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Serotonin (5-HT) acts as both a morphogenetic factor during early embryonic development and a neuromodulator of circuit plasticity in the mature brain. Dysregulation of serotonin signaling during critical periods is involved in developmental neurological disorders, such as schizophrenia and autism. In this study we focused on the consequences of defect reelin signaling for the development of the brainstem serotonergic raphe system. We observed that reelin signaling components are expressed by serotonergic neurons during the critical period of their lateral migration. Further, we found that reelin signaling is important for the normal migration of rostral, but not caudal hindbrain raphe nuclei and that reelin deficiency results in the malformation of the paramedian raphe nucleus and the lateral wings of the dorsal raphe nuclei. Additionally, we showed that serotonergic neurons projections to laminated brain structures were severely altered. With this study, we propose that the perturbation of canonical reelin signaling interferes with the orientation of tangentially, but not radially, migrating brainstem 5-HT neurons. Our results open the window for further studies on the interaction of reelin and serotonin and the pathogenesis of neurodevelopmental disorders. [ABSTRACT FROM AUTHOR]

Neuronal migration during embryonic development contributes to functional brain circuitry. Many neurons migrate in morphologically distinct stages that coincide with differentiation, requiring tight spatial regulation. It had been proposed that neurotransmitter-mediated activity could exert this control. Here, we demonstrate that intracellular calcium transients occur in cerebellar neurons of zebrafish embryos during migration. We show that depolarization increases and hyperpolarization reduces the speed of tegmental hindbrain neurons using optogenetic tools and advanced track analysis optimized for in vivo migration. Finally, we introduce a compound screening assay to identify acetylcholine (ACh), glutamate, and glycine as regulators of migration, which act regionally along the neurons’ route. We summarize our findings in a model describing how different neurotransmitters spatially interact to control neuronal migration. The high evolutionary conservation of the cerebellum and hindbrain makes it likely that polarization state-driven motility constitutes an important principle in building a functional brain. [ABSTRACT FROM AUTHOR]

In developing brain neuronal migration, dendrite outgrowth and dendritic spine outgrowth are controlled by Cdc42, a small GTPase of the Rho family, and its activators. Cdc42 function in promoting actin polymerization is crucial for glutamatergic synapse regulation. Here, we focus on GABAergic synapse-specific activator of Cdc42, collybistin (CB) and examine functional differences between its splice isoforms CB1 and CB2. We report that CB1 and CB2 differentially regulate GABAergic synapse formation in vitro along proximal-distal axis and adult-born neuron maturation in vivo. The functional specialization between CB1 and CB2 isoforms arises from their differential protein half-life, in turn regulated by ubiquitin conjugation of the unique CB1 C-terminus. We report that CB1 and CB2 negatively regulate Cdc42; however, Cdc42 activation is dependent on CB interaction with gephyrin. During hippocampal adult neurogenesis CB1 regulates neuronal migration, while CB2 is essential for dendrite outgrowth. Finally, using mice lacking Gabra2 subunit, we show that CB1 function is downstream of GABAARs, and we can rescue adult neurogenesis deficit observed in Gabra2 KO. Overall, our results uncover previously unexpected role for CB isoforms downstream of α2-containing GABAARs during neuron maturation in a Cdc42 dependent mechanism. [ABSTRACT FROM AUTHOR]

Actin is an important component of the cytoskeleton and its polymerization is delicately regulated by several kinases and phosphatases. Heterotrimeric protein phosphatase 2A (PP2A) is a potent phosphatase that is crucial for cell proliferation, apoptosis, tumorigenesis, signal transduction, cytoskeleton arrangement, and neurodegeneration. To facilitate these varied functions, different regulators determine the different targets of PP2A. Among these regulators of PP2A, the B subunits in particular may be involved in cytoskeleton arrangement. However, little is known about the role of PP2A in actin polymerization in vivo. Using sophisticated fly genetics, we demonstrated a novel function for the fly B subunit, twins, to promote actin polymerization in varied tissue types, suggesting a broad and conserved effect. Furthermore, our genetic data suggest that twins may act upstream of the actin-polymerized-proteins, Moesin and Myosin-light-chain, and downstream of Rho to promote actin polymerization. This work opens a new avenue for exploring the biological functions of a PP2A regulator, twins, in cytoskeleton regulation. [ABSTRACT FROM AUTHOR]

