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Principal investigator: Dr. Fadel TISSIR, Chercheur qualifié FNRS Co-investigator: André M. GOFFINET, MD, PhD Université catholique de Louvain Developmental Neurobiology 73, Av. E. Mounier, box DENE 73.82 B1200 Brussels, Belgium T: +32 – (0)2764 7384 F: +32 – (0)2764 7485 Email: Fadel.Tissir@uclouvain.be 102 Verslag – Rapport – Report 2012
Celsr genes in brain development and function Work progress During 2012, we have mainly focused on the role of Celsr1-3 in ependymal cilia polarity. In the mouse forebrain, ependymal cells lining cerebral ventricles bear at their apical surface multiple cilia that beat in concerted manner to assist the circulation of the cerebrospinal fluid (CSF) (Ibanez-Tallon, Pagenstecher et al. 2004; Lechtreck, Delmotte et al. 2008; Tissir, Qu et al. 2010). The base of each cilium displays a polarized organization best evidenced by the presence of asymmetric appendages extending from basal bodies (BB) particularly the basal foot, an arrowhead-like structure that points towards the active stroke of cilia beat. In differentiating ependymal cells, cilia appear randomly oriented (Guirao, Meunier et al. 2010). They subsequently rotate and progressively assume a common orientation, a process termed rotational polarity (Guirao, Meunier et al. 2010; Mirzadeh, Han et al. 2010). This polarity is also seen across the tissue in that cilia from adjacent cells point to the same direction. Furthermore, BBs that are initially widely scattered regroup into an off-centered patch at the anterior tier of the apical surface, a feature referred to as translational polarity (Mirzadeh, Han et al. 2010). Ependymal cells differentiate perinatally from radial glia (RG) progenitors which carry a single primary cilium (Merkle, Tramontin et al. 2004). Like motile multicilia, the primary cilium is displaced from the centre of the apical domain, and this anterior shift, shared by neighboring cells, is the first sign of tissue polarity. We first wondered whether PCP affects the polarity of the primary cilium and tested this hypothesis using the following mutants: Celsr1 -/- , Celsr2 -/- Celsr2 Dgen/Dgen , Celsr3 -/- , Fzd3 -/- and Vangl2 cKO . We analyzed “en face” preparations of newborn (P0–P1) lateral wall (LW), with emphasis on its anterior part to minimize regional variations. We found that, like in the wildtype (WT), RG cells had a primary cilium in all PCP mutants. We evaluated the relative distance between the centre of the apical cell surface and the BB and found that the primary cilium was displaced from the cell centre in all genotypes (WT=0.41±0.02; Celsr1 -/- =0.42±0.03, p=0.4101; Celsr2 -/- =0.43±0.02, p=0.1467; Celsr2 Dgen/Dgen =0.44±0.04, p=0.1208; Celsr3 -/- =0.44±0.02, p=0.0794; Fzd3 -/- =0.40±0.04, p=0.6857; Vangl2 cKO =0.43±0.02, p=0.2618). We analyzed the direction of BB displacement by drawing a vector from the cell center to the BB. Vectors from neighboring cells pointed to similar directions in WT, Celsr2 -/- , and Celsr3 -/- . In contrast, they were randomized in Celsr1 -/- , Celsr2 Dgen/Dgen , Fzd3 -/- , and Vangl2 cKO . Analysis by circular statistics showed that the angular deviation of individual vectors from the mean was comprised between -45° and +45° in WT, Celsr2 -/- , and Celsr3 -/- samples, but displayed broader distributions in Celsr1 -/- , Celsr2 Dgen/Dgen , Fzd3 -/- , and Vangl2 cKO mutants. These results show that PCP genes Celsr1, Fzd3 and Vangl2 coordinate the positioning of the primary cilium in radial progenitors. We have already shown that loss of function of Celsr2 and Celsr3 impairs rotational polarity of ependymal cilia and results in defective flow of CSF and lethal hydrocephalus (Tissir, Qu et al. 2010). Celsr2&3 mutant ependymal cilia never develop in normal numbers and display abnormalities in morphology, position, and planar organization. Ciliary basal feet are disoriented, and basal bodies were seen ectopically deep in the cytoplasm. The lateral plasma membrane localization of Vangl2 and Frizzled3 is disrupted in ependymal cells, indicating that Celsr2 and Celsr3 act via PCP to regulate the docking of basal bodies and the apical positioning of cilia. Rotational polarity is usually assessed by analyzing the orientation of the basal foot by transmission electron microscopy. This method is tedious and time consuming. To speed up the study of ependymal polarity in other PCP mutants, we sought to develop an alternative to electron microscopy. We test many markers and found that gamma tubulin and phosphorylated Verslag – Rapport – Report 2012 103
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ß-actin in neural crest cell migra
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mRNA metabolism at synapses and spi
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Role of DMRT transcription factors
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Developmental origin of multiple de
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