Fig. 5: The Cytoplasmic Domain of Sema4D is required for cell-autonomous migration and axon outgrowth.
From: Semaphorin heterodimerization in cis regulates membrane targeting and neocortical wiring
a Simultaneous downregulation of Semaphorin 4D by shRNA reverses Semaphorin 7A rescue of neuronal migration in Satb2-deficient neurons. Neuronal migration profiles from E14-18 IUEs, presented in the same format described in Fig. 1. GFP, Cre and Cre + Sema7A are reproduced from Fig. 1 here for comparison. Scalebar in the bottom of GFP IUE is 100μm. Cell distributions for the different conditions are shown overlaid on the right with mean cortical position per nbrains, along with the mean of means ±SD. One way ANOVA with Tukey’s multiple comparisons test. GFP vs Cre padjusted < 0.0001, GFP vs Cre+Sema7A padjusted = 0.0604, GFP vs Cre+Sema7A+shSema4D padjusted < 0.0001, Cre vs Cre+Sema7A padjusted < 0.0001, Cre vs Cre+Sema7A+shSema4D padjusted = 0.9816, Cre+Sema7A vs Cre+Sema7A+shSema4D padjusted < 0.0001. b Quantification of midline crossing after simultaneous downregulation of Semaphorin 4D in Sema7A-rescued Satb2-deficient neurons. GFP, Cre and Cre+Sema7A images and data are reproduced from Fig. 1. Brown-Forsythe ANOVA with Dunnett’s T3 multiple comparisons test. GFP vs Cre padjusted = 0.0328, GFP vs Cre+Sema7A padjusted = 0.0589, GFP vs Cre+Sema7A+shSema4D padjusted = 0.0326, Cre vs Cre+Sema7A padjusted = 0.0292, Cre vs Cre+Sema7A+shSema4D padjusted = 0.9592, Cre+Sema7A vs Cre+Sema7A+shSema4D padjusted = 0.0277. Migration nbrains GFP = 6, Cre = 5, Cre + Sema7A = 8, Cre + Sema7A+ shSema4D = 4. c shows Cre+Sema7A+shSema4D midline panorama example. Panorama scalebar = 500μm, magnification scalebar = 100μm. d Midline panoramas of Sema4D and Sema7A shRNA electroporations and magnifications. Control scrambled shRNA (shScr), shRNA against Sema4D, shRNA against Sema4D + Sema4D cDNA (Sema4D Overexpression, OE), or shRNA against Sema4D plus a version of Sema4D cDNA lacking the intracellular domain -IC (Sema4D-ΔIC) were introduced into the cerebral cortex at E14 and axons crossing the midline were observed at E18. Scale bar in panoramas is 500 μm, while scale bar in midline magnification is 100 μm. e Quantification of midline crossing of shRNA electroporations. Kruskal-Wallis with Dunn’s multiple comparison test. scrambled shRNA (shScr) vs shSema7A padjusted = 0.0404, shScr vs shSema4D padjusted = 0.0253, shScr vs shSema4D+Sema4D OE padjusted = 0.6127, shScr vs shSema4D + Sema4D-ΔIC padjusted = 0.0017. In box and whisker plots (b, e) whiskers represent min-max with box bounds at lower and upper quartiles and center line at the median. Midline nbrains GFP = 5, Cre = 4, Cre + Sema7A = 8, Cre + Sema7A + shSema4D = 4, shScr = 5, shSema7A = 4, shSema4D = 4, shSema4D + Sema4D OE = 7, shSema4D + Sema4D-ΔIC = 4. f Neuronal migration profiles of after downregulation of Sema4D from E14-18 IUEs, presented in the same format described in Fig. 1. Scalebar in the bottom of shScr IUE is 100μm. Representative electroporations are shown adjacent to raw data points and half violin plots of the total distribution with mean of means±SD cortical position on the right. One way ANOVA with Tukey’s multiple comparisons. scScr vs shSema7A padjusted = 0.0069, scScr vs shSema4D padjusted < 0.0001, shScr vs shSema4D+Sema4D OE padjusted = 0.8358, shScr vs shSema4D+Sema4D-ΔIC padjusted = 0.001, shSema4D vs shSema4D + Sema4D OE padjusted < 0.0001, shSema4D vs shSema4D+Sema4D-ΔIC padjusted = 0.3605, shSema4D-Sema4D OE vs shSema4D+Sema4D-ΔIC padjusted = 0.0115. Migration nbrains shScr = 8, shSema4D = 8, shSema7A = 4, shSema4D + Sema4D OE = 6, shSema4D + Sema4D-ΔIC = 3. For simplicity, panels denote p < 0.05 as *; p < 0.01 as **; and p < 0.001 as ***. All source data are provided in the Source Data file.
