Journal: Cell reports

Article Title: Organization of Purkinje cell development by neuronal MEGF11 in cerebellar granule cells.

doi: 10.1016/j.celrep.2023.113137

Figure Lengend Snippet: Figure 1. Establishment and validation of MEGF11-KD in GCs (A) Schematic of domain structures of MEGF11. (B) Positions and sequences of the three candi- date MEGF11 shRNAs. (C) Images of HEK 293T cells transfected with pEGFP or pEGFP-MEGF11-UTR together with shRNA expression plasmids. (D) Diagram of AAV-SicoR-shRNA (top), and a representative image of dT expression in an ICR mouse subjected to AAV-SicoR-shScr injection (bottom). (E and F) qPCR results of Megf11, Megf10, and Megf12 mRNA in the cerebellum of non-injected control (CTR) mice (n = 12), or mice expressing shScr (shS, n = 12), type 1 (T1, n = 14), type 2 (T2, n = 12), or type 3 (T3, n = 9) MEGF11 shRNA, obtained using TaqMan probes detecting exons 2 and 3 regions (E) or a primer pair detecting 30 UTR (F). Results of Megf11 in (E) are also shown in (F) for direct comparison. (G) Schematic diagram showing the injection of AAV-SicoR-shRNA into lobule IV/V of PV-Cre mice (left), and a representative image of resultant dT expression (right). (H) Experimental design to test Megf11 mRNA expression in PCs and MLIs, or in GCs by the TRAP technique. (I) qPCR results of Megf11 mRNA purified from PCs and MLIs (n = 5 for shScr, n = 6 for shMegf11), or from GCs (n = 7 for shScr, n = 8 for shMegf11), of PV-Cre mouse cerebella with shScr or shMegf11 expression. Data were normalized to those of CTR mice (E and F) or of mice expressing shScr (I). Error bars in this and subsequent figures indicate SEM. The roman numerals in the images of this and subse- quent figures indicate the cerebellar lobules. ***p < 0.001, **p < 0.01, *p < 0.05, one-way ANOVA followed by the Bonferroni test (E and F) or Stu- dent’s t test (I). The exact p values for the datasets in this and subsequent figures are shown in Table S1.

Article Snippet: TaqMan probes used for Megf11, Megf10, Megf12, and Gapdh were Mm00553032_m1, Mm01257625_m1, Mm00546891_m1, and Mm99999915_g1 (Thermo Fisher Scientific), respectively.

Techniques: Biomarker Discovery, Transfection, shRNA, Expressing, Injection, Control, Comparison

Journal: Cell reports

Article Title: Organization of Purkinje cell development by neuronal MEGF11 in cerebellar granule cells.

doi: 10.1016/j.celrep.2023.113137

Figure Lengend Snippet: Figure 2. Alteration of cerebellar structures and motor behavior caused by MEGF11-KD (A) Experimental time course for structural ana- lyses. (B) Representative images of calbindin, NeuN, vGluT2, or vGluT1 staining in cerebellar slices expressing shScr (top) or shMegf11 (bottom). (C) Averaged line-scan profiles of calbindin signals across the PCL and the ML of lobule IV/V. Cal- bindin intensity in S1 and S2 was used to calculate the calbindin localization index (S1/S2). (D–M) Comparison of the calbindin localization index (D), ML thickness (E), calbindin intensity normalized by lobules III and VI (F), size of PC somas (G), number of PC somas (H), number of GCs in the ML (I), GL thickness (J), density of GCs in the GL (K), vGluT2-positive area density in the ML (L), and vGluT1 intensity in the ML (M), analyzed in lobule IV/V, where AAV was injected, between slices expressing shScr (n = 7) and those expressing shMegf11 (n = 8). (N) Experimental time course for behavioral anal- ysis. (O–Q) Hindpaw width (O) and stride length (P) in footprint analyses, and latency to fall in the rotarod performance test (Q) (n = 11 , 9 , and 11 ). ***p < 0.001, **p < 0.01, *p < 0.05, Student’s t test (D–M), one-way ANOVA followed by the Bonfer- roni test (O and P), or repeated-measures two-way ANOVA followed by the Bonferroni test (Q).

