EMBO/SourceData huggingface dataset:
f4(a) Luciferase activity levels upon cotransfection of a luciferase construct containing wild-type (WT) or mutated (Mut.) 3′UTR of FoxO3 with scrambled (Scr) control (Ctrl), pre-miR-132 or pre-miR-212 (n=9).(b,c) Expression levels of FoxO3 on mRNA (b) and protein levels (c) in hearts of WT and α-MHC-miR-212/-132 transgenic (TG) mice. M: Size marker (n=9-13).(d) FoxO3 mRNA levels in neonatal rat cardiomyocytes transfected with Scr Ctrl, anti-miR-212 and anti-miR-132 after phenylephrine (PE, 10 μM) treatment (n=6-9; P-values against Scr+PE).(e-g) Expression levels of atrogin-1 (e) and Mcip1 (f) and calcineurin phosphatase activity levels (g) in hearts of WT and α-MHC-miR-212/132 TG mice (n=5-9).(h) Luciferase activity levels showing the NFAT transcriptional activity in cardiomyocytes transfected with Scr Ctrl, pre-miR-132 or pre-miR-212 (n=5).(i,j) Mcip1.4 (i) and atrogin-1 (j) mRNA levels in WT and miR-212/132 null (KO) mice 3 weeks after transaortic constriction (TAC) or Sham operation (n=5-7 per group). All values represent mean±s.e.m. *P0.05; **P0.01; ***P0.005. #P0.05 compared with WT TAC. FC, fold change.
f5(a) mRNA expression levels of autophagic marker genes in hearts of wild-type (WT) and α-MHC-miR-212/132 transgenic (TG) mice (n=9-10).(b,c) Ratio of LC3II to LC3I (b) and p62 protein levels (c) in WT, miR-212/132 null (KO) and α-MHC miR-212/132 TG mice. M: Size marker (n=4-12).(d,e) Representative images (d) and quantification (e) of LC3:mCherry puncta (in red) in control and miR-212/132-overexpressing TG H9c2 cells under normal and serum/glucose-deprivation conditions (n=30). Nuclei are stained in blue with Hoechst33342.(f,g) Representative electron microscopy images (f) and quantification (g) of autophagic vacuoles in control and miR-212/132-overexpressing TG H9c2 cells under normal and serum/glucose-deprivation conditions (n=20).(h,i) Representative FACS plots (h) and quantification (i) of percent increase of autophagic flux in control and miR-212/132-overexpressing TG H9c2 cells under normal and serum/glucose-deprivation conditions (n=3-4 experiments). All values represent mean±s.e.m. *P0.05; **P0.01; ***P0.005. Scale bars, 50 μm in d and 2 μm in f. FC, fold change.
f6(a) Schematic representation of starvation experiment in mice. The white and grey parts on the time line represent the day and night phases, respectively. The arrow shows the start and duration of the starvation and the indicated time points of starvation are when animals are scored for body conditioning index.(b) LC3II to LC3I ratios in wild-type (WT) mice fed with normal diet and WT, miR-212/132−/− null and α-MHC miR-212/132 transgenic (TG) mice under starvation for 31 h (n=4).(c) Electron microscopy from ultrathin sections of resin-embedded heart biopsies of fed and starved WT, miR-212/132 null (KO) and cardiomyocyte-specific miR-212/132-overexpressing (TG) mice. White spots around the mitochondria (dark grey structures) are autophagic vacuoles. The electron-dense black spots shown with white arrows are autophagosomes. Scale bars, 4 μm.(d) p-mTOR/mTOR ratios in WT and miR-212/132 TG H9c2 cells 24 h after normal and starvation (serum/glucose-deprived) conditions (n=6).(e) p-mTOR/mTOR ratios in hearts of WT and α-MHC miR-212/132 TG mice fed with normal diet or 31 h after starvation (n=4). M, size marker. All values represent mean±s.e.m. **P0.01; ***P0.005; #P=0.11.
