Dispersion of the neurons expressing layer specific markers in the reeler brain

Neurons with similar functions including neuronal connectivity and gene expression form discrete condensed structures within the vertebrate brain. This is exemplified within the circuitry formed by the cortical layers and the neuronal nuclei. It is well known that the Reelin protein is required for development of these neuronal structures in rodents and human, but the function of Reelin remains controversial. In this report, we used “layer‐specific markers” of the cerebral cortex to carry out detailed observations of spatial distribution of the neuronal subpopulations in the brain of the Reelin deficient mouse, reeler. We observed a spatially dispersed expression of the markers in the reeler cerebral cortex. These markers are expressed also in other laminated and non‐laminated structures of brain, in which we observed similar abnormal gene expression. Our observations suggest that neurons within the brain structures (such as the layers and the nuclei), which normally exhibit condensed distribution of marker expressions, loosen their segregation or scatter by a lack of Reelin.     

发展性阅读障碍相关基因KIAA0319对脑发育的影响——从动物到人

发展性阅读障碍是一种常见的学习障碍,KIAA0319是发展性阅读障碍相关基因,可能通过影响脑发育进而影响阅读能力。本文就发展性阅读障碍相关基因KIAA0319对鱼类、非灵长类哺乳动物、灵长类哺乳动物和人类大脑发育的影响进行了综述,发现该基因会对大脑语言及阅读相关脑结构如听觉通路、视觉通路和颞叶等的发育产生影响。听觉通路方面,KIAA0319基因可能会损伤内侧膝状体核从而影响听皮层的信息传入。视觉通路方面,KIAA0319基因可能影响外侧膝状体核内的大细胞,使得视觉信息无法正常传递到视皮层,影响背侧视觉通路。颞叶方面,KIAA0319基因的缺陷可能损害颞叶的灰质和白质,并影响颞叶的半球不对称以及颞叶和其他脑区的连接。不过阅读障碍机制复杂,不同阅读障碍相关基因之间、基因与环境之间存在相互影响,仍需进一步探讨。     

Application of in Utero Electroporation of G-Protein Coupled Receptor (GPCR) Genes,for Subcellular Localization of Hardly Identifiable GPCR in Mouse Cerebral Cortex

Lysophosphatidic acid (LPA) is a lipid growth factor that exerts diverse biological effects through its cognate receptors (LPA₁-LPA₆). LPA₁, which is predominantly expressed in the brain, plays a pivotal role in brain development. However, the role of LPA₁ in neuronal migration has not yet been fully elucidated. Here, we delivered LPA₁ to mouse cerebral cortex using in utero electroporation. We demonstrated that neuronal migration in the cerebral cortex was not affected by the overexpression of LPA₁. Moreover, these results can be applied to the identification of the localization of LPA₁. The subcellular localization of LPA₁ was endogenously present in the perinuclear area, and overexpressed LPA₁ was located in the plasma membrane. Furthermore, LPA₁ in developing mouse cerebral cortex was mainly expressed in the ventricular zone and the cortical plate. In summary, the overexpression of LPA₁ did not affect neuronal migration, and the protein expression of LPA₁ was mainly located in the ventricular zone and cortical plate within the developing mouse cerebral cortex. These studies have provided information on the role of LPA₁ in brain development and on the technical advantages of in utero electroporation.     

Mutation of the Variant α-Tubulin TUBA8 Results in Polymicrogyria with Optic Nerve Hypoplasia

The critical importance of cytoskeletal function for correct neuronal migration during development of the cerebral cortex has been underscored by the identities of germline mutations underlying a number of human neurodevelopmental disorders. The proteins affected include TUBA1A, a major α-tubulin isoform, and microtubule-associated components such as doublecortin, and LIS1. Mutations in these genes are associated with the anatomical abnormality lissencephaly, which is believed to reflect failure of neuronal migration. An important recent observation has been the dependence of cortical neuronal migration upon acetylation of α-tubulin at lysine 40 by the histone acetyltransferase Elongator complex. Here, we describe a recognizable autosomal recessive syndrome, characterized by generalized polymicrogyria in association with optic nerve hypoplasia (PMGOH). By autozygosity mapping, we show that the molecular basis for this condition is mutation of the TUBA8 gene, encoding a variant α-tubulin of unknown function that is not susceptible to the lysine 40 acetylation that regulates microtubule function during cortical neuron migration. Together with the unique expression pattern of TUBA8 within the developing cerebral cortex, these observations suggest a role for this atypical microtubule component in regulating mammalian brain development.     