Article Snippet: TaqMan probes used for Megf11, Megf10, Megf12, and Gapdh were Mm00553032_m1, Mm01257625_m1, Mm00546891_m1, and Mm99999915_g1 (Thermo Fisher Scientific), respectively.

Techniques: Staining, Expressing, Comparison, Injection

Journal: Cell reports

Article Title: Organization of Purkinje cell development by neuronal MEGF11 in cerebellar granule cells.

doi: 10.1016/j.celrep.2023.113137

Figure Lengend Snippet: Figure 3. Recovery of abnormal pheno- types by MEGF11 O/E in neurons (A) Representative calbindin images in slices ex- pressing shScr (top) or shMegf11 (bottom) with either GFP expression (G, left) or MEGF11 O/E (O/E, right) in neurons. (B) Averaged line-scan profiles of calbindin sig- nals. Results in this figure are a comparison of four groups provided at the top. (C–J) Comparison of the calbindin localization in- dex (C), ML thickness (D), calbindin intensity (E), size of PC somas (F), number of GCs in the ML (G), GL thickness (H), density of vGluT2-positive area in the ML (I), and vGluT1 intensity (J). n = 6 mice each for all groups. (K–M) Hindpaw width (K) and stride length (L) in foot print analyses, and latency to fall in the ro- tarod performance test (M) (n = 6 , 6 , 7 , 7 , and 7 ). ***p < 0.001, **p < 0.01, *p < 0.05, one-way ANOVA followed by the Bonferroni test (C–L), or repeated- measures two-way ANOVA followed by the Bon- ferroni test (M).

Article Snippet: TaqMan probes used for Megf11, Megf10, Megf12, and Gapdh were Mm00553032_m1, Mm01257625_m1, Mm00546891_m1, and Mm99999915_g1 (Thermo Fisher Scientific), respectively.

Techniques: Expressing, Comparison

Journal: Cell reports

Article Title: Organization of Purkinje cell development by neuronal MEGF11 in cerebellar granule cells.

doi: 10.1016/j.celrep.2023.113137

Figure Lengend Snippet: Figure 4. Timing of MEGF11-KD triggering abnormal cerebellar network structures (A) Experimental time course for the different tim- ings of MEGF11-KD. (B) Representative calbindin images in slices ex- pressing shScr (top) or shMegf11 (bottom) by AAV injection at P3 (left), P15 (middle), or P21 (right). (C) Effects of MEGF11-KD by AAV injection at P3, P6, P15, or P21, on eight structural features listed at the top of the graphs. The effects are presented as percentages of quantitative values in slices expressing shMegf11 (n = 9 , 7 , and 7 ) to those in control slices expressing shScr (n = 8 each ). The results for P6 were obtained by recalculating data used in Figure 2. (D) Experimental time course for the analysis with later MEGF11 O/E. The results in (E)–(G) are a comparison of four groups provided at the bottom. (E–G) Averaged line-scan profiles of calbindin staining (E), calbindin localization index (F), and number of PC somas (G) in the four groups (n = 6 , 7 , 8 , and 6 ). ***p < 0.001, **p < 0.01, *p < 0.05, Student’s t test (C, compared with results in shScr) or one-way ANOVA followed by the Bonferroni test (F and G).

Article Snippet: TaqMan probes used for Megf11, Megf10, Megf12, and Gapdh were Mm00553032_m1, Mm01257625_m1, Mm00546891_m1, and Mm99999915_g1 (Thermo Fisher Scientific), respectively.

