Analyses of Neurite and Spine Formation by In Utero Electroporation in Mice.

Dendrite morphology and dendritic spines are key features of the neuronal networks in the brain. Abnormalities in these features have been observed in patients with psychiatric disorders and mouse models of these diseases. In utero electroporation is an easy and efficient gene transfer system for developing mouse embryos in the uterus. By combining with the Cre-loxP system, the morphology of individual neurons can be clearly and sparsely visualized. Here, we describe how this labeling system can be applied to visualize and evaluate the dendrites and dendritic spines of cortical neurons.

Live Imaging of Migrating Neurons and Glial Progenitors Visualized by in Utero Electroporation.

During cortical development, both neurons and glial cells are generated in the germinal zone near the lateral ventricle, migrate in the correct direction, and settle in their appropriate locations. This developmental process can be clearly visualized by introducing fluorescent protein-expression vectors via in utero electroporation. In this chapter, we describe labeling methods for migrating neurons and glial progenitors, as well as methods for slice culture, and time-lapse imaging.

Time-Lapse Imaging of Migrating Neurons and Glial Progenitors in Embryonic Mouse Brain Slices.

During the development of the cerebral cortex, neurons and glial cells originate in the ventricular zone lining the ventricle and migrate toward the brain surface. This process is crucial for proper brain function, and its dysregulation can result in neurodevelopmental and psychiatric disorders after birth. In fact, many genes responsible for these diseases have been found to be involved in this process, and therefore, revealing how these mutations affect cellular dynamics is important for understanding the pathogenesis of these diseases. This protocol introduces a technique for time-lapse imaging of migrating neurons and glial progenitors in brain slices obtained from mouse embryos. Cells are labeled with fluorescent proteins using in utero electroporation, which visualizes individual cells migrating from the ventricular zone with a high signal-to-noise ratio. Moreover, this in vivo gene transfer system enables us to easily perform gain-of-function or loss-of-function experiments on the given genes by co-electroporation of their expression or knockdown/knockout vectors. Using this protocol, the migratory behavior and migration speed of individual cells, information that is never obtained from fixed brains, can be analyzed.

Interactions between neural cells and blood vessels in central nervous system development.

The sophisticated function of the central nervous system (CNS) is largely supported by proper interactions between neural cells and blood vessels. Accumulating evidence has demonstrated that neurons and glial cells support the formation of blood vessels, which in turn, act as migratory scaffolds for these cell types. Neural progenitors are also involved in the regulation of blood vessel formation. This mutual interaction between neural cells and blood vessels is elegantly controlled by several chemokines, growth factors, extracellular matrix, and adhesion molecules such as integrins. Recent research has revealed that newly migrating cell types along blood vessels repel other preexisting migrating cell types, causing them to detach from the blood vessels. In this review, we discuss vascular formation and cell migration, particularly during development. Moreover, we discuss how the crosstalk between blood vessels and neurons and glial cells could be related to neurodevelopmental disorders.

Expression analyses of C-terminal-binding protein 1 (CtBP1) during mouse brain development.

INTRODUCTION: CtBP1 (C-terminal-binding protein 1) is a multi-functional protein with well-established roles as a transcriptional co-repressor in the nucleus and a regulator of membrane fission in the cytoplasm. Although CtBP1 gene abnormalities have been reported to cause neurodevelopmental disorders, the physiological role and expression profile of CtBP1 remains to be elucidated. METHODS: In this study, we used biochemical, immunohistochemical and immunofluorescence methods to analyze the expression of CtBP1 during mouse brain development. RESULTS: Western blotting analyses revealed that CtBP1 appeared to be expressed mainly in the central nervous system throughout the developmental process. In immunohistochemical analyses, region-specific nuclear as well as weak cytoplasmic distribution of CtBP1 was observed in telencephalon at embryonic day (E)15 and E17. It is of note that CtBP1 was barely detected in axons, but observed in the nucleus of oligodendrocytes in the white matter at E17. As to cerebellum at postnatal day 30, CtBP1 appeared to be expressed in the nucleus and cytoplasm of Purkinje cells, the nucleus of granule cells and cells in the molecular layer (ML), and the ML per se where granule cell axons and Purkinje cell dendrites are enriched. In addition, CtBP1 was detected in the cerebellar nuclei. CONCLUSION: The obtained results suggest involvement of CtBP1 in brain function.

