Rap1 GTPases Are Master Regulators of Neural Cell Polarity in the Developing Neocortex.

During the development of the mammalian neocortex, the generation of neurons by neural progenitors and their migration to the final position are closely coordinated. The highly polarized radial glial cells (RGCs) serve both as progenitor cells to generate neurons and as support for the migration of these neurons. After their generation, neurons transiently assume a multipolar morphology before they polarize and begin their migration along the RGCs. Here, we show that Rap1 GTPases perform essential functions for cortical organization as master regulators of cell polarity. Conditional deletion of Rap1 GTPases leads to a complete loss of cortical lamination. In RGCs, Rap1 GTPases are required to maintain their polarized organization. In newborn neurons, the loss of Rap1 GTPases prevents the formation of axons and leading processes and thereby interferes with radial migration. Taken together, the loss of RGC and neuronal polarity results in the disruption of cortical organization.

Ephrin-B2 controls VEGF-induced angiogenesis and lymphangiogenesis.

In development, tissue regeneration or certain diseases, angiogenic growth leads to the expansion of blood vessels and the lymphatic vasculature. This involves endothelial cell proliferation as well as angiogenic sprouting, in which a subset of cells, termed tip cells, acquires motile, invasive behaviour and extends filopodial protrusions. Although it is already appreciated that angiogenesis is triggered by tissue-derived signals, such as vascular endothelial growth factor (VEGF) family growth factors, the resulting signalling processes in endothelial cells are only partly understood. Here we show with genetic experiments in mouse and zebrafish that ephrin-B2, a transmembrane ligand for Eph receptor tyrosine kinases, promotes sprouting behaviour and motility in the angiogenic endothelium. We link this pro-angiogenic function to a crucial role of ephrin-B2 in the VEGF signalling pathway, which we have studied in detail for VEGFR3, the receptor for VEGF-C. In the absence of ephrin-B2, the internalization of VEGFR3 in cultured cells and mutant mice is defective, which compromises downstream signal transduction by the small GTPase Rac1, Akt and the mitogen-activated protein kinase Erk. Our results show that full VEGFR3 signalling is coupled to receptor internalization. Ephrin-B2 is a key regulator of this process and thereby controls angiogenic and lymphangiogenic growth.

Dynamics of centrosome translocation and microtubule organization in neocortical neurons during distinct modes of polarization.

Neuronal migration and process formation require cytoskeletal organization and remodeling. Recent studies suggest that centrosome translocation is involved in initial axon outgrowth, while the role of centrosomal positioning is not clear. Here, we examine relations between centrosomal positioning, axonogenesis, and microtubule (MT) polarization in multipolar and bipolar neocortical neurons. We monitored dynamic movements of centrosomes and MT plus ends in migratory neurons in embryonic mouse cerebral slices. In locomoting bipolar neurons, the centrosome oriented toward the pia-directed leading process. Bipolar neurons displayed dense MT plus end dynamics in leading processes, while trailing processes showed clear bidirectional MTs. In migrating multipolar neurons, new processes emerged irrespective of centrosome localization, followed by centrosome reorientations toward the dominant process. Anterograde movements of MT plus ends occurred in growing processes and retrograde movements were observed after retraction of the distal tip. In multipolar neurons, axon formed by tangential extension of a dominant process and the centrosome oriented toward the growing axon, while in locomoting neurons, an axon formed opposite to the direction of migration and the centrosome localized to the base of the leading process. Our data suggest that MT organization may alter centrosomal localization and that centrosomal positioning does not necessarily direct process formation.

Shootin1 acts in concert with KIF20B to promote polarization of migrating neurons.

