Arginylation Regulates Neural Crest Migration Figure 2. Wnt1-Ate1 mice have palate defects. (A) Observation of the roof of the mouth of control (WT) and Wnt1-Ate1 (KO) newborn pups at P0 shows a shorter soft palate area (longer vertical arrow on the right in each image) and an enlarged nasopharynx entrance (two shorter perpendicular arrows on the left and bottom in each image). Heads have been contrasted with trypan blue dye for better observation. 5 wild-type and 5 Wnt1-Ate1 pups were analyzed. (B) Closer view of the nasopharynx entrance (arrow) and the surrounding area in a control (WT) and aWnt1- Ate1 (KO) newborn pup at P0 with the missing soft palate (arrowhead). (C) Sagittal sections through the palate areas of the control (WT) and Wnt1- Ate1 (KO) newborn pups at P0. While in control the soft palate (P) reaches all the way to the epiglottis (E), in the mutant the palate is shorter and leaves a large gap of exposed tissue at the back of the throat. Scale bar, 1 mm. 4 wild-type and 5 Wnt1-Ate1 pups were analyzed. (D) Quantification of the distance from the end of the palate to the epiglottis in all the examined pups at P0 (4 wild-type and 5 mutants). In wild-type, the average distance was 48.6+/228.9 (SEM), and in the mutants this number increased over 10-fold to 501.0+/293.7 (SEM), indicating a significant overall shortening of the tissue of the soft palate. doi:10.1371/journal.pgen.1000878.g002 sections of the Wnt1-Ate1 embryos at P0 revealed no prominent abnormalities in the major organs or structures throughout the body (data not shown). Lungs in the Wnt1-Ate1 mice appeared collapsed compared to the wild-type, with little or no air in the alveolae and hemorrhaging in the air passages, however no visible lung defects were observed in the E16.5, E18.5 and E19.5 embryos recovered before birth that did not have a chance to breathe (data not shown). Therefore, given that Wnt1-Ate1 pups have breathing abnormalities that may indirectly affect the lungs, it appears unlikely that these mice have an independent structural lung defect. Since neural crest cells contribute to the formation of bone and cartilage in the head, we next examined the skeletons of newborn Wnt1-Ate1 and control mice at P0 by staining with alizarin red S and alcian blue 8GS that interact with bones and cartilage to color them red and blue, respectively [41] (Figure 3). While the overall bone structure and skeletal architecture appeared normal in Wnt1- Ate1 mice (data not shown), prominent abnormalities were seen in the development of the frontal bones, the neural-crest-derived parts of craniofacial skeleton that contribute to the top of the skull [32,42]. In control mice, frontal bones came close together, leaving only a narrow slit along the top of the skull (Figure 3A, top images). In contrast, in the mutant mice, frontal bones appeared smaller and narrower and were unable to meet on top of the skull, leaving a wide gap that exposed the cranial cavity beneath (Figure 3A, bottom images). Measurements of the ratios between the width of the gap and the width of the skull showed that in the mutant the gap occupied on average almost 1/3 of the skull width, an area almost 4 times larger than in wild-type (Figure 3B). This defect was observed in all the analyzed mutants, even those that did not exhibit breathing defects at birth. To test whether frontal bone defects are also seen in the Wnt1- Ate1 mice that survive to adulthood, we euthanized several adult Wnt1-Ate1 animals and matching wild-type controls and stained their skeletons similarly to the way described above for the newborn mice. Consistent with the defects seen in the newborns, adult surviving Wnt1-Ate1 mice had short, deformed frontal bones that had altered shape and rougher outline and often appeared incompletely closed (arrows in Figure 3C) and unable to meet in the middle (arrowheads in Figure 3C). Nasal bones also appeared shortened, leading to the overall shortening of the snouts as seen in the intact animals (Figure 1C). Therefore, some neural crest- derived parts of the craniofacial skeletons are affected in Wnt1- Ate1 mice regardless of the severity of other phenotypic changes seen in these mice. Since, in addition to craniofacial structures, Wnt1-Ate1 mice also show prominent Ate1 deletion in the enteric neurons and some deletion in the peripheral nervous system (Figure S3 and Figure S4), we tested whether the mutants have abnormal distribution of peripheral nervous system structures and gut neurons that might indicate defects in gut innervation often associated with neural crest-related developmental abnormalities (reviewed in [43–45]) by staining embryo sections and whole mount guts excised from E16.5 Wnt1-Ate1 embryos with antibody to the neuron projection marker beta-III tubulin. This staining revealed no abnormalities in the peripheral nervous system (Figure S4) or gut neuronal network (Figure S5), suggesting that the peripheral nervous system and enteric neurons, despite being Ate1-deficient, were able to position normally during embryogenesis. Wnt1-Ate1 neural crest cells show defects in migration patterns during development To address the question whether Wnt1-Ate1 mice exhibit any defects in neural crest cell migration, we used the Wnt1-Ate1- R26R reporter conditional knockout line and analyzed the distribution of LacZ -expressing cells in wild-type and Wnt1- Ate1-R26R embryos at E9.5. Several litters, whose embryonic stage was determined by counting the somites, were analyzed, and the comparisons were made between embryos with comparable somite numbers. In the analyzed embryos, the somite numbers PLoS Genetics | www.plosgenetics.org 4 March 2010 | Volume 6 | Issue 3 | e1000878