Whisker-Related Axonal Patterns and Plasticity of Layer 2/3 Neurons in the Mouse Barrel Cortex

Sehara, Keisuke; Toda, Tatsuki; Iwai, Lena; Wakimoto, Mayu; Tanno, Kaori; Matsubayashi, Yutaka; Kawasaki, Hiroshi

doi:10.1523/jneurosci.6096-09.2010

Cited by 60 publications

(74 citation statements)

References 69 publications

(92 reference statements)

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“…It is well known that axons of layer 2/3 neurons run radially in the ipsilateral cortex to the white matter, and then run tangentially and project to the contralateral cortex. In the ipsilateral side, layer 2/3 axons normally extend branches projecting to layer 5 and form barrel nets, which are axonal trajectories made after the initial appearance of barrels (Sehara et al, 2010). In our study, layer 2/3 neurons lacking MKK7 did not extend axons running tangentially in the white matter or form contralateral projections, although the axons of these neurons did run radially in the ipsilateral cortex and formed branches and barrel nets (Fig.…”

Section: Role Of Mkk7 In Axon Elongationsupporting

confidence: 46%

Stress-Activated Protein Kinase MKK7 Regulates Axon Elongation in the Developing Cerebral Cortex

et al. 2011

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The c-Jun NH 2 -terminal protein kinase (JNK), which belongs to the mitogen-activated protein kinase family, plays important roles in a broad range of physiological processes. JNK is controlled by two upstream regulators, mitogen-activated protein kinase kinase (MKK) 7 and MKK4. To elucidate the physiological functions of MKK7, we used Nestin-Cre to generate a novel mouse model in which the mkk7 gene was specifically deleted in the nervous system (Mkk7 flox/flox Nestin-Cre mice). These mice were indistinguishable from their control littermates in gross appearance during embryogenesis but died immediately after birth without breathing. Histological examination showed that the mutants had severe defects in brain development, including enlarged ventricles, reduced striatum, and minimal axon tracts. Electron microscopy revealed abnormal accumulations of filamentous structures and autophagic vacuoles in Mkk7 flox/flox NestinCre brain. Further analysis showed that MKK7 deletion decreased numbers of TAG-1-expressing axons and delayed neuronal migration in the cerebrum. Neuronal differentiation was not altered. In utero electroporation studies showed that contralateral projection of axons by layer 2/3 neurons was impaired in the absence of MKK7. Moreover, MKK7 regulated axon elongation in a cell-autonomous manner in vivo, a finding confirmed in vitro. Finally, phosphorylation levels of JNK substrates, including c-Jun, neurofilament heavy chain, microtubule-associated protein 1B, and doublecortin, were reduced in Mkk7 flox/flox Nestin-Cre brain. Our findings demonstrate that the phenotype of Mkk7 flox/flox Nestin-Cre mice differs substantially from that of Mkk4 flox/flox Nestin-Cre mice, and establish that MKK7-mediated regulation of JNK is uniquely critical for both axon elongation and radial migration in the developing brain.

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Section: Role Of Mkk7 In Axon Elongationsupporting

confidence: 46%

Stress-Activated Protein Kinase MKK7 Regulates Axon Elongation in the Developing Cerebral Cortex

et al. 2011

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“…pCAG-EGxxFP-Satb2 contains a genomic fragment including the sgRNA target sequence deliver transgenes into the living rodent brain [7][8][9][10][11][12] , here we report a brain-specific gene knockout method, and demonstrate the power of our simple electroporation-based gene knockout in the living mouse brain.…”

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confidence: 76%

CRISPR/Cas9‐Mediated Gene Knockout in the Mouse Brain Using In Utero Electroporation