Shootin1 has been ascribed a role in regulating polarization of primary hippocampal neurons. To better understand the possible role of Shootin1 in the developing brain, we identified a member of the kinesin superfamily, KIF20B, as a novel Shootin1 interacting protein and a potential mediator of Shootin1 interaction with microtubules. KIF20B/Shootin1 binding was mapped to a 57 aa KIF20B sequence, which was used as a dominant-negative fragment. Direct interaction between that peptide (MBD) and Shootin1 was confirmed by surface plasmon resonance-based technology and the affinity was determined in the 10⁻7 m range. The proteins are expressed in the developing brain and formed a complex in vivo based on coimmunoprecipitation experiments and coimmunostaining in primary neurons. In primary hippocampal neurons Kif20b knockdown reduced Shootin1 mobilization to the developing axon, as evidenced by immunostaining and fluorescence recovery after photobleaching analysis, suggesting that Shootin1 is a novel KIF20B cargo. shRNA targeting of Shootin1 reduced PIP3 accumulation in the growth cone, as did Kif20b shRNA. In the developing mouse brain, Kif20b knockdown or expression of the KIF20B minimal binding domain inhibited neuronal migration, and in vivo migration assays suggested that Shootin1/Kif20b acts in the same genetic pathway. Time-lapse imaging of multipolar cells in the subventricular zone revealed that downregulating levels of either Shootin1 or Kif20b hindered the transition from multipolar to bipolar cells. Collectively, our data demonstrate the importance of the Shootin1/KIF20B interaction to the dynamic process of pyramidal neuronal polarization and migration.

WAVE2-Abi2 complex controls growth cone activity and regulates the multipolar-bipolar transition as well as the initiation of glia-guided migration.

Glia-guided migration (glia-guided locomotion) during radial migration is a characteristic yet unique mode of migration. In this process, the directionality of migration is predetermined by glial processes and not by growth cones. Prior to the initiation of glia-guided migration, migrating neurons transform from multipolar to bipolar, but the molecular mechanisms underlying this multipolar-bipolar transition and the commencement of glia-guided migration are not fully understood. Here, we demonstrate that the multipolar-bipolar transition is not solely a cell autonomous event; instead, the interaction of growth cones with glial processes plays an essential role. Time-lapse imaging with lattice assays reveals the importance of vigorously active growth cones in searching for appropriate glial scaffolds, completing the transition, and initiating glia-guided migration. These growth cone activities are regulated by Abl kinase and Cdk5 via WAVE2-Abi2 through the phosphorylation of tyrosine 150 and serine 137 of WAVE2. Neurons that do not display such growth cone activities are mispositioned in a more superficial location in the neocortex, suggesting the significance of growth cones for the final location of the neurons. This process occurs in spite of the "inside-out" principle in which later-born neurons are situated more superficially.

Periventricular notch activation and asymmetric Ngn2 and Tbr2 expression in pair-generated neocortical daughter cells.

To understand the cellular and molecular mechanisms regulating cytogenesis within the neocortical ventricular zone, we examined at high resolution the spatiotemporal expression patterns of Ngn2 and Tbr2. Individually DiI-labeled daughter cells were tracked from their birth in slice cultures and immunostained for Ngn2 and Tbr2. Both proteins were initially absent from daughter cells during the first 2 h. Ngn2 expression was then initiated asymmetrically in one of the daughter cells, with a bias towards the apical cell, followed by a similar pattern of expression for Tbr2, which we found to be a direct target of Ngn2. How this asymmetric Ngn2 expression is achieved is unclear, but gamma-secretase inhibition at the birth of daughter cells resulted in premature Ngn2 expression, suggesting that Notch signaling in nascent daughter cells suppresses a Ngn2-Tbr2 cascade. Many of the nascent cells exhibited pin-like morphology with a short ventricular process, suggesting periventricular presentation of Notch ligands to these cells.

Rac is involved in the interkinetic nuclear migration of cortical progenitor cells.

