Journal: bioRxiv

Article Title: Vascular signals coordinate cerebellar circuitry development

doi: 10.64898/2026.02.18.706533

Figure Lengend Snippet: ( A ) Wnt5a mRNA expression is decreased in bEnd.3 cells treated with Dab1 siRNA compared to siRNA control, assessed with quantitative PCR (n = 5). ( B ) Wnt5a mRNA expression is increased in bEnd.3 cells after 24 h stimulation with exogenous Reelin (Reln) protein, assessed by quantitative PCR (n =4-5). ( C ) Wnt5a immunostaining in cerebellar cortex at P8. Rectangles 1 and 2 are magnified on the right. Arrows indicate Wnt5a signal in a pial (1) and a cerebellar cortical vessel below the PCL (2). Blood vessels are detected with IB4 and Collagen IV (ColIV). ( D ) Wnt5a staining of control and Dab1 iΔEC cerebellar cortices at P8 after tamoxifen (Tmx) administration from P1 to P3. ( E ) Plot profile of Wnt5a signal intensity in (D). ( F ) Quantification of the Wnt5a intensity in the EGL and ML together with the PCL in (D) (n = 7-10 animals per genotype). ( G ) EdU and Pax6 labeling of granule cell progenitors (GCP) primary cell culture stimulated with exogenous Wnt5a. Arrows indicate proliferating GCP (EdU+ Pax6+). ( H ) Quantification of the proportion of proliferating GCP (EdU+ Pax6+) in (G) (n =6). ( I ) EdU and Pax6 labeling of GCP primary cell culture stimulated with exogenous Wnt5a and blockade of the Fzd2 receptor with anti-Fzd2 antibody. Arrows indicate proliferating GCP (EdU+ Pax6+). ( J ) Quantification of the proportion of proliferating GCP (EdU+ Pax6+) in (I) (n =11-12). ( K ) anti-pH3 immunostaining was used to determine the cell cycle stage of granule cell precursors in control and Wnt5a iΔEC cerebellar cortices at P8 after Tmx administration from P1 to P3. ( L ) Quantification of proliferating neurons (pH3+) per EGL area in (K) (n = 8-12 animals per genotype). ( M ) EdU staining of P6 control and Dab1 iΔEC organotypic cerebellar cortices cultured after Tmx administration from P1 to P3 and stimulated with exogenous Wnt5a. ( N ) Quantification of EdU+ cells per EGL area in (M) (n = 6-11 animals per genotype). Abbreviations: EGL = external granule layer, GCL = granule cell layer, ML = molecular layer and PCL = Purkinje cell layer. Scale bars: C, G and I = 30 µm; squares in C =10 µm; D and K = 50 µm; M = 20 µm. Data are shown as mean ± SEM. * P <0.05, *** P <0.001, ns = non significant.

Article Snippet: TaqMan Gene Expression probes for mouse Wnt5a (Thermo Fischer, Mm00437347_m1), mouse mKi67 (Mm05911137_s1), mouse GADPH (Thermo Fischer, Mm 99999915_g1) and mouse ß2m (Thermo Fischer, Mm00437762_m1), the last two served as endogenous control for GCP and bEnd.3 cells respectively.

Techniques: Expressing, Control, Real-time Polymerase Chain Reaction, Immunostaining, Staining, Labeling, Cell Culture