Mitotic spindle regulation by Nde1 controls cerebral cortical size

Ablation of the LIS1-interacting protein Nde1 (formerly mNudE) in mouse produces a small brain (microcephaly), with the most dramatic reduction affecting the cerebral cortex. While cortical lamination is mostly preserved, the mutant cortex has fewer neurons and very thin superficial cortical layers (II to IV). BrdU birthdating revealed retarded and modestly disorganized neuronal migration; however, more dramatic defects on mitotic progression, mitotic orientation, and mitotic chromosome localization in cortical progenitors were observed in Nde1 mutant embryos. The small cerebral cortex seems to reflect both reduced progenitor cell division and altered neuronal cell fates. In vitro analysis demonstrated that Nde1 is essential for centrosome duplication and mitotic spindle assembly. Our data show that mitotic spindle function and orientation are essential for normal development of mammalian cerebral cortex.     

Lissencephaly: Mechanistic insights from animal models and potential therapeutic strategies

Lissencephaly is a severe human neuronal migration defect characterized by a smooth cerebral surface, mental retardation and seizures. The two most common genes mutated in patients with lissencephaly are LIS1 and DCX. LIS1 was the first gene cloned that was important for neuronal migration in any organism, and heterozygous mutations or deletions of LIS1 are found in the majority of patients with lissencephaly, while DCX mutations were found in males with X-linked lissencephaly. In this review, we will discuss how an understanding of the molecular and cellular pathways disrupted in model organisms with Lis1 and Dcx mutations or knock-down not only provide insights into the normal processes of neuronal migration, including neurogenesis, but they also may lead to potential novel therapeutic strategies for these severe cortical malformations.     

Ablation of the 14‐3‐3gamma Protein Results in Neuronal Migration Delay and Morphological Defects in the Developing Cerebral Cortex

14‐3‐3 proteins are ubiquitously‐expressed and multifunctional proteins. There are seven isoforms in mammals with a high level of homology, suggesting potential functional redundancy. We previously found that two of seven isoforms, 14‐3‐3epsilon and 14‐3‐3zeta, are important for brain development, in particular, radial migration of pyramidal neurons in the developing cerebral cortex. In this work, we analyzed the function of another isoform, the protein 14‐3‐3gamma, with respect to neuronal migration in the developing cortex. We found that in utero 14‐3‐3gamma‐deficiency resulted in delays in neuronal migration as well as morphological defects. Migrating neurons deficient in 14‐3‐3gamma displayed a thicker leading process stem, and the basal ends of neurons were not able to reach the boundary between the cortical plate and the marginal zone. Consistent with the results obtained from in utero electroporation, time‐lapse live imaging of brain slices revealed that the ablation of the 14‐3‐3gamma proteins in pyramidal neurons slowed down their migration. In addition, the 14‐3‐3gamma deficient neurons showed morphological abnormalities, including increased multipolar neurons with a thicker leading processes stem during migration. These results indicate that the 14‐3‐3gamma proteins play an important role in radial migration by regulating the morphology of migrating neurons in the cerebral cortex. The findings underscore the pathological phenotypes of brain development associated with the disruption of different 14‐3‐3 proteins and will advance the preclinical data regarding disorders caused by neuronal migration defects. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 600–614, 2016     

PEA15 loss of function and defective cerebral development in the domestic cat

Cerebral cortical size and organization are critical features of neurodevelopment and human evolution, for which genetic investigation in model organisms can provide insight into developmental mechanisms and the causes of cerebral malformations. However, some abnormalities in cerebral cortical proliferation and folding are challenging to study in laboratory mice due to the absence of gyri and sulci in rodents. We report an autosomal recessive allele in domestic cats associated with impaired cerebral cortical expansion and folding, giving rise to a smooth, lissencephalic brain, and that appears to be caused by homozygosity for a frameshift in PEA15 (phosphoprotein expressed in astrocytes-15). Notably, previous studies of a Pea15 targeted mutation in mice did not reveal structural brain abnormalities. Affected cats, however, present with a non-progressive hypermetric gait and tremors, develop dissociative behavioral defects and aggression with age, and exhibit profound malformation of the cerebrum, with a 45% average decrease in overall brain weight, and reduction or absence of the ectosylvian, sylvian and anterior cingulate gyrus. Histologically, the cerebral cortical layers are disorganized, there is substantial loss of white matter in tracts such as the corona radiata and internal capsule, but the cerebellum is relatively spared. RNA-seq and immunohistochemical analysis reveal astrocytosis. Fibroblasts cultured from affected cats exhibit increased TNFα-mediated apoptosis, and increased FGFb-induced proliferation, consistent with previous studies implicating PEA15 as an intracellular adapter protein, and suggesting an underlying pathophysiology in which increased death of neurons accompanied by increased proliferation of astrocytes gives rise to abnormal organization of neuronal layers and loss of white matter. Taken together, our work points to a new role for PEA15 in development of a complex cerebral cortex that is only apparent in gyrencephalic species.

