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Sequential generation of neurons and glial cells during development is critical for the wiring and function of the cerebral cortex. This process requires accurate coordination of neural progenitor cell (NPC) fate decisions, by NPC-autonomous mechanisms as well as by negative feedback from neurons. Here, we show that neurogenesis is protracted and gliogenesis decreased in mice with mutations of genes Celsr3 and Fzd3. This phenotype is not due to gene inactivation in progenitors, but rather in immature cortical neurons. Mutant neurons are unable to upregulate expression of Jag1 in response to cortical Wnt7, resulting in blunted activation of Notch signalling in NPC. Thus, Celsr3 and Fzd3 enable immature neurons to respond to Wnt7, upregulate Jag1 and thereby facilitate feedback signals that tune the timing of NPC fate decisions via Notch activation.

The caudal migration of facial branchiomotor (FBM) neurons from rhombomere (r) 4 to r6 in the hindbrain is an excellent model to study neuronal migration mechanisms. Although several Wnt/Planar Cell Polarity (PCP) components are required for FBM neuron migration, only Celsr1, an atypical cadherin, regulates the direction of migration in mice. In Celsr1 mutants, a subset of FBM neurons migrates rostrally instead of caudally. Interestingly, Celsr1 is not expressed in the migrating FBM neurons, but rather in the adjacent floor plate and adjoining ventricular zone. To evaluate the contribution of different expression domains to neuronal migration, we conditionally inactivated Celsr1 in specific cell types. Intriguingly, inactivation of Celsr1 in the ventricular zone of r3-r5, but not in the floor plate, leads to rostral migration of FBM neurons, greatly resembling the migration defect of Celsr1 mutants. Dye fill experiments indicate that the rostrally-migrated FBM neurons in Celsr1 mutants originate from the anterior margin of r4. These data suggest strongly that Celsr1 ensures that FBM neurons migrate caudally by suppressing molecular cues in the rostral hindbrain that can attract FBM neurons.

The diaphanous homologue Diaph3 (aka mDia2) is a major regulator of actin cytoskeleton. Loss of Diaph3 has been constantly associated with cytokinesis failure ascribed to impaired accumulation of actin in the cleavage furrow. Here we report that Diaph3 is required before cell fission, to ensure the accurate segregation of chromosomes. Inactivation of the Diaph3 gene causes a massive loss of cortical progenitor cells, with subsequent depletion of intermediate progenitors and neurons, and results in microcephaly. In embryonic brain extracts, Diaph3 co-immunoprecipitates with BubR1, a key regulator of the spindle assembly checkpoint (SAC). Diaph3-deficient cortical progenitors have decreased levels of BubR1 and fail to properly activate the SAC. Hence, they bypass mitotic arrest and embark on anaphase in spite of incorrect chromosome segregation, generating aneuploidy. Our data identify Diaph3 as a major guard of cortical progenitors, unravel novel functions of Diaphanous formins and add insights into the pathobiology of microcephaly.

Cajal-Retzius cells (CRs), the first-born neurons in the developing cerebral cortex, coordinate crucial steps in the construction of functional circuits. CRs are thought to be transient, as they disappear during early postnatal life in both mice and humans, where their abnormal persistence is associated with pathological conditions. Embryonic CRs comprise at least three molecularly and functionally distinct subtypes: septum, ventral pallium/pallial-subpallial boundary (PSB), and hem. However, whether subtype-specific features exist postnatally and through which mechanisms they disappear remain unknown. We report that CR subtypes display unique distributions and dynamics of death in the postnatal mouse cortex. Surprisingly, although all CR subtypes undergo cell death, septum, but not hem, CRs die in a Bax-dependent manner. Bax-inactivated rescued septum-CRs maintain immature electrophysiological properties. These results underlie the existence of an exquisitely refined control of developmental cell death and provide a model to test the effect of maintaining immature circuits in the adult neocortex.

