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.

Chemokine receptor CXCR7 non-cell-autonomously controls pontine neuronal migration and nucleus formation.

Long distance tangential migration transports neurons from their birth places to distant destinations to be incorporated into neuronal circuits. How neuronal migration is guided during these long journeys is still not fully understood. We address this issue by studying the migration of pontine nucleus (PN) neurons in the mouse hindbrain. PN neurons migrate from the lower rhombic lip first anteriorly and then turn ventrally near the trigeminal ganglion root towards the anterior ventral hindbrain. Previously we showed that in mouse depleted of chemokine receptor CXCR4 or its ligand CXCL12, PN neurons make their anterior-to-ventral turn at posteriorized positions. However, the mechanism that spatiotemporally controls the anterior-to-ventral turning is still unclear. Furthermore, the role of CXCR7, the atypical receptor of CXCL12, in pontine migration has yet to be examined. Here, we find that the PN is elongated in Cxcr7 knockout due to a broadened anterior-to-ventral turning positions. Cxcr7 is not expressed in migrating PN neurons en route to their destinations, but is strongly expressed in the pial meninges. Neuroepithelium-specific knockout of Cxcr7 does not recapitulate the PN phenotype in Cxcr7 knockout, suggesting that CXCR7 acts non-cell-autonomously possibly from the pial meninges. We show further that CXCR7 regulates pontine migration by modulating CXCL12 protein levels.

Netrin-1 Derived from the Ventricular Zone, but not the Floor Plate, Directs Hindbrain Commissural Axons to the Ventral Midline.

Netrin-1 (Ntn1) emanating from the ventral midline has been thought to act as a long-range diffusible chemoattractant for commissural axons (CAs). However, CAs still grow towards the midline in the absence of the floor plate (FP), a glial structure occupying the midline. Here, using genetically loss-of-function approaches in mice, we show that Ntn1 derived from the ventricular zone (VZ), but not the FP, is crucial for CA guidance in the mouse hindbrain. During the period of CA growth, Ntn1 is expressed in the ventral two-thirds of the VZ, in addition to the FP. Remarkably, deletion of Ntn1 from the VZ and even from the dorsal VZ highly disrupts CA guidance to the midline, whereas the deletion from the FP has little impact on it. We also show that the severities of CA guidance defects found in the Ntn1 conditional mutants were irrelevant to their FP long-range chemoattractive activities. Our results are incompatible with the prevailing view that Ntn1 is an FP-derived long-range diffusible chemoattractant for CAs, but suggest a novel mechanism that VZ-derived Ntn1 directs CAs to the ventral midline by its local actions.

Distinct migratory behaviors of striosome and matrix cells underlying the mosaic formation in the developing striatum.

The striatum, the largest nucleus of the basal ganglia controlling motor and cognitive functions, can be characterized by a labyrinthine mosaic organization of striosome/matrix compartments. It is unclear how striosome/matrix mosaic formation is spatially and temporally controlled at the cellular level during striatal development. Here, by combining in vivo electroporation and brain slice cultures, we set up a prospective experimental system in which we differentially labeled striosome and matrix cells from the time of birth and followed their distributions and migratory behaviors. Our results showed that, at an initial stage of striosome/matrix mosaic formation, striosome cells were mostly stationary, whereas matrix cells actively migrated in multiple directions regardless of the presence of striosome cells. The mostly stationary striosome cells were still able to associate to form patchy clusters via attractive interactions. Our results suggest that the restricted migratory capability of striosome cells may allow them to cluster together only when they happen to be located in close proximity to each other and are not separated by actively migrating matrix cells. The way in which the mutidirectionally migrating matrix cells intermingle with the mostly stationary striosome cells may therefore determine the topographic features of striosomes. At later stages, the actively migrating matrix cells began to repulse the patchy clusters of striosomes, presumably enhancing the striosome cluster formation and the segregation and eventual formation of dichotomous homogeneous striosome/matrix compartments. Overall, our study reveals temporally distinct migratory behaviors of striosome/matrix cells, which may underlie the sequential steps of mosaic formation in the developing striatum. J. Comp. Neurol. 525:794-817, 2017. © 2016 Wiley Periodicals, Inc.

In vivo imaging of cortical interneurons migrating in the intermediate/subventricular zones.