The small GTPase Rac regulates neuronal behavior, but whether it also functions in neural progenitor cells has not yet been explored. Here we report that Rac contributes to the regulation of nuclear migration in neocortical progenitor cells. Rac1 is expressed by progenitor cells in a unique spatiotemporal pattern. Cross-sectional immunohistochemical examination revealed intense Rac1 immunoreactivity at the ventricular surface. Similar staining patterns were obtained by immunofluorescence for a Rac-activator, Tiam1, and by reactions to detect the GTP-bound (active) form of Rac. En face inspection of the ventricular surface revealed that apical Rac1 localization was most frequent in M-phase cells, and the endfeet of cells in other cell cycle phases also showed apical Rac1 distribution at lower frequencies. To ask whether progenitor cell behavior prior to and during M phase is Rac-dependent, we monitored individual DiI-labeled progenitor cells live in the presence of a Rac inhibitor, NSC23766. We observed significantly retarded adventricular nuclear migration, as well as cytokinesis failures. Similar inhibitory effects were obtained by forced expression of a dominant-negative Rac1. These results suggest that Rac may play a role in interkinetic nuclear migration in the developing mouse brain.

Semaphorin 6A-Plexin A2/A4 Interactions with Radial Glia Regulate Migration Termination of Superficial Layer Cortical Neurons.

Precise regulation of neuronal migration termination is crucial for the establishment of brain cytoarchitectures. However, little is known about how neurons terminate migration. Here we focused on interactions between migrating cortical neurons and their substrates, radial glial (RG) cells, and analyzed the role of Plexin A2 and A4 (PlxnA2/A4) receptors and their repulsive ligand, Semaphorin 6A (Sema6A), for this process. In both PlxnA2/A4 double-knockout and Sema6A mutant mice, the outermost cortical plate neurons ectopically invade layer 1 at a stage when they should reach their destinations. PlxnA2/A4 proteins are abundantly expressed on their leading processes, whereas Sema6A mRNA is enriched in RG cell somata. Cell-targeted gene expression and conditional knockouts indicate critical roles for these molecules. We hypothesize that the timely appearance of repulsive signaling mediated by Sema6A-PlxnA2/A4 weakens migrating neuron-RG cell interactions, leading to migration termination.

Dorsal-to-Ventral Cortical Expansion Is Physically Primed by Ventral Streaming of Early Embryonic Preplate Neurons.

Despite recent studies elucidating the molecular mechanisms underlying cortical patterning and map formation, very little is known about how the embryonic pallium expands ventrally to form the future cortex and the nature of the underlying force-generating events. We find that neurons born at embryonic day 10 (E10) in the mouse dorsal pallium ventrally stream until E13, thereby superficially spreading the preplate, and then constitute the subplate from E14. From E11 to E12, the preplate neurons migrate, exerting pulling and pushing forces at the process and the soma, respectively. At E13, they are morphologically heterogeneous, with ∼40% possessing corticofugal axons, which are found to be in tension. Ablation of these E10-born neurons attenuates both deflection of radial glial fibers (by E13) and extension of the cortical plate (by E14), which should occur ventrally, and subsequently shrinks the postnatal neocortical map dorsally. Thus, the preplate stream physically primes neocortical expansion and arealization.

Cry46Ab from Bacillus thuringiensis TK-E6 is a new mosquitocidal toxin with aerolysin-type architecture.

Cry46Ab is a Cry toxin derived from Bacillus thuringiensis TK-E6. Cry46Ab is not significantly homologous to other mosquitocidal Cry or Cyt toxins and is classified as an aerolysin-type pore-forming toxin based on structural similarity. In this study, the potency of Cry46Ab was assessed for its potential application to mosquito control. A synthetic Cry46Ab gene, cry46Ab-S1, was designed to produce recombinant Cry46Ab as a glutathione-S-transferase fusion in Escherichia coli. Recombinant Cry46Ab showed apparent toxicity to Culex pipiens larvae, with a 50% lethal dose of 1.02 µg/ml. In an artificial lipid bilayer, Cry46Ab activated by trypsin caused typical current transitions between open and closed states, suggesting it functions as a pore-forming toxin similar to other Cry and Cyt toxins. The single-channel conductance was 103.3 ± 4.1 pS in 150 mM KCl. Co-administration of recombinant Cry46Ab with other mosquitocidal Cry toxins, especially the combination of Cry4Aa and Cry46Ab, resulted in significant synergistic toxicity against C. pipiens larvae. Co-administration of multiple toxins exhibiting different modes of action is believed to prevent the onset of resistance in insects. Our data, taken in consideration with the differences in its structure, suggest that Cry46Ab could be useful in not only reducing resistance levels but also improving the insecticidal activity of Bt-based bio-insecticides.

