MOZ and its close homologue MORF (MOZ related factor, MYST4 or KAT6B) have been assigned specific roles in either hematopoietic or neural development, respectively [33, 34]. However, our previous observation that MozHAT−/− ESCs, unlike their Wt counterparts, did not extensively contribute to the formation of the brain in chimeric mice  suggests that MOZ, through its HAT activity, might also play a role in regulating the proliferation of NCS/Ps. To test this hypothesis, we first cultured cells isolated from the telencephalon of Wt and MozHAT−/− E14.5 embryos under clonogenic conditions to compare their potential to generate self-renewing neurospheres, a measurement of the number of cells with neural stem-like properties [35, 36]. MozHAT−/− embryos generated three times less neurospheres than Wt controls (Fig. 3A) suggesting that there are significantly fewer NSCs in the telencephalon of MozHAT−/− embryos. In addition, MozHAT−/− neurospheres displayed reduced expansion kinetics, producing fewer neurospheres at each passage, with an expansion index fivefold lower than Wt neurospheres (Fig. 3B), suggesting a reduced self-renewal potential. In fact, no neurospheres could be generated from the MOZHAT−/− cells after the third passage. In addition to this reduced expansion rate, MozHAT−/− neurospheres were, on average, smaller than their Wt counterparts (Fig. 3C), which could be indicative of cell-cycle arrest. BrdU incorporation analysis of MozHAT−/− secondary neurospheres revealed a reduced percentage of cells in the S-phase of the cell cycle and accumulation of cells at the G1 phase, similar to the phenotype observed for MozHAT−/− hematopoietic progenitors (Fig. 3D). Consistent with the acquisition of a senescent phenotype, cells from secondary MozHAT−/− neurospheres also displayed SA β-gal activity and higher p16INK4a expression levels than Wt controls (Fig. 3E, 3F). These in vitro findings were further substantiated in vivo by the observation that a lower percentage of cells expressing aldehyde dehydrogenase (ALDH), a marker of cells with stem-like properties [37, 38], was detected in brains of E14.5 MozHAT−/− embryos compared with Wt embryos (Fig. 4A). Furthermore, a reduction in the number of cells expressing the proliferation marker Ki67 (Fig. 4B) and cells incorporating BrdU (Fig. 4C) was also observed in the brains of E14.5 MozHAT−/− embryos. Finally, qPCR analysis of E14.5 telencephalons revealed an increased p16INK4a expression in the telencephalon of MozHAT−/− embryos (Fig. 4D). Altogether, these data demonstrate that, similarly to the hematopoietic system, MozHAT−/− NSC/Ps display proliferative defects ex vivo and in vivo, upregulate the expression of p16INK4a and readily enter into replicative senescence.
Figure 3. Telencephalon cells isolated from MozHAT−/− embryos display marked proliferative defects and signs of replicative senescence. (A): Frequency of primary neurospheres generated by E14.5 telencephalon cells. (B): Expansion index indicating the number of secondary neurospheres generated per primary neurosphere. (C): Morphology of Wt and MozHAT−/− day 8 tertiary neurospheres. Neurospheres are classified in different groups based on the diameter of the sphere. (<0.1 mm, 0.1–0.2 mm, and >0.2 mm). Bars represent mean ± SEM (n = 4). (D): Cell-cycle status of cells in neurospheres. Cells were isolated from day 6 secondary neurospheres. Percentage of cells in G1, S, and G2/M phases are indicated. Bars represent mean ± SEM (n = 3). (E): Senescence-associated β-galactosidase activity (pH: 6) in day 7 Wt and MozHAT−/− primary neurospheres. Magnification (×40). (F): Analysis of p16INK4a transcript levels in day 7 Wt, and MozHAT−/− neurospheres (n = 4 for each genotype). Abbreviations: 7AAD, 7-Aminoactinomycin D; BrdU, 5-bromo-2-deoxyuridine; HAT, histone acetyltransferase; SA β-gal, senescence-associated β-galactosidase; Wt, wild type.
Download figure to PowerPoint
Figure 4. Impaired proliferation and p16INK4a upregulation in MozHAT−/− neural stem cells and progenitors in vivo. (A): Flow cytometry plots of ALDH activity in Wt and MozHAT−/− telencephalon cells. Cells isolated from E14.5 telencephalons were incubated with the ALDH substrate aldefluor in the presence or absence of the ALDH inhibitor diethylaminobenzaldehyde. Bar graph reflects the average percentage of ALDH positive cells. (B): Coronal sections of Wt and MozHAT−/− E14.5 telencephalons stained with anti-Ki67 antibody. Scale bar = 0.2 mm. (C): Sections of Wt and MozHAT−/− E14.5 telencephalons stained with anti-BrdU antibody. Bar graph represents the percentage of cells stained with BrdU. (D): Analysis of p16INK4a transcript levels in telencephalon tissue (n = 24) isolated from Wt and MozHAT−/− E14.5 embryos. Scale bar = 0.2 mm. Abbreviations: ALDH, aldehyde dehydrogenase; BrdU, 5-bromo-2-deoxyuridine; DEAB, diethylaminobenzaldehyde; HAT, histone acetyltransferase; SSC, Side Scatter; Wt, wild type.
Download figure to PowerPoint