PAK3 activation promotes the tangential to radial migration switch of cortical interneurons by increasing leading process dynamics and disrupting cell polarity.

Mutations of PAK3, a p21-activated kinase, are associated in humans with cognitive deficits suggestive of defective cortical circuits and with frequent brain structural abnormalities. Most human variants no longer exhibit kinase activity. Since GABAergic interneurons express PAK3 as they migrate within the cortex, we here examined the role of PAK3 kinase activity in the regulation of cortical interneuron migration. During the embryonic development, cortical interneurons migrate a long distance tangentially and then re-orient radially to settle in the cortical plate, where they contribute to cortical circuits. We showed that interneurons expressing a constitutively kinase active PAK3 variant (PAK3-ca) extended shorter leading processes and exhibited unstable polarity. In the upper cortical layers, they entered the cortical plate and extended radially oriented processes. In the deep cortical layers, they exhibited erratic non-processive migration movements and accumulated in the deep pathway. Pharmacological inhibition of PAK3 kinase inhibited the radial migration switch of interneurons to the cortical plate and reduced their accumulation in the deep cortical layers. Interneurons expressing a kinase dead PAK3 variant (PAK3-kd) developed branched leading processes, maintained the same polarity during migration and exhibited processive and tangentially oriented movements in the cortex. These results reveal that PAK3 kinase activity, by promoting leading process shortening and cell polarity changes, inhibits the tangential processive migration of interneurons and favors their radial re- orientation and targeting to the cortical plate. They suggest that patients expressing PAK3 variants with impaired kinase activity likely present alterations in the cortical targeting of their GABAergic interneurons.

YIF1B mutations cause a post-natal neurodevelopmental syndrome associated with Golgi and primary cilium alterations.

Human post-natal neurodevelopmental delay is often associated with cerebral alterations that can lead, by themselves or associated with peripheral deficits, to premature death. Here, we report the clinical features of 10 patients from six independent families with mutations in the autosomal YIF1B gene encoding a ubiquitous protein involved in anterograde traffic from the endoplasmic reticulum to the cell membrane, and in Golgi apparatus morphology. The patients displayed global developmental delay, motor delay, visual deficits with brain MRI evidence of ventricle enlargement, myelination alterations and cerebellar atrophy. A similar profile was observed in the Yif1b knockout (KO) mouse model developed to identify the cellular alterations involved in the clinical defects. In the CNS, mice lacking Yif1b displayed neuronal reduction, altered myelination of the motor cortex, cerebellar atrophy, enlargement of the ventricles, and subcellular alterations of endoplasmic reticulum and Golgi apparatus compartments. Remarkably, although YIF1B was not detected in primary cilia, biallelic YIF1B mutations caused primary cilia abnormalities in skin fibroblasts from both patients and Yif1b-KO mice, and in ciliary architectural components in the Yif1b-KO brain. Consequently, our findings identify YIF1B as an essential gene in early post-natal development in human, and provide a new genetic target that should be tested in patients developing a neurodevelopmental delay during the first year of life. Thus, our work is the first description of a functional deficit linking Golgipathies and ciliopathies, diseases so far associated exclusively to mutations in genes coding for proteins expressed within the primary cilium or related ultrastructures. We therefore propose that these pathologies should be considered as belonging to a larger class of neurodevelopmental diseases depending on proteins involved in the trafficking of proteins towards specific cell membrane compartments.

Conserved rules in embryonic development of cortical interneurons.

This review will focus on early aspects of cortical interneurons (cIN) development from specification to migration and final positioning in the human cerebral cortex. These mechanisms have been largely studied in the mouse model, which provides unique possibilities of genetic analysis, essential to dissect the molecular and cellular events involved in cortical development. An important goal here is to discuss the conservation and the potential divergence of these mechanisms, with a particular interest for the situation in the human embryo. We will thus cover recent works, but also revisit older studies in the light of recent data to better understand the developmental mechanisms underlying cIN differentiation in human. Because cIN are implicated in severe developmental disorders, understanding the molecular and cellular mechanisms controlling their differentiation might clarify some causes and potential therapeutic approaches to these important clinical conditions.

