Isolation of genetically manipulated neural progenitors and immature neurons from embryonic mouse neocortex by FACS

Abstract

The embryonic mammalian neocortex includes neural progenitors and neurons at various stages of differentiation. The regulatory mechanisms underlying multiple aspects of neocortical development-including cell division, neuronal fate commitment, neuronal migration, and neuronal differentiation-have been explored using in utero electroporation and virus infection. Here, we describe a protocol for investigation of the effects of genetic manipulation on neural development through direct isolation of neural progenitors and neurons from the mouse embryonic neocortex by fluorescence-activated cell sorting. For complete details on the use and execution of this protocol, please refer to Tsuboi et al. (2018) and Sakai et al. (2019).

Keywords: Cell Biology; Cell Differentiation; Cell isolation; Developmental biology; Flow Cytometry/Mass Cytometry; Gene Expression; Molecular Biology; Neuroscience; Stem Cells.

Graphical abstract

Figure 1

Gating strategies for isolation of CD133^high neural progenitors from the embryonic neocortex after in utero electroporation with a plasmid encoding GFP Plasmids encoding GFP and either control (shLuc) or Hmga2 (shHmga2) shRNAs were introduced into neural progenitors of mouse embryos at E11 by in utero electroporation as described previously (Sakai et al., 2019). Neocortical cells were dissociated at E13, stained with APC-conjugated antibodies to CD133, and subjected to FACS. (A) Debris and doublets were removed as described in steps 24a and 24b. (B and C) GFP and CD133 profiles of negative control (B) and electroporated and stained (C) samples. For electroporated samples, the CD133^high population (top 20% of CD133-expressing cells) was defined with the use of the GFP^neg fraction, and the gating was then applied to the GFP^pos fraction, as described in steps 24c to 24e. This approach allows appropriate comparison between neural progenitors of control and shHmga2 samples.

Figure 2

Gating strategies for isolation of neural progenitors and neurons from the neocortex of Nestin-d4Venus or wild-type embryos (A) Neocortical cells of Nestin-d4Venus^–/– or Nestin-d4Venus^+/– embryos at E14 were dissociated and subjected to FACS analysis as described previously (Sakai et al., 2019). The gates for dVenus-high, -medium, -low, and -negative cells are shown. (B) Neocortical cells of wild-type embryos at E14 were dissociated, stained (or not) with PE-conjugated antibodies to CD133, and subjected to FACS analysis as described previously (Sakai et al., 2019). The gates for CD133-high, -medium, -low, and -negative cells are shown. (C) Neocortical cells of C57BL/6 embryos at E15 were dissociated, stained (or not) with both APC-conjugated antibodies to CD133 and PE-conjugated antibodies to CD24, and subjected to FACS analysis. Nonneural cells determined as those negative for both CD133 and CD24 were removed. The gate for CD133-high cells is shown.

Figure 3

Quality check for neural progenitors and neurons isolated from the neocortex of Nestin-d4Venus embryos (A and B) Isolated d4Venus-high, -medium, -low, and -negative cells at E14 (A) or E16 (B) were subjected to RT-qPCR analysis of the indicated mRNAs. Data were normalized by the amount of Gapdh mRNA, expressed relative to the higher value for each mRNA, and are means + SEM from 11 (Sox2, Pax6, Eomes, Neurod1, Tubb3, and Tbr1 at E14) and four (E16 and Nestin at E14) independent experiments independent experiments with the exception of Nestin mRNA (four independent experiments).

Figure 4

Quality check for neural progenitors and neurons isolated from the neocortex of wild-type embryos on the basis of CD133 staining (A–D) Isolated CD133-high, -medium, -low, or -negative cells at E12 (A), E14 (B), E16 (C), or E18 (D) were subjected to RT-qPCR analysis of the indicated mRNAs. Data were normalized by the amount of Gapdh mRNA, are expressed relative to the higher value for each mRNA, and are means + SEM from three (E12, E18, and Pax6 and Tbr1 at E14) or four (E16 and Eomes, Neurod1, and Tubb3 at E14) independent experiments.