The basic helix-loop-helix (bHLH) transcription factors exert multiple functions in mammalian cerebral cortex development. The aryl hydrocarbon receptor (AhR), a member of the bHLH-Per-Arnt-Sim subfamily, is a ligand-activated transcription factor reported to regulate nervous system development in both invertebrates and vertebrates, but the functions that AhR signaling pathway may have for mammalian cerebral cortex development remains elusive. Although the endogenous ligand involved in brain developmental process has not been identified, the environmental pollutant dioxin potently binds AhR and induces abnormalities in higher brain function of laboratory animals. Thus, we studied how activation of AhR signaling influences cortical development in mice. To this end, we produced mice expressing either constitutively active-AhR (CA-AhR), which has the capacity for ligand-independent activation of downstream genes, or AhR, which requires its ligands for activation. In brief, CA-AhR-expressing plasmid and AhR-expressing plasmid were each transfected into neural stems cells in the developing cerebrum by in utero electroporation on embryonic day 14.5. On postnatal day 14, mice transfected in utero with CA-AhR, but not those transfected with AhR, exhibited drastically reduced dendritic arborization of layer II/III pyramidal neurons and impaired neuronal positioning in the developing somatosensory cortex. The effects of CA-AhR were observed for dendrite development but not for the commissural fiber projection, suggesting a preferential influence on dendrites. The present results indicate that over-activation of AhR perturbs neuronal migration and morphological development in mammalian cortex, supporting previous observations of impaired dendritic structure, cortical dysgenesis, and behavioral abnormalities following perinatal dioxin exposure. [ABSTRACT FROM AUTHOR]

Long distance migratory birds find their way by sensing and integrating information from a large number of cues in their environment. These cues are essential to navigate over thousands of kilometers and reach the same breeding, stopover, and wintering sites every year. The semipalmated sandpiper (Calidris pusilla) is a long-distance migrant that breeds in the arctic tundra of Canada and Alaska and winters on the northeast coast of South America. Its fall migration includes a 5,300-kilometer nonstop flight over the Atlantic Ocean. The avian hippocampus has been proposed to play a central role in the integration of multisensory spatial information for navigation. Hippocampal neurogenesis may contribute to hippocampal function and a variety of factors including cognitive activity, exercise, enrichment, diet and stress influence neurogenesis in the hippocampus. We quantified hippocampal neurogenesis and volume in adult migrating and wintering semipalmated sandpipers using stereological counts of doublecortin (DCX) immunolabeled immature neurons. We found that birds captured in the coastal region of Bragança, Brazil during the wintering period had more DCX positive neurons and larger volume in the hippocampus than individuals captured in the Bay of Fundy, Canada during fall migration. We also estimate the number of NeuN immunolabeled cells in migrating and wintering birds and found no significant differences between them. These findings suggest that, at this time window, neurogenesis just replaced neurons that might be lost during the transatlantic flight. Our findings also show that in active fall migrating birds, a lower level of adult hippocampal neurogenesis is associated with a smaller hippocampal formation. High levels of adult hippocampal neurogenesis and a larger hippocampal formation found in wintering birds may be late occurring effects of long distance migratory flight or the result of conditions the birds experienced while wintering. [ABSTRACT FROM AUTHOR]

In the developing neocortex, cells in the ventricular/subventricular zone are largely multipotent neural stem cells and neural progenitor cells. These cells undergo self-renewal at the early stage of embryonic development to amplify the progenitor pool and subsequently differentiate into neurons. It is thus of considerable interest to investigate mechanisms controlling the switch from neural stem cells or neural progenitor cells to neurons. Here, we present evidence that Kif2a, a member of the Kinesin-13 family, plays a role in regulating the proliferation and differentiation of neural stem cells or neural progenitor cells at embryonic day 13.5. Silencing Kif2a by use of in utero electroporation of Kif2a shRNA reduced neural stem cells proliferation or self-renewal but increased neuronal differentiation. We further found that knockdown of Kif2a decreased the protein level of β-catenin, which is a critical molecule for neocortical neurogenesis. Together, these results reveal an important function of Kif2a in embryonic neocortical neurogenesis. [ABSTRACT FROM AUTHOR]