SummaryGyrification is the neurodevelopmental process in certain mammalian species during which the cerebral cortex expands and folds resulting in the classic wrinkled appearance of the brain. Abnormalities in this process underlie many congenital malformations of the brain. However, unlike many other human malformations, genetic insight into gyrification is not possible in laboratory mice because rodents have a lissencephalic or smooth cerebral cortex. We identified a pathogenic variant in domestic cats that likely causes failure of the cerebral cortex to expand and fold properly, and discovered that the pathogenic variant impairs production of a protein, PEA15 (phosphoprotein expressed in astrocytes-15), involved in intracellular signaling. Affected cats have profound abnormalities in brain development, with minimal changes in their superficial behavior and neurologic function. Additional studies of tissue and cultured cells from affected animals suggest a pathophysiologic mechanism in which increased death of neurons accompanied by increased cell division of astrocytes gives rise to abnormal organization of neuronal layers and loss of white matter. These results provide new insight into a developmental process that is unique to animals with gyrencephalic brains.     

Development of the cerebral cortex in rodents and man

Studies mainly in rodents and man have contributed to new vistas on mammalian cerebral cortex development. Due to the much longer development in man and the larger size of the human brain, particular features (such as the existence of the subplate and tangential migration) were first detected in the human cortex. In addition, experimental techniques that can only be applied in nonhuman mammals revealed the pattern of neuronal generation, and demonstrated the different ways of neuronal migration and the formation of neuronal pathways. In this short review the present vistas on neuronal generation and migration, and the occurrence of transient layers are summarized.     

Cortical deficiency of laminin γ1 impairs the AKT/GSK-3β signaling pathway and leads to defects in neurite outgrowth and neuronal migration

Laminins have dramatic and varied actions on neurons in vitro. However, their in vivo function in brain development is not clear. Here we show that knockout of laminin γ1 in the cerebral cortex leads to defects in neuritogenesis and neuronal migration. In the mutant mice, cortical layer structures were disrupted, and axonal pathfinding was impaired. During development, loss of laminin expression impaired phosphorylation of FAK and paxillin, indicating defects in integrin signaling pathways. Moreover, both phosphorylation and protein levels of GSK-3β were significantly decreased, but only phosphorylation of AKT was affected in the mutant cortex. Knockout of laminin γ1 expression in vitro, dramatically inhibited neurite growth. These results indicate that laminin regulates neurite growth and neuronal migration via integrin signaling through the AKT/GSK-3β pathway, and thus reveal a novel mechanism of laminin function in brain development.     

Smooth,rough and upside-down neocortical development

Lissencephaly, which means 'smooth cortex', is caused by defective neuronal migration during development of the cerebral cortex and has devastating clinical consequences. 'Classical' lissencephaly seems to reflect mutations in regulators of the microtubule cytoskeleton, whereas 'cobblestone' lissencephaly is caused by mutations in genes needed for the integrity of the basal lamina of the central nervous system. Reelin, which is mutated in a third type of lissencephaly, may represent a unifying link because it encodes an extracellular protein that regulates neuronal migration and may also regulate the microtubule cytoskeleton.     

Cytoskeletal-associated proteins in the migration of cortical neurons

Neuronal migration is a hallmark of cerebral cortical development as neurons born deep within the brain migrate to the surface in a highly choreographed process. The cytoskeleton extends throughout the cell, mediating the dramatic morphological changes that accompany migration. On a cellular level, proper migration is accompanied by polarization of the cytoskeleton and cellular contents and by dynamic reorganization that generates the force for cell locomotion. Genetic analyses of human brain malformations, as well as genetically engineered mouse mutants, have highlighted a number of cytoskeletal-associated proteins underlying these functions, which are necessary for proper cortical development. While these proteins are involved in diverse molecular mechanisms, disruption during development results in the ectopic placement of neurons in the cortex. We review key cytoskeletal events and the critical cytoskeletal-associated proteins involved in cortical neuronal migration.     

