Discussion Our data show that knockout of the arginyltransferase Ate1 in the cells of the neural crest lineage results in multiple morphogenic defects and perinatal lethality in mice. It has been previously shown that complete Ate1 knockout in mice leads to embryonic lethality and defects in cardiovascular development and angio- genesis [9] that are reminiscent of the defects seen in mouse models with knockout of genes implicated in cell adhesion and migration during embryogenesis [10]. Here we show for the first time that Ate1 deletion in the migratory subpopulations of the neural crest cells leads to delayed development and reduced size of the neural crest-derived organs and tissues, suggesting that Ate1- dependent migration of the neural crest cells is essential for normal embryogenesis. We have previously shown that Ate1 knockout embryonic fibroblasts have leading edge defects that arise from abnormalities in the non-arginylated actin cytoskeleton [24]. Here we found that in addition to the abnormal leading edge Ate1 knockout cells also have impaired adhesion to the extracellular matrix and that the rate of their motility is significantly delayed due to the impaired intracellular machinery rather than to the extracellular environ- ment. It is possible that the defects seen in Wnt1-Ate1 embryos may have additional underlying mechanisms, including impaired epithelio-mesenchimal transformation that could hinder the size of the migratory cell population, or abnormal responses to the signals that coordinate the migratory events on the organismal level. However, the intracellular changes observed in response to Ate1 knockout are likely sufficient to induce major morphogenic defects even without perturbations in other important developmental processes. Wnt1-Ate1 mice show defects of varying severity, with a small fraction of mice surviving to adulthood. Our data show that the impairments of neural crest cell migration patterns observed in Wnt1-Ate1 knockout are variable from embryo to embryo, suggesting that local variations in the migration speeds of the Ate1 knockout cells can contribute to the phenotype variability. Our data on co-culture of wild-type and Ate1 knockout cells further suggest that Ate1 knockout cells during migration and tissue expansion can ‘ride’ on the neighboring expanding wild-type tissues, reaching further destinations in the embryo than they could reach on their own. This seems to be especially likely in the case of the enteric neurons that migrate as individual cells on the walls of the expanding gut. Indeed, the migrating speeds of these neurons have been calculated to exceed the average speed of migrating mesenchymal cells in culture and in situ by at least a factor of 2 [10], suggesting that the expanding tissues greatly aid them along in reaching their destination and covering the entire gut wall. Consistent with this fact, Wnt1-Ate1 mice have no defects in gut innervation, while having significant defects in craniofacial migratory neural crest tissues, suggesting that gut neuron precursors in Wnt1-Ate1 knockout reach their destinations mostly by ‘riding’, which is not affected by Ate1 knockout–related impairments in cell migration speeds. In addition to the neural crest-derived craniofacial structures, Wnt1-expressing cells in the neural tube give rise to the peripheral nervous system and large parts of the brain, including the entire midbrain. In Wnt1-Ate1 mice parts of the peripheral nervous system and the midbrain region are prominently devoid of Ate1 expression (Figure S3 and Figure S4). While we observed no morphological defects in the midbrain or the layout of the peripheral nervous system in the mutant mice, the Wnt1-Ate1 pups that survived for 1–3 weeks past birth exhibited severe behavioral retardation, delayed growth, and apparent difficulty Arginylation Regulates Neural Crest Migration with feeding. While some of these defects, especially the feeding abnormalities, could result from the shortened palates that prevent normal food intake, others could result from the neurological problems, such as reduced innervation leading to the impaired neuromuscular functions and reduced expansion in the thoracic cage (which may result in breathing abnormalities and lung collapse), or from the defects in the sensory organs that are controlled by the midbrain, and possibly from other midbrain- related physiological abnormalities. It has been previously suggested that Ate1-dependent arginylation plays an important role in nerve growth and regeneration [17,47], leading to an interesting possibility that some of the Wnt1-Ate1 mice may have neurological abnormalities of varying severity originated from the developmental defects not directly related to the neural crest cell migration. This possibility requires further study. Despite prominent Wnt1 expression in the head and chest area and somites, we saw no visible reduction in Ate1 staining in such neural crest-derived structures as teeth, endocrine glands, and heart, and no abnormalities were detected in these structures in Wnt1-Ate1 knockout mice. It is possible that in these areas Wnt1- expressing cells exist in a mixture with other cell populations, making them difficult to distinguish on tissue sections. In such case, their migration through the embryo could be heavily aided by surrounding tissues and impairment in their migratory machinery would be less visible. It is also possible that these cells normally express lower levels of Ate1, making the Ate1 knockout and the corresponding migratory defects difficult to detect. Further investigation of these differences may lead to important discoveries of the role of arginylation in the development of these organs. Our data show that uniform impairment of the intracellular arginylation-dependent mechanisms of cell migration produces different effects in different Wnt1-expressing migratory cell subpopulations during development. These differences may be explained by the diverse mechanisms by which these subpopula- tions migrate in situ (Figure 8). Cells of the cranial neural crest migrate in large groups from the upper trunk region into the craniofacial areas and contribute to the soft palate, frontal bones, and other craniofacial structures. Vagal and sacral neural crest cells migrate individually along the developing gut from the back and the front, to give rise to the enteric nervous system. Trunk neural crest cells migrate over relatively short distances forming pigment cells, ganglia, and some other structures. Based on the timing of the corresponding developmental events and the distances these cells cover overall, it has been calculated that migration in these subpopulations occurs at different speeds [10], and therefore such migration on the organismal level would be differentially affected by impairment of the same intracellular mechanisms. In the trunk, where cells migrate slowly over short distances, 2–4-fold reduction in the motility rate would be practically unnoticeable on the developmental scale. Consistent with this, we did not observe significant defects in the structures that develop from these cells due to Ate1 knockout. In the gut, cells migrate individually and while the migration speed of these cells is believed to be the fastest, up until now it has been unknown how much of this speed is contributed by the movement of the expanding tissues rather than the migrating cells themselves. Our data that enteric neurons are positioned normally despite the Ate1 knockout in these cells points to a possibility that their migration is heavily aided by tissue growth and gut elongation and that the cells themselves do not need to move fast to reach their final position. Cells contributing to craniofacial skeleton migrate as large subpopulations, with predicted speeds close to those experimen- tally observed for neural crest and other mesenchymal cells in culture and in situ (,40 mm/hour, see [10] for review). Our data PLoS Genetics | www.plosgenetics.org 10 March 2010 | Volume 6 | Issue 3 | e1000878