Arginylation Regulates Neural Crest Migration Figure 3. Wnt1-Ate1 mice have defects in the frontal bones. (A) Top view of the skulls of the control (WT, top) and Wnt1-Ate1 (KO, bottom) pups at P0 stained with alizarin red S and alcian blue 8GS show a significant gap on top of the skull in the mutant pups, due to the significantly reduced frontal bones. Left images show the same pictures as those shown on right with the edges of the gap outlined. Arrowheads indicate the point of the opening of the gap used for the measurements shown in (B). (B) Measurement of the ratio of the gap width to skull width along the same axis (gap/skull) at P0 show that in the mutant (KO) unlike the control (WT) the gap occupies on average more than 1/3 of the skull (ratio 0.334+/ 20.018 (SEM, n = 5) in the mutant vs. 0.076+/20.016 (SEM, n = 12) in wild-type). (C) Skulls of adult Wnt1-Ate1 mice compared to their littermate controls show frontal bone abnormalities, including abnormal shape, incomplete cranial suture (arrows), and abnormal suture between the frontal bones (arrowheads). 2 wild-type and 2 mutant animals were analyzed. doi:10.1371/journal.pgen.1000878.g003 ranged between 22 and 26 in wild-type and between 21 to 28 in Wnt1-Ate1, consistent with the expected somite numbers at this stage. While the X-gal staining of the migrating neural crest cells in these embryos, especially with the larger somite numbers, was heavily masked by staining of other organs originating from Wnt1- expressing cells, such as midbrain (Figure S14), cell migration patterns could be clearly observed in the somite regions and near the pharyngeal arches, where the migrating cells appeared as streams of LacZ-expressing ‘dots’ arranged in different patterns from the back to the front of the embryo (Figure 4). In wild-type (Figure 4A and 4B), prominent populations of cells migrated from back to front as continuous lines (from the third pharyngeal arch down to the somites) and as triangular ‘streams’ directed toward the third and fourth pharyngeal arches and along each of the somites. In Wnt1-Ate1, several of these migration patterns were affected with different degrees of severity. Embryos with 24+ somites had altered cell distribution in the streams migrating toward the third and fourth pharyngeal arches and in the line migrating over the upper area of the trunk, which appeared diffuse, with fewer cells present in those areas (Figure 4C and 4D). In some embryos (Figure 4E), migration toward the pharyngeal arches appeared normal, but X-gal staining in the upper trunk area appeared weaker than control, indicating a reduced number of migrating cells in that area. In the embryos at a slightly earlier developmental stage (21–23 somites, Figure 4F–4H) these differences were more obvious, resulting in much lower overall levels of the X-gal-stained cells visible in these areas. Such extremely affected embryos also appeared smaller than wild-type embryos or knockout embryos with less severe phenotypes (see Figure S14). To further confirm this result, we performed in situ hybridiza- tion of whole mount embryos for the neural crest marker Sox10. Analysis of embryos at E9.5 and E10.5 showed similar changes in the migration patterns in Wnt1-Ate1 mice compared to control (Figure 4I and 4J and Figure S15). Finally, to obtain an independent confirmation of altered migration in Ate1 knockout neural crest cells, we isolated neural crest explants from E8.5 Wnt1-Ate1-R26R mice, and observed the migratory patterns of LacZ-expressing cells after incubation for 48 hourrs in culture (Figure S6). While in control explants (Figure S6, left panels), many of the LacZ-expressing cells during this time emigrated from the original cell mass and traveled to the periphery of the expanding explant as groups or individual cells, in the knockout LacZ-expressing cells traveled to a shorter distance as streams of cells mostly connected to the original explant, without venturing out on their own (Figure S6, right panels). All of the examined explants behaved consistently with each other, suggesting that this altered migration pattern occurs universally in response to Ate1 knockout. Therefore, consistent with the situation in situ, Ate1 knockout in the neural crest cells results in impairment of their migration in culture. Since most of the developmental defects observed in Wnt1-Ate1 knockout mice are related to the size reduction of the structures that are normally derived from the neural crest cells (such as palate or frontal bones), it is possible that in addition to decreased cell migration some of these defects are due to other reasons, such as decreased proliferation or increased apoptosis in the Ate1 knockout neural crest cells. To test for possible increase in apoptosis, we used TUNEL assay to stain E9.5 Wnt1-Ate1 embryos (Figure S7) or E12.5 Ate1 knockout embryos (not shown) and found no differences in the amount or distribution of the stain in the wild- type and knockout littermates. We also stained sections of Wnt1- Ate1 embryos with antibodies to the apoptotic cell marker cleaved caspase 3 and found no differences in the staining intensity or patterns between wild-type and Wnt1-Ate1 embryos (see Figure S8 for representative images). To test for possible changes in cell proliferation, we stained embryo sections for the cell proliferation marker phospho-histone PLoS Genetics | www.plosgenetics.org 5 March 2010 | Volume 6 | Issue 3 | e1000878