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First published online 21 July 2004
doi: 10.1242/dev.01215

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Telomere shortening and chromosomal instability abrogates proliferation of adult but not embryonic neural stem cells

Sacri Ferrón^1,*, Helena Mira^1,*,, Sonia Franco^2,*, Marifé Cano-Jaimez¹, Elena Bellmunt¹, Carmen Ramírez¹, Isabel Fariñas^1, and María A. Blasco²

¹ Departamento de Biología Celular, Universidad de Valencia, 46100 Burjassot, Spain
² Spanish National Cancer Center (CNIO), 28029 Madrid, Spain

View larger version (47K): [in a new window]   Fig. 1. Analysis of the phenotype of the subventricular zone (SVZ) of G2 and G4 Terc–/– adult mice in vivo. (A) Schematic diagram of (left) a sagittal section through an adult mouse brain, in which the location and extent of the lateral ventricle (LV) and of the rostral migratory stream (rms) are indicated, and (right) a coronal section through an anterior portion (a) of the lateral ventricle where the SVZ is indicated. OB, olfactory bulb; CB, cerebellum; aob, accessory olfactory bulb; ov, olfactory ventricle; rms, rostral migratory stream; st, striatum; Ctx, cerebral cortex; CC, corpus callosum. The lower boxed area indicates the region shown in B,C. The upper boxed area is the dorsolateral corner of the rms, shown in D and E. (B-E) Coronal sections through the walls of the lateral ventricle (LV) at an anterior level from wild-type (WT) and mutant (G4 Terc–/–) mice. (B,C) Detection of BrdU after repetitive pulses over a 12-hour period. Notice the dramatic decrease in the number of cells incorporating the analogue in the mutant, probably the result of reduced proliferation. (D,E) Immunofluorescent images of the rms stained with antibodies to PSA-NCAM (green) and GFAP (red) showing a reduction of labeling in the mutant. (F) Quantification of the total number of BrdU-positive cells per section in the walls of the lateral ventricle in coronal serial sections containing the entire extent of the SVZ. Data are shown as the mean number of immunopositive cells per section ± s.e.m. of 6 wild-type (black bars), 2 G4 Terc–/– mutants (gray bars) and 2 G4 Terc–/– mutants (white bars). The length of the SVZ and the size of the BrdU-immunopositive nuclei were the same in all genotypes. Determinations in anterior (a) and posterior levels (p), were done separately because of rostrocaudal differences in proliferating cell frequency. Notice that proliferation is significantly reduced in the mutants at both levels (one-tailed Student's t-test: ***P<0.001; **P<0.01). (G) Bar chart of the volume of the region (see scheme in A) that is rostrocaudally between the accessory olfactory bulb (aob) and of the olfactory ventricle (ov) and the volume of the striatum in wild-type (WT) and mutant (G4 Terc–/–) mice. Mutants have a significantly smaller olfactory bulb (one-tailed Student's t-test: *P<0.05) but no differences were seen in striatal volume.

View larger version (64K): [in a new window]   Fig. 2. Analysis of dorsal root ganglia (DRG) and ganglionic eminences (GE) in wild-type (WT) and Terc mutant E12.5 embryos of the fifth generation (G5 Terc–/–). (A) Schematic diagram of a sagittal section through an E12.5-E14.5 embryo showing the location of lumbar DRGs and ganglionic eminences. (B-F) Analysis of DRG development at the peak of the neurogenic period. (B,C) BrdU immunocytochemical detection in DRGs counterstained with Hematoxylin. (D,E) DRGs immunostained with anti-neurofilament (NF) 150 kDa-specific antibodies. (F) Quantification of the total number of cells, and of the number of neurofilament-positive, BrdU-positive, and apoptotic cells in wild-type (black bars) and G5 Terc–/– embryos (white bars). Bars indicate the means ± s.e.m. obtained from the number of independent embryos shown in parenthesis. DRG development is normal in G5 Terc–/– embryos in terms of precursor proliferation, neuronal differentiation and survival. (G-I) Analysis of proliferation in wild-type and mutant ganglionic eminences. Immunodetection of BrdU after a 1 hour pulse in (G) WT and (H) G5 Terc–/– embryos, and (I) quantification of the numbers of BrdU-positive cells relative to the total number of cells, as determined in a number of randomly chosen regions of equal size from three wild-type (solid bar) and three G5 Terc–/– (white bar) embryos (I). Notice that there are no differences in BrdU labeling index. Scale bar: 100 µm.