Journal: bioRxiv

Article Title: Vascular signals coordinate cerebellar circuitry development

doi: 10.64898/2026.02.18.706533

Figure Lengend Snippet: ( A ) Representative immunoblot showing enhanced Wnt5a protein expression in bEnd.3 cells after 48h stimulation with exogenous Reelin (Reln) protein. Pan-cadherin was used as a loading control. ( B ) Relative protein levels quantification of Wnt5a long isoform and short isoform versus pan-cadherin housekeeping protein in (A) (n = 3). ( C ) Wnt5a mRNA detected with fluorescence in situ hybridization in cerebellar cortex at P8. Blood vessels are labelled with isolectinB4 (IB4). Higher magnification images are shown on the right. Arrows indicate Wnt5a signal in a pial (1) and a cerebellar cortical vessel below the Purkinje cell layer (PCL) (2). ( D ) Mki67 mRNA expression is increased in cerebellar granule cell progenitors (GCP) after 24 h stimulation with exogenous Wnt5a protein, assessed by quantitative PCR (n =6). ( E ) TUNEL and anti-Pax6 immunostaining of primary cell culture of GCP stimulated with exogenous Wnt5a protein. Arrows indicate apoptotic cells (TUNEL+ Pax6+). ( F ) Quantification of the proportion of apoptotic GCP in (E) (n =4). ( G ) Fzd2 mRNA labelled by fluorescent in situ hybridization in cerebellar cortex at P8. Higher magnification images show granule cell progenitors immunolabeled with anti-Pax6 and Purkinje cells with anti-Calbindin (Cb) respectively. Abbreviations: EGL = external granule layer, GCL = granule cell layer, ML = molecular layer and PCL = Purkinje cell layer. Scale bars: C and E = 30 µm; G = 25 µm; squares in C and G =10 µm. Data are shown as mean ± SEM. * P <0.05, ** P <0.01, *** P <0.001, ns = non significant.

Article Snippet: TaqMan Gene Expression probes for mouse Wnt5a (Thermo Fischer, Mm00437347_m1), mouse mKi67 (Mm05911137_s1), mouse GADPH (Thermo Fischer, Mm 99999915_g1) and mouse ß2m (Thermo Fischer, Mm00437762_m1), the last two served as endogenous control for GCP and bEnd.3 cells respectively.

Techniques: Western Blot, Expressing, Control, Fluorescence, In Situ Hybridization, Real-time Polymerase Chain Reaction, TUNEL Assay, Immunostaining, Cell Culture, Immunolabeling

Journal: bioRxiv

Article Title: Vascular signals coordinate cerebellar circuitry development

doi: 10.64898/2026.02.18.706533

Figure Lengend Snippet: ( A ) Purkinje cells (PC) stained with anti-Calbindin (Cb) antibody in control and Dab1 iΔEC cerebellar cortices at P8 after tamoxifen (Tmx) administration from P1 to P3. ( B ) Quantification of length (L)/width (W) PC soma in (A) (n = 8-9 animals per genotype). ( C ) Quantification of the mean molecular layer length in (A) (n = 4 animals per genotype). ( D ) Purkinje cells (PC) stained with an antibody against Calbindin (Cb) in control and Dab1 iΔEC cerebellar cortices in 4-week-old mice after Tmx administration from P1 to P3. ( E ) Quantification of the distance from the first branch point in the main dendrite to PC soma in D (orange dashed line) (n = 5-6 animals per genotype). ( F ) Quantification of number of branch points measured up to the tertiary dendrite in D (n = 9 neurons, 3 animals per genotype). ( G ) Cb immunostaining of PC primary cell culture stimulated with Wnt5a after 5-days (D) post culture (DPC). ( H - I ) Quantifications of the dendritic length (H) and number of dendritic branches (I) in (G) (n = 7-8 neurons). ( J ) Purkinje cells (PC) stained with anti-Calbindin (Cb) antibody in control and Wnt5a iΔEC cerebellar cortices at P8 after Tmx administration from P1 to P3. ( K ) Quantification of length (L)/width (W) PC soma in (J) (n = 8-12 animals per genotype). ( L ) Quantification of the mean molecular layer length in (J) (n = 5 animals per genotype). ( M ) Purkinje cells (PC) stained with an antibody against Calbindin (Cb) in control and Wnt5a iΔEC cerebellar cortices in 4-week-old mice after Tmx administration from P1 to P3. ( N ) Quantification of the distance from the first branch point in the main dendrite to PC soma in M (orange dashed line) (n = 6 animals per genotype). ( O ) Quantification of number of branch points measured up to the tertiary dendrite in M (n = 9 neurons, 3 animals per genotype). Abbreviations: ML = molecular layer, PCL = Purkinje cell layer. Scale bars: A = 20 µm; D and M = 50 µm; G and J = 30 µm. Data are shown as mean ± SEM. * P <0.05, ** P <0.01, *** P <0.001.

Article Snippet: TaqMan Gene Expression probes for mouse Wnt5a (Thermo Fischer, Mm00437347_m1), mouse mKi67 (Mm05911137_s1), mouse GADPH (Thermo Fischer, Mm 99999915_g1) and mouse ß2m (Thermo Fischer, Mm00437762_m1), the last two served as endogenous control for GCP and bEnd.3 cells respectively.

Techniques: Staining, Control, Immunostaining, Cell Culture