Histological Analysis of a Mouse Model of the 22q11.2 Microdeletion Syndrome.

22q11.2 deletion syndrome (22q11.2DS) is associated with a high risk of developing various psychiatric and developmental disorders, including schizophrenia and early-onset Parkinson's disease. Recently, a mouse model of this disease, Del(3.0Mb)/+, mimicking the 3.0 Mb deletion which is most frequently found in patients with 22q11.2DS, was generated. The behavior of this mouse model was extensively studied and several abnormalities related to the symptoms of 22q11.2DS were found. However, the histological features of their brains have been little addressed. Here we describe the cytoarchitectures of the brains of Del(3.0Mb)/+ mice. First, we investigated the overall histology of the embryonic and adult cerebral cortices, but they were indistinguishable from the wild type. However, the morphologies of individual neurons were slightly but significantly changed from the wild type counterparts in a region-specific manner. The dendritic branches and/or dendritic spine densities of neurons in the medial prefrontal cortex, nucleus accumbens, and primary somatosensory cortex were reduced. We also observed reduced axon innervation of dopaminergic neurons into the prefrontal cortex. Given these affected neurons function together as the dopamine system to control animal behaviors, the impairment we observed may explain a part of the abnormal behaviors of Del(3.0Mb)/+ mice and the psychiatric symptoms of 22q11.2DS.

Expression Analyses of Rich2/Arhgap44, a Rho Family GTPase-Activating Protein, during Mouse Brain Development.

Rho family small GTPases, such as Rho, Rac, and Cdc42, play essential roles during brain development, by regulating cellular signaling and actin cytoskeletal reorganization. Rich2/Arhgap44, a Rac- and Cdc42-specific GTPase-activating protein, has been reported to be a key regulator for dendritic spine morphology and synaptic function. Given the essential roles of Rac and Cdc42 in brain development, Rich2 is supposed to take part in brain development. However, not only the molecular mechanism involved but also the expression profile of Rich2 during neurodevelopment has not yet been elucidated. In this study, we carried out expression analyses of Rich2 by focusing on mouse brain development. In immunoblotting, Rich2 exhibited a tissue-dependent expression profile in the young adult mouse, and the expression was increased during brain development. In immunohistochemical analyses, Rich2 was observed in the cytoplasm of cortical neurons at postnatal day (P) 0 and then came to be enriched in the nucleus with moderate distribution in neuropils at P7. Later at P30, a complex immunostaining pattern of Rich2 was observed; Rich2 was distributed in the nucleus, cytoplasm, and neuropils in many cortical neurons, whereas other neurons frequently displayed little expression. In the hippocampus at P7, Rich2 was distributed mainly in the cytoplasm of excitatory neurons in the cornu ammonis regions, while it was moderately detected in the nucleus in the dentate granule cells. Notably, Rich2 was distributed in excitatory synapses of the cornu ammonis 1 region at P30. Biochemical fractionation analyses also detected Rich2 in the postsynaptic density. Taken together, Rich2 is found to be expressed in the central nervous system in a developmental stage-dependent manner and may be involved in synapse formation/maintenance in cortical neurons.

Erratic and blood vessel-guided migration of astrocyte progenitors in the cerebral cortex.

Astrocytes are one of the most abundant cell types in the mammalian brain. They play essential roles in synapse formation, maturation, and elimination. However, how astrocytes migrate into the gray matter to accomplish these processes is poorly understood. Here, we show that, by combinational analyses of in vitro and in vivo time-lapse observations and lineage traces, astrocyte progenitors move rapidly and irregularly within the developing cortex, which we call erratic migration. Astrocyte progenitors also adopt blood vessel-guided migration. These highly motile progenitors are generated in the restricted prenatal stages and differentiate into protoplasmic astrocytes in the gray matter, whereas postnatally generated progenitors do not move extensively and differentiate into fibrous astrocytes in the white matter. We found Cxcr4/7, and integrin ß1 regulate the blood vessel-guided migration, and their functional blocking disrupts their positioning. This study provides insight into astrocyte development and may contribute to understanding the pathogenesis caused by their defects.