Mid-hindbrain malformations can occur during embryogenesis through a disturbance of transient and localized gene expression patterns within these distinct brain structures. Rho guanine nucleotide exchange factor (ARHGEF) family members are key for controlling the spatiotemporal activation of Rho GTPase, to modulate cytoskeleton dynamics, cell division, and cell migration. We identified, by means of whole exome sequencing, a homozygous frameshift mutation in the ARHGEF2 as a cause of intellectual disability, a midbrain-hindbrain malformation, and mild microcephaly in a consanguineous pedigree of Kurdish-Turkish descent. We show that loss of ARHGEF2 perturbs progenitor cell differentiation and that this is associated with a shift of mitotic spindle plane orientation, putatively favoring more symmetric divisions. The ARHGEF2 mutation leads to reduction in the activation of the RhoA/ROCK/MLC pathway crucial for cell migration. We demonstrate that the human brain malformation is recapitulated in Arhgef2 mutant mice and identify an aberrant migration of distinct components of the precerebellar system as a pathomechanism underlying the midbrain-hindbrain phenotype. Our results highlight the crucial function of ARHGEF2 in human brain development and identify a mutation in ARHGEF2 as novel cause of a neurodevelopmental disorder. [ABSTRACT FROM AUTHOR]

BIG1, an activator protein of the small GTPase, Arf, and encoded by the Arfgef1 gene, is one of candidate genes for epileptic encephalopathy. To know the involvement of BIG1 in epileptic encephalopathy, we analyzed BIG1-deficient mice and found that BIG1 regulates neurite outgrowth and brain development in vitro and in vivo. The loss of BIG1 decreased the size of the neocortex and hippocampus. In BIG1-deficient mice, the neuronal progenitor cells (NPCs) and the interneurons were unaffected. However, Tbr1+ and Ctip2+ deep layer (DL) neurons showed spatial-temporal dependent apoptosis. This apoptosis gradually progressed from the piriform cortex (PIR), peaked in the neocortex, and then progressed into the hippocampus from embryonic day 13.5 (E13.5) to E17.5. The upper layer (UL) and DL order in the neocortex was maintained in BIG1-deficient mice, but the excitatory neurons tended to accumulate before their destination layers. Further pulse-chase migration assay showed that the migration defect was non-cell autonomous and secondary to the progression of apoptosis into the BIG1-deficient neocortex after E15.5. In BIG1-deficient mice, we observed an ectopic projection of corticothalamic axons from the primary somatosensory cortex (S1) into the dorsal lateral geniculate nucleus (dLGN). The thalamocortical axons were unable to cross the diencephalon–telencephalon boundary (DTB). In vitro, BIG1-deficient neurons showed a delay in neuronal polarization. BIG1-deficient neurons were also hypersensitive to low dose glutamate (5 μM), and died via apoptosis. This study showed the role of BIG1 in the survival of DL neurons in developing embryonic brain and in the generation of neuronal polarity. [ABSTRACT FROM AUTHOR]

The regulation of cell migration is essential to animal development and physiology. Heparan sulfate proteoglycans shape the interactions of morphogens and guidance cues with their respective receptors to elicit appropriate cellular responses. Heparan sulfate proteoglycans consist of a protein core with attached heparan sulfate glycosaminoglycan chains, which are synthesized by glycosyltransferases of the exostosin (EXT) family. Abnormal HS chain synthesis results in pleiotropic consequences, including abnormal development and tumor formation. In humans, mutations in either of the exostosin genes EXT1 and EXT2 lead to osteosarcomas or multiple exostoses. Complete loss of any of the exostosin glycosyltransferases in mouse, fish, flies and worms leads to drastic morphogenetic defects and embryonic lethality. Here we identify and study previously unavailable viable hypomorphic mutations in the two C. elegans exostosin glycosyltransferases genes, rib-1 and rib-2. These partial loss-of-function mutations lead to a severe reduction of HS levels and result in profound but specific developmental defects, including abnormal cell and axonal migrations. We find that the expression pattern of the HS copolymerase is dynamic during embryonic and larval morphogenesis, and is sustained throughout life in specific cell types, consistent with HSPGs playing both developmental and post-developmental roles. Cell-type specific expression of the HS copolymerase shows that HS elongation is required in both the migrating neuron and neighboring cells to coordinate migration guidance. Our findings provide insights into general principles underlying HSPG function in development. [ABSTRACT FROM AUTHOR]