Distribution of N-cadherin in human cerebral cortex during prenatal development

An important subgroup of adhesion molecules is the superfamily of cadherins, which takes part in cell recognition and differentiation during development. To our knowledge only one study describing N-cadherin expression in developing human brain has been performed so far. Our aim is to identify N-cadherin expression to establish a relationship between its expression and function in human cerebral cortex during prenatal development. In the present study, localization and intensity of N-cadherin was investigated in developing cerebral cortex. Fetuses from spontaneous abortions (n=13) were obtained from first, second, and third trimesters. Western blot analysis revealed three bands and the third trimester samples showed the strongest bands for N-cadherin. Cell processes, axon bundles, and some of the developing neurons revealed immunoreactivity for N-cadherin throughout pregnancy. The immunoreactivity increased in the developing neocortex and expanded from the ventricular layer toward the marginal zone as development progressed. Moreover, the immunoreactivity was strong in vascular endothelium during all three trimesters. We conclude that N-cadherin is dynamically related to the organization of cerebral cortex layers during prenatal development. The dynamic expression pattern implicates N-cadherin as a potential regulator of cell migration, axon extension and fasciculation, the establishment of synaptic contacts, and neurovascular angiogenesis in the developing human cerebral cortex.     

Dab2ip Regulates Neuronal Migration and Neurite Outgrowth in the Developing Neocortex

Dab2ip (DOC-2/DAB2 interacting protein) is a member of the Ras GTPase-activating protein (GAP) family that has been previously shown to function as a tumor suppressor in several systems. Dab2ip is also highly expressed in the brain where it interacts with Dab1, a key mediator of the Reelin pathway that controls several aspects of brain development and function. We found that Dab2ip is highly expressed in the developing cerebral cortex, but that mutations in the Reelin signaling pathway do not affect its expression. To determine whether Dab2ip plays a role in brain development, we knocked down or over expressed it in neuronal progenitor cells of the embryonic mouse neocortex using in utero electroporation. Dab2ip down-regulation severely disrupts neuronal migration, affecting preferentially late-born principal cortical neurons. Dab2ip overexpression also leads to migration defects. Structure-function experiments in vivo further show that both PH and GRD domains of Dab2ip are important for neuronal migration. A detailed analysis of transfected neurons reveals that Dab2ip down- or up-regulation disrupts the transition from a multipolar to a bipolar neuronal morphology in the intermediate zone. Knock down of Dab2ip in neurons ex-vivo indicates that this protein is necessary for proper neurite development and for the expression of several major neuronal microtubule associated proteins (MAPs), which are important for neurite growth and stabilization. Thus, our study identifies, for the first time, a critical role for Dab2ip in mammalian cortical development and begins to reveal molecular mechanisms that underlie this function.     

Doublecortin is a microtubule-associated protein and is expressed widely by migrating neurons.

Doublecortin (DCX) is required for normal migration of neurons into the cerebral cortex, since mutations in the human gene cause a disruption of cortical neuronal migration. To date, little is known about the distribution of DCX protein or its function. Here, we demonstrate that DCX is expressed in migrating neurons throughout the central and peripheral nervous system during embryonic and postnatal development. DCX protein localization overlaps with microtubules in cultured primary cortical neurons, and this overlapping expression is disrupted by microtubule depolymerization. DCX coassembles with brain microtubules, and recombinant DCX stimulates the polymerization of purified tubulin. Finally, overexpression of DCX in heterologous cells leads to a dramatic microtubule phenotype that is resistant to depolymerization. Therefore, DCX likely directs neuronal migration by regulating the organization and stability of microtubules.     