Crosstalk between Blood Vessels and Glia during the Central Nervous System Development.

The formation of proper blood vessel patterns in the central nervous system (CNS) is crucial to deliver oxygen and nutrient to neurons efficiently. At the same time, neurons must be isolated from the outer blood circulation by a specialized structure, the blood-brain barrier (BBB), to maintain the microenvironment of brain parenchyma for the survival of neurons and proper synaptic transmission. To develop this highly organized structure, glial cells, a major component of the brain, have been reported to play essential roles. In this review, the crosstalk between the macroglia, including astrocytes and oligodendrocytes, and endothelial cells during the development of CNS will be discussed. First, the known roles of astrocytes in neuro-vascular unit and its development, and then, the requirements of astrocytes for BBB development and maintenance are shown. Then, various genetic and cellular studies revealing the roles of astrocytes in the growth of blood vessels by providing a scaffold, including laminins and fibronectin, as well as by secreting trophic factors, including vascular endothelial growth factor (VEGF) and transforming growth factor-ß (TGF-ß) are introduced. Finally, the interactions between oligodendrocyte progenitors and blood vessels are overviewed. Although these studies revealed the necessity for proper communication between glia and endothelial cells for CNS development, our knowledge about the detailed cellular and molecular mechanisms for them is still limited. The questions to be clarified in the future are also discussed.

Variant-specific changes in RAC3 function disrupt corticogenesis in neurodevelopmental phenotypes.

Variants in RAC3, encoding a small GTPase RAC3 which is critical for the regulation of actin cytoskeleton and intracellular signal transduction, are associated with a rare neurodevelopmental disorder with structural brain anomalies and facial dysmorphism. We investigated a cohort of 10 unrelated participants presenting with global psychomotor delay, hypotonia, behavioural disturbances, stereotyped movements, dysmorphic features, seizures and musculoskeletal abnormalities. MRI of brain revealed a complex pattern of variable brain malformations, including callosal abnormalities, white matter thinning, grey matter heterotopia, polymicrogyria/dysgyria, brainstem anomalies and cerebellar dysplasia. These patients harboured eight distinct de novo RAC3 variants, including six novel variants (NM_005052.3): c.34G > C p.G12R, c.179G > A p.G60D, c.186_188delGGA p.E62del, c.187G > A p.D63N, c.191A > G p.Y64C and c.348G > C p.K116N. We then examined the pathophysiological significance of these novel and previously reported pathogenic variants p.P29L, p.P34R, p.A59G, p.Q61L and p.E62K. In vitro analyses revealed that all tested RAC3 variants were biochemically and biologically active to variable extent, and exhibited a spectrum of different affinities to downstream effectors including p21-activated kinase 1. We then focused on the four variants p.Q61L, p.E62del, p.D63N and p.Y64C in the Switch II region, which is essential for the biochemical activity of small GTPases and also a variation hot spot common to other Rho family genes, RAC1 and CDC42. Acute expression of the four variants in embryonic mouse brain using in utero electroporation caused defects in cortical neuron morphology and migration ending up with cluster formation during corticogenesis. Notably, defective migration by p.E62del, p.D63N and p.Y64C were rescued by a dominant negative version of p21-activated kinase 1. Our results indicate that RAC3 variants result in morphological and functional defects in cortical neurons during brain development through variant-specific mechanisms, eventually leading to heterogeneous neurodevelopmental phenotypes.

Impaired Function of PLEKHG2, a Rho-Guanine Nucleotide-Exchange Factor, Disrupts Corticogenesis in Neurodevelopmental Phenotypes.

