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Figures
- Fig. 1.
Effects of altering SHH signaling in vitro: synergism between SHH and EGF signaling and induction of gene expression. (A) Proliferation response of plated nsps to different concentrations of EGF with (darker bars) or without (lighter bars) added SHH. The total number of BrdU+ cells per well, 1 week after the initiation of EGF/SHH treatment, is shown. P values from comparing control and +SHH samples are: 0.05 ng/ml EGF, P<0.001; 0.25 ng/ml EGF, P<0.01; 0.5 ng/ml EGF, P=0.001; 2.5 ng/ml EGF, P<0.001; and 5ng/ml EGF, P<0.5. (B) Quantification of BrdU+ cells in a 24-hour cell culture assay in the presence of 1 ng/ml of EGF and varying concentrations of SHH. P values from comparing control and +EGF samples are: 0.1 nM SHH, P=0.61; 0.5nM SHH, P=0.58; 1 nM SHH, P<0.5; 5 nM SHH, P=0.013; and 25 nM SHH, P=0.596. Similar results were obtained with 48-hour cultures. (C) Cloning dilution assay (at 1 cell/μl, 2000 cells per well in 1/3 conditioned media and 10 ng/ml of EGF in uncoated 6-well plates) after a 1 week treatment of nsps in 2.5 ng/ml EGF, in the presence of either 5nM SHH or 5 μM cyc. The total number of clones per well were counted in triplicate (P<0.0001). (D) RT-PCR analyses of E15.5 nsps, treated for 48 hours or 6 days in the presence of 5 nM SHH or 10 μM cyc. (E,G) Comparison of the percentage of BrdU+ cells at different concentrations of cyc, in the presence of 10ng/ml EGF, at E15.5 (E) and P2 (G) after 24 hours. (F,H) 48-hour cloning assays in presence of 10 ng/ml of EGF and cyc as indicated. (F) 12.5 E15.5 cells/μl plated in 1 ml media in uncoated 12-well plates (n=3) and (H) 25 P2 cells/μl plated in 2 ml media in uncoated 6-well plates (n=6). The experiments in A-H were performed with cells selected from the beginning in EGF only. Similar differences were obtained counting clones after one week in culture. (I) RT-PCR analyses of fresh dissected cortex of control and cyc-treated embryos. Regulation of Egfr expression by SHH was also seen at E17.5 and P3 (not shown). All RT-PCR assays were repeated 2-3 times. (J) Quantification of BrdU incorporation in acutely dissociated cultures plated in the absence of growth factors from HBC- (control) or cyc-treated neocorteces in vivo. Cells were grown in the presence of one-third conditioned media without additional cyc. Histograms in all figures show mean±s.e.m.
- Fig. 2.
Precursor proliferation and neurosphere-forming cells in Shh null brains. (A) Morphology of wild-type (left, dorsal view) and Shh-/- (right, side view) E18.5 dissected brains. Anterior is to the top. (B-E) Comparison of wild-type (B,D) and Shh-/- (C,E) cortical nsp, obtained at E15.5 (B,C) or E18.5 (D,E). (F,G) BrdU incorporation assay on E18.5 attached nsps. (H-K) Differentiation of Nestin+ Shh-/- nsps (H) into Tuj1+ neurons (I), GFAP+ astrocytes (J) and O4+ oligodendrocytes (K). (L) RT-PCR analyses of E18.5 wild-type and Shh null nsp cultures. (M) Quantification of wild-type versus Shh-/- E15.5 and E18.5 nsp size in clonal dilution assays (E15.5 wild type, n=12; Shh null, n=11; E18.5 wild type, n=11; Shh null, n=14; P<0.001 for E15.5; P<0.05 for E18.5). (N) Quantification of the number of BrdU+ cells (after 1 week in culture for both E15.5 and E18.5) after a 7 hour pulse in wild-type versus Shh-/- nsp (E15.5, P=0.006; E18.5, P<0.001). (O) Quantification of E15.5 and E18.5 wild-type versus Shh-/- nsp number (P<0.001 for both). Scale bar in E: 620 μm for A; 90 μm for B,C,E; 70 μm for D; 45 μm for F,G; 15 μm for H-K.
- Fig. 3.