The assembly of neuronal circuits depends on the correct wiring of axons and dendrites. Studies in our laboratory revealed a critical role of seven-pass atypical cadherin Celsr3, a member of planar cell polarity (PCP) proteins, in the development of axonal tracts in the central nervous system, such as the anterior commissure, internal capsule and corticospinal tract. Celsr3 deficiency does not alter axonal growth, but affects axon guidance in cell-autonomous or non-cell-autonomous manners, causing axon stalling at intermediate targets or rerouting. Notably, all axon guidance defects in Celsr3−/− were observed in mice bearing mutations in the PCP gene Fzd3, and some errors were reported in mice with mutations of Vangl2, another PCP gene. Despite their unequivocally role, underlying molecular mechanisms remain elusive. Furthermore, their functions in the peripheral nervous system are still largely unexplored. Here we show that Celsr3 cooperates with Fzd3 in spinal motor neurons to mediate pathfinding of motor axons innervating the dorsal limb. Celsr3 is expressed in postmitotic neurons in the developing spinal cord. Specific inactivation of Celsr3 in spinal motor neurons severely perturbs peroneal nerve development, leading to absent innervation of the tibialis anterior muscle and stiff hindlimb. Deletion of Celsr3 affects neither the specification of motor neurons nor neuronal survival or neurite outgrowth. Celsr3-deficient axons of the peroneal nerve segregate from those of the tibial nerve but fail to extend dorsally, and they stall just after the branch point of the sciatic nerve. Mutant axons respond to repulsive ephrinA-EphA forward signaling and attractive glial cell–derived neurotrophic factor (GDNF). However, they are insensitive to attractive EphA-ephrinA reverse signaling. In transfected cells, Celsr3 immunoprecipitates with ephrinA2, ephrinA5, Ret, GDNF family receptor a1 (GFRa1) and Fzd3. The function of Celsr3 in motor axons is Fzd3 dependent but Vangl2, (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2015

The striatum is a large brain nucleus with an important role in the control of movement and emotions. Medium spiny neurons (MSNs) are striatal output neurons forming prominent descending axon tracts that target different brain nuclei. However, how MSN axon tracts in the forebrain develop remains poorly understood. Here, we implicate the Wnt binding receptor Frizzled3 in several uncharacterized aspects of MSN pathway formation [i.e., anterior–posterior guidance of MSN axons in the striatum and their subsequent growth into the globus pallidus (GP), an important (intermediate) target]. In Frizzled3 knock-out mice, MSN axons fail to extend along the anterior–posterior axis of the striatum, and many do not reach the GP. Wnt5a acts as an attractant for MSN axons in vitro, is expressed in a posterior high, anterior low gradient in the striatum, and Wnt5a knock-out mice phenocopy striatal anterior–posterior defects observed in Frizzled3 mutants. This suggests that Wnt5a controls anterior–posterior guidance of MSN axons through Frizzled3. Axons that reach the GP in Frizzled3 knock-out mice fail to enter this structure. Surprisingly, entry of MSN axons into the GP non–cell-autonomously requires Frizzled3, and our data suggest that GP entry may be contingent on the correct positioning of “corridor” guidepost cells for thalamocortical axons by Frizzled3. Together, these data dissect MSN pathway development and reveal (non)cell-autonomous roles for Frizzled3 in MSN axon guidance. Further, they are the first to identify a gene that provides anterior–posterior axon guidance in a large brain nucleus and link Frizzled3 to corridor cell development.

The cadherin Celsr3 regulates the directional growth and targeting of axons in the CNS, but whether it acts in collaboration with or in parallel to other guidance cues is unknown. Furthermore, the function of Celsr3 in the peripheral nervous system is still largely unexplored. Here we show that Celsr3 mediates pathfinding of motor axons innervating the hindlimb. In mice, Celsr3-deficient axons of the peroneal nerve segregate from those of the tibial nerve but fail to extend dorsally, and they stall near the branch point. Mutant axons respond to repulsive ephrinA-EphA forward signaling and glial cell-derived neurotrophic factor (GDNF). However, they are insensitive to attractive EphA-ephrinA reverse signaling. In transfected cells, Celsr3 immunoprecipitates with ephrinA2, ephrinA5, Ret, GDNF family receptor α1 (GFRα1) and Frizzled3 (Fzd3). The function of Celsr3 is Fzd3 dependent but Vangl2 independent. Our results provide evidence that the Celsr3-Fzd3 pathway interacts with EphA-ephrinA reverse signaling to guide motor axons in the hindlimb., info:eu-repo/semantics/published