Shinmyo

Kawasaki

2017

CP Neuroscience

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The CRISPR/Cas9 system has recently been adapted for generating knockout mice to investigate physiological functions and pathological mechanisms. Here, we report a highly efficient procedure for brain-specific disruption of genes of interest in vivo. We constructed pX330 plasmids expressing humanized Cas9 and single-guide RNAs (sgRNAs) against the Satb2 gene, which encodes an AT-rich DNA-binding transcription factor and is responsible for callosal axon projections in the developing mouse brain. We first confirmed that these constructs efficiently induced double-strand breaks (DSBs) in target sites of exogenous plasmids both in vitro and in vivo. We then found that the introduction of pX330-Satb2 into the developing mouse brain using in utero electroporation led to a dramatic reduction of Satb2 expression in the transfected cerebral cortex, suggesting DSBs had occurred in the Satb2 gene with high efficiency. Furthermore, we found that Cas9-mediated targeting of the Satb2 gene induced abnormalities in axonal projection patterns, which is consistent with the phenotypes previously observed in Satb2 mutant mice. Introduction of pX330-NeuN using our procedure also resulted in the efficient disruption of the NeuN gene. Thus, our procedure combining the CRISPR/Cas9 system and in utero electroporation is an effective and rapid approach to achieve brain-specific gene knockout in vivo.Technologies for disrupting the genome in living animals are essential for elucidating the physiological mechanisms of our body and the pathological mechanisms of diseases, and for contributing to the discovery of new therapeutic interventions for human diseases. One technique which has been widely utilized is homologous recombination-based gene targeting in embryonic stem (ES) cells 1 . In addition, conditional mutagenesis using ES cells that relies on the DNA recombinase Cre and its recognition site loxP has become a valuable tool to achieve gene targeting in selected cell types 2 . Although this gene targeting technology using ES cells is extensively utilized for generating various kinds of genetically modified mice, it is relatively arduous, costly and time-consuming.Recently, a simple and efficient genome targeting technology has been developed based on the microbial type II clustered, regularly interspaced, short palindromic repeats (CRISPR)/associated protein (Cas) adaptive immune systems derived from Streptococcus pyogenes 3 . Cas9 endonuclease guided by a duplex of mature CRISPR RNA (crRNA) and transactivating crRNA (tracrRNA) cleaves trespassing DNA from bacteriophages or plasmids in a sequence-specific manner 3 . In mammalian cells, it has been demonstrated that a combination of Cas9 and sgRNA, which is an artificial chimera of crRNA and tracrRNA, caused site-specific DSBs, and as a result, genome targeting via insertions and deletions (indels) was caused by error-prone non-homologous end-joining (NHEJ) 4,5 . These features of the CRISPR/Cas9 system facilitated the generation of mice carrying mutations in specific genes 6 . Indeed,...

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“…By P7, cortical neurons are set in their final layers; however, their morphology and synaptic organization is still immature (Foeller and Feldman, 2004). Growth of tangential connections within infragranular and supragranular layers of the S1 cortex has already begun but continues until the third week (Miller et al, 2001;Dagnew et al, 2003;Sehara et al, 2010). This protracted development correlates with extended plasticity in layers II/III (Fox, 2002).…”

Section: Introductionmentioning

confidence: 99%

Functional Development of Large-Scale Sensorimotor Cortical Networks in the Brain

Quairiaux

Mégevand

Kiss

et al. 2011

Journal of Neuroscience

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Large-scale neuronal networks integrating several cortical areas mediate the complex functions of the brain such as sensorimotor integration. Little is known about the functional development of these networks and the maturational processes by which distant networks become functionally connected. We addressed this question in the postnatal rat sensorimotor system. Using epicranial multielectrode grids that span most of the cortical surface and intracortical electrodes, we show that sensory evoked cortical responses continuously maturate throughout the first 3 weeks with the strongest developmental changes occurring in a very short time around postnatal day 13 (P13). Before P13, whisker stimulation evokes slow, initially surface-negative activity restricted mostly to the lateral parietal area of the contralateral hemisphere. In a narrow time window of ϳ48 h around P13, a new early, sharp surface-positive component emerges that coincides with subsequent propagation of activity to sensory and motor areas of both hemispheres. Our data show that this new component developing at the end of the second week corresponds principally to functional maturation of the supragranular cortical layers and appears to be crucial for the functional associations in the large-scale sensorimotor cortical network. It goes along with the onset of whisking behavior, as well as major synaptic and functional changes within the S1 cortex that are known to develop during this period.

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Whisker-Related Axonal Patterns and Plasticity of Layer 2/3 Neurons in the Mouse Barrel Cortex

Cited by 60 publications

References 69 publications

Stress-Activated Protein Kinase MKK7 Regulates Axon Elongation in the Developing Cerebral Cortex

Stress-Activated Protein Kinase MKK7 Regulates Axon Elongation in the Developing Cerebral Cortex

CRISPR/Cas9‐Mediated Gene Knockout in the Mouse Brain Using In Utero Electroporation

Functional Development of Large-Scale Sensorimotor Cortical Networks in the Brain

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