Auditory cortex interneuron development requires cadherins operating hair-cell mechanoelectrical transduction.

Many genetic forms of congenital deafness affect the sound reception antenna of cochlear sensory cells, the hair bundle. The resulting sensory deprivation jeopardizes auditory cortex (AC) maturation. Early prosthetic intervention should revive this process. Nevertheless, this view assumes that no intrinsic AC deficits coexist with the cochlear ones, a possibility as yet unexplored. We show here that many GABAergic interneurons, from their generation in the medial ganglionic eminence up to their settlement in the AC, express two cadherin-related (cdhr) proteins, cdhr23 and cdhr15, that form the hair bundle tip links gating the mechanoelectrical transduction channels. Mutant mice lacking either protein showed a major decrease in the number of parvalbumin interneurons specifically in the AC, and displayed audiogenic reflex seizures. Cdhr15- and Cdhr23-expressing interneuron precursors in Cdhr23-/- and Cdhr15-/- mouse embryos, respectively, failed to enter the embryonic cortex and were scattered throughout the subpallium, consistent with the cell polarity abnormalities we observed in vitro. In the absence of adhesion G protein-coupled receptor V1 (adgrv1), another hair bundle link protein, the entry of Cdhr23- and Cdhr15-expressing interneuron precursors into the embryonic cortex was also impaired. Our results demonstrate that a population of newborn interneurons is endowed with specific cdhr proteins necessary for these cells to reach the developing AC. We suggest that an "early adhesion code" targets populations of interneuron precursors to restricted neocortical regions belonging to the same functional area. These findings open up new perspectives for auditory rehabilitation and cortical therapies in patients.

The role of primary cilia in corpus callosum formation is mediated by production of the Gli3 repressor.

Agenesis of the corpus callosum (AgCC) is a frequent brain disorder found in over 80 human congenital syndromes including ciliopathies. Here, we report a severe AgCC in Ftm/Rpgrip1l knockout mouse, which provides a valuable model for Meckel-Grüber syndrome. Rpgrip1l encodes a protein of the ciliary transition zone, which is essential for ciliogenesis in several cell types in mouse including neuroepithelial cells in the developing forebrain. We show that AgCC in Rpgrip1l(-/-) mouse is associated with a disturbed location of guidepost cells in the dorsomedial telencephalon. This mislocalization results from early patterning defects and abnormal cortico-septal boundary (CSB) formation in the medial telencephalon. We demonstrate that all these defects primarily result from altered GLI3 processing. Indeed, AgCC, together with patterning defects and mispositioning of guidepost cells, is rescued by overexpressing in Rpgrip1l(-/-) embryos, the short repressor form of the GLI3 transcription factor (GLI3R), provided by the Gli3(Δ699) allele. Furthermore, Gli3(Δ699) also rescues AgCC in Rfx3(-/-) embryos deficient for the ciliogenic RFX3 transcription factor that regulates the expression of several ciliary genes. These data demonstrate that GLI3 processing is a major outcome of primary cilia function in dorsal telencephalon morphogenesis. Rescuing CC formation in two independent ciliary mutants by GLI3(Δ699) highlights the crucial role of primary cilia in maintaining the proper level of GLI3R required for morphogenesis of the CC.

N-cadherin sustains motility and polarity of future cortical interneurons during tangential migration.

