GoKawiilMouse experiments
All mice were handled in accordance with NIH guidelines and protocols approved by the UCSF Institutional Animal Care and Use Committee. Mice were housed under specific pathogen-free conditions in individually ventilated cages in a barrier facility on a 12 h:12 h light:dark cycle, with controlled temperature (20–26 °C) and humidity (30–70%). Housing density did not exceed five adult mice per cage; breeding cages (one male and up to two females) were maintained in a dedicated high-barrier area. Cages were changed weekly under laminar flow hoods, access was restricted with required PPE and colony health was monitored using sentinel mice. Both sexes were used, no sex-specific differences were observed and mice were randomly assigned to experimental groups.
C57BL/6 wild-type mice were obtained from the Jackson Laboratory (JAX:000664).
The Emx1-Cre line (B6.129S2Emx1tm1(cre)Krj/J, JAX:005628) has been previously described49. These mice were crossed with Atf4 floxed mice to delete Atf4 specifically in the early embryonic cortex. To assess phenotypes after blocking cell death, Emx1-Cre mice were also crossed with Atf4 floxed and p53-null animals. Emx1-Cre mice were crossed with LSL-H2B-GFP mice for lineage tracing of EMX1+ cortical cells across developmental stages.
The Atf4fl/fl line (C57BL/6-Atf4tm1.1Cmad/J, JAX:033380) carries loxP sites flanking exons 2–3, which include the ATG start codon of the Atf4 gene. These mice have been described previously50 and were crossed with Emx1-Cre and/or p53-null mice to knock out the Atf4 expression.
The p53-null (p53−/−) line (B6.129S2-Trp53tm1Tyj/J, JAX:002101) carries a neomycin cassette replacing exons 2–6 (including the start codon) of the Trp53 gene. This line has been previously described51. These mice were crossed with Emx1-Cre and Atf4 floxed mice to block p53-dependent cell death.
The LSL-H2B-GFP line (B6.Cg-Gt(ROSA)26Sortm8(CAG-HIST1H2BB/EGFP)Zjh/J JAX:036761) has a targeted mutation in the Gt(ROSA)26Sor locus with a loxP-flanked STOP cassette preventing transcription of a CAG promoter-driven enhanced green fluorescent protein (EGFP). EGFP expression occurs only after Cre-mediated recombination. This line has been previously described52 and was crossed with Emx1-Cre mice for lineage-tracing experiments.
Antibodies
For the immunostaining: GFP was detected with antibody GFP-1020 (Aves) at 1:1,000 dilution; mouse PAX6 was detected with antibody AB2237 (Millipore) at 1:500 dilution; mouse TBR2 was detected with antibody ab23345 (Abcam) at 1:500 dilution; mouse TBR1 was detected with antibody ab31940 (Abcam) at 1:1,000 dilution; mouse TUJ1 was detected with antibody T2200 (Sigma) at 1:1,000 dilution; mouse SATB2 was detected with antibody ab51502 (Abcam) at 1:500 dilution; mouse CTIP2 was detected with antibody ab18465 (Abcam) at 1:1,000 dilution; mouse CUX1 + CUX2 was detected with antibody ab309139 (Abcam) at 1:500 dilution; mouse calretinin was detected with antibody MAB1568 (Millipore) at 1:500 dilution; mouse parvalbumin was detected with antibody MAB1572 (Millipore) at 1:500 dilution; mouse calbindin was detected with antibody CB38a (Swant) at 1:500 dilution; human PAX6 was detected with antibody 901301 (Biolegend) at 1:500 dilution; mouse γH2A.X was detected with antibody ab2893 (abcam) at 1:500 dilution or 05-636 (Millipore) at 1:500 dilution; mouse CC3 was detected with antibody 9661 (Cell Signaling Technology) at 1:400 dilution; mouse Phospho-KAP-1 (Ser824) was detected with antibody A300-767A (Bethyl Laboratories) at 1:1,000 dilution; mouse p53 was detected with antibody 2524 (Cell Signaling Technology) at 1:500 dilution; mouse PCNA was detected with antibody 2586 (Cell Signaling Technology) at 1:500 dilution; mouse Ki67 was detected with antibody 550609 (BD Biosciences) at 1:500 dilution; mouse 53BP1 was detected with antibody NB100-304 (Novus Biologicals) at 1:500 dilution; mouse DNA-RNA Hybrid S9.6 was detected with antibody ENH001 (Kerafast) at 1:500 dilution; mouse p-ATM(Ser1981) was detected with antibody 05-740 (Millipore Sigma) at 1:500 dilution; mouse PHH3 was detected with Phospho-Histone H3 (Ser10) Antibody 9701 (Cell Signaling Technology at 1:500 dilution; mouse Nestin was detected with antibody MAB353 (Millipore Sigma) at 1:500 dilution; and mouse SOX2 was detected with antibody ab92494 (Abcam) at 1:500 dilution. All secondary antibodies for immunostaining were used at a dilution of 1:1,000. For immunoblotting: β-actin was detected with antibody 66009-1-Ig (Proteintech) at 1:1,000 dilution; EBF1 was detected with antibody AB10523 (Millipore) at 1:500 dilution; UBA52 was detected with antibody 18039-1-AP (Proteintech) at 1:500 dilution; CIRBP was detected with antibody 10209-2-AP (Proteintech) at 1:500 dilution; and ATF4 was detected with antibody 11815 (Cell Signaling Technology) at 1:500 dilution. All secondary antibodies used for immunoblotting were applied at a dilution of 1:20,000. For ChIP-qPCR, ATF4 antibody 11815 (Cell Signaling Technology) used at 1:50 dilution.
In utero electroporation
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