Analysis of the role of primary cilium in the dopaminergic neurogenesis in mouse embryonic midbrain

Primary cilia are whip-like structures, usually 1-3μm long, protruding from the cell membrane of almost all mammalian cells. During the last decades, it has been uncovered that primary cilium is crucial for Hedgehog (Hh) signalling. Its involvement in the appropriate function of Hh signalling pathway indicated by extension its importance in developmental processes. Midbrain dopaminergic neurons are responsible for the regulation of voluntary movements, as well as for cognitive functions such as emotion and reward. Studies have revealed that midbrain dopaminergic (mDA) neurons are dysfunctional in a number of neuropsychiatric or neurodegenerative diseases. Nowadays it is also widely known that Hh signalling is necessary for the generation and specification of dopaminergic (DA) neurons in the ventral midbrain. This thesis, through a collaboration with PD Dr. Sandra Blaess and her colleagues at the University of Bonn, investigates the role of primary cilia in the development of mDA neurons by using two mouse mutants defective in intraflagellar transport protein 88kDA (Ift88) or kinesin family member 3a (Kif3a), two genes important for the genesis and maintenance of the primary cilium. This is the first time that the role of primary cilia in the generation of mDA is examined. Study of conditional inactivation of Ift88 after embryonic day (E) 9.0 resulted in a progressive loss of primary cilia that was completed by E10.5 and a significant reduction of both mDA progenitors and mDA neurons at later stages, as observed by immunostainings against tyrosine hydroxylase (TH) – a typical marker for dopaminergic neurons and forkhead box A2 (FoxA2), a transcription factor induced by Sonic hedgehog (Shh) and commonly used as a floor plate marker. These observations show that functional primary cilia are necessary for the patterning of the ventral midbrain. Additionally, in situ hybridizations for glioma-associated oncogene 1 and -3 (Gli1 and Gli3) revealed that in the ventral midbrain of Ift88 knock-down (cko) mutants Shh signalling was inactivated. Shh pathway inactivation was attributed to the loss of primary cilia. Moreover, employment of a mouse model in which Smoothened (Smo) was constitutively active demonstrated that the constitutive activation of Shh signalling in these mice led to the expansion of the mDA precursor domain. However, this was not the case in the absence of Ift88; hence in the absence of primary cilia. This indicates that Smo acts downstream of Ift88 and by extension of primary cilia in Shh signalling. Surprisingly, conditional inactivation of Kif3a did not produce a similar phenotype of the mDA neurons. However, this could be attributed to the slightly delayed loss of primary cilia and the delayed inactivation of Shh signalling in Kif3a cko in comparison to Ift88 cko. Taken together, these results uncover the importance of the relationship between primary cilia and Shh signalling and identify for the first time a crucial window at which the two are critical for the induction of mDA neurons