540 flox/flox nestin-cre mice) we also observed evidence of this linkage, with a parallel underrepresentation of N-myc flox/ WT; c-myc flox/flox cre-negative (healthy controls) and doubly homozygously floxed nestin-cre (DKO) mice. This was evident at some early embryonic stages examined (Fig. 1a), where only 1/6 mice (instead of one fourth) were N-myc flox/WT; c-myc flox/flox, and only 1/6 were N-myc flox/flox; c-myc flox/flox nestin-cre+. However, by age E17.5 doubly homozygously floxed nestin-cre embryos (DKO) began to be underrepresented even compared to N- myc flox/WT; c-myc flox/flox control embryos, with which they were earlier balanced in numbers. This specific underrepresentation was by ratios of approximately 2-fold or more indicating lethality, which became progressively more severe with age. By P21, there was a 2.5-fold underrepresentation of DKO compared with N-myc flox/ WT; c-myc flox/flox control embryos (Fig. 1). By 4– 6 weeks of age, only extraordinarily rare DKO animals survived (<5%), while N-myc flox/WT; c-myc flox/flox cre- negative mice were surviving normally and appeared healthy. After birth, surviving DKO animals were outwardly small and had severe ataxia. Brain growth was severely impaired in the DKO mice, likely explaining their lethality. The few DKO animals surviving to maturity had a 60% reduction in total brain growth and a near complete absence of cerebellar growth (Fig. 1b). Mice heterozygous for c-myc, but homozygously for N-myc were not produced in the breeding in Fig. 1a, however they exhibited a phenotype almost as severe with a 53% reduction in brain growth. Surprisingly, mice heterozygous for N-myc, but homozygously for c- myc had a brain growth phenotype only slightly more severe than homozygously c-myc animals with WT N- myc (24% vs 18% reductions in brain growth), indicating that knocking out one allele of N-myc has little effect even in the context of absent c-myc (Fig. 1b). Loss of c- and N-myc Strongly Impairs Early Cerebellar Development At E17.5, cerebellar growth was strongly impaired in the DKO (Fig. 2a) as was overall brain growth (not shown). By P6, the cerebellar phenotype became more severe (Fig. 2b). DKO cerebellar size at P6 was only a fewfold increased compared with the DKO at E17.5 indicating minimal cerebellar growth and development occurs in the DKO during that approximately 8 day period of time. The morphology of the E17.5 and P6 DKO cerebella were also quite similar. While the DKO cerebella did grow from E17.5- P6 moderately, it is notable that the thickness of the EGL did not significantly change. In contrast, in the control, the EGL became substantially thicker. These data indicate that loss of c- and N-myc almost completely impairs EGL thickening. A 60 50 40 r e b um30 N 20 10 0 s s a m n i a r b T W f o % B Cerebellum (2010) 9:537–547 Cre- N-myc fl/wt c-myc fl/fl Cre+ N-myc fl/wt c-myc fl/fl Cre- N-myc fl/fl c-myc fl/fl Cre+ N-myc fl/fl c-myc fl/fl 12 26 24 19.5 12 16% 35% 32% 61% E12.5 6 6 8 6 6.5 23% 23% 31% 32% E14.5 Age (Days) 28 43 48 12 33 14 26 18 8 16.5 21% 39% 27% 12% E17.5 21% 33% 37% 9% P21 c-myc N-myc WT WT WT* Het WT WT WT Het KO WT KO Het WT KO Het KO KO KO Fig. 1. Myc DKO results in lethality beginning as early as E17.5 and impaired brain growth. a Ratios of the four genotypes of embryos or pups of the indicated age produced from N-myc fl/fl; c-myc fl/fl mice crossed with N-myc fl/wt; c-myc fl/fl nestin-cre+ mice. Note a genetic linkage between the WT N-myc allele and nestin-cre transgene results in an approximately 2-fold underrepresentation of N-myc fl/wt c-myc fl/fl Cre− (control) and N-myc fl/fl c-myc fl/fl Cre+ (DKO) animals independent of phenotype. DKOs are further underrepresented from E17.5 onward indicating lethality. Numbers above bars are absolute numbers of mice produced, italicized numbers above horizontal bars are the expected number based on Mendelian ratios, and numbers within bars are% of total pups/embryos. b Brain mass as a percentage of WT. Asterisk Cre+ The myc DKO phenotypes in the cerebellum at E17.5 and P6 (Fig. 2a) were far more severe than that observed with either single KO alone [16, 17]. These data indicate that both c- and N-myc are essential for NSC function that directs cerebellar genesis, or that N-myc is the central player but its function can be compensated for by c-myc. Interestingly, despite the apparent lethality of the nestin-cre Myc DKO and the severe cerebellar phenotype, no elevation of apoptosis was evident in the E17.5 in the cerebellum (Electronic supplementary material, Fig. S1) or brain as a whole (data shown). Therefore, c- and N-myc are not essential for NSC survival.