The endoplasmic reticulum (ER), including the nuclear envelope, is a continuous and intricate membrane-bound organelle responsible for various cellular functions. In neurons, the ER network is found in cell bodies, axons, and dendrites. Recent studies indicate the involvement of the ER network in neuronal development, such as neuronal migration and axonal outgrowth. However, the regulation of neural development by ER-localized proteins is not fully understood. We previously reported that the multi-transmembrane protein Dpy19L1 is required for neuronal migration in the developing mouse cerebral cortex. A Dpy19L family member, Dpy19L2, which is a causative gene for human Globozoospermia, is suggested to act as an anchor of the acrosome to the nuclear envelope. In this study, we found that the patterns of exogenous Dpy19L1 were partially coincident with the ER, including the nuclear envelope in COS-7 cells at the level of the light microscope. The reticular distribution of Dpy19L1 was disrupted by microtubule depolymerization that induces retraction of the ER. Furthermore, Dpy19L1 showed a similar distribution pattern with a ER marker protein in embryonic mouse cortical neurons. Finally, we showed that Dpy19L1 knockdown mediated by siRNA resulted in decreased neurite outgrowth in cultured neurons. These results indicate that transmembrane protein Dpy19L1 is localized to the ER membrane and regulates neurite extension during development. [ABSTRACT FROM AUTHOR]

Collective migration depends on cell-cell interactions between neighbors that contribute to their overall directionality, yet the mechanisms that control the coordinated migration of neurons remains to be elucidated. During hindbrain development, facial branchiomotor neurons (FBMNs) undergo a stereotypic tangential caudal migration from their place of birth in rhombomere (r)4 to their final location in r6/7. FBMNs engage in collective cell migration that depends on neuron-to-neuron interactions to facilitate caudal directionality. Here, we demonstrate that Cadherin-2-mediated neuron-to-neuron adhesion is necessary for directional and collective migration of FBMNs. We generated stable transgenic zebrafish expressing dominant-negative Cadherin-2 (Cdh2ΔEC) driven by the islet1 promoter. Cell-autonomous inactivation of Cadherin-2 function led to non-directional migration of FBMNs and a defect in caudal tangential migration. Additionally, mosaic analysis revealed that Cdh2ΔEC-expressing FBMNs are not influenced to migrate caudally by neighboring wild-type FBMNs due to a defect in collective cell migration. Taken together, our data suggest that Cadherin-2 plays an essential cell-autonomous role in mediating the collective migration of FBMNs. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The planar cell polarity (PCP) pathway is a cell-contact mediated mechanism for transmitting polarity information between neighboring cells. PCP “core components” (Vangl, Fz, Pk, Dsh, and Celsr) are essential for a number of cell migratory events including the posterior migration of facial branchiomotor neurons (FBMNs) in the plane of the hindbrain neuroepithelium in zebrafish and mice. While the mechanism by which PCP signaling polarizes static epithelial cells is well understood, how PCP signaling controls highly dynamic processes like neuronal migration remains an important outstanding question given that PCP components have been implicated in a range of directed cell movements, particularly during vertebrate development. Here, by systematically disrupting PCP signaling in a rhombomere-restricted manner we show that PCP signaling is required both within FBMNs and the hindbrain rhombomere 4 environment at the time when they initiate their migration. Correspondingly, we demonstrate planar polarized localization of PCP core components Vangl2 and Fzd3a in the hindbrain neuroepithelium, and transient localization of Vangl2 at the tips of retracting FBMN filopodia. Using high-resolution timelapse imaging of FBMNs in genetic chimeras we uncover opposing cell-autonomous and non-cell-autonomous functions for Fzd3a and Vangl2 in regulating FBMN protrusive activity. Within FBMNs, Fzd3a is required to stabilize filopodia while Vangl2 has an antagonistic, destabilizing role. However, in the migratory environment Fzd3a acts to destabilize FBMN filopodia while Vangl2 has a stabilizing role. Together, our findings suggest a model in which PCP signaling between the planar polarized neuroepithelial environment and FBMNs directs migration by the selective stabilization of FBMN filopodia. [ABSTRACT FROM AUTHOR]