Celsr3 and Fzd3, members of "core planar cell polarity" (PCP) genes, were shown previously to control forebrain axon guidance and wiring by acting in axons and/or guidepost cells. Here we show that Celsr2 acts redundantly with Celsr3, and that their combined mutation mimics that of Fzd3. The phenotypes generated upon inactivation of Fzd3 in different forebrain compartments are similar to those in conditional Celsr2-3 mutants, indicating that Fzd3 and Celsr2-3 act in the same population of cells. Inactivation of Celsr2-3 or Fzd3 in thalamus does not affect forebrain wiring, and joint inactivation in cortex and thalamus adds little to cortical inactivation alone in terms of thalamocortical projections. On the other hand, joint inactivation perturbs strongly the formation of the barrel field which is unaffected upon single cortical or thalamic inactivation, indicating a role for interactions between thalamic axons and cortical neurons in cortical arealization. Unexpectedly, forebrain wiring is normal in mice defective in Vangl1 and Vangl2,showing that, contrary to epithelial PCP, axon guidance can be Vangl-independent in some contexts. Our results suggest thatCelsr2-3 and Fzd3 regulate axonal navigation in the forebrain by using mechanisms different than classical epithelial PCP, and require interacting partners other than Vangl1-2, that remain to be identified.

The oviduct is an important organ in reproduction where fertilization occurs, and through which the fertilized eggs are carried to the uterus in mammals. This organ is highly polarized, where the epithelium forms longitudinal folds along the ovary-uterus axis, and the epithelial multicilia beat towards the uterus to transport the ovulated ova. Here, we analyzed the postnatal development of mouse oviduct and report that multilevel polarities of the oviduct are regulated by a planar cell polarity (PCP) gene, Celsr1. In the epithelium, Celsr1 is concentrated in the specific cellular boundaries perpendicular to the ovary-uterus axis from postnatal day 2. We found a new feature of cellular polarity in the oviduct - the apical surface of epithelial cells is elongated along the ovary-uterus axis. In Celsr1-deficient mice, the ciliary motion is not orchestrated along the ovary-uterus axis and the transport ability of beating cilia is impaired. Epithelial cells show less elongation and randomized orientation, and epithelial folds show randomized directionality and ectopic branches in the mutant. Our mosaic analysis suggests that the geometry of epithelial cells is primarily regulated by Celsr1 and as a consequence the epithelial folds are aligned. Taken together, we reveal the characteristics of the multilevel polarity formation processes in the mouse oviduct epithelium and suggest a novel function of the PCP pathway for proper tissue morphogenesis.

This thesis describes the work that I have carried out in the Laboratory of Neurophysiolgy at the Université Libre de Bruxelles, under the supervision of Prof. Serge Schiffmann, in collaboration with Prof. Stéphane Schurmans of Université of Liège.The work is divided in two distinct but related projects and the results section is thus divided into two main chapters. The results described are presented in the form of two manuscripts, the first chapter is named “Ciliary phosphoinositides regulation by INPP5E controls Shh signaling by allowing trafficking of Gpr161 in neural stem cells primary cilium”.The second is named “Regulation of phosphoinositides ciliary levels controls trafficking and ciliogenesis in ependymal cells”.Since both manuscripts are comprehensive regarding the results, and methods, these are inserted as such into the thesis.An expanded introduction to the field, placing the results into context, precedes these two chapters. An extended discussion section follows each chapter; it presents some elements of discussion not included in the manuscripts, the implications of the results and the scope for further research., Doctorat en Sciences biomédicales et pharmaceutiques, info:eu-repo/semantics/nonPublished