f7(a-c) Heart-to-body-weight ratios (a), cardiomyocyte diameters (b) and cardiac fibrosis (c) in Sham-operated mice and mice treated with intravenous injection of either scrambled control (Scr) or miR-132 inhibitors (Ant-132) after TAC. These mice were analysed 3 weeks after TAC (n=4-11). PSR, picrosirius red staining.(d-f) Echocardiography analysis of cardiac dimensions and function in Sham-operated mice and mice treated with intravenous injection of either control (Scr) or miR-132 inhibitors (Ant-132) after TAC. These mice were analysed 3 weeks after TAC for (d) fractional shortening, (e) end-diastolic area and (f) end-systolic area (n=4-9).(g-i) Cardiac FoxO3 protein levels in mice treated with intravenous injection of either control (Scr) or miR-132 inhibitors (Ant-132) 3 weeks after TAC and treatment (n=4-8). M: size marker. All values represent mean±s.e.m. *P0.05; **P0.01; ***P0.005; #P0.05 against TAC-control; ##P0.01 against TAC-control; ###P0.005 against TAC-control. Scale bars, 50 μm. DAPI, 4′,6-diamidino-2-phenylindole; FC, fold change; WGA, wheat germ agglutinin.(g-i) calcineurin activity (h) and Mcip1.4 mRNA levels (i) in mice treated with intravenous injection of either control (Scr) or miR-132 inhibitors (Ant-132) 3 weeks after TAC and treatment (n=4-8). M: size marker. All values represent mean±s.e.m. *P0.05; **P0.01; ***P0.005; #P0.05 against TAC-control; ##P0.01 against TAC-control; ###P0.005 against TAC-control. Scale bars, 50 μm. DAPI, 4′,6-diamidino-2-phenylindole; FC, fold change; WGA, wheat germ agglutinin.
Figure 3(A) Decrease in the fraction of chromosome arms exhibiting conservative (Consrv) DNA replication of the entire telomere length or part of the telomere (E+P) in U2OS cells after depletion of POLD3 or POLD4 by siRNA. si Ctrl, control siRNA; NE, cells expressing normal levels of cyclin E; OE, cells overexpressing cyclin E for four days. Bars represent means and standard errors of the mean from two independent experiments. For statistical comparisons the effects of PolD3 and Pold4 depletion were examined after grouping together the data from the NE and OE cells. PolD3 depletion reduced the fraction of conservatively replicated telomeres at a level of P=0.072; whereas for PolD4 depletion the decrease was significant at a level of P<0.025, as calculated using the paired t test.(B) Decreased telomere length following PolD3 or PolD4 depletion in U2OS cells expressing normal levels of cyclin E (NE) or overexpressing cyclin E (OE). The cells were transfected with control siRNA (si Ctrl) or siRNAs targeting POLD3 or POLD4 and, five days later, fixed and examined for telomere length by Q-FISH. Bars indicate means and standard error of the mean from more than 2,600 telomeres examined per condition. The differences between control and PolD3 or PolD4-depleted cells were significant (P<10-6) for both NE and OE cells, as determined by unpaired t tests.(C) Decreased telomere length following PolD3 or PolD4 depletion in parental U2OS cells. The cells were transfected with control siRNA (si Ctrl) or siRNAs targeting POLD3 or POLD4 and, five days later, fixed and examined for telomere length by Q-FISH. Bars indicate means and standard error of the mean from more than 2,800 telomeres examined per condition. The differences between control and PolD3 or PolD4-depleted cells were significant (P<10-6), as determined by unpaired t tests.(D) Increased frequency of chromosome end-to-end fusions following POLD3 or POLD4 depletion. U2OS cells expressing normal levels of cyclin E (NE) or overexpressing cyclin E for four days (OE) were transfected with control siRNA (siCtrl) os siRNAs targeting POLD3 or POLD4. For each condition, 25 metaphases were examined (representing about 1,900 high quality chromosome arms per condition); the percentage of chromosome fusions was determined for each metaphase and used to calculate means and standard errors of the mean. For statistical analysis, the number of chromosome end-to-end fusions were compared by chi square tests among the various conditions. Cyclin E overexpression led to a higher number of fusions (P<0.0005); PolD3 and PolD4 depletion also led to a higher number of fusions (P<0.001 and P<0.025, respectively).