Techniques: Injection, Expressing, Control, Comparison, Staining

Journal: Cell reports

Article Title: Organization of Purkinje cell development by neuronal MEGF11 in cerebellar granule cells.

doi: 10.1016/j.celrep.2023.113137

Figure Lengend Snippet: Figure 5. MEGF11-KD-induced abnormal phenotypes in PCs observed earlier than those in GCs (A) Experimental time course for the analysis at P10 or P13. This figure shows results of four groups provided on the right. (B) Representative dT images in P10 (left) and P13 (right) slices expressing shMegf11. (C) Representative images of calbindin, NeuN, vGluT2, or vGluT1 staining in cerebellar slices of P10 or P13 PV-Cre mice expressing shScr or shMegf11. (D–K) Quantification of the calbindin localization index (D), ML thickness (E), calbindin intensity (F), size of PC somas (G), number of GCs in the ML (H), GL thickness (I), density of vGluT2-positive area in the ML (J), and vGluT1 intensity (K) (n = 6 mice each for all groups). ***p < 0.001, **p < 0.01, *p < 0.05, Student’s t test.

Article Snippet: TaqMan probes used for Megf11, Megf10, Megf12, and Gapdh were Mm00553032_m1, Mm01257625_m1, Mm00546891_m1, and Mm99999915_g1 (Thermo Fisher Scientific), respectively.

Techniques: Expressing, Staining

Journal: Cell reports

Article Title: Organization of Purkinje cell development by neuronal MEGF11 in cerebellar granule cells.

doi: 10.1016/j.celrep.2023.113137

Figure Lengend Snippet: Figure 6. Abnormal PF-PC synaptic trans- mission by MEGF11-KD (A and B) Representative traces and quantification of PF-EPSCs elicited by electrical stimulation at 36 mA and recorded from PCs in slices of non-in- jected CTR (black) or in slices expressing shScr (gray) or shMegf11 (dark red) at P15 (A, n = 11 , 14 , and 14 ) or P10 (B, n = 8 , 12 , and 11 ). The right panels show the PPR obtained by a pair of PF stimuli with different in- tervals (50–250 ms), and the inset shows the representative traces (50-ms interval; calibration: 100 pA, 20 ms). (C) Representative traces and quantification of mEPSCs recorded from PCs in slices expressing shScr (n = 14) or shMegf11 (n = 18) at P10. (D) Representative traces and quantification of PF- EPSCs induced by electrical stimulation at 36 mA and recorded at P10 in 1 mM extracel- lular Ca2+ concentration (n = 14 and 12 ). The reduced EPSC amplitudes in 1 mM Ca2+ are expressed as a percentage of EPSC amplitudes in 2 mM Ca2+ (bottom right). ***p < 0.001, **p < 0.01, *p < 0.05, two-way ANOVA followed by the Bonferroni test (A and B, right panel), one-way ANOVA followed by the Bonfer- roni test (A and B, other panels), or Student’s t test (D).

Article Snippet: TaqMan probes used for Megf11, Megf10, Megf12, and Gapdh were Mm00553032_m1, Mm01257625_m1, Mm00546891_m1, and Mm99999915_g1 (Thermo Fisher Scientific), respectively.

Techniques: Expressing, Concentration Assay

Journal: Cell reports

Article Title: Organization of Purkinje cell development by neuronal MEGF11 in cerebellar granule cells.

doi: 10.1016/j.celrep.2023.113137

Figure Lengend Snippet: Figure 7. MEGF11-KD-induced abnormal PC maturation through impaired PF-PC synaptic transmission (A) Experimental time course for the analysis with the blockade of synaptic transmission. The following results are a comparison of four groups provided at the bottom. (B) Representative calbindin images in slices ex- pressing shScr (left) or shMegf11 (right) with the expression of either GFP or TeTx. (C–J) Quantification of the calbindin localization index (C), ML thickness (D), calbindin intensity (E), size of PC somas (F), number of GCs in the ML (G), GL thickness (H), density of vGluT2-positive area in the ML (I), and vGluT1 intensity (J) in lobule IV/V (n = 6 , 9 , 6 , and 7 ). ***p < 0.001, **p < 0.01, *p < 0.05, one-way ANOVA followed by the Bonferroni test.

Article Snippet: TaqMan probes used for Megf11, Megf10, Megf12, and Gapdh were Mm00553032_m1, Mm01257625_m1, Mm00546891_m1, and Mm99999915_g1 (Thermo Fisher Scientific), respectively.

Techniques: Transmission Assay, Comparison, Expressing