Arginylation Regulates Neural Crest Migration Figure 4. Wnt1-Ate1 mice have defects in neural crest cell migration. (A–H) X-gal staining of E9.5 control (A,B) and Wnt1-Ate1 (C–E) embryos derived from Wnt1-Ate1-R26R mouse line grouped by somite count (indicated on the top left for each embryo). A, lower magnification image of a control embryo at 24 somite stage. (B–E), back areas of different wild-type and Wnt1-Ate1 embryos with higher somite count, corresponding to the region boxed in (A), showing different neural crest migration patterns as described in the text. For Wnt1-Ate1, three littermates are shown, illustrating different pattern and severity of defects as described in the text; asterisk indicates the embryo, for which somite count was not performed and the staging relied on the comparison with its littermates shown on both sides. (F–H), back areas of wild-type and Wnt1-Ate1 embryos with lower somite count. Bar, 1 mm, for the images shown in (B–H). 10 wild-type and 10 mutant embryos were analyzed. See Figure S14 for whole embryo views. (I,J) In situ hybridization of E9.5 (I) and E10.5 (J) control and Wnt1-Ate1 embryos using Sox10 neural crest marker. 2 wild-type and 2 Wnt1-Ate1 embryos were analyzed for each developmental stage. See Figure S15 for whole embryo views. doi:10.1371/journal.pgen.1000878.g004 H3 and found no differences in the staining intensity or distribution between wild-type and Wnt1-Ate1 embryos (Figure S9). To further test this possibility, we isolated neural crest explants from E8.5 and E9.5 Ate1 knockout littermate embryos and, after two days in culture, labeled the actively proliferating cells with 5-bromo-2-deoxyuridine (BrdU). No difference was found between the numbers or distribution of actively proliferating cells in wild-type and Ate1 knockout explants (Figure S10). These results suggest that Ate1 knockout does not cause changes in the rates of cell proliferation or apoptosis during embryogenesis, pointing to the fact that the defects observed in Wnt1-Ate1 knockout mice are due primarily to the impairment in neural crest cell migration. Ate1 knockout cells of mesenchymal morphology migrate abnormally in culture To further characterize impairments in cell migration induced by Ate1 knockout, we analyzed the motility of cultured Ate1 knockout fibroblasts, derived from the back portion of the E12.5 Ate1 knockout embryos and immortalized by continuous passaging in culture as described in [9,24]. This model was chosen as one of the closest cell culture models of neural crest cell migration. PLoS Genetics | www.plosgenetics.org 6 March 2010 | Volume 6 | Issue 3 | e1000878