Author Summary Formation of many organs during development depends on the coordinated migration of individual cells and cell layers throughout the embryo. The majority of migrating cells originate from the neural crest lineage that gives rise to peripheral neurons, ganglia, pigment cells, and cranio- facial structures, as well as parts of other organs in the body. Recent studies have implicated arginylation—a poorly understood protein modification—in the regulation of basic mechanisms that underlie cell migration. Here we test the role of arginylation in neural crest cell migration during mouse development by constructing and examin- ing a mouse model with arginylation-deficient neural crest cells. We find that these mice die at or soon after birth and exhibit severe defects in the development of distinct neural crest-derived structures. Our findings uncover a previously unknown mechanism of the regulation of neural crest cell migration during development, and shed light on general principles of neural crest migration in vivo. cultured with wild-type tend to ‘ride’ on the migrating cells rather than move on their own. Taken together, our data indicate that arginylation regulates tissue and organ morphogenesis by affecting the intracellular mechanisms that drive the migration of the mesenchymal cells of the neural crest lineage. Results Generation of Wnt1-Ate1 mice To produce an Ate1 conditional knockout we first generated an ‘Ate1-floxed’ mouse line with the first three exons of the Ate1 gene flanked by LoxP sites (Figure S1). We have previously shown that exons 1 and 2 are essential for the formation of all four Ate1 isoforms [25] and that deletion of the region encoded by these exons from the Ate1 sequence leads to the abolishment of Ate1 activity in yeast complementation assays [26]. Control experiments (data not shown) confirmed that deletion of the exons 1–3 using the Cre-driven recombination in the Ate1-floxed line resulted in embryonic lethality similarly to the previously described Ate1 knockout [9,27]. To produce a neural crest-specific Ate1 knockout, we crossed the Ate1- floxed line with a commercial mouse strain (Wnt1-Cre) where Cre recombinase is expressed under neural crest-inducing Wnt1 promoter, resulting in Ate1 deletion in subsequently derived neural crest and some other cell types. These mice, termed Wnt1-Ate1 mice, were used in the present study. To confirm the efficiency of the Ate1 knockout in these mice, we analyzed the Ate1 protein expression by immunohistochemistry of the sagittal sections of E12.5 and E16.5 Wnt1-Ate1 embryos probed with rat monoclonal antibody that recognizes all four Ate1 isoforms (see Figure S2 for antibody characterization and Figure S3 and Figure S4 for embryo staining). In control embryos Ate1 expression was observed in most tissues and organs (data not shown), consistent with our previous data that Ate1 is expressed throughout the embryo [9,25]. In contrast, Ate1 expression in the conditional knockout mice was prominently excluded from the midbrain region and the enteric nerves, as well as parts of the peripheral nervous system (Figure S3 and Figure S4), the structures that are derived from Wnt1 expressing cells [6,28]. No prominent areas with missing Ate1 expression were observed anywhere else in the embryo, however it is possible that Wnt1- expressing Ate1 knockout cells in other areas (such as, the somite regions along the back) mixed with other cell populations, making them difficult to detect. It is also possible that in such areas the Arginylation Regulates Neural Crest Migration normal levels of Ate1 are reduced, making the knockout cells poorly distinguishable from the background. To address this possibility and further confirm the efficiency of the Cre transgene expression, we crossed Wnt1-Cre mice to the R26R Rosa reporter mouse strain, in which prominent LacZ expression occurs in all Cre-expressing tissues [29–32]. X-gal staining of Wnt1-Cre embryos at E9.5 showed that Cre expression occurred as expected, with the majority of staining observed in the head and the somite region (Figure S14). Wnt1-Ate1 mice exhibit perinatal lethality Unlike the complete Ate1 knockout mice that die just past mid- gestation (E12.5–E14.5) [9,27], Wnt1-Ate1 mice survived until birth and exhibited perinatal lethality (Table 1). Over 60% of the pups died on the day of birth (P0), and another 24% died during the following 3 weeks, with less than 13% of mice surviving to adulthood. Such a variability in the mortality rate could be explained by the variability in the migratory patterns of neural crest cells between individual embryos (see below). The conditional knockout mice that died at P0 had severe breathing problems. Unlike their littermates they breathed frequently and irregularly and appeared to be gasping as if from the lack of air (Video S1). Within a few hours, these mice became bloated, with abnormally enlarged stomachs, and died (Figure 1A, left). Post-mortem dissection showed large amounts of air in their stomach (Figure 1A, right), suggesting that the air was misdirected there from the respiratory tract, possibly through the digestive system, and accumulated due to extensive breathing. Wnt1-Ate1 mice that died at later stages after birth (mostly within the first week, Table 1) showed no visibly abnormal breathing, however these mice appeared severely retarded, with delayed growth and significantly smaller body size compared to their littermates (Figure 1B) and apparent inability to actively move and explore the surrounding space (Video S2). Their appearance and behavior could be explained by malnutrition caused by non-lethal alterations in their ability to breathe and feed compared to their wild-type littermates. It is also possible, however, that these defects were caused by additional physiolog- ical and/or neurological abnormalities, caused by Ate1 deletion in other neural crest cell populations. The remaining 12.8% of Wnt1-Ate1 mice were able to grow to adulthood. These mice were fertile and able to produce healthy, surviving pups, however they were consistently smaller than their littermates and had visible facial abnormalities (short snouts and abnormally shaped skulls, Figure 1C). Thus, despite the variations in the severity of the Wnt1-Ate1 phenotype, over 87% of the conditional knockout mice died at or soon after birth, and all of them exhibited varying degrees of defects. Wnt1-Ate1 mice have craniofacial defects It has been previously found that mouse knockouts of the genes implicated in neural crest cell migration and neural crest- Table 1. Death/survival rates of Wnt1-Ate1 mice. Numbers (%) of Wnt1-Ate1 mice born 125 (100.0) died at P0 79 (63.2) doi:10.1371/journal.pgen.1000878.t001 P1–P21 30 (24.0) survived 16 (12.8) PLoS Genetics | www.plosgenetics.org 2 March 2010 | Volume 6 | Issue 3 | e1000878