We developed an imaging system that enables migrating cortical interneurons (CIs) through the lower intermediate zone/subventricular zone (IZ/SVZ) in mouse embryos. CIs were labeled by in utero electroporation performed at embryonic day (E) 11.5 and were observed, through the skull of living embryos, detached from the dam with the umbilical cord remain attached. To identify imaged cell locations, we used GAD67-GFP mice and GFP fluorescence was photo-bleached after the recording. We found that CIs in the IZ/SVZ migrated medially straight toward the midline on the tangential plane, while those in the marginal zone migrated in all directions.

Evidence That the Laminar Fate of LGE/CGE-Derived Neocortical Interneurons Is Dependent on Their Progenitor Domains.

Neocortical interneurons show tremendous diversity in terms of their neurochemical marker expressions, morphology, electrophysiological properties, and laminar fate. Allocation of interneurons to their appropriate regions and layers in the neocortex is thought to play important roles for the emergence of higher functions of the neocortex. Neocortical interneurons mainly originate from the medial ganglionic eminence (MGE) and the caudal ganglionic eminence (CGE). The diversity and the laminar fate of MGE-derived interneurons depend on the location of their birth and birthdate, respectively. However, this relationship does not hold for CGE-derived interneurons. Here, using the method of in utero electroporation, which causes arbitrary occurrence of labeled progenitor domains, we tracked all descendants of the lateral ganglionic eminence (LGE)/CGE progenitors in mice. We provide evidence that neocortical interneurons with distinct laminar fate originate from distinct progenitor domains within the LGE/CGE. We find layer I interneurons are predominantly labeled in a set of animals, whereas other upper layer neurons are predominantly labeled in another set. We also find distinct subcortical structures labeled between the two sets. Further, interneurons labeled in layer I show distinct neurochemical properties from those in other layers. Together, these results suggest that the laminar fate of LGE/CGE-derived interneurons depends on their spatial origin. SIGNIFICANCE STATEMENT: Diverse types of neocortical interneurons have distinct laminar fate, neurochemical marker expression, morphology, and electrophysiological properties. Although the specifications and laminar fate of medial ganglionic eminence-derived neocortical interneurons depend on their location of embryonic origin and birthdate, no similar causality of lateral/caudal ganglionic eminence (LGE/CGE)-derived neocortical interneurons is known. Here, we performed in utero electroporation on mouse LGE/CGE and found two groups of animals, one with preferential labeling of layer I and the other with preferential labeling of other layers. Interneurons labeled in these two groups show distinct neurochemical properties and morphologies and are associated with labeling of distinct subcortical structures. These findings suggest that the laminar fate of LGE/CGE-derived neocortical interneurons depends on their spatial origin.

From migration to settlement: the pathways, migration modes and dynamics of neurons in the developing brain.

Neuronal migration is crucial for the construction of the nervous system. To reach their correct destination, migrating neurons choose pathways using physical substrates and chemical cues of either diffusible or non-diffusible nature. Migrating neurons extend a leading and a trailing process. The leading process, which extends in the direction of migration, determines navigation, in particular when a neuron changes its direction of migration. While most neurons simply migrate radially, certain neurons switch their mode of migration between radial and tangential, with the latter allowing migration to destinations far from the neurons' site of generation. Consequently, neurons with distinct origins are intermingled, which results in intricate neuronal architectures and connectivities and provides an important basis for higher brain function. The trailing process, in contrast, contributes to the late stage of development by turning into the axon, thus contributing to the formation of neuronal circuits.

Association of astrocytes with neurons and astrocytes derived from distinct progenitor domains in the subpallium.

Astrocytes play pivotal roles in metabolism and homeostasis as well as in neural development and function in a manner thought to depend on their region-specific diversity. In the mouse spinal cord, astrocytes and neurons, which are derived from a common progenitor domain (PD) and controlled by common PD-specific transcription factors, migrate radially and share their final positions. However, whether astrocytes can only interact with neurons from common PDs in the brain remains unknown. Here, we focused on subpallium-derived cells, because the subpallium generates neurons that show a diverse mode of migration. We tracked their fate by in utero electroporation of plasmids that allow for chromosomal integration of transgenes or of a Cre recombinase expression vector to reporter mice. We also used an Nkx2.1(Cre) mouse line to fate map the cells originating from the medial ganglionic eminence and preoptic area. We find that although neurons and astrocytes are labeled in various regions, only neurons are labeled in the neocortex, hippocampus and olfactory bulb. Furthermore, we find astrocytes derived from an Nkx 2.1-negative PD are associated with neurons from the Nkx2.1(+) PD. Thus, forebrain astrocytes can associate with neurons as well as astrocytes derived from a distinct PD.

