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First published online 2 January 2008
doi: 10.1242/dev.010801

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Impaired generation of mature neurons by neural stem cells from hypomorphic Sox2 mutants

Maurizio Cavallaro^1,*, Jessica Mariani^1,*, Cesare Lancini^1,*, Elisa Latorre¹, Roberta Caccia¹, Francesca Gullo¹, Menella Valotta¹, Silvia DeBiasi², Laura Spinardi^1,3, Antonella Ronchi¹, Enzo Wanke¹, Silvia Brunelli^4,5, Rebecca Favaro¹, Sergio Ottolenghi¹ and Silvia K. Nicolis^1,

¹ Dipartimento di Biotecnologie e Bioscienze, Università di Milano-Bicocca, Piazza della Scienza 2, 20126 Milano, Italy.
² Dipartimento di Scienze Biomolecolari e Biotecnologie, Università degli Studi di Milano, Via Celoria 26, 20133 Milano, Italy.
³ Direzione Scientifica Fondazione IRCCS Ospedale Maggiore Policlinico, Mangiagalli e Regina Elena, Via Francesco Sforza 28, 20122 Milano, Italy.
⁴ Dipartimento di Medicina Sperimentale, Facoltà di Medicina, Università degli Studi di Milano-Bicocca, Via Cadore 48, 20052 Monza, Italy.
⁵ Stem Cell Research Institute, DIBIT H San Raffaele, Via Olgettina 58, 20132 Milano, Italy.

View larger version (40K): [in this window] [in a new window]   Fig. 1. Sox2 expression during in vitro neural stem cell differentiation. (A) In vitro neural stem cell differentiation scheme. (B) Specificity of the anti-Sox2 antibodies used in immunocytochemistry. Differentiation day 1 and 9 of wild-type (wt) and Sox2 conditionally deleted (null) cells are shown. Left, R&D antibody; right, Chemicon antibody (see also Fig. S1 in the supplementary material). A clear nuclear signal is visible in wild-type, but not in Sox2-null, cells. A slight cytoplasmic staining can be seen with the rabbit antibody (Chemicon) in wild-type and null cells, thus likely representing a nonspecific background. (C) Sox2 and nestin immunofluorescence on differentiation day 1. We used Chemicon's anti-Sox2 antibody, confirming with R&D antibody. (D) RT-PCR of Sox2 expression in undifferentiated neurospheres (Undiff. NSC), day 9 differentiated cells (diff. NSC) and P0 cortical cells. Top: cDNA dilutions from undifferentiated NSC (0.1, 0.25, 0.5, 1) allow an estimate of Sox2 expression levels in differentiated (diff. NSC) and cortical cells. Bottom: 18S RNA PCR, for normalization. (E) Western blot of Sox2 (R&D antibody) in normal (+/+) and mutant (MUT) undifferentiated neurospheres. Upper band: ubiquitous CP2 transcription factor (loading control). Sox2 protein in the mutant is 15-25% of normal by densitometry.

View larger version (61K): [in this window] [in a new window]   Fig. 2. Immunofluorescence for Sox2, neuronal and glial markers at differentiation day 9. (A) Sox2 and β-tubulin in normal cells. β-Tubulin-expressing cells show relatively high Sox2 positivity. (B) Sox2 and MAP2. Top: normal; bottom: mutant. MAP2-positive cells show significant Sox2 levels in both normal and mutant. (C) Sox2 and GALC, marking oligodendrocytes. (D) Sox2 and GFAP.

View larger version (47K): [in this window] [in a new window]   Fig. 3. β-Tubulin-positive cells are abnormal in differentiated Sox2 mutant cell cultures from adult mouse. (A) β-Tubulin immunofluorescence of normal (left) and mutant (right) day 9-differentiated cells. Bottom: DAPI. Many of the mutant poorly arborized, less intensely stained cells are barely visible in this low-magnification image. (B) Higher magnification of normal and mutant β-tubulin staining. In mutant, the arrowhead indicates a cell with well-developed neuronal morphology and long arborizations; arrows indicate abnormal cells with short processes and often weak β-tubulin staining typical of the mutant. (C) Time course of β-tubulin expression during differentiation. `Mut, well developed' indicates cells with long arborizations (B, wt or arrowhead in mutant); `mut, total': total β-tubulin-positive cells (including those indicated by arrows in B, mut). The abnormal phenotype is already observed at day 5, the earliest stage when significant numbers of β-tubulin-positive cells appear.