In the developing brain, cortical GABAergic interneurons migrate long distances from the medial ganglionic eminence (MGE) in which they are generated, to the cortex in which they settle. MGE cells express the cell adhesion molecule N-cadherin, a homophilic cell-cell adhesion molecule that regulates numerous steps of brain development, from neuroepithelium morphogenesis to synapse formation. N-cadherin is also expressed in embryonic territories crossed by MGE cells during their migration. In this study, we demonstrate that N-cadherin is a key player in the long-distance migration of future cortical interneurons. Using N-cadherin-coated substrate, we show that N-cadherin-dependent adhesion promotes the migration of mouse MGE cells in vitro. Conversely, mouse MGE cells electroporated with a construct interfering with cadherin function show reduced cell motility, leading process instability, and impaired polarization associated with abnormal myosin IIB dynamics. In vivo, the capability of electroporated MGE cells to invade the developing cortical plate is altered. Using genetic ablation of N-cadherin in mouse embryos, we show that N-cadherin-depleted MGEs are severely disorganized. MGE cells hardly exit the disorganized proliferative area. N-cadherin ablation at the postmitotic stage, which does not affect MGE morphogenesis, alters MGE cell motility and directionality. The tangential migration to the cortex of N-cadherin ablated MGE cells is delayed, and their radial migration within the cortical plate is perturbed. Altogether, these results identify N-cadherin as a pivotal adhesion substrate that activates cell motility in future cortical interneurons and maintains cell polarity over their long-distance migration to the developing cortex.

[Cilia and neuronal migrations]. / Cils et migrations neuronales.

In a landmark paper published in 1977, G. Albrecht-Buehler described a primary cilium on the surface of migrating fibroblasts, and noticed that cilia are oriented parallel to the direction of migration of fibroblasts. While the presence of a primary cilium on neural progenitors and on post-mitotic neurons was noted long ago, it has been observed on migrating cortical interneurons only recently. As in fibroblasts, the cilium of interneurons controls the directionality of migration. It plays an important role in the reorientation of cortical interneurons towards the cortical plate. The morphogen Shh, which is expressed in the migratory pathway of interneurons, is one of the signals that control this reorientation.

CXCL12 targets the primary cilium cAMP/cGMP ratio to regulate cell polarity during migration.

Directed cell migration requires sustained cell polarisation. In migrating cortical interneurons, nuclear movements are directed towards the centrosome that organises the primary cilium signalling hub. Primary cilium-elicited signalling, and how it affects migration, remain however ill characterised. Here, we show that altering cAMP/cGMP levels in the primary cilium by buffering cAMP, cGMP or by locally increasing cAMP, influences the polarity and directionality of migrating interneurons, whereas buffering cAMP or cGMP in the apposed centrosome compartment alters their motility. Remarkably, we identify CXCL12 as a trigger that targets the ciliary cAMP/cGMP ratio to promote sustained polarity and directed migration. We thereby uncover cAMP/cGMP levels in the primary cilium as a major target of extrinsic cues and as the steering wheel of neuronal migration.

Primary cilium-dependent cAMP/PKA signaling at the centrosome regulates neuronal migration.

The primary cilium (PC) is a small centrosome-assembled organelle, protruding from the surface of most eukaryotic cells. It plays a key role in cell migration, but the underlying mechanisms are unknown. Here, we show that the PC regulates neuronal migration via cyclic adenosine 3'-5' monosphosphate (cAMP) production activating centrosomal protein kinase A (PKA). Biosensor live imaging revealed a periodic cAMP hotspot at the centrosome of embryonic, postnatal, and adult migrating neurons. Genetic ablation of the PC, or knockdown of ciliary adenylate cyclase 3, caused hotspot disappearance and migratory defects, with defective centrosome dynamics and altered nucleokinesis. Delocalization of PKA from the centrosome phenocopied the migratory defects. Our results show that the PC and centrosome form a single cAMP signaling unit dynamically regulating migration, further highlighting the centrosome as a signaling hub.

SpiCee: A Genetic Tool for Subcellular and Cell-Specific Calcium Manipulation.

Calcium is a second messenger crucial to a myriad of cellular processes ranging from regulation of metabolism and cell survival to vesicle release and motility. Current strategies to directly manipulate endogenous calcium signals lack cellular and subcellular specificity. We introduce SpiCee, a versatile and genetically encoded chelator combining low- and high-affinity sites for calcium. This scavenger enables altering endogenous calcium signaling and functions in single cells in vitro and in vivo with biochemically controlled subcellular resolution. SpiCee paves the way to investigate local calcium signaling in vivo and directly manipulate this second messenger for therapeutic use.