View larger version (56K): [in a new window]   Fig. 3. In vitro growth kinetics of wild-type (WT) and G4 Terc–/– adult NSCs (n=3 independent cultures). (A) G4 Terc–/– primary and secondary spheres were much smaller than wild-type spheres as shown by a shift to the left in the size distribution (left). Phase contrast micrographs and mean average diameter (in µm ± s.e.m.) of floating neurospheres (right). Diameter is significantly lower (one-tailed Student's t-test, P<0.001) in G4 Terc–/– NSCs when compared to wild-type NSCs. (B) Fold increase in the number of cells 7 DIV after passage 2 (P2) and 3 (P3) is dramatically reduced in G4 Terc–/– (white bars) relative to wild-type (black bars) cells (one-tailed Student's t-test, P<0.05). (C) There are less BrdU-positive cells (red) relative to the total number of cells (DAPI stain) in mutant spheres (see quantifications in Fig. 5). Scale bars: A, 200 µm; C,D, 50 µm.

View larger version (42K): [in a new window]   Fig. 5. (A) Telomere length distribution by Q-FISH analysis in interphase nuclei of wild-type and Terc–/– G4 adult- and G5 embryo-derived cells in early cultures. Measurements are given in arbitrary units of fluorescence (a.u.f.). A shift to the left in the size distribution of telomerase-deficient compared to wild-type cells indicates a significantly higher proportion of telomeres with lower fluorescence signals. An average reduction in telomere length of around 60% is observed in both adult and embryonic telomerase-deficient cells. (B) Photomicrographs showing immunofluorescent detection of p53 (green) and DAPI staining (blue) in adult wild-type (WT) and G4 Terc–/– neurospheres (left) and in embryonic wild-type (WT) and G5 Terc–/– neurospheres (right). Wild-type neurospheres did not express detectable levels of p53 after 15 DIV. However, primary neurospheres derived from G4 Terc–/– adult mice cultured for the same time showed nuclear p53 staining. No signs of apoptosis were observed. p53 immunostaining of embryonic wild-type and G5 Terc–/– neurospheres after 15 DIV showed no detectable p53 protein. (C) Percentages of p53 and BrdU immunopositive cells in the adult neurospheres of both genotypes showing an inverse relationship. Immunodetections were performed separately because p53 could not be detected by the primary antibody after the acid treatment needed for BrdU immunocytochemistry. Parallel staining in spheres obtained from p53 null mice yielded no fluorescence at all (data not shown).

View larger version (49K): [in a new window]   Fig. 4. In vitro growth kinetics of wild-type (WT) and G5 Terc–/– embryonic NSCs (n=3 independent cultures). (A) Size distribution of secondary spheres formed after 6 DIV showing no differences between wild-type and G5 Terc–/– cultures. (B) Cell growth in passages 2-8 (early cultures) is the same in both genotypes. (C,D) Phase contrast micrographs and mean average diameter of secondary neurospheres from wild-type and G5 Terc–/– embryonic cultures. (E,F) Representative photomicrographs showing immunofluorescent detection of BrdU incorporation (red) and DAPI staining (blue) in wild-type (WT) and G5 Terc–/– secondary neurospheres. (G) Percentages of secondary spheres formed out of an equivalent number (10,000 cells) of dissociated wild-type (black bars) and G5 Terc–/– (white bars) in early cultures, showing no apparent differences between the two genotypes. (H) Proliferation rates for wild-type (black bars) and G5 Terc–/– (white bars). Mutant NSCs have a normal proliferation rate. Scale bars: C,D, 200 µm; E,F, 50 µm.

View larger version (33K): [in a new window]   Fig. 6. Telomere length distribution by Q-FISH analysis in wild-type (A) and G5 Terc–/– (B) embryo-derived metaphases at different times in culture (DIV, days in vitro; PD, population doublings). Measurements are given in a.u.f. A shift to the left in the size distribution of telomerase-deficient cells with replication in vitro indicates a significantly higher proportion of telomeres with lower fluorescence signals. Notice the increase in the frequency of undetectable telomeres that show no fluorescent signal (sensitivity of the assay, around 200 bp; arrows; see E for quantification). (C,D) Representative photomicrographs of wild-type and aneuploid G5 Terc–/– DAPI-stained metaphases (blue) after hybridization with a Cy3-labeled telomeric probe (yellow). Notice decreased signal intensity as well as undetectable telomeres (red arrows) in G5 Terc–/– chromosomes. (E) Cytogenetic analysis in embryonic wild-type and G5 Terc–/– NSCs during expansion in vitro. Table shows the number of cytogenetic abnormalities per metaphase. Notice their increase with time in culture in the absence of telomerase (see example of a Robertsonian fusion in D, yellow arrows).

View larger version (47K): [in a new window]   Fig. 7. (A) Photomicrographs showing immunofluorescent detection of BrdU incorporation (red), p53 (green) and DAPI staining (blue) in embryonic wild-type (WT) and G5 Terc–/– NSCs cultured for 110 DIV. Most nuclei in mutant neurospheres were immunoreactive for p53 while only a few wild type were positive. In spite of high levels of p53 protein, most G5 Terc–/– cells cultured for 110 DIV incorporated BrdU (see quantification in C). No signs of apoptosis are observed. (B) Cell growth in passages 15 to 20 (late cultures) is the same in both genotypes. Scale bar: 100 µm.

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