Chemokine Signaling Controls Integrity of Radial Glial Scaffold in Developing Spinal Cord and Consequential Proper Position of Boundary Cap Cells.

Radial glial cells are the neural progenitors of the developing CNS and have long radial processes that guide radially migrating neurons. The integrity of the radial glial scaffold, in particular proper adhesion between the endfeet of radial processes and the pial basement membrane (BM), is important for the cellular organization of the CNS, as indicated by evidence emerging from the developing cortex. However, the mechanisms underlying the maintenance of radial glial scaffold integrity during development, when the neuroepithelium rapidly expands, are still poorly understood. Here, we addressed this issue in the developing mouse spinal cord. We show that CXCR4, a receptor of chemokine CXCL12, is expressed in spinal cord radial glia. Conditional knock-out of Cxcr4 in radial glia caused disrupted radial glial scaffold with gaps at the pial endfeet layer and consequentially led to an invasion of boundary cap (BC) cells into the spinal cord. Because BC cells are PNS cells normally positioned at the incoming and outgoing axonal roots, their invasion into the spinal cord suggests a compromised CNS/PNS boundary in the absence of CXCL12/CXCR4 signaling. Both disrupted radial glial scaffold and invasion of BC cells into the CNS were also present in mice deficient in CXCR7, a second receptor of CXCL12. We further show that CXCL12 signaling promotes the radial glia adhesion to BM components and activates integrin ß1 avidity. Our study unravels a novel molecular mechanism that deploys CXCL12/CXCR4/CXCR7 for the maintenance of radial glial scaffold integrity, which in turn safeguards the CNS/PNS boundary during spinal cord development.

Specific labeling of climbing fibers shows early synaptic interactions with immature Purkinje cells in the prenatal cerebellum.

During development, growing axons must locate target cells to form synapses. This is not easy, since target cells are also growing and even actively migrating. In some brain regions, such axons have been reported to wait for the timing when target cells become mature, without invading their target region. However, in the cerebellum climbing fibers (CFs), major afferent axons, arrive near their target neurons, Purkinje cells, when the neurons are still actively migrating. We, therefore, examined whether synaptic contacts are established at such early stages. To specifically label CFs, we introduced by in utero electroporation a mixture of genes encoding for Ptf1a-enhancer-driven Cre recombinase and Cre-dependent fluorescent protein into the mouse hindbrain at embryonic day (E) 10.5 and observed them during development. The earliest stages at which labeled CFs were observed in the cerebellar primordium were E15.5-E16.5. These fibers were fasciculated in the dorsal region and entered the cerebellar primordium. Some fibers defasciculated and reached the caudal region. At E17.5 and E18.5, fasciculated fibers were also found in the mantle region, and some grew toward the surface of the primordium to penetrate a mass of Purkinje cells. Interestingly, as early as E16.5, labeled fibers were found to run in close apposition to Purkinje cell dendrites and to express a presynaptic marker. These observations suggest that CFs form synapses with Purkinje cells as soon as the fibers enter the cerebellum.

Calm1 signaling pathway is essential for the migration of mouse precerebellar neurons.

The calcium ion regulates many aspects of neuronal migration, which is an indispensable process in the development of the nervous system. Calmodulin (CaM) is a multifunctional calcium ion sensor that transduces much of the signal. To better understand the role of Ca(2+)-CaM in neuronal migration, we investigated mouse precerebellar neurons (PCNs), which undergo stereotyped, long-distance migration to reach their final position in the developing hindbrain. In mammals, CaM is encoded by three non-allelic CaM (Calm) genes (Calm1, Calm2 and Calm3), which produce an identical protein with no amino acid substitutions. We found that these CaM genes are expressed in migrating PCNs. When the expression of CaM from this multigene family was inhibited by RNAi-mediated acute knockdown, inhibition of Calm1 but not the other two genes caused defective PCN migration. Many PCNs treated with Calm1 shRNA failed to complete their circumferential tangential migration and thus failed to reach their prospective target position. Those that did reach the target position failed to invade the depth of the hindbrain through the required radial migration. Overall, our results suggest the participation of CaM in both the tangential and radial migration of PCNs.