Modes and mishaps of neuronal migration in the mammalian brain.

The ability of neurons to migrate to their appropriate positions in the developing brain is critical to brain architecture and function. Recent research has elucidated different modes of neuronal migration and the involvement of a host of signaling factors in orchestrating the migration, as well as vulnerabilities of this process to environmental and genetic factors. Here we discuss the role of cytoskeleton, motor proteins, and mechanisms of nuclear translocation in radial and tangential migration of neurons. We will also discuss how these and other events essential for normal migration of neurons can be disrupted by genetic and environmental factors that contribute to neurological disease in humans.

Topographical cues control the morphology and dynamics of migrating cortical interneurons.

In mammalian embryos, cortical interneurons travel long distances among complex three-dimensional tissues before integrating into cortical circuits. Several molecular guiding cues involved in this migration process have been identified, but the influence of physical parameters remains poorly understood. In the present study, we have investigated in vitro the influence of the topography of the microenvironment on the migration of primary cortical interneurons released from mouse embryonic explants. We found that arrays of PDMS micro-pillars of 10 µm size and spacing, either round or square, influenced both the morphology and the migratory behavior of interneurons. Strikingly, most interneurons exhibited a single and long leading process oriented along the diagonals of the square pillared array, whereas leading processes of interneurons migrating in-between round pillars were shorter, often branched and oriented in all available directions. Accordingly, dynamic studies revealed that growth cone divisions were twice more frequent in round than in square pillars. Both soma and leading process tips presented forward directed movements within square pillars, contrasting with the erratic trajectories and more dynamic movements observed among round pillars. In support of these observations, long interneurons migrating in square pillars displayed tight bundles of stable microtubules aligned in the direction of migration. Overall, our results show that micron-sized topography provides global spatial constraints promoting the establishment of different morphological and migratory states. Remarkably, these different states belong to the natural range of migratory behaviors of cortical interneurons, highlighting the potential importance of topographical cues in the guidance of these embryonic neurons, and more generally in brain development.

Nocodazole-induced changes in microtubule dynamics impair the morphology and directionality of migrating medial ganglionic eminence cells.

We have shown previously that actomyosin contractility plays an important role in controlling nuclear movements in future interneurons born in the medial ganglionic eminence (MGE) [Bellion et al.: J Neurosci 2005;25:5691-5699]. Because microtubules are known to control the structural and motile properties of migrating neurons, we asked whether alterations in the dynamic instability of microtubules would impair MGE cell migration. Migration was analyzed in flat cocultures in which green-fluorescent-protein-expressing MGE cells migrate on cortical cells from their explant of origin. A low (100 nM) concentration of nocodazole shortened the leading process of MGE cells that nevertheless continued to migrate at the same rate but frequently changed their direction of migration relative to control cells. MGE cells treated with a higher (1 muM) concentration of nocodazole that strongly destabilized microtubules took on multipolar morphology. They extended thin and labile processes. MGE cells no longer exhibited directional migration and migration velocity slowed 2-fold. These results suggest that microtubule stability is crucial for maintaining polarity and controlling the directional migration of MGE cells, whereas additional mechanisms are required to control cell motility.

In Vitro Models to Analyze the Migration of MGE-Derived Interneurons.

In the developing brain, MGE-derived interneuron precursors migrate tangentially long distances to reach the cortex in which they later establish connections with the principal cortical cells to control the activity of adult cortical circuits. Interneuron precursors exhibit complex morphologies and migratory properties, which are difficult to study in the heterogeneous and uncontrolled in vivo environment. Here, we describe two in vitro models in which the migration environment of interneuron precursors is significantly simplified and where their migration can be observed for one to 3 days. In one model, MGE-derived interneuron precursors are cultured and migrate on a flat synthetic substrate. In the other model, fluorescent MGE-derived interneuron precursors migrate on a monolayer of dissociated cortical cells. In both models, cell movements can be recorded by time-lapse microscopy for dynamic analyses.

