Mice were housed, bred, and treated according to the guidelines approved by the Home Office under the Animal (Scientific Procedures) Act 1986. Protocols detailing the generation and genotyping of the genetically modified mice used in this article have been described Selleck Obeticholic Acid previously for NexCre mice ( Goebbels et al., 2006) and are described in Figure S1A and Supplemental Experimental Procedures for Ascl1flox/flox mice. PCR genotyping of Ascl1 ( Guillemot et al., 1993) and Neurog2 ( Fode et al., 1998) mutant and wild-type
alleles was described previously. In utero electroporation, cell counting, and statistical analyses were performed as described previously (Nguyen et al., 2006), with minor modifications as explained in the Supplemental Experimental Procedures. Embryonic brains were dissected in 1 × phosphate buffered saline (PBS) and fixed overnight in 4% paraformaldehyde (PFA)/1 × PBS at 4°C. Fixed samples were cryoprotected overnight in 20% sucrose/1 × PBS at 4°C, mounted in OCT Compound (VWR International), and sectioned coronally with a cryostat (Leica). Nonradioactive RNA in situ hybridizations on frozen sections of brains were performed with digoxigenin-labeled riboprobes as described previously (Cau et al., 1997). The full-length coding sequence for mouse Rnd3 was PCR cloned into pBluescript SK to generate an antisense probe for mouse Rnd3. Probes for mouse Rnd2 ( Heng et al., 2008)
and LacZ ( Seo et al., 2007) were prepared as previously described. Immunolabelings were performed with standard protocols by using find more the following primary antibodies: mouse anti-Ascl1 (1/200, gift from D.J. Anderson),
rat anti-BrdU (1/1000, AbD Serotec), rabbit anti-Caspase-3 (1/1000, R&D Systems), goat anti-EEA1 (1/50, Santa Cruz), chicken anti-GFP (1/700, Millipore), mouse anti-Flag (1/250, Sigma), mouse anti-LAMP1 (1/100, Developmental Studies Hybridoma Bank), mouse anti N-cadherin (1/200, BD Biosciences), mouse anti-Nestin (1/100, Millipore), mouse anti-Rab7 (1/500, Abcam), rabbit anti-Rnd2 (1/50, Santa Cruz), mouse anti-Rnd3 (1/500, Abcam), rabbit anti-Rnd3 (1/50, Abcam), science and mouse anti-transferrin receptor (1/100, Zymed). Cells or sections were then incubated with appropriate fluorescent secondary antibodies. Pretreatment with 2 N HCl for 30 min at 37°C prior to preincubation with primary antibody was performed to detect BrdU. For in vivo FRET analysis, cortices were coelectroporated in utero at E14.5 with the FRET probes for RhoA pRaichu-1298x or pRaichu-1293x (0.25 μg/μl) and control shRNA-RFP, Rnd2 shRNA-RFP, or Rnd3 shRNA-RFP (1 μg/μl). RhoA FRET efficiency was analyzed 1 day later in fixed brain sections. For FRET analysis in dissociated cortical cells, cortices were coelectroporated ex vivo at E14.5 with the same constructs and sliced with a vibratome immediately after electroporation and sections were cultured overnight.