C3G/Rapgef1 Is Required in Multipolar Neurons for the Transition to a Bipolar Morphology during Cortical Development.

The establishment of a polarized morphology is essential for the development and function of neurons. During the development of the mammalian neocortex, neurons arise in the ventricular zone (VZ) from radial glia cells (RGCs) and leave the VZ to generate the cortical plate (CP). During their migration, newborn neurons first assume a multipolar morphology in the subventricular zone (SVZ) and lower intermediate zone (IZ). Subsequently, they undergo a multi-to-bipolar (MTB) transition to become bipolar in the upper IZ by developing a leading process and a trailing axon. The small GTPases Rap1A and Rap1B act as master regulators of neural cell polarity in the developing mouse neocortex. They are required for maintaining the polarity of RGCs and directing the MTB transition of multipolar neurons. Here we show that the Rap1 guanine nucleotide exchange factor (GEF) C3G (encoded by the Rapgef1 gene) is a crucial regulator of the MTB transition in vivo by conditionally inactivating the Rapgef1 gene in the developing mouse cortex at different time points during neuronal development. Inactivation of C3G results in defects in neuronal migration, axon formation and cortical lamination. Live cell imaging shows that C3G is required in cortical neurons for both the specification of an axon and the initiation of radial migration by forming a leading process.

Neuronal polarization in the developing cerebral cortex.

Cortical neurons consist of excitatory projection neurons and inhibitory GABAergic interneurons, whose connections construct highly organized neuronal circuits that control higher order information processing. Recent progress in live imaging has allowed us to examine how these neurons differentiate during development in vivo or in in vivo-like conditions. These analyses have revealed how the initial steps of polarization, in which neurons establish an axon, occur. Interestingly, both excitatory and inhibitory cortical neurons establish neuronal polarity de novo by undergoing a multipolar stage reminiscent of the manner in which polarity formation occurs in hippocampal neurons in dissociated culture. In this review, we focus on polarity formation in cortical neurons and describe their typical morphology and dynamic behavior during the polarization period. We also discuss cellular and molecular mechanisms underlying polarization, with reference to polarity formation in dissociated hippocampal neurons in vitro.

Pioneering axons regulate neuronal polarization in the developing cerebral cortex.

The polarization of neurons, which mainly includes the differentiation of axons and dendrites, is regulated by cell-autonomous and non-cell-autonomous factors. In the developing central nervous system, neuronal development occurs in a heterogeneous environment that also comprises extracellular matrices, radial glial cells, and neurons. Although many cell-autonomous factors that affect neuronal polarization have been identified, the microenvironmental cues involved in neuronal polarization remain largely unknown. Here, we show that neuronal polarization occurs in a microenvironment in the lower intermediate zone, where the cell adhesion molecule transient axonal glycoprotein-1 (TAG-1) is expressed in cortical efferent axons. The immature neurites of multipolar cells closely contact TAG-1-positive axons and generate axons. Inhibition of TAG-1-mediated cell-to-cell interaction or its downstream kinase Lyn impairs neuronal polarization. These results show that the TAG-1-mediated cell-to-cell interaction between the unpolarized multipolar cells and the pioneering axons regulates the polarization of multipolar cells partly through Lyn kinase and Rac1.

Microtubule dynamics in neuronal morphogenesis.