Requirement of adenylate cyclase 1 for the ephrin-A5-dependent retraction of exuberant retinal axons.

The calcium-stimulated adenylate cyclase 1 (AC1) has been shown to be required for the refinement of the retinotopic map, but the mechanisms involved are not known. To investigate this question, we devised a retinotectal coculture preparation that reproduces the gradual acquisition of topographic specificity along the rostrocaudal axis of the superior colliculus (SC). Temporal retinal axons invade the entire SC at 4 d in vitro (DIV) and eliminate exuberant branches caudally by 12 DIV. Temporal and nasal axons form branches preferentially in the rostral or caudal SC, respectively. Retinal explants from AC1-deficient mice, AC1(brl/brl), maintain exuberant branches and lose the regional selectivity of branching when confronted with wild-type (WT) SC. Conversely, WT retinas correctly target AC1(brl/brl) collicular explants. The effects of AC1 loss of function in the retina are mimicked by the blockade of ephrin-A5 signaling in WT cocultures. Video microscopic analyses show that AC1(brl/brl) axons have modified responses to ephrin-A5: the collapse of the growth cones occurs, but the rearward movement of the axon is arrested. Our results demonstrate a presynaptic, cell autonomous role of AC1 in the retina and further indicate that AC1 is necessary to enact a retraction response of the retinal axons to ephrin-A5 during the refinement of the retinotopic map.

Nucleokinesis in tangentially migrating neurons comprises two alternating phases: forward migration of the Golgi/centrosome associated with centrosome splitting and myosin contraction at the rear.

During rodent cortex development, cells born in the medial ganglionic eminence (MGE) of the basal telencephalon reach the embryonic cortex by tangential migration and differentiate as interneurons. Migrating MGE cells exhibit a saltatory progression of the nucleus and continuously extend and retract branches in their neuritic arbor. We have analyzed the migration cycle of these neurons using in vitro models. We show that the nucleokinesis in MGE cells comprises two phases. First, cytoplasmic organelles migrate forward, and second, the nucleus translocates toward these organelles. During the first phase, a large swelling that contains the centrosome and the Golgi apparatus separates from the perinuclear compartment and moves rostrally into the leading neurite, up to 30 mum from the waiting nucleus. This long-distance migration is associated with a splitting of the centrioles that line up along a linear Golgi apparatus. It is followed by the second, dynamic phase of nuclear translocation toward the displaced centrosome and Golgi apparatus. The forward movement of the nucleus is blocked by blebbistatin, a specific inhibitor of nonmuscle myosin II. Because myosin II accumulates at the rear of migrating MGE cells, actomyosin contraction likely plays a prominent role to drive forward translocations of the nucleus toward the centrosome. During this phase of nuclear translocation, the leading growth cone either stops migrating or divides, showing a tight correlation between leading edge movements and nuclear movements.

Inhibition of SRC family kinases and non-classical protein kinases C induce a reeler-like malformation of cortical plate development.

During development, most cortical neurons migrate to the cortical plate (CP) radially. CP development is abnormal in reeler and other mutant mice with defective Reelin signaling. Reelin is secreted by Cajal-Retzius cells and binds to the very low density lipoprotein receptor and apolipoprotein E receptor type 2 receptors on the surface of CP cells, inducing tyrosine phosphorylation of the intracellular Dab1 adapter. As with Reelin receptors, the identification of Reelin signaling partners is hampered by genetic redundancy. Using a new in vitro embryonic slice culture system, we demonstrate that chemical inhibitors of Src family kinases and Abl, but not inhibitors of Abl alone, generate a reeler-like malformation and that inhibitors of protein kinases C induce a malformation of cortical development that is also reminiscent of reeler. Our observations demonstrate a key role for these enzymes in radial migration to the cortical plate, possibly via interference with Reelin signaling.