Figure 3A. The levels of Sig1R in the lumbar spinal cord (LSC) or brain of Sig1R−/−, Sig1R+/−, or Sig1R+/+ mice at 5 months old. Representative immunoblots obtained from three independent experiments are shown. Asterisk denotes non-specific bands.B and C. Sig1R-deficient SOD1G85Rmice (SOD1G85R/Sig1R−/−) exhibited accelerated onset of the disease (B) and shortened survival time (C) compared to SOD1G85Rmice with one or two copies of Sig1R (SOD1G85R/Sig1R+/−, SOD1G85R/Sig1R+/+). Open circles indicate Sig1R knockout mice (Sig1R−/−) that never developed motor neuron disease but that were sacrificed at 396 days of age (n = 8). Mean onset or survival times of mice with ±SD are shown on the top of Kaplan-Meyer curves. **: p < 0.01, *: p < 0.05, log-rank test. n = 14 (SOD1G85R/Sig1R+/+), 29 (SOD1G85R/Sig1R+/−), 17 (SOD1G85R/Sig1R−/−).D and E. Body weights were lost earlier both in male (D) and female (E) of SOD1G85R/ Sig1R−/−mice. Data are shown as mean ± SD. *: p < 0.05 in SOD1G85R/Sig1R+/+ vs. SOD1G85R/Sig1R−/−, †: p < 0.05 in SOD1G85R/Sig1R+/− vs. SOD1G85R/Sig1R−/−. n = 8 (SOD1G85R/Sig1R+/+), 13 (SOD1G85R/Sig1R+/−), 8 (SOD1G85R/Sig1R−/−) in male (D), and n = 6 (SOD1G85R/Sig1R+/+), 16 (SOD1G85R/Sig1R+/−), 9 (SOD1G85R/Sig1R−/−) in female (E). The used animals were the same in the panels B and C. The animals used in the panels D and E were the same in the panels B and C. *: p < 0.05 in SOD1G85R/Sig1R+/+ vs. SOD1G85R/Sig1R−/−, †: p < 0.05 in SOD1G85R/Sig1R+/− vs. SOD1G85R/Sig1R−/−. Two-way ANOVA with subsequent post hoc Tukey's test.F. Decreased performance on the rotarod test was observed in SOD1G85R/ Sig1R−/−mice. The test was performed at 0-30 rpm with 0.1 rpm/s acceleration every week. Data are shown as mean ± SD. **: p < 0.0001 in SOD1G85R/Sig1R+/+ vs. SOD1G85R/Sig1R−/−, † †: p < 0.0001 in SOD1G85R/Sig1R+/− vs. SOD1G85R/Sig1R−/−. The used animals were the same in the panels B-E. Two-way ANOVA with subsequent post hoc Tukey's test.G and H. Motor function of WT (Sig1R+/+) and Sig1R knockout (Sig1R−/−) mice at 5 or 12 months old was evaluated by the rotarod test (0-30 rpm, accelerated at 0.1 rpm/s). Average time on the rotating rod was plotted. Error bars denote SD. Unpaired t-tests.
Figure 4A and B. N2a cells expressing SOD1WT, SOD1G85R or SOD1G93A (A), or spinal cords of WT or mutant SOD1 transgenic mice at end-stage (B) were fractionated as described in Materials and methods section (Cyto: cytoplasm, P1: nuclei and debris, Mito: mitochondria, MAM: mitochondria-associate membrane, P3: microsomal fraction). Proper fractionation was confirmed by the fraction specific markers as indicated, and also shown in Fig EV1 A-E. An asterisk denotes non-specific bands.C. Tissue and cell-type specific accumulation of mutant SOD1 at the MAM. Brain, liver, or primary astrocytes from SOD1G93Amice were fractionated and blotted as in Figures 4A and B (Upper panel). Quantification of SOD1 proteins at the MAM relative to ones in the cytoplasm was performed from immunoblotting data with an anti-human SOD1 antibody in (B) and the top panel of (C), and the data are plotted as mean ± SEM from three independent experiments (Lower panel). **: p < 0.01 vs. SOD1WTspinal cord. A one-way ANOVA with subsequent post hoc Tukey's test.Proper fractionation of these samples was confirmed in Fig EV 1F-H.D. Mutant SOD1 prevented association of ER with mitochondria. Isolated ER (P3: microsomal fraction) and mitochondria of N2a cells expressing wild-type (WT) or G85R (85) SOD1 for 48 h were mixed and incubated. Mitochondrial pellets after centrifugation were analyzed by immunoblotting using anti-PDI (ER marker) and anti-VDAC (mitochondrial marker), respectively. All these results were confirmed by three independent experiments.