Microtubules (MTs) are essential for neuronal morphogenesis in the developing brain. The MT cytoskeleton provides physical support to shape the fine structure of neuronal processes. MT-based motors play important roles in nucleokinesis, process formation and retraction. Regulation of MT stability downstream of extracellular cues is proposed to be critical for axonogenesis. Axons and dendrites exhibit different patterns of MT organization, underlying the divergent functions of these processes. Centrosomal positioning has drawn the attention of researchers because it is a major clue to understanding neuronal MT organization. In this review, we focus on how recent advances in live imaging have revealed the dynamics of MT organization and centrosome positioning during neural development.

TAG-1-assisted progenitor elongation streamlines nuclear migration to optimize subapical crowding.

Neural progenitors exhibit cell cycle-dependent interkinetic nuclear migration (INM) along the apicobasal axis. Despite recent advances in understanding its underlying molecular mechanisms, the processes to which INM contributes mechanically and the regulation of INM by the apicobasally elongated morphology of progenitors remain unclear. We found that knockdown of the cell-surface molecule TAG-1 resulted in retraction of neocortical progenitors' basal processes. Highly shortened stem-like progenitors failed to undergo basalward INM and became overcrowded in the periventricular (subapical) space. Surprisingly, the overcrowded progenitors left the apical surface and migrated into basal neuronal territories. These observations, together with the results of in toto imaging and physical tests, suggest that progenitors may sense and respond to excessive mechanical stress. Although, unexpectedly, the heterotopic progenitors remained stem-like and continued to sequentially produce neurons until the late embryonic period, histogenesis was severely disrupted. Thus, INM is essential for preventing overcrowding of nuclei and their somata, thereby ensuring normal brain histogenesis.

Reelin-dependent ApoER2 downregulation uncouples newborn neurons from progenitor cells.

Reelin and its receptor machinery are well known to be required for the migration and positioning of neocortical projection neurons. More recently, reelin has been shown both necessary and sufficient to determine the rate of neocortical neurogenesis. The molecular links underlying its seemingly distinct proliferative and post-proliferative functions remain unknown. Here we reveal an enriched expression of functional reelin receptors, largely of Apolipoprotein E Receptor 2 (ApoER2), in radial glia basal processes and intermediate progenitor cells during mid/late cortical development. In vivo, ApoER2 overexpression inhibits neuronal migration. In contrast, precluding excessive levels of ApoER2 in reelin-deficient cortices, by either ApoER2 knock-down or the transgenic expression of reelin in neural progenitor cells, improves neuronal migration and positioning. Our study provides groundwork for the highly orchestrated clearance of neocortical neurons from their birth site, suggesting that a reelin-dependent ApoER2 downregulation mechanism uncouples newborn neurons from progenitor cells, thereby enabling neurons to migrate.

Local application of neurotrophins specifies axons through inositol 1,4,5-trisphosphate, calcium, and Ca2+/calmodulin-dependent protein kinases.

Neurons are highly polarized cells that have structurally distinct processes-the axons and dendrites-that differentiate from common immature neurites. In cultured hippocampal neurons, one of these immature neurites stochastically initiates rapid extension and becomes an axon, whereas the others become dendrites. Various extracellular and intracellular signals contribute to axon specification; however, the specific intracellular pathways whereby particular extracellular stimuli lead to axon specification remain to be delineated. Here, we found that the neurotrophins brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) were required for axon specification in an autocrine or a paracrine fashion. Using local application with a micropipette to selectively stimulate individual neurites, we found that stimulation of a selected neurite by BDNF or NT-3 induced neurite outgrowth and subsequent axon formation. NT-3 induced a rapid increase in calcium ions (Ca(2+)) in an inositol 1,4,5-trisphosphate (IP(3))-dependent fashion as well as local activation of the Ca(2+) effector Ca(2+)/calmodulin-dependent protein kinase kinase (CaMKK) in the growth cone. Inhibition of neurotrophin receptors or CaMKK attenuated NT-3-induced axon specification in cultured neurons and axon formation in cortical neurons in vivo. These results identify a role for IP(3)-Ca(2+)-CaMKK signaling in axon specification.