View larger version (64K): [in this window] [in a new window]   Fig. 4. Cells expressing mature neuronal markers are very reduced in differentiated Sox2 mutant cultures. Neuronal markers in normal and mutant cells at differentiation day 9 (NeuN/β-tubulin, rows 1, 2; MAP2/β-tubulin, rows 3, 4; PSA-NCAM, row 5). Most β-tubulin-positive cells in normal are positive for mature markers NeuN or MAP2; by contrast, very few mutant cells are positive for these markers. Histograms show percentage of cells positive for NeuN/β-tubulin, rows 1, 2; MAP2/β-tubulin, rows 3, 4; PSA-NCAM, row 5, with wild-type average of 100%. Results from n=4 normal and n=4 mutant mice (see Table S1 in the supplementary material).

View larger version (45K): [in this window] [in a new window]   Fig. 5. Cells expressing GABAergic markers are very reduced in differentiated Sox2 mutant cultures. Double-immunofluorescence with general neuronal markers (β-tubulin, rows 1, 2; MAP2, rows 3, 6; red), GABA (rows 1-4) and calretinin (5-6), in normal and mutant day 9-differentiated cultures. Histograms: percentage of positive cells, with wild-type average of 100%. Most β-tubulin-positive cells in normal (top) are GABA positive. In mutant (second row), two immature-looking β-tubulin-positive cells are very weakly GABA positive (or negative) (arrows), in contrast to the adjacent well-arborized GABA-positive cell. In normal cultures, most GABA- and virtually all calretinin-positive cells (rows 3, 5) express the mature neuronal marker MAP2; these cells are extremely reduced in mutant cultures (rows 4, 6 and histogram). Results from n=4 normal and n=4 mutant mice (see Table S1 in the supplementary material).

View larger version (57K): [in this window] [in a new window]   Fig. 6. Co-expression of neuronal and glial markers in individual cells in Sox2 mutant cultures. Double immunofluorescence (β-tubulin and GFAP) of normal (wt) and mutant (mut) day 9-differentiated cells. Typical wild-type neurons (β-tubulin positive) show extensive arborization, are closely associated with glia (which are GFAP positive), and are GFAP negative (top row). Rare cells with a very undifferentiated morphology are weakly positive for both markers (top, arrowhead). In mutant, various arborized cells are positive for both β-tubulin and GFAP (second row, arrowhead; third row, two arborized cells). Well-developed astrocytes are GFAP positive, but β-tubulin negative (arrows, rows 2, 4). In mutant, some intensely β-tubulin stained cells with neuronal morphology are also present (fourth row, arrowhead); these cells are GFAP-negative, as in wild type.

View larger version (70K): [in this window] [in a new window]   Fig. 7. Rescue of neuronal maturation in mutant cells by lentiviral Sox2 expression at early stages of in vitro differentiation. (A) Immunofluorescence for Sox2 (red) (R&D) and GFP (green), encoded by Sox2-IRES-GFP lentivirus, in cells infected at day 1 (d1) or day 4 (d4), compared with non-infected (ni) control. Immunofluorescences were performed the day after infection. Efficient infection (high proportion of GFP-positive cells) is coupled to clear Sox2 overexpression, which is observed at variable levels in transduced cells. (B) β-tubulin- and GFP immunofluorescence, at differentiation day 9, of mutant cells transduced with Sox2-GFP lentivirus at day 1 (d1), or day 4 (d4), compared with non-infected (ni) control, or the control infected with GFP-only transducing virus. Abundant well-arborized β-tubulin-positive cells (arrowheads indicate two of them) are observed in cultures transduced at day 1 with the Sox2-expressing virus, but not in cells transduced at day 4, or in controls. (C) GFP (green) and β-tubulin (red, top) or MAP2 (red, bottom) immunofluorescence shows that well-arborized neuronal cells (arrowheads) are always double-positive for the neuronal marker and for GFP, indicating that they derive from a Sox2-transduced cell. By contrast, some poorly developed neuronal cells (arrow) are not green, thus presumably originating from non-transduced cells. (D) Fold-increase in numbers of MAP2-positive and well-arborized β-tubulin-positive cells in mutant cells infected with Sox2-lentivirus at differentiation day 1, when compared with infection at day 4, or with control virus (day 1) expressing GFP but not Sox2. Values represent fold increase in numbers of MAP2-positive or well-arborized β-tubulin-positive cells (arrowheads in B,C for examples) relative to non-infected control. In day 1 transduced cells, numbers of well-arborized β-tubulin-positive and of MAP2-positive cells were 3.7% and 4.3%, respectively. In a parallel experiment using wild-type control cells mock-treated in the same way with a non-Sox2-expressing virus, the corresponding values were 5.7 and 6.2%. Data from two experiments in duplicate.