Planar cell polarity (PCP) signaling controls tissue morphogenesis by coordinating collective cell behaviors. We show a critical role for the core PCP proteins Celsr1 and Vangl2 in the complex morphogenetic process of intraluminal valve formation in lymphatic vessels. We found that valve-forming endothelial cells undergo elongation, reorientation, and collective migration into the vessel lumen as they initiate valve leaflet formation. During this process, Celsr1 and Vangl2 are recruited from endothelial filopodia to discrete membrane domains at cell-cell contacts. Celsr1- or Vangl2-deficient mice show valve aplasia due to failure of endothelial cells to undergo rearrangements and adopt perpendicular orientation at valve initiation sites. Mechanistically, we show that Celsr1 regulates dynamic cell movements by inhibiting stabilization of VE-cadherin and maturation of adherens junctions. These findings reveal a role for PCP signaling in regulating adherens junctions and directed cell rearrangements during vascular development.

BACKGROUND: P73 belongs to the p53 family of cell survival regulators with the corresponding locus Trp73 producing the N-terminally distinct isoforms, TAp73 and DeltaNp73. Recently, two studies have implicated the murine Trp73 in the modulation in phospho-tau accumulation in aged wild type mice and in young mice modeling Alzheimer's disease (AD) suggesting that Trp73, particularly the DeltaNp73 isoform, links the accumulation of amyloid peptides to the creation of neurofibrillary tangles (NFTs). Here, we reevaluated tau pathologies in the same TgCRND8 mouse model as the previous studies. RESULTS: Despite the use of the same animal models, our in vivo studies failed to demonstrate biochemical or histological evidence for misprocessing of tau in young compound Trp73+/- + TgCRND8 mice or in aged Trp73+/- mice analyzed at the ages reported previously, or older. Secondly, we analyzed an additional mouse model where the DeltaNp73 was specifically deleted and confirmed a lack of impact of the DeltaNp73 allele, either in heterozygous or homozygous form, upon tau pathology in aged mice. Lastly, we also examined human TP73 for single nucleotide polymorphisms (SNPs) and/or copy number variants in a meta-analysis of 10 AD genome-wide association datasets. No SNPs reached significance after correction for multiple testing and no duplications/deletions in TP73 were found in 549 cases of AD and 544 non-demented controls. CONCLUSION: Our results fail to support P73 as a contributor to AD pathogenesis.

In humans, a large number of diseases and lesions of the CNS, leading to abnormalities of locomotion have been described. Locomotor activity is regulated at three levels in the CNS. Motor areas receive information that will be transmitted to the spinal cord via the pyramidal or extrapyramidal tracts, resulting in an appropriate motor response by effector muscles. In addition, the cerebellum and basal ganglia are involved in the coordination, planning and execution of motor programs. It is important to understand the cells, structures and circuits involved in locomotion in order to better understand the mechanisms of these lesions and diseases and to apply them in humans. The family of the Onecut transcription factors comprises three members in mammals, namely HNF-6, OC-2 and OC-3. They play important roles in the CNS including cell differentiation, maintenance of cell identity and cell migration. Hnf6-/- mice display a paralysis of the hindlimbs at birth. The goal of my thesis was to understand the origin of the paralysis in these mice. To assess locomotor defects in adult Hnf6-/- mice, we performed behavioral tests. These mice display a reduction of muscular strength and an abnormal positioning of the hindlimbs, and a defect in the coordination and in the balance. These defects suggest that the neuromuscular junction and the cerebellum are altered in Hnf6-/- mice. Therefore, we aimed at identifying abnormalities of the neuromuscular junctions and of the cerebellum in mice Hnf6-/- and to characterize the roles of HNF-6 in these two structures. The morphology of the neuromuscular junctions and the localization of synaptophysin were abnormal in Hnf6-/- mice. HNF-6 regulates, directly or indirectly, the expression of agrin and neuregulin, two genes necessary for proper formation and maintenance of the neuromuscular junction. In addition, the organization of Purkinje cells in the cerebellum was abnormal and characterized by a superposition of cells, a localization of ce, Chez l’homme, il existe un grand nombre de pathologies et de lésions du SNC qui conduisent à des altérations de locomotion. L’activité locomotrice est régulée à trois niveaux dans le SNC. Les aires motrices reçoivent des informations nerveuses qui vont être transmises à la moelle épinière par la voie pyramidale ou extrapyramidale, induisant une réponse motrice appropriée de la part des muscles effecteurs. De plus, le cervelet et les ganglions de la base sont des structures qui sont impliqués dans la coordination, la planification et l’exécution des ordres moteurs. Il est important de mieux comprendre les cellules, les structures et les circuits impliqués dans la locomotion afin de mieux comprendre ce qui se passe dans ces pathologies et de pouvoir l’appliquer chez l’homme à des fins thérapeutiques. La famille de facteurs de transcription Onecut comprend trois membres chez les mammifères, HNF-6 (OC-1), OC-2 et OC-3 et sont décrits comme ayant un rôle important dans la différenciation de certaines populations neuronales, le maintien de leur identité cellulaire et dans la migration de ces cellules. Les souris Hnf6-/- ont la particularité de présenter à la naissance une paralysie des pattes postérieures. Le but de ce travail a été de comprendre l’origine de cette paralysie. Afin d’évaluer les défauts locomoteurs présents à l’âge adulte chez ces souris, nous avons réalisé des tests comportementaux. Nous avons montré que les souris Hnf6-/- présentent, une diminution de la force musculaire, un positionnement anormal des pattes postérieures et un défaut de coordination et d’équilibre. Ce type de défaut locomoteur suggère que les motoneurones et/ou les jonctions neuromusculaires (JNM) soient à l’origine des anomalies motrices et que le cervelet celles de la coordination et de l’équilibre. Mon objectif a donc été d’identifier les anomalies présentes au sein des JNM ainsi que dans le cervelet chez les souris Hnf6-/- afin d’identifier les rôles du facteur HNF-6 dans ces deux st, (SBIM 3) -- UCL, 2013