Homozygosity of the p.Arg204Trp variation in the Pleckstrin homology and RhoGEF domain containing G2 (PLEKHG2) gene, which encodes a Rho family-specific guanine nucleotide-exchange factor, is responsible for microcephaly with intellectual disability. However, the role of PLEKHG2 during neurodevelopment remains unknown. In this study, we analyzed mouse Plekhg2 function during cortical development, both in vitro and in vivo. The p.Arg200Trp variant in mouse (Plekhg2-RW), which corresponds to the p.Arg204Trp variant in humans, showed decreased guanine nucleotide-exchange activity for Rac1, Rac3, and Cdc42. Acute knockdown of Plekhg2 using in utero electroporation-mediated gene transfer did not affect the migration of excitatory neurons during corticogenesis. On the other hand, silencing Plekhg2 expression delayed dendritic arbor formation at postnatal day 7 (P7), perhaps because of impaired Rac/Cdc42 and p21-activated kinase 1 signaling pathways. This phenotype was rescued by expressing an RNAi-resistant version of wildtype Plekhg2, but not of Plekhg2-RW. Axon pathfinding was also impaired in vitro and in vivo in Plekhg2-deficient cortical neurons. At P14, knockdown of Plekhg2 was observed to cause defects in dendritic spine morphology formation. Collectively, these results strongly suggest that PLEKHG2 has essential roles in the maturation of axon, dendrites, and spines. Moreover, impairment of PLEKHG2 function is most likely to cause defects in neuronal functions that lead to neurodevelopmental disorders.

Expression Analyses of Rac3, a Rho Family Small GTPase, during Mouse Brain Development.

Rac3 is a member of Rho family small GTPases which regulate cellular signaling and cytoskeletal dynamics. The RAC3 gene abnormalities have been shown to cause neurodevelopmental disorders with structural brain anomalies, including polymicrogyria/dysgyria, callosal abnormalities, brainstem anomalies, and cerebellar dysplasia. Although this evidence indicates that Rac3 is essential in brain development, not only its molecular mechanism but also the expression profile is yet to be elucidated. In this study, we carried out expression analyses of Rac3 with mouse brain tissues. In immunoblotting, Rac3 exhibited a tissue-dependent expression profile in the young adult mouse and was expressed in a developmental stage-dependent manner in brain. In primary cultured hippocampal neurons, while Rac3 was distributed mainly in the cytoplasm, it was visualized in axon and dendrites with partial localization at synapses, in consistent with the observation in biochemical fractionation analyses. In immunofluorescence analyses with brain slices, Rac3 was distributed strongly and moderately in the axon and cytoplasm, respectively, of cerebral cortex at postnatal day (P) 2 and P18. Similar distribution profile was also observed in hippocampus. Taken together, the results obtained strongly suggest that Rac3 plays an important physiological role in neuronal tissues during corticogenesis, and defects in the Rac3 function induce structural brain anomalies leading to pathogenesis of neurodevelopmental disorders.

Expression analyses of PLEKHG2, a Rho family-specific guanine nucleotide exchange factor, during mouse brain development.

Abnormalities of PLEKHG2 gene, encoding a Rho family-specific guanine nucleotide exchange factor, are involved in microcephaly with intellectual disability. However, not only the role of PLEKHG2 in the developmental process but also its expression profile is unknown. In this study, we prepared a specific antibody against PLEKHG2 and carried out expression analyses with mouse tissues. In western blotting, PLEKHG2 exhibited a tissue-dependent expression profile in adult mouse and was expressed in a developmental stage-dependent manner in brain. Then, in immunohistochemical analyses, while PLEKHG2 was observed in the cortical plate and ventricular zone surface of the cerebral cortex at embryonic day 14, it came to be distributed throughout the cerebral cortex in layer II/III and V during corticogenesis. PLEKHG2 was also detected mainly in the nucleus of neurons in the hippocampal CA regions and dentate gyrus at P7. Notably, the nuclear accumulation disappeared at P30 and PLEKHG2 came to be located at the axons and/or dendrites at this time point. Moreover, in vitro immunofluorescence revealed that PLEKHG2 was at least partially localized at both excitatory and inhibitory synapses in primary cultured hippocampal neurons. These results suggest roles of PLEKHG2 in the development of the central nervous tissue and synaptic function.

