loss using neurofilaments as a neuronal marker. In the current study, we examined whether the decrease in the mDA neurons could be due to apoptosis. I performed immunohistochemical studies using antiserum against activated Caspase-3, which is considered the central apoptotic effector enzyme responsible for many of the biochemical and morphological feature of apoptosis. I observed comparable differences in the number of apoptotic cells between the control and cKO mice.
Our results considerably contributed on the understanding of the molecular mechanisms underlying development of mDA with focus on the signaling molecules Shh and TGF-β.