PMC

Apoptosis is increased in the adult FA SVZ. (A) Pycnotic nuclei and cleaved-caspase-3 staining (white arrows) are shown in the SVZ of adult fancg^−/− mice. Colocalization of pycnotic nuclei and cleaved-caspase-3 staining is showed in an enlargement (lower panel). (B) Quantifications of cleaved-caspase-3-positive cells per SVZ cross section showed significant elevated apoptosis in the SVZ of fanca^−/− and fancg^−/− compared with fanca^+/+ and fancg^+/+ controls. The number of mice is indicated within the bars. V, ventricle. ^***P<0.005.

Proliferation is decreased in the SVZ of adult FA mice. (A) Proliferation of NSPCs was examined after BrdU incorporation for 3 h in fanca^−/− and fanca^+/+ control mice. (B, C) Quantifications of BrdU-positive and Ki67-positive cells in the SVZ showed a decreased percentage of proliferating cells in the fanca^−/− SVZ compared with fanca^+/+ controls. (D) γ-H3-histone immunostaining showed a decreased number of mitotic cells in the fanca^−/− and fancg^−/− SVZ in comparison with fanca^+/+ and fancg^+/+ controls. Data were obtained with 3–5 mice, as indicated within the bars, representing from 3024 to 5671 nuclei. ^*P<0.05; ^***P<0.005.

NSPC self-renewal in vitro is altered in FA mice. Growth, proliferation, and apoptosis were examined in neurosphere cultures from embryonic (A–C) and adult (D–F) NSPCs. (A) Population doublings of fanca^−/− embryonic NSPCs were reduced in comparison with fanca^+/+ controls. (B, E) Proliferating NSPCs were determined by flow cytometry after BrdU incorporation (30 min or 1 h for embryonic or adult NSPCs, respectively). (C, F) Analysis of cleaved-caspase-3 by flow cytometry revealed increased level of apoptosis in fanca^−/− and fancg^−/− embryonic and adult NSPCs in comparison with WT controls. (D) Population doublings of adult NSPCs were drastically reduced compared with WT controls. The number of mice is indicated within the bars. ^**P<0.01.

Apoptosis is increased in FA VZ. (A) DAPI and cleaved-caspase-3 immunostaining are shown in a fanca^−/− E14.5 neocortex section. Arrows pointed out the apoptotic cells in the VZ. Nonspecific staining was observed on blood vessels (stars) and on meninges (dotted lines). Colocalization of pycnotic nuclei and cleaved-caspase-3 staining is shown in the enlargement of the white box (A′). White arrows pointed out the isolated apoptotic cells and red arrows pointed out a doublet of apoptotic cells. (B) Quantifications of pycnotic nuclei and (C) cleaved-caspase-3-positive cells in the VZ of E13.5 and E14.5 embryonic brains show a significant increase of apoptosis in fanca^−/− and fancg^−/− compared with fanca^+/+ and fancg^+/+ controls. (D) Apoptotic cells in the fancg^−/− neocortex express nestin, an NSPC marker. The number of mice is indicated within the bars. V, ventricle. Scale bars, 10 μm. ^*P<0.05; ^***P<0.005.

FA NSPCs display chromosomic aberrations. (A) γ-H2AX foci were numerous in the VZ of fancg^−/− embryos, but infrequent in fancg^+/+ controls. Cytogenetic analyses were performed on embryonic NSPCs in culture. Figures of abnormal metaphases are shown to illustrate the most frequent aberrations that were observed in fanca^−/− NSPC cultures; (B) the arrow points a chromatid break; (C) a complex rearrangement is surrounded (C′); and an endoreplication is shown (arrowheads pointed out a pair of chromosomes) (D). (E) Quantifications were performed on 300 metaphases obtained from four embryos at the first and second passage in culture, and they showed increased levels of chromatid breaks and rearrangements (radial formation, endoreplication) in fanca^−/− compared with fanca^+/+ controls. ^**P<0.01; ^***P<0.005.

FA embryos have a reduced neocortex size. Embryonic brains (E14.5) were cut into coronal sections at different rostro-caudal levels (I, II, III) as shown in the sagittal representation. (A) A typical DAPI staining of caudal sections (III) is shown for fancg^+/+ and fancg^−/− brains. (A′) βIII-Tubulin (red) and nestin (green) immunostainings corresponding to white boxes on the left panel. (B, C) Neocortices of fanca^−/− and fancg^−/− showed a size reduction in the dorsal pallium (DP boxes in A′) compared with WT controls at each rostro-caudal level. (D, E) The thickness of the βIII-tubulin zone, containing neurons, was decreased in dorsal pallium of the fanca^−/− and fancg^−/− neocortex compared with WT controls at each rostro-caudal level. The number of mice is indicated within the bars. V, ventricle. Scale bars, 100 μm. ^*P<0.05; ^**P<0.01; ^***P<0.005.

Exhaustion of the NSPC pool in the SVZ of FA mice is accentuated with ageing. (A) Quantification of NG2/Ki67-double-positive cells in the adult SVZ shows decreased level of proliferating progenitors in the SVZ of young (2–3 months) and old (10–12 months) fanca^−/− adults compared with fanca^+/+ age-matched controls. (B) BrdU-label-retaining cells in the adult SVZ, recognized as slowly cycling neural stem cells, have a reduced level in the SVZ of young (2–3 months) and old (10–12 months) fanca^−/− adults compared with fanca^+/+ age-matched controls. (C) The NSPC pool was determined by culturing freshly harvested SVZ in a semi-solid medium that allows discrimination between neural stem cell-derived colonies and neural progenitor-derived colonies. The number of neural progenitor-derived colonies was decreased in the SVZ of young (2–3 months) fanca^−/− and fancg^−/− adults compared with fanca^+/+ and fancg^+/+ age-matched controls. Both neural stem cell- and progenitor-derived colonies were profoundly reduced in old fancg^−/− mice. Data were obtained with 2–6 mice, as indicated within or below (n=) the bars, representing in panels A and B from 1908 to 2894 nuclei (except the number of nuclei for NG2/Ki67 in old fanca^−/− mice, which was 798). ^*P<0.05; ^**P<0.01; ^***P<0.005.