molecular mechanisms that regulate their assembly into functional neuronal circuits are currently unknown. Here we report that the planar cell polarity (PCP) genes Celsr3 and Fzd3 are required during murine embryogenesis to specifically control the guidance and growth of enteric neuronal projections relative to the longitudinal and radial gut axes. Ablation of these genes disrupts the normal organization of nascent neuronal projections, leading to subtle changes of axonal tract configuration in the mature enteric nervous system (ENS), but profound abnormalities in gastrointestinal motility. Our data argue that PCP-dependent modules of connectivity established at early stages of enteric neurogenesis control gastrointestinal function in adult animals and provide the first evidence that developmental deficits in ENS wiring may contribute to the pathogenesis of idiopathic bowel disorders.

Planar cell polarity (PCP) signaling controls tissue morphogenesis by coordinating collective cell behaviours. Here we show a critical role for the core PCP proteins Celsr1 and Vangl2 in the complex morphogenetic process of intraluminal valve formation in lymphatic vessels. We show that valve-forming endothelial cells undergo elongation, reorientation and collective migration into the vessel lumen as they initiate valve leaflet formation. During this process, Celsr1 and Vangl2 are recruited from endothelial filopodia to discrete membrane domains at cell-cell contacts. Celsr1 or Vangl2 deficient mice show valve aplasia due to failure of endothelial cells to undergo rearrangements and adopt perpendicular orientation at valve initiation sites. Mechanistically, we show that Celsr1 regulates dynamic cell movements by inhibiting stabilization of VE-cadherin and maturation of adherens junctions. These findings reveal a novel role for PCP signaling in regulating adherens junctions and directed cell rearrangements during vascular development.

Planar cell polarity (PCP) is complementary to the intrinsic polarization of single cells and refers to the global coordination of cell behaviour in the plane of a tissue, and by extension to the signalling pathways that control it. PCP is most evident in cell sheets and research into PCP was for years confined to studies in Drosophila. However, PCP has more recently emerged as an important phenomenon in vertebrates where it regulates various developmental processes and is associated with multiple disorders. In particular, core PCP genes are crucial for the development and function of the nervous system. They are involved in neural tube closure, ependymal polarity, neuronal migration, dendritic growth and axon guidance.