In vivo knockdown of ckit impairs neuronal migration and axonal extension in the cerebral cortex

The tyrosine kinase receptor cKit and its ligand stem cell factor (SCF) are well known mediators in proliferation, survival, and positive chemotaxis of different cell types in the hematopoietic system. However, and in spite of previous reports showing robust expression of cKit and SCF in the brain during development, their possible function in the cerebral cortex has not been clarified. In this study, embryonic knockdown expression of cKit in the rat cortex by in utero electroporation of specific RNAi resulted in delayed radial migration of cortical neurons. In conditional Nestin‐cKit KO homozygous mutants, radial migration in the cortex was also delayed. The opposite phenotype was observed after overexpressing cKit in the cortex: radial migration was accelerated. Callosal fibers electroporated with cKit RNAi were also delayed in their extension within the contralateral cortex and eventually failed to innervate their target area. In vitro experiments showed that, whereas SCF was able to promote migration of cortical neurons, it had no effect on cortical neurite outgrowth. In summary, our results demonstrate that (1) cKit is necessary for radial migration of cortical neurons, probably through SCF binding and (2) cKit is necessary for the correct formation of the callosal projection, most likely by a mechanism not involving SCF. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 73: 871–887, 2013     

The Effect of Hypothermia Therapy on Cortical Laminar Disruption following Ischemic Injury in Neonatal Mice

Hypothermia has been proposed as a treatment for reducing neuronal damage in the brain induced by hypoxic ischemia. In the developing brain, hypoxic ischemia-induced injury may give rise to cerebral palsy (CP). However, it is unknown whether hypothermia might affect the development of CP. The purpose of this study was to investigate whether hypothermia would have a protective effect on the brains of immature, 3-day old (P3) mice after a challenge of cerebral ischemia. Cerebral ischemia was induced in P3 mice with a right common carotid artery ligation followed by hypoxia (6% O₂, 37°C) for 30 min. Immediately after hypoxic ischemia, mice were exposed to hypothermia (32°C) or normothermia (37°C) for 24 h. At 4 weeks of age, mouse motor development was tested in a behavioral test. Mice were sacrificed at P4, P7, and 5 weeks to examine brain morphology. The laminar structure of the cortex was examined with immunohistochemistry (Cux1/Ctip2); the number of neurons was counted; and the expression of myelin basic protein (MBP) was determined. The hypothermia treatment was associated with improved neurological outcomes in the behavioral test. In the normothermia group, histological analyses indicated reduced numbers of neurons, reduced cortical laminar thickness in the deep, ischemic cortical layers, and significant reduction in MBP expression in the ischemic cortex compared to the contralateral cortex. In the hypothermia group, no reductions were noted in deep cortical layer thickness and in MBP expression in the ischemic cortex compared to the contralateral cortex. At 24 h after the hypothermia treatment prevented the neuronal cell death that had predominantly occurred in the ischemic cortical deep layers with normothermia treatment. Our findings may provide a preclinical basis for testing hypothermal therapies in patients with CP induced by hypoxic ischemia in the preterm period.     

Formation and preservation of cortical layers in slice cultures

During cortical development, neurons generated at the same time in the ventricular zone migrate out into the cortical plate and form a cortical layer (Berry and Eayrs, 1963, Nature 197:984–985; Berry and Rogers, 1965, J. Anat. 99:691–709). We have been studying both the formation and maintenance of cortical layers in slice cultures from rat cortex. The bromodexyuridine (BrdU) method was used to label cortical neurons on their birthday in vivo. When slice cultures were prepared from animals at different embryonic and postnatal ages, all cortical layers that have already been established in vivo remained preserved for several weeks in vitro. In slice cultures prepared during migration in the cortex, cells contiuned to migrate towards the pial side of the cortical slice, however, migration ceased after about 1 week in culture. Thus, cortical cells reached their final laminar position only in slice cultures from postnatal animals, whereas in embryonic slices, migrating cells became scattered throughout the cortex. Previous studies demonstrated that radial glia fibers are the major substrate for migrating neurons (Rakic, 1972, J. Comp. Neurol. 145:61–84; Hatten and Mason, 1990, Experientia 46:907–916). Using antibodies directed against the intermediate filament Vimentin, radial glial cells were detected in all slice cutures where cell migration did occur. Comparable to the glia development in vivo, radial glial fibers disappeared and astrocytes containing the glia fibrillary-associated protein (GFAP) differentiated in slice cultures from postnatal cortex, after the neurons have completed their migration. In contrast, radial glial cells were detected over the whole culture period, and very few astrocytes differentiated in embryonic slices, where cortical neurons failed to finish their migration. The results of this study indicate that the local environment is sufficient to sustain the layered organization of the cortex and support the migration of cortical neurons. In addition, our results reveal a close relationship between cell migration and the developmental status of glial cells. © 1992 John Wiley & Sons, Inc.