Fig. 2.

Mosaic knockout of Ars2 during early neural development. (A,B) Dorsal view of wild-type (control) and hGFAP-Cre; Ars2^fl/fl brains. The mutant shows reduced cortex (bracketed region), exposing the thalamus (TH) and midbrain (MB), normally not visible in the control hGFAP-Cre; Ars2^fl/+ brain. The cerebellum (CB) spheres are also reduced. (C,D) Hematoxylin and Eosin staining of coronal sections of P5 hGFAP-Cre; Ars2^fl/+ (Control) and Ars2^fl/fl brains. Arrows indicate enlarged lateral ventricle (LV) and damaged white matter tract. (E-G) DAPI staining on sagittal sections of P15 cerebellar vermis from Ars2^fl/fl mouse shows abnormal cerebellum foliation with varied severity (F,G) compared with control (E). The arrowhead highlights the appearance of the precentral fissure and lobule I. n>20 (strong phenotype) and n>15 (weak phenotype) animals were analyzed. Note that initial crosses yielded ∼4:1 strong:weak phenotypes, but the weak Cre-dependent effect was segregatable to yield mostly the weak cKO phenotypic class for several generations. (H) Strategy to generate hGFAP-Cre; Ai9[ROSA-flox-stop-flox-tdTomato] reporter mice. (I,J) Coronal sections of P0 hGFAP-Cre; Ai9 mice show variable Cre activity. (K-M) DAPI staining on coronal brain sections of P12 mouse with Ars2 knockout using ‘weak’ hGFAP-Cre line (L,L′) compared with control (K,K′). K′ and L′ show magnifications of the boxed areas in K and L, respectively. Arrowheads indicate the irregular hippocampal CA area. The size of the hippocampus is defined as the least upper bound. n=4, two-tailed t-test; data are mean±s.d. (N) Western blot validation of new Ars2 antibodies following Ars2 overexpression in 293 T cells. (O) P9 hGFAP-Cre; Ars2^fl/f; Ai9 hippocampal CA1 region stained for tdTomato (red) and Ars2 (green). Dotted lines highlight the areas with Cre-induced tdTomato that consistently lack Ars2.