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. 2017 Jul 12;6:e24660. doi: 10.7554/eLife.24660

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© 2017, Semerci et al

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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Figure 5. — (A) Left panel: Lfng-eGFP NSCs express GFAP⁺ radial processes originating from the Sox2⁺ cell nuclei located in the SGZ. Right panel: The total number of GFAP⁺ Sox2⁺ NSCs in 4-month-old wild-type mice does not differ from the total number of eGFP⁺ NSCs in 4 month-old Lfng-eGFP (N = 3 per group; Student’s t-test, p=0.99). Scale bar = 10 µm. (B) The total number of NSCs in 2-month-old wild-type, Lfng heterozygote (Lfng^−/+), and homozygote (Lfng^−/−) knockout mice shows Lfng dose-dependent decrease in the NSC population (N = 3–4 per group; One-way ANOVA p<0.00001, Tukey HSD post-hoc test: p<0.0001 for wild-type vs Lfng^−/+ or Lfng^−/−, p=0.0484 for Lfng^−/+ vs Lfng^−/−). (C) Lack of Lfng promotes increased division of NSCs. Left panel: NSCs lacking Lfng have a higher ratio of cycling Ki67⁺ NSCs compared to wild-type (N = 4 per group; Student’s t-test p=0.0221). Middle panel: NSCs lacking Lfng have a higher ratio of actively dividing, BrdU⁺ NSCs compared to wild-type (N = 4 per group; Student’s t-test p=0.0017). Right panel: Lfng acts cell-autonomously and in a dose-dependent manner to control the NSC division (N = 4 per group). The ratio of BrdU⁺ NSCs was compared between iLfng^fl/fl mutant clones (tdTomato⁺), iLfng^fl/fl wild type clones (tdTomato^- GFAP⁺ Sox2⁺), and Lfng-CreER^T2; RCL-tdT control mice (tdTomato⁺ GFAP⁺ Sox2⁺). N = 4; p=0.9978 for control vs iLfng^fl/fl tdTom^- clones; p=0.0049 for control vs iLfng^fl/fl tdTom⁺ clones; p=0.0045 for iLfng^fl/fl tdTom^- clones vs iLfng^fl/fl tdTom⁺ clones. (D) NSCs lacking Lfng are hyper-activated in response to ECS treatment (N = 3–4 per group; Student’s t-test, p=0.0178). (E) Lack of Lfng decreases Notch signal intensity in mutant NSCs. Relative intensities of NICD1 staining are significantly lower in iLfng^fl/fl mutant NSCs compared to control (N = 4 for control; N = 3 for iLfng^fl/fl; Student’s t-test, p=0.0004). (F) NSCs lacking Lfng spend less time in the active state than wild-type NSCs. Lfng absence is associated with decreased S-phase re-entry 3 (left panel) and 7 (middle panel) days following the initial division compared to the wild-type NSCs (N = 4 per group; Student’s t-test, p=0.0024 for 3d, p=0.0003 for 7d). CldU⁺ IdU⁺ cells represent NSCs that underwent first division at the time of CldU injection (day 0) and were in S-phase at the time of IdU injection (day 3 or day 7). Right panel: In iLfng^fl/fl mice, no NSCs were found that re-entered S-phase 7 days after the initial division. CldU⁺ IdU⁺ cells represent NSCs that were induced at day 0, underwent first division 1 day post-induction (CldU⁺) and were in S-phase 7 days post-induction (IdU⁺; N = 3–4 per group; Student’s t-test, p=0.0014). (G) NSCs lacking Lfng mostly exit cell cycle within a week of first division. CldU⁺ Ki67⁺ NSCs represent NSCs that are actively cycling 7 days following the CldU injection (N = 4; Student’s t-test, p=0.0105). (H) NSCs lacking Lfng give rise to astrocyte-like cells. Left panel: The number of BrdU-retaining NSCs 7 days after the BrdU injection is significantly reduced in Lfng^−/+ mice compared to wild-type (N = 4 per timepoint; Student’s t-test, p=0.0003). Middle panel: In iLfng^fl/fl mice, significantly more tdTomato⁺ astrocyte-like cells accumulate 7 and 30 days following induction compared to controls, while the number of tdTomato⁺ ANPs significantly decreases (N = 4 per group, p=0.0003 and p<0.0001 for astrocyte-like cells, p=0.0024 and p=0.0049 for ANPs). Right panel: The number of BrdU-retaining astrocyte-like cells 7 days after the BrdU injection is significantly higher in iLfng^fl/fl mice compared to controls, but the difference is lost at 30 days (N = 4 per group; Student’s t-test, p=0.0302 for 7 days, p=0.4412 for 30 days). Bars represent mean ± SEM. *p<0.05, **p<0.001, ***p<0.0001. See Figure 5—figure supplement 1 for further details.