Migration, early axonogenesis, and Reelin-dependent layer-forming behavior of early/posterior-born Purkinje cells in the developing mouse lateral cerebellum.

BACKGROUND: Cerebellar corticogenesis begins with the assembly of Purkinje cells into the Purkinje plate (PP) by embryonic day 14.5 (E14.5) in mice. Although the dependence of PP formation on the secreted protein Reelin is well known and a prevailing model suggests that Purkinje cells migrate along the 'radial glial' fibers connecting the ventricular and pial surfaces, it is not clear how Purkinje cells behave in response to Reelin to initiate the PP. Furthermore, it is not known what nascent Purkinje cells look like in vivo. When and how Purkinje cells start axonogenesis must also be elucidated. RESULTS: We show that Purkinje cells generated on E10.5 in the posterior periventricular region of the lateral cerebellum migrate tangentially, after only transiently migrating radially, towards the anterior, exhibiting an elongated morphology consistent with axonogenesis at E12.5. After their somata reach the outer/dorsal region by E13.5, they change 'posture' by E14.5 through remodeling of non-axon (dendrite-like) processes and a switchback-like mode of somal movement towards a superficial Reelin-rich zone, while their axon-like fibers remain relatively deep, which demarcates the somata-packed portion as a plate. In reeler cerebella, the early born posterior lateral Purkinje cells are initially normal during migration with anteriorly extended axon-like fibers until E13.5, but then fail to form the PP due to lack of the posture-change step. CONCLUSIONS: Previously unknown behaviors are revealed for a subset of Purkinje cells born early in the posteior lateral cerebellum: tangential migration; early axonogenesis; and Reelin-dependent reorientation initiating PP formation. This study provides a solid basis for further elucidation of Reelin's function and the mechanisms underlying the cerebellar corticogenesis, and will contribute to the understanding of how polarization of individual cells drives overall brain morphogenesis.

Mechanism of polarized protrusion formation on neuronal precursors migrating in the developing chicken cerebellum.

Directed cell migration results from the polarization of the cellular motile apparatus by integration of extracellular signals, which are presented in a three-dimensional, spatiotemporal manner in living organisms. To investigate the mechanism underlying the highly polarized and directional nature of migration in vivo, we have developed an imaging system for observing rhombic lip cell migration in the developing chicken cerebellum. First, we show that Cdc42 is the central regulator of the overall polarity, morphology and protrusion formation in these cells. However, perturbation of canonical polarity effectors of Cdc42, e.g. the Par6-Par3-aPKC complex, does not disrupt the cell asymmetry, whereas it affects orientation of the tip of the leading process. In contrast to Cdc42, Rac is required for the generation of protrusions but not the overall polarity. Function interference of class IA phosphoinositide 3-kinase abrogates both directional extension and maintenance of the long leading process, whereas PTEN modulates the size of the protrusion. Actomyosin contractility is important for coordinated spreading of the tip of the leading process in situ. Finally, ErbB4 functions in the generation of protrusions on the rhombic lip cells. These results suggest that polarized protrusion formation on neuronal precursors may occur by a more divergent and complex mechanism than that seen in studies of other cell types growing on planar substrates.

Effect of pressure on electrophoretic mobility of polystyrene latex particles.

A capillary electrophoresis system that can apply arbitrary helium gas pressures at both inlet and outlet reservoirs was constructed. The system was used to investigate the effect of pressure on electrophoretic behavior of polystyrene latex particles. The electrophoretic mobility of latex particles was increased with the application of pressure (< 3.0 kgf/cm2). The shrinkage of particle diameter under pressurization was observed using a microscope, however, the magnitude of shrinkage was not enough to explain the increase in electrophoretic mobility. Therefore, the application of pressure might increase the electric charge of the latex particle. Since methanol inhibited the enhancement in the electrophoretic mobility of the latex particles, water might play an important role in increasing mobility.