How growth cones detect small concentration differences of guidance cues for correct steering remains a long-standing puzzle. Commissural axons engage planar cell polarity (PCP) signaling components to turn anteriorly in a Wnt gradient after midline crossing. We found here that Frizzled3, a Wnt receptor, undergoes endocytosis via filopodia tips. Wnt5a increases Frizzled3 endocytosis, which correlates with filopodia elongation. We discovered an unexpected antagonism between Dishevelleds, which may function as a signal amplification mechanism in filopodia where PCP signaling is activated: Dishevelled2 blocks Dishevelled1-induced Frizzled3 hyperphosphorylation and membrane accumulation. A key component of apical-basal polarity (A-BP) signaling, aPKC, also inhibits Dishevelled1-induced Frizzled3 hyperphosphorylation. Celsr3, another PCP component, is required in commissural neurons for anterior turning. Frizzled3 hyperphosphorylation is increased in Celsr3 mutant mice, where PCP signaling is impaired, suggesting Frizzled3 hyperphosphorylation does correlate with loss of PCP signaling in vivo. Furthermore, we found that the small GTPase, Arf6, which is required for Frizzled3 endocytosis, is essential for Wnt-promoted outgrowth, highlighting the importance of Frizzled3 recycling in PCP signaling in growth cone guidance. In a Wnt5a gradient, more Frizzled3 endocytosis and activation of atypical protein kinase C was observed on the side of growth cones facing higher Wnt5a concentration, suggesting that spatially controlled Frizzled3 endocytosis is part of the key mechanism for growth cone steering.

Cadherin EGF LAG seven-pass G-type receptors 1, 2, and 3 (Celsr1-3) form a family of three atypical cadherins with multiple functions in epithelia and in the nervous system. During the past decade, evidence has accumulated for important and distinct roles of Celsr1-3 in planar cell polarity (PCP) and brain development and maintenance. Although the role of Celsr in PCP is conserved from flies to mammals, other functions may be more distantly related, with Celsr working only with one or a subset of the classical PCP partners. Here, we review the literature on Celsr in PCP and neural development, point to several remaining questions, and consider future challenges and possible research trends.

The patterning of cortical areas is controlled by a combination of intrinsic factors that are expressed in the cortex and external signals such as inputs from the thalamus. EphA7 is a guidance receptor that is involved in key aspects of cortical development and is expressed in gradients within developing cortical areas. Here, we identified a regulatory element of the EphA7 promoter, named pA7, that can recapitulate salient features of the pattern of expression of EphA7, including cortical gradients. Using a pA7-Green fluorescent Protein (GFP) mouse reporter line, we isolated cortical neuron populations displaying different levels of EphA7/GFP expression. Transcriptome analysis of these populations enabled to identify many differentially expressed genes, including 26 transcription factors with putative binding sites in the pA7 element. Among these, Pbx1 was found to bind directly to the EphA7 promoter in the developing cortex. All genes validated further were confirmed to be expressed differentially in the developing cortex, similarly to EphA7. Their expression was unchanged in mutant mice defective for thalamocortical projections, indicating a transcriptional control largely intrinsic to the cortex. Our study identifies a novel repertoire of cortical neuron genes that may act upstream of, or together with EphA7, to control the patterning of cortical areas.

The primary cilium, a signal transduction organelle, is present on the cell bodies of adult-born dentate gyrus granule cells as they begin maturation. In its absence, their maturation and integration are impaired.

Planar cell polarity (PCP) refers to the collective orientation of cells within the epithelial plane. We show that progenitor cells forming the ducts of the embryonic pancreas express PCP proteins and exhibit an active PCP pathway. Planar polarity proteins are acquired at embryonic day 11.5 synchronously to apicobasal polarization of pancreas progenitors. Loss of function of the two PCP core components Celsr2 and Celsr3 shows that they control the differentiation of endocrine cells from polarized progenitors, with a prevalent effect on insulin-producing beta cells. This results in a decreased glucose clearance. Loss of Celsr2 and 3 leads to a reduction of Jun phosphorylation in progenitors, which, in turn, reduces beta cell differentiation from endocrine progenitors. These results highlight the importance of the PCP pathway in cell differentiation in vertebrates. In addition, they reveal that tridimensional organization and collective communication of cells are needed in the pancreatic epithelium in order to generate appropriate numbers of endocrine cells.