Figure 5A and B. Time course analyses of mutant SOD1 levels at the MAM in SOD1G93Amousespinal cords (A) and brains (B). Immunoblots show levels of mutant SOD1 protein in the indicated fractions at various time points (left panels). Quantitative data at the right were plotted as mean ± SEM of three independent experiments. Arrows indicate a rapid reduction in SOD1 level at the MAM just following the onset of disease.C and D. Time course analyses of mutant SOD1 accumulation at the MAM in SOD1G85R (C) and SOD1G37R (D) mousespinal cords. Quantitative data from three independent experiments were plotted (bottom). The arrows in the bottom panel are the same as in A and B.E. Time course analyses of MAM-specific proteins at MAM and in the whole lysates of SOD1G93Amousespinal cords.
Figure 6A-I. Immunofluorescence staining of spinal cords and brains from non-transgenic (Non-Tg), SOD1 transgenic, or Sig1R−/−mice. Transverse sections of mousespinal cords (A, D-I) or sagittal sections of mouse brains (B, C) were stained using anti-Sig1R (white), βIII-tubulin (red) and IP3R3 (green) antibodies. Note that Sig1R and IP3R3 are co-localized in the motor neurons of the anterior horn (A) and the hypoglossal nucleus (B), and IP3R3 was not expressed in hippocampal neurons (C). Mutant SOD1 induced aggregation of Sig1R and mislocalization of IP3R3 in anterior horn neurons (D-G), while their abnormalities were not observed in SOD1WTmotor neurons (H). Mislocalization of IP3R3 was also observed in Sig1R−/−mouse spinal cords (I). Scale bars: 50 µm.J and K. Representative electron micrographs of the MAM (J) (double-headed arrows) in motor neurons of 12 months-old Non-Tg, SOD1G85R or Sig1R−/−mice. Note that ER-mitochondria contacted areas were reduced in both SOD1G85R or Sig1R−/−mice. Quantification of the mitochondria surface associated to ER was calculated in K. For quantification, 13-19 motor neurons and 224-316 mitochondria with MAM in each animal (n = 2) were analyzed. Data are expressed as mean ± SEM. Double asterisk means p < 0.0001 vs Non-Tg. A one-way ANOVA with subsequent post hoc Tukey's test. Scale bars: 300 nm.L. The levels for IP3R3 and calreticulin were decreased in the MAM fractions of Sig1R−/−mouse brains. MAM fractions and whole tissue lysates of Sig1R+/+ or Sig1R−/−mouse brains were immunoblotted. Representative blots from three independent experiments are shown.
Figure 7A. N2a cells were transfected with WT or mutant SOD1 in the presence or absence of IP3R3. Plotted viability of the cells measured by a neurotoxicity assay revealed that IP3R3 is involved in cell vulnerability against mutant SOD1. Data are expressed as mean ± SEM from three independent experiments, triplicated in each experiment. **: p < 0.01, *: p < 0.05.B-G. Calpain activity (B-D) were measured in N2a-IP3R3 cells expressing Sig1R-FLAG (B and E), SOD1 (C and F), or both (D and G). Mean ± SEM from three independent experiments is plotted. *: p < 0.05.B-G. intracellular ATP levels (E-G) were measured in N2a-IP3R3 cells expressing Sig1R-FLAG (B and E), SOD1 (C and F), or both (D and G). Mean ± SEM from three independent experiments is plotted. *: p < 0.05.H and I. Calpain activity in vivo was determined by the cleavage of spectrin αII. Calpain-cleaved 150 kDa fragment of spectrin αII in mouselumbar spinal cord or brain (H) was normalized to β-actin (I). Quantitative data in immunoblotting analyses are plotted as mean ± SEM from three independent experiments. **: p < 0.01.
Figure 8A and B. Cytoplasmic (A) or mitochondrial (B) Ca2+ flux was measured in N2a-IP3R3 cells treated with PRE-084 (5 µM) or NE-100 (5 µM) for 1 h prior to fluorescent imaging. Cytoplasmic and mitochondrialCa2+ flux were detected by fluo-4 and Case12-mito, respectively. The fluorescent intensity was normalized by the resting state at 0 s, and plotted as mean ± SD. n = 10 each. **: p < 0.01, *: p < 0.05. Two-way ANOVA with subsequent post hoc Tukey's test.C and D. Calpain activity were measured in N2a-IP3R3 cells treated with PRE-084 (5 µM) or NE-100 (5 µM) for 24 h. Data are plotted as mean ± SEM from three independent experiments. *: p < 0.05 vs. mock control. A one-way ANOVA with subsequent post hoc Tukey's test.C and D. intracellular ATP levels were measured in N2a-IP3R3 cells treated with PRE-084 (5 µM) or NE-100 (5 µM) for 24 h. Data are plotted as mean ± SEM from three independent experiments. *: p < 0.05 vs. mock control. A one-way ANOVA with subsequent post hoc Tukey's test.E and F. SOD1G93Amice were intraperitoneally administered with saline or PRE-084 (0.25 mg/kg, 3 times per week) from postnatal day 35 to 95. Lumbar spinal cord sections were stained by using anti-Sig1R (white), IP3R3 (green), and βIII-tubulin (red) at 95 days old. Arrowheads indicate the neurons with normal distribution of Sig1R and IP3R3. Scale bars: 50 µm.
