Classic Cadherins Mediate Selective Intracortical Circuit Formation in the Mouse Neocortex

Wakimoto, Mayu; Sehara, Keisuke; Ebisu, Haruka; Hoshiba, Yoshio; Tsunoda, Shin‐ichi; Ichikawa, Yoshie; Kawasaki, Hiroshi

doi:10.1093/cercor/bhu197

“…We transfected layer 2/3 neurons with 0.5 mg/ml pCAG-EGFP and 2.5 mg/ml pX330-Satb2-2129 using in utero electroporation at E15.5 and examined the projection patterns of EGFP-positive axons derived from transfected neurons 10 12 . As expected, in control animals transfected with the pX330 vector, EGFP-positive axons extended to the contralateral cortex ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Achieving organ-specific gene knockout using the CRISPR/Cas9 system has been an important challenge because if a gene is knocked out throughout the body, it often leads to embryonic lethality. By combining the CRISPR/Cas9 system and in utero electroporation, which is a recently invented, rapid and efficient technique to 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.…”

mentioning

confidence: 95%

CRISPR/Cas9-mediated gene knockout in the mouse brain using in utero electroporation

Shinmyo

¹

,

Tanaka

²

,

Tsunoda

³

et al. 2016

Sci Rep

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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.

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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.…”

mentioning

confidence: 76%

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

Shinmyo

¹

,

Kawasaki

²

2017

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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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“…In utero electroporation (IUE) procedure. In utero electroporation using mice was performed as described previously with slight modifications (Fukuchi-Shimogori and Grove, 2001;Tabata and Nakajima, 2001;Saito, 2006;Wakimoto et al, 2015;Hoshiba et al, 2016). Briefly, pregnant ICR mice were anesthetized, and the uterine horns were exposed.…”

Section: Methodsmentioning

confidence: 99%

FGF Signaling Directs the Cell Fate Switch from Neurons to Astrocytes in the Developing Mouse Cerebral Cortex

Duong

¹

,

Hoshiba

²

,

Saito

³

et al. 2019

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During mammalian neocortical development, neural precursor cells generate neurons first and astrocytes later. The cell fate switch from neurons to astrocytes is a key process generating proper numbers of neurons and astrocytes. Although the intracellular mechanisms regulating this cell fate switch have been well characterized, extracellular regulators are still largely unknown. Here, we uncovered that fibroblast growth factor (FGF) regulates the cell fate switch from neurons to astrocytes in the developing cerebral cortex using mice of both sexes. We found that the FGF signaling pathway is activated in radial glial cells of the ventricular zone at time points corresponding to the switch in cell fate. Our loss-and gain-of-function studies using in utero electroporation indicate that activation of FGF signaling is necessary and sufficient to change cell fates from neurons to astrocytes. We further found that the FGF-induced neuron-astrocyte cell fate switch is mediated by the MAPK pathway. These results indicate that FGF is a critical extracellular regulator of the cell fate switch from neurons to astrocytes in the mammalian cerebral cortex.Although the intracellular mechanisms regulating the neuron-astrocyte cell fate switch in the mammalian cerebral cortex during development have been well studied, their upstream extracellular regulators remain unknown. By using in utero electroporation, our study provides in vivo data showing that activation of FGF signaling is necessary and sufficient for changing cell fates from neurons to astrocytes. Manipulation of FGF signaling activity led to drastic changes in the numbers of neurons and astrocytes. These results indicate that FGF is a key extracellular regulator determining the numbers of neurons and astrocytes in the mammalian cerebral cortex, and is indispensable for the establishment of appropriate neural circuitry.

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Classic Cadherins Mediate Selective Intracortical Circuit Formation in the Mouse Neocortex

Cited by 23 publications

References 59 publications

CRISPR/Cas9-mediated gene knockout in the mouse brain using in utero electroporation

CRISPR/Cas9-mediated gene knockout in the mouse brain using in utero electroporation

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

FGF Signaling Directs the Cell Fate Switch from Neurons to Astrocytes in the Developing Mouse Cerebral Cortex

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