DOI: http://dx.doi.org/10.7554/eLife.24660.009

Figure 5—figure supplement 1. — (A) Left panel: Lfng-eGFP NSCs express GFAP⁺ radial processes originating from the Sox2⁺ cell nuclei located in the SGZ. Right panel: The total number of GFAP⁺ Sox2⁺ NSCs in 4-month-old wild-type mice does not differ from the total number of eGFP⁺ NSCs in 4 month-old Lfng-eGFP (N = 3 per group; Student’s t-test, p=0.99). Scale bar = 10 µm. (B) The total number of NSCs in 2-month-old wild-type, Lfng heterozygote (Lfng^−/+), and homozygote (Lfng^−/−) knockout mice shows Lfng dose-dependent decrease in the NSC population (N = 3–4 per group; One-way ANOVA p<0.00001, Tukey HSD post-hoc test: p<0.0001 for wild-type vs Lfng^−/+ or Lfng^−/−, p=0.0484 for Lfng^−/+ vs Lfng^−/−). (C) Lack of Lfng promotes increased division of NSCs. Left panel: NSCs lacking Lfng have a higher ratio of cycling Ki67⁺ NSCs compared to wild-type (N = 4 per group; Student’s t-test p=0.0221). Middle panel: NSCs lacking Lfng have a higher ratio of actively dividing, BrdU⁺ NSCs compared to wild-type (N = 4 per group; Student’s t-test p=0.0017). Right panel: Lfng acts cell-autonomously and in a dose-dependent manner to control the NSC division (N = 4 per group). The ratio of BrdU⁺ NSCs was compared between iLfng^fl/fl mutant clones (tdTomato⁺), iLfng^fl/fl wild type clones (tdTomato^- GFAP⁺ Sox2⁺), and Lfng-CreER^T2; RCL-tdT control mice (tdTomato⁺ GFAP⁺ Sox2⁺). N = 4; p=0.9978 for control vs iLfng^fl/fl tdTom^- clones; p=0.0049 for control vs iLfng^fl/fl tdTom⁺ clones; p=0.0045 for iLfng^fl/fl tdTom^- clones vs iLfng^fl/fl tdTom⁺ clones. (D) NSCs lacking Lfng are hyper-activated in response to ECS treatment (N = 3–4 per group; Student’s t-test, p=0.0178). (E) Lack of Lfng decreases Notch signal intensity in mutant NSCs. Relative intensities of NICD1 staining are significantly lower in iLfng^fl/fl mutant NSCs compared to control (N = 4 for control; N = 3 for iLfng^fl/fl; Student’s t-test, p=0.0004). (F) NSCs lacking Lfng spend less time in the active state than wild-type NSCs. Lfng absence is associated with decreased S-phase re-entry 3 (left panel) and 7 (middle panel) days following the initial division compared to the wild-type NSCs (N = 4 per group; Student’s t-test, p=0.0024 for 3d, p=0.0003 for 7d). CldU⁺ IdU⁺ cells represent NSCs that underwent first division at the time of CldU injection (day 0) and were in S-phase at the time of IdU injection (day 3 or day 7). Right panel: In iLfng^fl/fl mice, no NSCs were found that re-entered S-phase 7 days after the initial division. CldU⁺ IdU⁺ cells represent NSCs that were induced at day 0, underwent first division 1 day post-induction (CldU⁺) and were in S-phase 7 days post-induction (IdU⁺; N = 3–4 per group; Student’s t-test, p=0.0014). (G) NSCs lacking Lfng mostly exit cell cycle within a week of first division. CldU⁺ Ki67⁺ NSCs represent NSCs that are actively cycling 7 days following the CldU injection (N = 4; Student’s t-test, p=0.0105). (H) NSCs lacking Lfng give rise to astrocyte-like cells. Left panel: The number of BrdU-retaining NSCs 7 days after the BrdU injection is significantly reduced in Lfng^−/+ mice compared to wild-type (N = 4 per timepoint; Student’s t-test, p=0.0003). Middle panel: In iLfng^fl/fl mice, significantly more tdTomato⁺ astrocyte-like cells accumulate 7 and 30 days following induction compared to controls, while the number of tdTomato⁺ ANPs significantly decreases (N = 4 per group, p=0.0003 and p<0.0001 for astrocyte-like cells, p=0.0024 and p=0.0049 for ANPs). Right panel: The number of BrdU-retaining astrocyte-like cells 7 days after the BrdU injection is significantly higher in iLfng^fl/fl mice compared to controls, but the difference is lost at 30 days (N = 4 per group; Student’s t-test, p=0.0302 for 7 days, p=0.4412 for 30 days). Bars represent mean ± SEM. *p<0.05, **p<0.001, ***p<0.0001. See Figure 5—figure supplement 1 for further details.

DOI: http://dx.doi.org/10.7554/eLife.24660.009