Knockdown of Son, a mouse homologue of the ZTTK syndrome gene, causes neuronal migration defects and dendritic spine abnormalities.

Zhu-Tokita-Takenouchi-Kim (ZTTK) syndrome, a rare congenital anomaly syndrome characterized by intellectual disability, brain malformation, facial dysmorphism, musculoskeletal abnormalities, and some visceral malformations is caused by de novo heterozygous mutations of the SON gene. The nuclear protein SON is involved in gene transcription and RNA splicing; however, the roles of SON in neural development remain undetermined. We investigated the effects of Son knockdown on neural development in mice and found that Son knockdown in neural progenitors resulted in defective migration during corticogenesis and reduced spine density on mature cortical neurons. The induction of human wild-type SON expression rescued these neural abnormalities, confirming that the abnormalities were caused by SON insufficiency. We also applied truncated SON proteins encoded by disease-associated mutant SON genes for rescue experiments and found that a truncated SON protein encoded by the most prevalent SON mutant found in ZTTK syndrome rescued the neural abnormalities while another much shorter mutant SON protein did not. These data indicate that SON insufficiency causes neuronal migration defects and dendritic spine abnormalities, which seem neuropathological bases of the neural symptoms of ZTTK syndrome. In addition, the results support that the neural abnormalities in ZTTK syndrome are caused by SON haploinsufficiency independent of the types of mutation that results in functional or dysfunctional proteins.

Spatiotemporally Dependent Vascularization Is Differently Utilized among Neural Progenitor Subtypes during Neocortical Development.

To facilitate efficient oxygen and nutrient delivery, blood vessels in the brain form three-dimensional patterns. However, little is known about how blood vessels develop stereographically in the neocortex and how they control the expansion and differentiation of neural progenitors during neocortical development. We show that highly vascularized and avascular regions are strictly controlled in a spatially and temporally restricted manner and are associated with distinct cell populations. Dividing basal progenitors and oligodendrocyte precursors preferentially contact honeycomb vessels, but dividing apical progenitors are localized in avascular regions without Flt1-positive endothelial cells but directly contact with sprouting neovascular tip cells. Therefore, not all blood vessels are associated equally with neural progenitors. Furthermore, a disruption of normal vascular patterning can induce abnormalities in neural development, whereas the impaired features of neural progenitors influenced angiogenesis patterning. These results indicate that close association between the nervous and vascular systems is essential for neocortex assembly.

Expression Analyses of POGZ, A Responsible Gene for Neurodevelopmental Disorders, during Mouse Brain Development.

POGZ is a heterochromatin protein 1 α-binding protein and regulates gene expression. On the other hand, accumulating pieces of evidence indicate that the POGZ gene abnormalities are involved in various neurodevelopmental disorders. In this study, we prepared a specific antibody against POGZ, anti-POGZ, and carried out biochemical and morphological characterization with mouse brain tissues. Western blotting analyses revealed that POGZ is expressed strongly at embryonic day 13 and then gradually decreased throughout the brain development process. In immunohistochemical analyses, POGZ was found to be enriched in cerebrocortical and hippocampal neurons in the early developmental stage. The nuclear expression was also detected in Purkinje cells in cerebellum at postnatal day (P)7 and P15 but disappeared at P30. In primary cultured hippocampal neurons, while POGZ was distributed mainly in the nucleus, it was also visualized in axon and dendrites with partial localization at synapses in consistency with the results obtained in biochemical fractionation analyses. The obtained results suggest that POGZ takes part in the regulation of synaptic function as well as gene expression during brain development.

Role of Per3, a circadian clock gene, in embryonic development of mouse cerebral cortex.

Per3 is one of the primary components of circadian clock system. While circadian dysregulation is known to be involved in the pathogenesis of several neuropsychiatric diseases. It remains largely unknown whether they participate in embryonic brain development. Here, we examined the role of clock gene Per3 in the development of mouse cerebral cortex. In situ hybridization analysis revealed that Per3 is expressed in the developing mouse cortex. Acute knockdown of Per3 with in utero electroporation caused abnormal positioning of cortical neurons, which was rescued by RNAi-resistant Per3. Per3-deficient cells showed abnormal migration phenotypes, impaired axon extension and dendritic arbor formation. Taken together, Per3 was found to play a pivotal role in corticogenesis via regulation of excitatory neuron migration and synaptic network formation.