Directed neuroblast and neuronal migration is important in the proper development of nervous systems. In C. elegans the bilateral Q neuroblasts QR (on the right) and QL (on the left) undergo an identical pattern of cell division and differentiation but migrate in opposite directions (QR and descendants anteriorly and QL and descendants posteriorly). EGL-20/Wnt, via canonical Wnt signaling, drives the expression of MAB-5/Hox in QL but not QR. MAB-5 acts as a determinant of posterior migration, and mab-5 and egl-20 mutants display anterior QL descendant migrations. Here we analyze the behaviors of QR and QL descendants as they begin their anterior and posterior migrations, and the effects of EGL-20 and MAB-5 on these behaviors. The anterior and posterior daughters of QR (QR.a/p) after the first division immediately polarize and begin anterior migration, whereas QL.a/p remain rounded and non-migratory. After ~1 hour, QL.a migrates posteriorly over QL.p. We find that in egl-20/Wnt, bar-1/β-catenin, and mab-5/Hox mutants, QL.a/p polarize and migrate anteriorly, indicating that these molecules normally inhibit anterior migration of QL.a/p. In egl-20/Wnt mutants, QL.a/p immediately polarize and begin migration, whereas in bar-1/β-catenin and mab-5/Hox, the cells transiently retain a rounded, non-migratory morphology before anterior migration. Thus, EGL-20/Wnt mediates an acute inhibition of anterior migration independently of BAR-1/β-catenin and MAB-5/Hox, and a later, possible transcriptional response mediated by BAR-1/β-catenin and MAB-5/Hox. In addition to inhibiting anterior migration, MAB-5/Hox also cell-autonomously promotes posterior migration of QL.a (and QR.a in a mab-5 gain-of-function). [ABSTRACT FROM AUTHOR]

The zebrafish posterior lateral line is formed during early development by the deposition of neuromasts from a migrating primordium. The molecular mechanisms regulating the regional organization and migration of the primordium involve interactions between Fgf and Wnt/-catenin signaling and the establishment of specific cxcr4b and cxcr7b cytokine receptor expression domains. Itch has been identified as a regulator in several different signaling pathways, including Wnt and Cxcr4 signaling. We identified two homologous itch genes in zebrafish, itcha and itchb, with generalized expression patterns. By reducing itchb expression in particular upon morpholino knockdown, we demonstrated the importance of Itch in regulating lateral line development by perturbing the patterns of cxcr4b and cxcr7b expression. Itch knockdown results in a failure to down-regulate Wnt signaling and overexpression of cxcr4b in the primordium, slowing migration of the posterior lateral line primordium and resulting in abnormal development of the lateral line. [ABSTRACT FROM AUTHOR]

Apolipoprotein E Receptor 2 (ApoER2) and the tyrosine kinase Fyn are both members of the Reelin pathway, a signaling pathway essential for the laminar formation of the cortex during development and proper dendritic spine density and long-term potential (LTP) in the adult brain. In the presence of extracellular Reelin, ApoER2 binds the intracellular protein Dab1, an adaptor protein that is phosphorylated by Fyn. However, direct interactions between ApoER2 and Fyn are not well defined. Here, we show that total levels of ApoER2 and surface levels of ApoER2 are increased by active Fyn. Via a separate mechanism, ApoER2 is also phosphorylated by Fyn, an event that peaks in the postnatal cortex at day 5 and can occur at multiple ApoER2 tyrosine residues. Dab1 is also involved in this phosphorylation, promoting the phosphorylation of ApoER2 by Fyn when it is itself phosphorylated. These results elucidate some of the intracellular mechanisms that give rise to a functional Reelin pathway. [ABSTRACT FROM AUTHOR]