Evidence that dendritic mitochondria negatively regulate dendritic branching in pyramidal neurons in the neocortex.

The precise branching patterns of dendritic arbors have a profound impact on information processing in individual neurons and the brain. These patterns are established by positive and negative regulation of the dendritic branching. Although the mechanisms for positive regulation have been extensively investigated, little is known about those for negative regulation. Here, we present evidence that mitochondria located in developing dendrites are involved in the negative regulation of dendritic branching. We visualized mitochondria in pyramidal neurons of the mouse neocortex during dendritic morphogenesis using in utero electroporation of a mitochondria-targeted fluorescent construct. We altered the mitochondrial distribution in vivo by overexpressing Mfn1, a mitochondrial shaping protein, or the Miro-binding domain of TRAK2 (TRAK2-MBD), a truncated form of a motor-adaptor protein. We found that dendritic mitochondria were preferentially targeted to the proximal portion of dendrites only during dendritic morphogenesis. Overexpression of Mfn1 or TRAK2-MBD depleted mitochondria from the dendrites, an effect that was accompanied by increased branching of the proximal portion of the dendrites. This dendritic abnormality cannot be accounted for by changes in the distribution of membrane trafficking organelles since the overexpression of Mfn1 did not alter the distributions of the endoplasmic reticulum, Golgi, or endosomes. Additionally, neither did these constructs impair neuronal viability or mitochondrial function. Therefore, our results suggest that dendritic mitochondria play a critical role in the establishment of the precise branching pattern of dendritic arbors by negatively affecting dendritic branching.

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.

Development of cerebellar neurons and glias revealed by in utero electroporation: Golgi-like labeling of cerebellar neurons and glias.

Cerebellar cortical functions rely on precisely arranged cytoarchitectures composed of several distinct types of neurons and glias. Studies have indicated that cerebellar excitatory and inhibitory neurons have distinct spatial origins, the upper rhombic lip (uRL) and ventricular zone (VZ), respectively, and that different types of neurons have different birthdates. However, the spatiotemporal relationship between uRL/VZ progenitors and their final phenotype remains poorly understood due to technical limitations. To address this issue, we performed in utero electroporation (IUE) of fluorescent protein plasmids using mouse embryos to label uRL/VZ progenitors at specific developmental stages, and observed labeled cells at maturity. To overcome any potential dilution of the plasmids caused by progenitor division, we also utilized constructs that enable permanent labeling of cells. Cerebellar neurons and glias were labeled in a Golgi-like manner enabling ready identification of labeled cells. Five types of cerebellar neurons, namely Purkinje, Golgi, Lugaro and unipolar brush cells, large-diameter deep nuclei (DN) neurons, and DN astrocytes were labeled by conventional plasmids, whereas plasmids that enable permanent labeling additionally labeled stellate, basket, and granule cells as well as three types of glias. IUE allows us to label uRL/VZ progenitors at different developmental stages. We found that the five types of neurons and DN astrocytes were labeled in an IUE stage-dependent manner, while stellate, basket, granule cells and three types of glias were labeled regardless of the IUE stage. Thus, the results indicate the IUE is an efficient method to track the development of cerebellar cells from uRL/VZ progenitors facing the ventricular lumen. They also indicate that while the generation of the five types of neurons by uRL/VZ progenitors is regulated in a time-dependent manner, the progenitor pool retains multipotency throughout embryonic development.

Four-dimensional analysis of nucleogenesis of the pontine nucleus in the hindbrain.

Nuclei in the central nervous system are 3D aggregates of neurons that have common physiological properties, functionalities, and connectivities. To form specific nuclei, neurons migrate from their birthplace towards the presumptive nuclear region where they change their dynamics to aggregate and rearrange into a distinct 3D structure, a process that we term nucleogenesis. Nuclei, together with the laminar structure, form the basic cytoarchitectonic unit for information processing. However, in contrast to much-studied laminar structures, the neuronal dynamics that contribute to the aggregation process to form nuclei are poorly understood. Here, we analyze nucleogenesis by observing the mouse precerebellar pontine nucleus (PN), and provide the first 4D view of nucleogenesis by tracking neuronal behaviors along the three spatial axes over time. Early- and late-born PN neurons were labeled by in utero electroporation and their behaviors on cultured brain slices were recorded by time-lapse imaging. We find that when PN neurons migrate medially into the nuclear region, many of them switch to migrate radially and laterally, to populate the dorsal and lateral PN regions, respectively. The tendency to switch to radial migration is much less in later-born neurons, whereas that to switch to lateral migration is comparable between the two groups. In contrast to the radial and mediolateral axes, very few PN neurons switch to migrate rostrocaudally. These results could thus provide a framework for understanding the mechanisms that regulate this complex yet important process.

