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

The kinetochore protein, CENPF, is mutated in human ciliopathy and microcephaly phenotypes

Background: Mutations in microtubule-regulating genes are associated with disorders of neuronal migration and microcephaly. Regulation of centriole length has been shown to underlie the pathogenesis of certain ciliopathy phenotypes. Using a next-generation sequencing approach, we identified mutations in a novel centriolar disease gene in a kindred with an embryonic lethal ciliopathy phenotype and in a patient with primary microcephaly. Methods and results Whole exome sequencing data from a non-consanguineous Caucasian kindred exhibiting mid-gestation lethality and ciliopathic malformations revealed two novel non-synonymous variants in CENPF, a microtubule-regulating gene. All four affected fetuses showed segregation for two mutated alleles [IVS5-2A>C, predicted to abolish the consensus splice-acceptor site from exon 6; c.1744G>T, p.E582X]. In a second unrelated patient exhibiting microcephaly, we identified two CENPF mutations [c.1744G>T, p.E582X; c.8692 C>T, p.R2898X] by whole exome sequencing. We found that CENP-F colocalised with Ninein at the subdistal appendages of the mother centriole in mouse inner medullary collecting duct cells. Intraflagellar transport protein-88 (IFT-88) colocalised with CENP-F along the ciliary axonemes of renal epithelial cells in age-matched control human fetuses but did not in truncated cilia of mutant CENPF kidneys. Pairwise co-immunoprecipitation assays of mitotic and serum-starved HEKT293 cells confirmed that IFT88 precipitates with endogenous CENP-F. Conclusions: Our data identify CENPF as a new centriolar disease gene implicated in severe human ciliopathy and microcephaly related phenotypes. CENP-F has a novel putative function in ciliogenesis and cortical neurogenesis

Nuclear FOXO1 promotes lymphomagenesis in germinal center B cells

Forkhead box class O1 (FOXO1) acts as a tumor suppressor in solid tumors. The oncogenic phosphoinositide-3-kinase (PI3K) pathway suppresses FOXO1 transcriptional activity by enforcing its nuclear exclusion upon AKT-mediated phosphorylation. We show here abundant nuclear expression of FOXO1 in Burkitt lymphoma (BL), a germinal center (GC) B-cell-derived lymphoma whose pathogenesis is linked to PI3K activation. Recurrent FOXO1 mutations, which prevent AKT targeting and lock the transcription factor in the nucleus, are used by BL to circumvent mutual exclusivity between PI3K and FOXO1 activation. Using genome editing in human and mouse lymphomas in which MYC and PI3K cooperate synergistically in tumor development, we demonstrate proproliferative and antiapoptotic activity of FOXO1 in BL and identify its nuclear localization as an oncogenic event in GC B-cell-derived lymphomagenesis

Primary cilia are critical for Sonic hedgehog-mediated dopaminergic neurogenesis in the embryonic midbrain

AbstractMidbrain dopaminergic (mDA) neurons modulate various motor and cognitive functions, and their dysfunction or degeneration has been implicated in several psychiatric diseases. Both Sonic Hedgehog (Shh) and Wnt signaling pathways have been shown to be essential for normal development of mDA neurons. Primary cilia are critical for the development of a number of structures in the brain by serving as a hub for essential developmental signaling cascades, but their role in the generation of mDA neurons has not been examined. We analyzed mutant mouse lines deficient in the intraflagellar transport protein IFT88, which is critical for primary cilia function. Conditional inactivation of Ift88 in the midbrain after E9.0 results in progressive loss of primary cilia, a decreased size of the mDA progenitor domain, and a reduction in mDA neurons. We identified Shh signaling as the primary cause of these defects, since conditional inactivation of the Shh signaling pathway after E9.0, through genetic ablation of Gli2 and Gli3 in the midbrain, results in a phenotype basically identical to the one seen in Ift88 conditional mutants. Moreover, the expansion of the mDA progenitor domain observed when Shh signaling is constitutively activated does not occur in absence of Ift88. In contrast, clusters of Shh-responding progenitors are maintained in the ventral midbrain of the hypomorphic Ift88 mouse mutant, cobblestone. Despite the residual Shh signaling, the integrity of the mDA progenitor domain is severely disturbed, and consequently very few mDA neurons are generated in cobblestone mutants. Our results identify for the first time a crucial role of primary cilia in the induction of mDA progenitors, define a narrow time window in which Shh-mediated signaling is dependent upon normal primary cilia function for this purpose, and suggest that later Wnt signaling-dependent events act independently of primary cilia