Fig 5. Tmem2-depleted NCCs are defective in FA formation and migration in HA-rich environments both in vitro and in vivo.

(A) Cell-based hyaluronidase assay. Tmem2-depleted and control O9-1 cells were cultured for 48 h on glass coverslips coated with fluoresceinated HA (FA-HA). HA degrading activity is revealed as dark areas in the fluorescent background. The level of HA degradation was also quantitatively compared between Tmem2-depleted and control O9-1 cells as described in Materials and Methods (bar graph). Data represent mean ± SD of the fluorescence intensity underneath a cell relative to that in cell-free area (n > 50 cells per condition pooled from three independent experiments). ***p < 0.001 by unpaired Student’s t-test. (B) O9-1 cells degrade substrate-bound HA at FAs. Cell-based hyaluronidase assays were performed for 16 h and cells were stained for vinculin. In control O9-1 cells, HA degradation occurs coincident with vinculin-positive FAs. In Tmem2-depleted O9-1 cells, HA degradation and FA formation are greatly diminished. The number of FAs per cell was quantitatively compared between Tmem2-depleted and control O9-1 cells (bar graph). Data represent mean ± SD (n >30 cells per condition pooled from three independent experiments). ***p < 0.001 by unpaired Student’s t-test. (C) Representative images of the migration of Tmem2-depleted and control O9-1 cells into a cell-free gap on Col1/HA mixed substrates. Top panels show images of gaps immediately after removal of the ibidi 2-well Culture-Insert. Other panels show images of gaps after a 24 h or 48 h incubation. Data are representative of three independent experiments. Bar graph shows the quantitative analysis of cell migration. Data represent the mean ± SD of the gap area covered by migratory cells relative to the area of the original gap (n = 5 per condition). ***p < 0.001 by two-way ANOVA with Bonferroni’s multiple comparison test. (D, E) Transverse sections of the neural tube in the caudal and cranial regions of E9.5 Tmem2^CKO;ZsGreen and control embryos were stained for vinculin (D) or N-cadherin (E). (D) Vinculin accumulation in the cellular cortex is reduced in NCCs in Tmem2^CKO;ZsGreen embryos. Insets show enlarged images of migrating NCCs in the boxed areas. (E) Cell surface expression of N-cadherin is reduced in NCCs in Tmem2^CKO;ZsGreen embryos. Lower panels show enlarge images of the boxed areas. nt, neural tube. Scale bars, 25 μm in A; 2.5 μm in B; 200 μm in C; 150 μm in D and E.