Role of neuropilin-2 in the ipsilateral growth of midbrain dopaminergic axons.

Axonal projections in the CNS can be categorized as either crossed or uncrossed. Crossing and uncrossing of axons has been explained by attractive and repulsive molecules like Netrin-1 and Slits, which are secreted by midline structures. However, uncrossed projections can be established even in double knockout mice of slit1 and slit2 or of roundabout1 (robo1) and robo2, two receptors for Slits. Here, we found that a novel mechanism mediated by Neuropilin-2 (Nrp2) contributes to the formation of uncrossed projections of midbrain dopaminergic neurons (mDANs). Nrp2 transcriptional activities were detected in a subset of mDANs, and its protein was expressed in mDAN axons growing through the ipsilateral diencephalon. In nrp2(lac) (Z) (/lac) (Z) mice, mDAN axons aberrantly grew toward the ventral midline and even crossed it, suggesting that Nrp2 is necessary for the development of mDAN ipsilateral projections. We investigated the involvement of Semaphorin 3B (Sema3B) and Sema3F, two ligands of Nrp2, by analysing mDAN axon trajectories in single or double knockout mice. In both cases, mDAN axons still projected ipsilaterally, suggesting the involvement mechanisms independent of these Sema3s. Nrp2-deficient mDAN axons retained their responsiveness to Slit2, demonstrating that aberrant mDAN axons in nrp2(lac) (Z) (/lac) (Z) mice were not indirectly mediated by alterations in Slit/Robo signaling. Taken together, our results indicate that a novel mechanism mediated by Nrp2 contributes to the establishment of uncrossed projections by mDAN axons.

Dynamics of the leading process, nucleus, and Golgi apparatus of migrating cortical interneurons in living mouse embryos.

Precisely arranged cytoarchitectures such as layers and nuclei depend on neuronal migration, of which many in vitro studies have revealed the mode and underlying mechanisms. However, how neuronal migration is achieved in vivo remains unknown. Here we established an imaging system that allows direct visualization of cortical interneuron migration in living mouse embryos. We found that during nucleokinesis, translocation of the Golgi apparatus either precedes or occurs in parallel to that of the nucleus, suggesting the existence of both a Golgi/centrosome-dependent and -independent mechanism of nucleokinesis. Changes in migratory direction occur when the nucleus enters one of the leading process branches, which is accompanied by the retraction of other branches. The nucleus occasionally swings between two branches before translocating into one of them, the occurrence of which is most often preceded by Golgi apparatus translocation into that branch. These in vivo observations provide important insight into the mechanisms of neuronal migration and demonstrate the usefulness of our system for studying dynamic events in living animals.

Chemokine CXCL12 and its receptors in the developing central nervous system: emerging themes and future perspectives.

Homeostatic chemokine CXCL12 (also known as SDF-1) and its receptor CXCR4 are indispensable for the normal development of the nervous system. This chemokine system plays a plethora of functions in numerous neural developmental processes, from which the underlying molecular and cellular mechanisms are beginning to be unravelled. Recent identification of CXCR7 as a second receptor for CXCL12 provides opportunities to gain deeper insights into how CXCL12 operates in the nervous system. Here, we review the diverse roles of CXCL12 in the developing central nervous system, summarize the recent progress in uncovering CXCR7 functions, and discuss the emerging common themes from these works and future perspectives.

Formation of axon-dendrite polarity in situ: initiation of axons from polarized and non-polarized cells.

Neurons are polarized cells that extend a single axon and several dendrites. Historically, how neurons establish their axon-dendrite polarity has been extensively studied using dissociated hippocampal cells in culture. Although such studies have identified the cellular and molecular mechanisms underlying axon-dendrite polarization, the conclusions have been limited to in vitro conditions. Recent progress using live imaging has enabled us to directly observe axon formation in situ, revealing distinct cellular mechanisms that regulate axon-dendrite polarization in vivo. In this review, we compare the cellular events during axon formation studied in various systems both in vivo and in vitro and discuss possible common mechanisms underlying the axon-dendrite polarization.