Morphology and gene expression in Gli2 null brains. (A) Dorsal view of the morphology of wild-type (left) and Gli2-/- (right) dissected E18.5 brains. Anterior is to the top. (B,C) Comparison of lateral views of dissected cortex, and dorsal views of tectum and cerebellum in wild-type (B) versus Gli2-/- (C) brains. Arrows point to the posterior cortex in the two hemispheres, tectum and cerebellum. Gli2-/- mutant mice have an apparently normal choroid plexus (not shown). The ballooning of the telencephalic vesicle may be due to the inability of the tissue, which is thinner than normal, to sustain the same degree of intravesicular pressure. (D) Comparison of wild-type and Gli2-/- cortices seen in parietal sagittal sections stained for Hematoxylin and Eosin. The intermediate zone and cortical plate layers appear of normal size and cell density. (E-G) BrdU incorporation in wild-type (F) and mutant (G) cortices, and quantification of cell proliferation (E). The mean number of BrdU+ cells per section from wild-type and Gli2-/- animals is shown. For simplicity, the vz was considered as the zone in between the ventricle and ∼5-cell diameters away, and the svz as that in between ∼5- and 10-cell diameters from the ventricle (svz cells, P<0.05; vz cells, P<0.01). (H,I) Comparison of Neurod1 expression by in situ hybridization in the cerebellum of wild-type (H) and Gli2-/- (I) E18.5 animals. (J-S) Images of in situ hybridization analyses of sagittal sections from E18.5 (J-Q), and coronal sections from E15.5 (R,S), wild type (J,L,N,P,R) and Gli2-/- (K,M,O,Q,S) animals probed with Gli1 (J,K), Gli2 (L,M), Gli3 (N,O), Neurod1 (P,Q) or clone 53 (R,S). Neocortical Gli1 expression is absent in Gli2-/- animals, but a domain of its expression in the striatum was still present (inset, K). Note the small hippocampus in the Gli2-/- brain (Q, arrow), compared with in wild type (P). Forebrain pattern appeared largely unchanged as expression of the Pax6 anteroposterior gradient and of Dlx2 ventrally were not grossly affected (not shown). (T,U) Quantification of the number of Neurod1+ (T) and clone 53+ (U) cells in the dorsal telencephalon of wild-type versus Gli2-/- animals (Neurod1+, P<0.001; clone 53+, P<0.001). cb, cerebellum; cp, cortical plate; ctx, cortex; h, hippocampus; iz, intermediate zone; Med, medulla; st, striatum; svz, subventricular zone; tct, tectum; vz, ventricular zone. Scale bar in A: 800μ m for A; 1.3 mm for B,C; 75 μm for D; 50 μm for F,G,H,I; 130 μm for J-Q; 300 μm for R,S.
- Fig. 4.
Neurosphere cultures from Gli2 null brains. (A-D) Phase contrast images of representative cortical nsps cultured from wild-type (A,B) and Gli2-/- (C,D) animals at E18.5. (E) RT-PCR analyses of cortical nsps. Note the loss of Gli1 expression, the shift in the Gli2 mutant allele band (arrows), the reduced Gli3 expression, and the induction of Ihh and to a lesser extent Dhh in Gli2-/- cells. Hprt is shown as a control. (F-I) Expression of nestin in precursors of Gli2-/- nsps (F), of TuJ1 in neurons (G), of GFAP in astrocytes (H) and of O4 in oligodendrocytes (I) after differentiation. Nuclei were counterstained with DAPI. (J-M) Quantification of nsp size at E15.5 (J) and E18.5 (K). An average of 20 nsps from two independent experiments is shown (E15.5, P<0.05; E18.5, P<0.001). Given their rarity at E18.5, single Gli2-/- nsps were measured. (L,M) Quantification of nsps obtained in cloning assays. Three independent experiments are shown for E15.5 (P<0.001) and one for E18.5. Scale bar: 75 μm for A-D; 10 μm for F-I.
- Fig. 5.
Morphology and neurosphere cultures from Gli3 null brains. (A) Dorsal morphology of wild-type (left) and Gli3-/- (right) dissected brains at E15.5. Anterior is to the top. (B,C,E,F) Phase contrast images of representative primary cultures of wild-type (B,E) and Gli3-/- (C,F) animals at E15.5 (B,C) and E18.5 (E,F). Insets (E,F) correspond to TuJ1+ immunostaining of primary cultures. (D) RT-PCR analyses of E15.5 first passage nsps representing the rare survivors. Hprt was used as a loading control. RT-PCR analyses at E18.5 were not possible due to the lack of nsps.
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