Figure 1A,B, Immunostaining for CD31+ blood vessels in control (ctrl, A) and Gpr124KO (B) forebrains. Note the normal vasculature in the cortical hem (asterisk) of Gpr124KO mice.C,D, Immunostaining for Pax6 in control (C) and Gpr124KO (D) forebrains to reveal the lateral extension of the neocortex (dashed line) between the cortical hem (arrowhead) and the lateral ganglionic eminence (arrow). E, Quantification of the lateral extension of the cortex in control and Gpr124KO brains (mean±SEM; N=10; *** p<0.001).F,G Staining for EdU (red) and Ki67 (green) in control (F) and Gpr124KO (G) cortices at E13.5, 24 hours after EdU injection. H, Quantification of Ki67+ NPCs in the VZ (blue), SVZ/CP (grey) and of Ki67- neurons (cyan) generated from EdU-labeled NPCs in control and Gpr124KO embryos (mean±SEM; N=4 (control) and N=3 (Gpr124KO); * p<0.05, ** p<0.01). Full, dotted and dashed lines indicate basal and apical boundaries of the cortex or boundaries of the cortical zones, respectively. CP, cortical plate; IZ, intermediate zone; SVZ, subventricular zone; VZ, ventricular zone. Scale bar: 100 µmI,J, Immunostaining for Tbr2 (red) and Pax6 (green ) in control (I) and Gpr124KO (J) cortices. K, Quantification of newborn BPs in the VZ of control and Gpr124KO cortices (mean±SEM; N=4; * p<0.05). Full, dotted and dashed lines indicate basal and apical boundaries of the cortex or boundaries of the cortical zones, respectively. CP, cortical plate; IZ, intermediate zone; SVZ, subventricular zone; VZ, ventricular zone. Scale bar: 100 µmL,M, Immunostaining for Tbr2 (red) and Ngn1 (green) in control (L) and Gpr124KO (M) cortices. N,O Quantification of neurogenic (Ngn1+) RGs and BPs (N) or expanding (Tbr2- Ngn1-) RGs (O) in control and Gpr124KO cortices (mean±SEM; N=4; * p<0.05, *** p<0.001). Note that the fraction of total BPs is significantly reduced (Fig 1K) while the fraction of Ngn1+BPs is insignificantly reduced (Fig 1N), because the proportion of Ngn1+Tbr2+ cells within all BPs is slightly, though statisically insignificantly higher in the Gpr124KObrains (see Fig S2M). Full, dotted and dashed lines indicate basal and apical boundaries of the cortex or boundaries of the cortical zones, respectively. CP, cortical plate; IZ, intermediate zone; SVZ, subventricular zone; VZ, ventricular zone. Scale bar: 100 µmP,Q Stainings for Tbr2 (red) and for Tis21-GFP expression (green) in control (P) and Gpr124KO (Q) cortices. R,S Quantification of neurogenic (Tis21-GFP+) RGs and BPs (R) or expanding (Tbr2- Tis21-GFP-) RGs (S) in control and Gpr124KO cortices (mean±SEM; N=4; *** p<0.001). Full, dotted and dashed lines indicate basal and apical boundaries of the cortex or boundaries of the cortical zones, respectively. CP, cortical plate; IZ, intermediate zone; SVZ, subventricular zone; VZ, ventricular zone. Scale bar: 100 µm
Figure 2A,B, Staining for Isolectin B4+ blood vessels in control (A) and Gpr124ECcKO (B) forebrains. Asterisks mark the lateral ventricles.C,D, Staining for pimonidazole (PIMO, green) and Isolectin B4+ blood vessels (red) in ctrl (C) and Gpr124ECcKO (D) cortices.E,F, Immunostaining for Pax6 in control (E) and Gpr124ECcKO (F) forebrain sections to reveal the lateral extension of the neocortex (dashed line) between the cortical hem (arrowhead) and the lateral ganglionic eminence (arrow). G, Quantification the lateral extension