Rho family GTPases, Rac and Cdc42, control the localization of neonatal dentate granule cells during brain development.

The hippocampus is generally considered as a brain center for learning and memory. We have recently established an electroporation-mediated gene transfer method to investigate the development of neonatal dentate granule cells in vivo. Using this new technique, we introduced knockdown vectors against Rac1 small GTPase into precursors for dentate granule cells at postnatal day 0. After 21 days, Rac1-deficient cells were frequently mispositioned between the granule cell layer (GCL) and hilus. About 60% of these mislocalized cells expressed a dentate granule cell marker, Prox1. Both the dendritic spine density and the ratio of mature spine were reduced when Rac1 was silenced. Notably, the deficient cells have immature thin processes during migrating in the early neonatal period. Knockdown of another Rac isoform, Rac3, also resulted in mislocalization of neonatally born dentate granule cells. In addition, knockdown of Cdc42, another Rho family protein, also caused mislocalization of the cell, although the effects were moderate compared to Rac1 and 3. Despite the ectopic localization, Rac3- or Cdc42-disrupted mispositioned cells expressed Prox1. These results indicate that Rho signaling pathways differentially regulate the proper localization and differentiation of dentate granule cells.

Isozyme-Specific Role of SAD-A in Neuronal Migration During Development of Cerebral Cortex.

SAD kinases regulate presynaptic vesicle clustering and neuronal polarization. A previous report demonstrated that Sada-/- and Sadb-/- double-mutant mice showed perinatal lethality with a severe defect in axon/dendrite differentiation, but their single mutants did not. These results indicated that they were functionally redundant. Surprisingly, we show that on a C57BL/6N background, SAD-A is essential for cortical development whereas SAD-B is dispensable. Sada-/- mice died within a few days after birth. Their cortical lamination pattern was disorganized and radial migration of cortical neurons was perturbed. Birth date analyses with BrdU and in utero electroporation using pCAG-EGFP vector showed a delayed migration of cortical neurons to the pial surface in Sada-/- mice. Time-lapse imaging of these mice confirmed slow migration velocity in the cortical plate. While the neurites of hippocampal neurons in Sada-/- mice could ultimately differentiate in culture to form axons and dendrites, the average length of their axons was shorter than that of the wild type. Thus, analysis on a different genetic background than that used initially revealed a nonredundant role for SAD-A in neuronal migration and differentiation.

Biochemical and Morphological Characterization of a Neurodevelopmental Disorder-Related Mono-ADP-Ribosylhydrolase, MACRO Domain Containing 2.

MACRO Domain Containing 2 (MacroD2) is a neurodevelopmental disorder-related mono-ADP-ribosylhydrolase. Molecular features of this protein in neural tissues are largely unknown. In this study, we generated a specific antibody against MacroD2, and carried out expression and morphological analyses of the molecule during mouse brain development. In Western blotting, 2 MacroD2 isoforms with molecular masses of ∼70 and ∼75 kDa started to be expressed at embryonic day 16.5, reached the maximal level at postnatal day 8, and then gradually decreased through P30. In contrast, other isoforms with molecular masses of ∼110 and ∼140 kDa gradually increased during embryonic to postnatal development. In immunohistochemical analyses, MacroD2 was strongly detected in cortical neurons in layer II-V at P0 and P7, while the protein expression decreased significantly in the neurons at P30. Immunofluorescence analyses revealed that MacroD2 was mainly distributed in the soma and to a lesser extent in the axon and dendrite of immature primary cultured mouse hippocampal neurons. On the other hand, in the matured hippocampal neurons, while MacroD2 was detected in the soma, it displayed in dendrites a punctate distribution pattern with a partial colocalization with synaptic markers, synaptophysin, and PSD95. The obtained results indicate that MacroD2 is expressed and may have a physiological role in the central nervous system during brain development.