Early regionalisation of the neocortex and the medial ganglionic eminence.

The two major functional classes of neurons that build the cerebral cortex are generated in two distinct parts of the telencephalon. Excitatory long distance projecting neurons are produced dorsally in the pallium, whereas local inhibitory interneurons are mainly produced in the medial ridge of the ventral telencephalon. These two parts of the telencephalon are molecularly regionalized from early embryonic stages, but cellular indices of regionalisation are observed only at later stages of development. We have looked for cellular indices of regionalisation in the cortical anlage at early embryonic stages, when the first efferent cortical neurons are generated. Similarly, we have looked for functional regionalisation of the medial ganglionic eminence at the same stages, when the future cortical interneurones are generated. Here, we summarize data showing that two regions in the mouse cortex embryo, the lateral and dorsal cortex, differ strongly in their early neurogenesis. Moreover, the two domains differ in their capacity to produce GABAergic neurons in vitro; this capacity is only observed in the dorsal cortex. The differentiation of the two domains appears to be independent of the laterorostral to mediocaudal gradient of maturation of the cortex. In the basal telencephalon too, the capacity to differentiate GABAergic neurons is not uniformly distributed across the medial ganglionic eminence. The neurogenesis of future cortical interneurons is seen to be highly active in a small area located in the rostral MGE, at mid dorso-ventral level.

Cortical interneurons migrating on a pure substrate of N-cadherin exhibit fast synchronous centrosomal and nuclear movements and reduced ciliogenesis.

The embryonic development of the cortex involves a phase of long distance migration of interneurons born in the basal telencephalon. Interneurons first migrate tangentially and then reorient their trajectories radially to enter the developing cortex. We have shown that migrating interneurons can assemble a primary cilium, which maintains the centrosome to the plasma membrane and processes signals to control interneuron trajectory (Baudoin et al., 2012). In the developing cortex, N-cadherin is expressed by migrating interneurons and by cells in their migratory pathway. N-cadherin promotes the motility and maintains the polarity of tangentially migrating interneurons (Luccardini et al., 2013). Because N-cadherin is an important factor that regulates the migration of medial ganglionic eminence (MGE) cells in vivo, we further characterized the motility and polarity of MGE cells on a substrate that only comprises this protein. MGE cells migrating on a N-cadherin substrate were seven times faster than on a laminin substrate and two times faster than on a substrate of cortical cells. A primary cilium was much less frequently observed on MGE cells migrating on N-cadherin than on laminin. Nevertheless, the mature centriole (MC) frequently docked to the plasma membrane in MGE cells migrating on N-cadherin, suggesting that plasma membrane docking is a basic feature of the centrosome in migrating MGE cells. On the N-cadherin substrate, centrosomal and nuclear movements were remarkably synchronous and the centrosome remained near the nucleus. Interestingly, MGE cells with cadherin invalidation presented centrosomal movements no longer coordinated with nuclear movements. In summary, MGE cells migrating on a pure substrate of N-cadherin show fast, coordinated nuclear and centrosomal movements, and rarely present a primary cilium.

Cilia: traffic directors along the road of cortical development.

While the presence of a primary cilium on neural progenitors and on post-mitotic neurons was noted long ago, a primary cilium has been observed on migrating cortical interneurons only recently. As in fibroblasts, the cilium of interneurons controls the directionality of migration. It plays an important role in the reorientation of cortical interneurons toward the cortical plate. The morphogen Shh, which is expressed in the migratory pathway of interneurons, is one of the signals that control this reorientation. After a short description of the migratory pathways of cortical interneurons, we focus on cellular mechanisms that allow interneurons to reorient their trajectory during their long-distance migration. Then we examine the role of the primary cilium in cell migration and how ciliogenesis might be related to the migration cycle in interneurons. Finally, we review the molecular mechanisms at the basis of the sensory function of the primary cilium and examine how Shh signals could influence the migratory behavior of cortical interneurons. These novel data provide a cellular basis to further understanding cognitive deficits associated with human ciliopathies.