View larger version (48K): [in this window] [in a new window]   Fig. 8. Sox2 regulates GFAP expression and directly interacts with upstream regulatory DNA sequences of the GFAP gene in vitro and in neural cells chromatin. (A) Sox2 overexpression in differentiating cells represses endogenous GFAP expression. Double immunofluorescence (confocal microscopy) of day 9-differentiated cells transduced with Sox2-expressing lentivirus (Sox2-GFP; left) or control lentivirus (GFP; right) at day 1 (d1) or 4 (d4), with antibodies against GFP (green, revealing Sox2-IRES-GFP, or GFP for control virus), and the astroglial marker GFAP (red). Sox2-lentivirus-transduced cells show no, or very little, GFAP expression, whereas strongly GFAP-positive cells in the same field are Sox2-GFP-negative (left). By contrast, in cells transduced with control virus, GFP and GFAP colocalize within most cells. (B) Double immunofluorescence for GFAP and astrocytic markers S-100 (left) or connexin 43 (CX43; right) (Nagy and Rash, 2000) in differentiation day 9 cells; not transduced (nt) or day 1 transduced with Sox2-GFP-expressing lentivirus (d1). Virtually all cells positive for GFAP co-express S-100 or CX43 in non-transduced cells. In Sox2-transduced cells, numerous cells can be seen which have low or absent GFAP expression; and are positive for S-100 (left) or for CX43 (right), confirming their astroglial identity. (C) Putative Sox2-binding sites within a 0.6 kb region (0.6GFAP) just upstream to a previously investigated 2.5 kb GFAP promoter/enhancer. The sequence highlights the Sox2 consensus sequences investigated (red). Gfap is the oligonucleotide used in EMSA experiments in E; MutGfap is its mutated version (nucleotide substitutions in green). CDS: coding sequence. (D) Co-transfection experiments in P19 cells. Activity of a luciferase reporter gene driven by the 0.6 GFAP region linked to a TK minimal promoter (0.6GfapTK), or by the TK promoter only (TK), when co-transfected with Sox2 expression vector, or control `empty' vector (as indicated). Asterisk indicates a statistically significant difference (paired t-test, P<0.005). Results are average of n=4 transfections in duplicate. (E) EMSA with probes (indicated below the panels) encompassing the Sox2 consensus binding sites in the 0.6 GFAP region (Gfap), or the same probe mutated as in 8B (MutGfap), or a control probe carrying a Sox2-binding site from an Oct4 gene enhancer (Oct4). Nuclear extracts (P19; SOX2/COS, COS cells transfected with Sox2 expression vector; COS, untransfected COS cells), and competitor oligonucleotides with the molar excesses used for the competition experiments in the right panel, are indicated above the figure. (F) ChIP with anti-SOX2 antibodies of the 0.6 Gfap region in P19 and E12.5 spinal cord cell chromatin, compared with control SRR2 (which is bound by Sox2 in P19, but not in E12.5 spinal cord cell chromatin) (Miyagi et al., 2006) or nestin (bound by Sox2 in P19 and E12.5 spinal cord cell chromatin) (Tanaka et al., 2004; Miyagi et al., 2006) regulatory regions. The anti-Sox2 antibody precipitates both GFAP and SRR2 chromatin in P19 cells, but only GFAP chromatin in spinal cord cells, as expected. Antibodies are indicated above the panels; cell types and amplified DNA regions are indicated below the panels. Arrowheads indicate the positions of PCR bands corresponding to amplified target regions. Low-intensity diffused bands at the bottom are non-reacted primers. Results are representative of three experiments. unrel, unrelated control antibody against SV40 large-T antigen; Input chrom, input chromatin (not immunoprecipitated) - a positive control for the PCR reaction.

