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Figure 6. DS-epi1 has a tissue-autonomous role in cranial neural crest cell migration, adherence to fibronectin and cell polarization. (A,A’) Schemes for transplantation experiments. A CNC explant from an embryo injected with 300 pg GFP mRNA was homotypically grafted at stage 17. MOs were injected into the donor (A) or host embryo (A’). (B-E) Lateral view of embryos at stage 26. Grafted GFP+ CNC cells migrate ventrally when derived from control-MO- (B); however, they do not properly migrate when derived from Dse-MO-injected embryos (C). The CNC cell migration was normal when the host embryo was injected with control-MO or Dse-MO (D,E). Three independent experiments (n=3). (F) Scheme illustrating the culture of stage 17 morphant CNC explants on fibronectincoated plates.(G,G’) Two hours after plating (G), the control-MO-injected CNC explant exhibits collective cell migration in one direction (arrow). The inset shows a magnification of spread cells. After 4 hours (G’), the cells migrate in distinct streams (stars). (H,H’) Cells of Dse-MO-injected CNC explants detach from each other and fail to adhere to the fibronectin substrate. The inset depicts a magnification of the spherical cells. (I-K) Confocal microscopy of fixed CNC cells after 5 hours of explant culture on fibronectin. Phalloidin-488 and DAPI label F-actin and cell nuclei, respectively. The Dse-5MM-MO-injected control cell (I) exhibits lamellipodia at the leading edge (arrowhead) and stress fibers in the inner regions of the cell (arrow in inset). Dsemorphant cells (J) exhibit cortical networks of stress fibers and lack polarized protrusions. Co-injection of Dse-MO and 1 ng Dse* mRNA per embryo (K) restores the normal cytoskeleton and cell shape. (L,M) Quantification of cell spreading (L) and formation of polarized cell protrusions (M) in dissociated single, phalloidin-stained cells of CNC explants following 5 hours of culture on fibronectin. Cell spreading and polarized protrusions were quantified by calculating the cell size as the square number of pixels (ImageJ) and determining the percentage of cells with lamellipodia or filopodia, respectively. Uninjected and Dse- 5MM-MO-injected explants exhibit a similar extent of cell spreading and formation of polarized protrusions. The reduction in the cell size and the lack of lamellipodia and filopodia are rescued by the co-injection of Dse* mRNA in Dse-morphant explants. A minimum of 100 cells per sample were evaluated in each experiment. Number of independent experiments (n=3). (N) Cell-matrix adhesion of dissociated single CNC cells on fibronectin- or BSA coated plates. Following the co-injection of MO and 300 pg GFP mRNA, CNC explants from stage 17 embryos were dissociated in Ca2+/Mg2+-free medium and cultured for 45 min on fibronectin or BSA. The Dse-morphant cells exhibit decreased adhesion to fibronectin compared with the control and Dse-5MM-MO-injected cells. None of the analyzed cell samples exhibited significant cell adhesion to BSA. At least three independent experiments for each sample (n=3).BSA, bovine serum albumin. Indicated phenotypes are shown as follows: B, 10/12; C, 11/13; D, 7/7; E, 9/9; G, 30/34; H, 26/28. The scale is 100 um in G-H’ and 10 um in I-K. Statistical significance was determined using ordinary one-way ANOVA multiple comparisons test with Tukey correction**, p<0.01, ***, p<0.001, ****, p<0.0001. Image published in: Gouignard N et al. (2016) © 2016. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution license Larger Image Printer Friendly View

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