of the cortex in control and Gpr124ECcKObrains (mean±SEM; N=6 for ctrl and N=8 for Gpr124ECcKO; * p<0.05).H,I, Staining for EdU (red) and Ki67 (green) in control (H) and Gpr124ECcKO (I) cortices at E13.5, 24 hours after EdU injection. J, Quantification of Ki67+ NPCs in the in VZ (blue), SVZ/CP (grey) and of Ki67- neurons (cyan) generated from EdU-labeled NPCs in control and Gpr124ECcKO embryos (mean±SEM; N=4; * p<0.05, ** P<0.01). Full, dotted and dashed lines indicate basal and apical boundaries of the cortex or boundaries of the cortical zones, respectively. CP, cortical plate; IZ, intermediate zone; SVZ, subventricular zone; VZ, ventricular zone. Scale bar: 100 µmK,L, Immunostaining for Tbr2 (red) and Ngn1 (green) in control (K) and Gpr124KO (L) cortices. M-O, Quantification of neurogenic (Ngn1+) RGs and BPs (M), newborn BPs (N) or expanding (Tbr2- Ngn1-) RGs (O) in control and Gpr124ECcKO cortices (mean±SEM; N=4; * p<0.05, ** p<0.01). Full, dotted and dashed lines indicate basal and apical boundaries of the cortex or boundaries of the cortical zones, respectively. CP, cortical plate; IZ, intermediate zone; SVZ, subventricular zone; VZ, ventricular zone. Scale bar: 100 µm
Figure 3A, qRT-PCR analysis of NSC-specific markers (Fabp7, Hes5) and neuronal markers (Dcx, Tubb3) in the Prom1+ fraction, presented as fold change compared to the Prom1- fraction) from WT cortices at E14.5 (mean±SEM; N=3 for Dcx; N=6 for all other genes).F, Western blot for HIF-1 (top), HIF-2 (middle) and a-tubulin as loading control (bottom) using forebrain lysates from E13.5 control (left) or Gpr124KOembryos (middle). HIF-1/2-transfected HEK293 cells served as positive control (right). G, Quantification of HIF-1 band intensities shown in panel F (mean±SEM; N=3; ** p<0.01).H,I, Immunostaining for HIF-1α (red) and DAPI (blue) in control (H, H', H'') and Gpr124KO (I,I',I'') cortices. Panels H',H'',I' and I'' are magnifications of the boxed areas in panels I and J, showing HIF-1α signal alone (H',I') or together with DAPI (H'',I''). Asterisks indicate autofluorescent blood cells. The dashed line indicates the basal boundary of the VZ. Scale bar = 100 µm. Full, dotted and dashed lines indicate basal and apical boundaries of the cortex or boundaries of the cortical zones, respectively. CP, cortical plate; IZ, intermediate zone; SVZ, subventricular zone; VZ, ventricular zone. Scale bar: 100 µmJ, qRT-PCR analysis of HIF-1 target gene expression in VZ cells from the cortex of control and Gpr124KO E13.5 embryos (mean±SEM; N=4; *** p<0.001). SVZ, subventricular zone; VZ, ventricular zone.
Figure 4A-F, Staining for Isolectin B4 (A-C) and oxidized thiols (D-F) in WT cortices at E10.5 (A,D), E11.5 (B,E) and E12.5 (C,F). Panels D-F are pseudocolor conversions of staining intensity (from low [purple] to high [red]). Arrowheads denote the position of the vascular front.G-I, Immunostaining for HIF-1α (red) and DAPI (blue) in WT cortices at E10.5 (G-G''), E11.5 (H-H'') and E12.5 (I-I''). Panels G',G'',H',H'',I' and I'' are magnifications of the boxed areas in their respective original panels. Arrows point to the border of lateral and dorsal cortex.J-L, Immunostaining for Glut1 of WT cortices at E10.0 (J), E11.5 (K) and E12.5 (L). Dashed lines indicate the apical border of the cortex. Scale bar: 100 µm (D,F,G,H,I, J-L) or 50 µm (I''), respectively.