Adult NSCs Require Sustained IGF-II Production

Badea et al., 2003 Badea T.C.

Wang Y.

Nathans J. A noninvasive genetic/pharmacologic strategy for visualizing cell morphology and clonal relationships in the mouse. Haley et al., 2012 Haley V.L.

Barnes D.J.

Sandovici I.

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Hassan A.B. Igf2 pathway dependency of the Trp53 developmental and tumour phenotypes. fl/fl and either Cre+ or Cre−. Administering tamoxifen over 5 consecutive days beginning at postnatal day 21 (p21) resulted in death of Igf2 knockout (KO) mice (see below regarding this lethal phenotype); therefore, we administered tamoxifen (75 mg/kg) every 3 days for the studies on the CNS (fl/flCre+/−) mice versus wild-type (WT) (Igf2fl/fl) ( Silver and Livingston, 2001 Silver D.P.

Livingston D.M. Self-excising retroviral vectors encoding the Cre recombinase overcome Cre-mediated cellular toxicity. Figure 1 Tamoxifen-Induced Removal of Igf2 Show full caption fl/fl Cre+/−) with no tamoxifen or in WT mice (Igf2fl/fl Cre−/−) or for the recombined allele at 384 bp in iKO mice (Igf2fl/fl Cre+/−) plus tamoxifen (B). qPCR for Igf2 mRNA in choroid plexus (C) and hippocampus (D) from samples collected 2 weeks post tamoxifen administration. See also ∗p < 0.05 using ANOVA and Tukey’s post hoc test, n = 5 WT and n = 12 iKO mice. Error bars represent SEM. Scale bar, 1 mm. Schematic for tamoxifen dosing in C57BL/6 mice (A). PCR for the floxed Igf2 allele at 449 bp in iKO mice (Igf2Cre) with no tamoxifen or in WT mice (Igf2Cre) or for the recombined allele at 384 bp in iKO mice (Igf2Cre) plus tamoxifen (B). qPCR for Igf2 mRNA in choroid plexus (C) and hippocampus (D) from samples collected 2 weeks post tamoxifen administration. See also Figure S1 . Representative sections of Luxol fast blue and H&E stains at the level of the septal nuclei and the third ventricle of WT and iKO mice (E and F). Statistical significance:p < 0.05 using ANOVA and Tukey’s post hoc test, n = 5 WT and n = 12 iKO mice. Error bars represent SEM. Scale bar, 1 mm. To determine whether IGF-II is necessary for adult stem cell homeostasis, we mated floxed Igf2 mice with a tamoxifen-inducible Rosa 26 CreER driver mouse line () to generate littermates for experimental animals that were all Igf2and either Creor Cre. Administering tamoxifen over 5 consecutive days beginning at postnatal day 21 (p21) resulted in death of Igf2 knockout (KO) mice (see below regarding this lethal phenotype); therefore, we administered tamoxifen (75 mg/kg) every 3 days for the studies on the CNS ( Figure 1 A). PCR analysis 2 weeks after tamoxifen administration showed a recombined band of the expected size indicating excision of exons 4–6 of the Igf2 gene ( Figure 1 B). Consistent with these results, mRNA levels of Igf2 in choroid plexus and hippocampus were reduced by 90% in heterozygous Cre (Igf2Cre) mice versus wild-type (WT) (Igf2) ( Figures 1 C and 1D). Since Igf2 mRNA levels were similarly reduced in mice heterozygous and homozygous for Cre ( Figure S1 ), heterozygous Cre mice were used for all in vivo studies to exclude possible Cre toxicity (). Luxol fast blue with H&E staining from adult mice 3 months after Igf2 removal revealed no gross anatomical changes in the brain structures surrounding the SVZ or SGZ of the Igf2-inducible KO (iKO) mice ( Figures 1 E and 1F). Additionally, no differences were observed in organ weights of the heart, lung, liver, kidney, and spleen adjusted to total body weight for WT and iKO mice (data not shown).

Zhao et al., 2008 Zhao C.

Deng W.

Gage F.H. Mechanisms and functional implications of adult neurogenesis. + proliferating cells, we performed studies to determine whether the Igf2 iKO mice had deficits in hippocampal function using the Morris water maze to test spatial learning and memory and the open field test to evaluate exploratory activity and anxiety ( Several studies have correlated increased or decreased cell proliferation in the SGZ with improvements or deficits in learning and memory, respectively (for a review see). Since Igf2 deletion altered the numbers of dual nucleotide label-retaining cells and increased the number of CldUproliferating cells, we performed studies to determine whether the Igf2 iKO mice had deficits in hippocampal function using the Morris water maze to test spatial learning and memory and the open field test to evaluate exploratory activity and anxiety ( Figures 2 E–2H). WT mice initially required 50 s to find the hidden platform in the Morris water maze, but quickly learned the location of the platform, and their latency to find the platform decreased from 50 s to 10 s. By contrast, the iKO mice were impaired in learning and displayed longer latencies to find the platform (repeated-measures ANOVA, n = 16 iKO and n = 13 WT) ( Figure 2 E). To test memory retention, we tested mice in a probe trial. WT mice swam significantly longer (50%) in the target quadrant than the iKO mice (35%) ( Figure 2 F). There was no difference between genotypes in the ability to find a visible platform (data not shown).

We further assessed the effects of losing IGF-II on exploratory and anxiety behavior using an open field test. Mice were placed into an open field test apparatus and were evaluated across six 5-min intervals. Overall the total distance traveled was unaltered between WT and iKO mice (data not shown). However, the iKO mice traveled less distance than the WT mice in the first two intervals (iKO 1,683 ± 121 and 1,103 ± 104 versus WT 2,072 ± 129 and 1,426 ± 94), and the total time that the iKO mice were immobile was significantly increased compared with WT mice (iKO 955.4 ± 47.7 versus WT 761 ± 64.8). What emerged most clearly across trials was that the iKO mice avoided the center of the arena ( Figures 2 G and 2H) (n = 13 iKO and n = 10 WT), indicating increased anxiety in the iKO mice.