Celsr1-3 (for Cadherin EGF Laminin Seven Pass Receptor), the mammalian orthologs of Drosophila Flamingo/starry night, encode proteins of the cadherin superfamily. Celsr1-3 belong to a group named ‘core PCP (Planar Cell Polarity) genes’. All three Celsr genes are expressed in the developing Central nervous system (CNS) in mice. Our lab is interested in studying their functions in mouse CNS development. In this program, I specifically addressed two issues, namely facial branchiomotor neuron (FBM neuron) migration and ependymal ciliogenesis. During development, neurons are generated in ventricular zones, and postmitotic neurons usually need to migrate over variable distances to reach their final destination. Among the many neuronal migration events, that of FBM neuron in the hindbrain is unique and complex. In zebrafish, mutations in PCP genes such as van gogh-like2, celsr2, frizzled3a, prickle1a and prickle1b are known to affect normal FBM neuron caudal migration. This prompted us to investigate the role of Celsr1-3, and Fzd3 in FBM neuron migration in mice. During mouse hindbrain development, FBM neurons migrate caudally from medial rhombomere (r) 4 to lateral r6. Celsr1 is expressed in FBM neuron precursors and the floor plate, but not in FBM neurons themselves. In Celsr1 constitutive mutants, caudal migration was compromised and neurons often migrated rostrally into r2 and r3, as well as laterally. Consistent with the expression profile, when we specially deleted Celsr1 in FBM neurons using Isl1- Cre, no rostral migration stream was observed. In contrast, when Celsr1 was deleted in FBM neuron precursors by Nkx6.2-Cre, abnormal rostral migration was seen in Celsr1|Nkx6.2 mutants. Thus, Celsr1 regulates FBM neuron migration direction in a non-FBM neuron-autonomous manner. In Celsr2 mutants, FBM neurons initiated caudal migration but moved prematurely into lateral r4 and r5. This phenotype was enhanced by inactivation of Celsr3 in FBM neurons and mimicked by inactivat, (SBIM 3) -- UCL, 2011

The p73 locus encodes two types of transcription factors: full length pro-apoptotic isoforms (TAp73), and N-terminally truncated anti-apoptotic proteins (DeltaNp73). To study the function of DeltaNp73 in vivo, we generated mutant mice in which DeltaNp73 is inactivated, but TAp73 expression is intact. In addition, we knocked in the locus the Cre recombinase, and the enhanced green fluorescent protein (EGFP). Using this allele, we refined the expression of DeltaNp73 during brain development and emphasized the importance of the thalamic eminence, a transient source that contributes neurons to the telencephalon. We showed that DeltaNp73 inactivation increases apoptosis in neurons. (1) We also investigated the role of DeltaNp73 in carcinogenesis by inducing tumors with methylcholanthrene in mutant and control mice, and found that mutant females, but not males, have decreased propensity to tumor development. Both effects on neuronal apoptosis and tumor development were milder than predicted from in vitro studies.

How much neocortical development depends on connections remains elusive. Here, we show that Celsr3|Dlx mutant mice have no extrinsic neocortical connections yet survive to postnatal day 20, acquire a basic behavioral repertoire, and display spontaneous hyperactivity, with abnormal light/dark activity cycling. Except for hallmarks related to thalamic input, such as barrels in somatosensory cortex, cortical arealization and laminar maturation proceeded normally. However, the tangential extension of the mature cortex was diminished, with radial thickness less severely affected. Deep layer neurons were reduced in number, and their apical and basal dendritic arbors were blunted, with reduced synapse density. Interneurons reached the cortex, and their density was comparable with wild type. The excitability of mutant pyramidal neurons, measured in vitro in patch-clamp experiments in acute slices, was decreased. However, their firing activity in vivo was quite similar to the wild type, except for the presence of rapid firing exhaustion in some mutant neurons. Local field potential and electrocorticogram showed similar range of oscillations, albeit with higher frequency peaks and reduced left-right synchrony in the mutant. Thus, "protomap" formation, namely cortical lamination and arealization, proceed normally in absence of extrinsic connections, but survival of projection neurons and acquisition of mature morphological and some electrophysiological features depend on the establishment of normal cortical-subcortical relationships.