View larger version (46K): [in this window] [in a new window]   Fig. 9. Neurons expressing GABAergic markers are reduced in Sox2 mutant neonatal brains. (A,B) GABA (A) and calretinin (B) immunofluorescence of P0 cortical neurons (normal, left; mutant, right). Lower panels are counterstained with DAPI. (C) Percentage of GABA- or calretinin-positive cells in normal or mutant P0 cortical neurons. Results from n=3 normal and n=3 mutant mice.

View larger version (133K): [in this window] [in a new window]   Fig. 10. Abnormal calretinin- and GABA-positive neurons in E17.5 mutant brain. Calretinin (A-H) or GABA (I-N) immunohistochemistry in sections from normal (A-D,I-K) and mutant (E-H,L-N) forebrains. (A,E,I,L) General views of normal and mutant forebrain sections (dorsal region). Lower panels show progressively more enlarged details. (B,F,J,M) Details of the cortical region. The boxed regions in B and F are shown in C,D and G,H, respectively. Arrows in B indicate calretinin-positive neurons that reached the more external cortical layers following migration. Neurons in these positions are much rarer in the corresponding mutant section (F). C shows neurons that reached deep layers of the cortical plate; in the corresponding region of the mutant (G), no cells are seen. (D) Subcortical fiber bundles (along which calretinin-positive cells migrate from ganglionic eminences to cortex at earlier stages); no cells are seen here in the wild type. In the corresponding region of the mutant (H), calretinin-positive cells are still seen along this migratory route. (K,N) Enlarged details of J and M. In mutant (N), general disorganization of the GABA-positive neurons and of their arborizations is seen. V, ventricle; VZ, ventricular zone; CP, cortical plate.

View larger version (176K): [in this window] [in a new window]   Fig. 11. Decreased frequency and arborization of calretinin-positive neurons in adult mutant somatosensory cortex. (A,C) Calretinin immunohistochemistry reveals lower frequency of calretinin-positive neurons in mutant (C) versus wild-type (A) mice. (B,D) Higher magnification shows reduction of dendritic arborizations and of axonal varicosities (the swellings where transmitter-containing vesicles accumulate) in calretinin-positive neurons (asterisks) of mutant (D) versus wild-type (B) brains. Insets in B show, on the left, two vertically oriented varicose processes (arrows) and on the right a highly ramified calretinin-positive neuron (asterisk). Inset in D shows a poorly ramified calretinin-positive neuron (asterisk) with a vertically oriented smooth process (arrow).

View larger version (46K): [in this window] [in a new window]   Fig. 12. Impaired neuronal maturation in adult olfactory bulb of Sox2 mutant mice. (A) Immunofluorescence of BrdU/NeuN-double positive (red and green, yellow in overlay; first row) and BrdU-single-positive (red only; second row) cells in olfactory bulb sections. Histograms: percentage of BrdU/NeuN double-positive cells within the total BrdU-positive population in normal (WT) and mutant (MUT) olfactory bulb, in the entire bulb (TOT) or specifically in the granule layer (GL) and periglomerular layer (PGL) neuronal populations. Results from wild-type (n=4) and mutant mice (n=6). (B) Calretinin-positive cells (green) in olfactory bulb. Histograms: quantitation of calretinin-positive cells in normal (WT) and mutant (MUT) olfactory bulb within the periglomerular layer (four wild type, six mutants). (C) Confocal microscopy of calretinin-positive cells in the olfactory bulb reveals very limited arborization of mutant (mut) cells compared with wild type (wt). This morphology was clearly detected in two out of the four mutant mice analyzed.

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