Cellular and subcellular imaging of motor protein-based behavior in embryonic rat brain

Baffet, Alexandre D.; Carabalona, Aurelie; Dantas, Tiago J.; Doobin, David D.; Hu, Daniel Jun-Kit; Vallee, Richard B.

doi:10.1016/bs.mcb.2015.06.013

Cited by 20 publications

(18 citation statements)

References 24 publications

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“…1b). E15 mouse embryonic brains were sliced and cultured in modified cortical culture medium (M-CCM) (Baffet et al, 2016) and brain slices were infected for 2 h with either virus produced on C6/36 mosquito cells (6.10 5 FFU). Viral input was then removed and brain slices cultivated for 24 to 48 h at 37 °C (Fig.…”

Section: Resultsmentioning

confidence: 99%

Comparative Analysis Between Flaviviruses Reveals Specific Neural Stem Cell Tropism for Zika Virus in the Mouse Developing Neocortex

et al. 2016

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The recent Zika outbreak in South America and French Polynesia was associated with an epidemic of microcephaly, a disease characterized by a reduced size of the cerebral cortex. Other members of the Flavivirus genus, including West Nile virus (WNV), can cause encephalitis but were not demonstrated to cause microcephaly. It remains unclear whether Zika virus (ZIKV) and other flaviviruses may infect different cell populations in the developing neocortex and lead to distinct developmental defects. Here, we describe an assay to infect mouse E15 embryonic brain slices with ZIKV, WNV and dengue virus serotype 4 (DENV-4). We show that this tissue is able to support viral replication of ZIKV and WNV, but not DENV-4. Cell fate analysis reveals a remarkable tropism of ZIKV infection for neural stem cells. Closely related WNV displays a very different tropism of infection, with a bias towards neurons. We further show that ZIKV infection, but not WNV infection, impairs cell cycle progression of neural stem cells. Both viruses inhibited apoptosis at early stages of infection. This work establishes a powerful comparative approach to identify ZIKV-specific alterations in the developing neocortex and reveals specific preferential infection of neural stem cells by ZIKV.

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Section: Resultsmentioning

confidence: 99%

Comparative Analysis Between Flaviviruses Reveals Specific Neural Stem Cell Tropism for Zika Virus in the Mouse Developing Neocortex

et al. 2016

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“…The uterus was returned to the abdominal cavity, the incision site closed by suture of the abdominal rectus muscle and closure of the skin via abdominal wound clips. The rats were monitored every day post surgery until the desired developmental time point, and buprenorphine (0.05 mg kg −1 ) was provided every 8–12 h for the first 48 h of recovery for pain management55. All animal protocols were approved by the Institutional Animal Use and Care Committee at Columbia University.…”

Section: Methodsmentioning

confidence: 99%

“…The dissected rat brains were embedded in 4% low melting agarose diluted in artificial cerebrospinal fluid55 and were sliced into 300 μm coronal sections as described above. The slices were placed on 0.4 μm, 30 mm diameter Millicell-CM inserts (Millipore) in cortical culture medium containing 25% Hanks balanced salt solution, 47% basal MEM, 25% normal horse serum, 1 × penicillin/streptomycin/glutamine (GIBCO BRL), and 30% glucose.…”

Section: Methodsmentioning

confidence: 99%

Severe NDE1-mediated microcephaly results from neural progenitor cell cycle arrests at multiple specific stages

et al. 2016

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Microcephaly is a cortical malformation disorder characterized by an abnormally small brain. Recent studies have revealed severe cases of microcephaly resulting from human mutations in the NDE1 gene, which is involved in the regulation of cytoplasmic dynein. Here using in utero electroporation of NDE1 short hairpin RNA (shRNA) in embryonic rat brains, we observe cell cycle arrest of proliferating neural progenitors at three distinct stages: during apical interkinetic nuclear migration, at the G2-to-M transition and in regulation of primary cilia at the G1-to-S transition. RNAi against the NDE1 paralogue NDEL1 has no such effects. However, NDEL1 overexpression can functionally compensate for NDE1, except at the G2-to-M transition, revealing a unique NDE1 role. In contrast, NDE1 and NDEL1 RNAi have comparable effects on postmitotic neuronal migration. These results reveal that the severity of NDE1-associated microcephaly results not from defects in mitosis, but rather the inability of neural progenitors to ever reach this stage.

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“…Organoid cultures do not allow for the study of neuropathologies in the late developing brain, are difficult to consistently manufacture, and are characterized by immature neuronal connectivity; therefore, they are far more simplistic than the brain (19). Organotypic brain slice cultures are widely used to understand neuronal connectivity and neurodegenerative disorders (20)(21)(22) and have enriched our understanding of autosomal recessive microcephaly and brain development (23,24). Furthermore, organotypic brain slice cultures from embryonic mice are a genetically amenable system to study cZVS and the ability of the virus to infect neural cells (neurotropism), as the virus inoculum is placed directly on target cells of the cultures.…”

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confidence: 99%

Replication of early and recent Zika virus isolates throughout mouse brain development

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Doobin

Warren

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Fetal infection with Zika virus (ZIKV) can lead to congenital Zika virus syndrome (cZVS), which includes cortical malformations and microcephaly. The aspects of cortical development that are affected during virus infection are unknown. Using organotypic brain slice cultures generated from embryonic mice of various ages, sites of ZIKV replication including the neocortical proliferative zone and radial columns, as well as the developing midbrain, were identified. The infected radial units are surrounded by uninfected cells undergoing apoptosis, suggesting that programmed cell death may limit viral dissemination in the brain and may constrain virus-associated injury. Therefore, a critical aspect of ZIKV-induced neuropathology may be defined by death of uninfected cells. All ZIKV isolates assayed replicated efficiently in early and midgestation cultures, and two isolates examined replicated in late-gestation tissue. Alteration of neocortical cytoarchitecture, such as disruption of the highly elongated basal processes of the radial glial progenitor cells and impairment of postmitotic neuronal migration, were also observed. These data suggest that all lineages of ZIKV tested are neurotropic, and that ZIKV infection interferes with multiple aspects of neurodevelopment that contribute to the complexity of cZVS.

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Cellular and subcellular imaging of motor protein-based behavior in embryonic rat brain

Cited by 20 publications

References 24 publications

Comparative Analysis Between Flaviviruses Reveals Specific Neural Stem Cell Tropism for Zika Virus in the Mouse Developing Neocortex

Comparative Analysis Between Flaviviruses Reveals Specific Neural Stem Cell Tropism for Zika Virus in the Mouse Developing Neocortex

Severe NDE1-mediated microcephaly results from neural progenitor cell cycle arrests at multiple specific stages

Replication of early and recent Zika virus isolates throughout mouse brain development

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