During hindbrain development, facial branchiomotor neurons (FBM neurons) migrate from medial rhombomere (r) 4 to lateral r6. In zebrafish, mutations in planar cell polarity genes celsr2 and frizzled3a block caudal migration of FBM neurons. Here, we investigated the role of cadherins Celsr1-3, and Fzd3 in FBM neuron migration in mice. In Celsr1 mutants (knock-out and Crash alleles), caudal migration was compromised and neurons often migrated rostrally into r2 and r3, as well as laterally. These phenotypes were not caused by defects in hindbrain patterning or neuronal specification. Celsr1 is expressed in FBM neuron precursors and the floor plate, but not in FBM neurons. Consistent with this, conditional inactivation showed that the function of Celsr1 in FBM neuron migration was non-cell autonomous. In Celsr2 mutants, FBM neurons initiated caudal migration but moved prematurely into lateral r4 and r5. This phenotype was enhanced by inactivation of Celsr3 in FBM neurons and mimicked by inactivation of Fzd3. Furthermore, Celsr2 was epistatic to Celsr1. These data indicate that Celsr1-3 differentially regulate FBM neuron migration. Celsr1 helps to specify the direction of FBM neuron migration, whereas Celsr2 and 3 control its ability to migrate.

Monoaminergic neurons [serotonergic (5-HT) and dopaminergic (mdDA)] in the brainstem project axons along the anterior-posterior axis. Despite their important physiological functions and implication in disease, the molecular mechanisms that dictate the formation of these projections along the anterior-posterior axis remain unknown. Here we reveal a novel requirement for Wnt/planar cell polarity signaling in the anterior-posterior organization of the monoaminergic system. We find that 5-HT and mdDA axons express the core planar cell polarity components Frizzled3, Celsr3, and Vangl2. In addition, monoaminergic projections show anterior-posterior guidance defects in Frizzled3, Celsr3, and Vangl2 mutant mice. The only known ligands for planar cell polarity signaling are Wnt proteins. In culture, Wnt5a attracts 5-HT but repels mdDA axons, and Wnt7b attracts mdDA axons. However, mdDA axons from Frizzled3 mutant mice are unresponsive to Wnt5a and Wnt7b. Both Wnts are expressed in gradients along the anterior-posterior axis, consistent with their role as directional cues. Finally, Wnt5a mutants show transient anterior-posterior guidance defects in mdDA projections. Furthermore, we observe during development that the cell bodies of migrating descending 5-HT neurons eventually reorient along the direction of their axons. In Frizzled3 mutants, many 5-HT and mdDA neuron cell bodies are oriented abnormally along the direction of their aberrant axon projections. Overall, our data suggest that Wnt/planar cell polarity signaling may be a global anterior-posterior guidance mechanism that controls axonal and cellular organization beyond the spinal cord.

The development of axonal tracts requires interactions between growth cones and the environment. Major bundles, particularly in the internal capsule, are completely defective in mice with constitutive mutation of Celsr3. In order to understand better how Celsr3 controls axonal tract formation, we generated a conditional allele that allowed inactivation of Celsr3 in different sectors of the forebrain. Effects of Celsr3 inactivation specifically in the telencephalon, in the ventral forebrain, or in the cortex, demonstrate essential roles for the gene, both in the neurons that project their axons to subcerebral targets such as the spinal cord, as well as in cells that guide projecting axons through the ventral forebrain. These observations provide unequivocal in vivo evidence that heterotypic interactions between axons and guidepost cells govern axonal path formation in mammals, and that Celsr3 plays a key role in this process. In absence of cortico-subcortical connections, mice can survive up to P20, allowing studies of behavior and cortical maturation. Mutant mice with defective corticospinal tracts survive normally and provide a model to evaluate in vivo the role of this tract in motor function in rodents.