suggest that the migration of these cells depends heavily on the intracellular mechanical components that drive cell adhesion and movement, and that this cell population is affected the most by the impairment in these mechanisms. This is consistent with the results of other studies, where mouse knockouts of the cell migration- related genes (such as fibronectin, cell adhesion molecules, etc., see [10] for review) leads to similar defects. Recent data suggest that many proteins involved in different physiological events are arginylated [23] and the list of identified arginylation targets is by far not complete. A prominent subset of the identified arginylated proteins (such as actin, etc.) are directly implicated in cell migration and it is very easy to suggest how lack of arginylation could lead to the defects in these proteins and affect cell migration by impairing the intracellular cytoskeleton-related machinery. For example, talin, a major focal adhesion protein, has been found to be arginylated on Ala1903, and the present study suggests that talin-containing focal adhesions are significantly reduced in Ate1 knockout, pointing to a possibility that talin arginylation is one of the key players in the Ate1 regulation of cell adhesion. Studies of the regulation of these, and other proteins by arginylation and its contribution to Ate1-dependent cell migration constitute exciting directions of further research. Materials and Methods Transgenic mice Mice with the exons 1–3 of the Ate1 gene flanked by LoxP sites (Ate1-floxed mice) were generated by introducing a targeting construct into the corresponding genome region in a cassette containing the floxed allele of the Ate1 genomic region fused with an frt-flanked Neo gene. The targeting vector was constructed using recombineering technique as described in [48]. A 12,375 bp genomic DNA fragment (position Chr 7: 130,302,694–130,315,068 Mouse Feb 2006 Assembly) containing exon 1–4 of the gene was retrieved from BAC clone RP23-92D13. A loxP sequence was inserted 657 bp upstream of exon 1 and A frt-neo-frt-loxP cassette was inserted into the intron 3, 505 bp downstream of exon 3. Thus a fragment of 5,019 bp genomic DNA containing exons 1–3 of the Ate1 gene was floxed (see Figure S1A). For ES cell targeting, the targeting vector was linearized with Not1 and electroporated into D1 ES cells derived from F1 hybrid blastocysts of 129S6 x C57BL/6J by Gene Targeting & Transgenic Facility at University of Connecticut Health Center. 192 G418 resistant ES colonies were isolated, and 32 clones were screened by nested PCR using primers outside the construct paired with primers inside the neo cassette. The sequences for primers used for ES cell screening were as follows: 59 arm forward primers: ATE Scr F1 (59- GTCTCAC- TTCCCTTCCTTAG -39) and ATE Scr F2 (59- ATTACCA- GTGCTCGGTGCTT -39). Reverse primers: Loxp scrR1 (59– GAGGGACCTAATAACTTCGT-39) and loxp scrR2 (59-GGA- ATTGGGCTGCAGGAATT-39). 39 arm forward primers: frt scr F1 (59-TTCTGAGGCGGAAAGAACCA-39) and frt scr F2, (59- CGAAGTTATTAGGTGGATCC-39); Reverse primers: ATE Scr R1 (59- tcagtggttctcaacctgtg -39) and ATE Scr R2 (59- caggggttacc- taagaccat -39); 7 out of 32 clones were PCR positive for both arms and were expanded. The genotypes were confirmed after ES cell expansion. For chimera generation and F1 mice genotype analysis three clones (1B3, 1A4 and 1H2) were aggregated with 8-cell embryos of CD-1 strain. The aggregated embryos were transferred to pseudopregnant recipients and allowed to develop to term. 25 chimeric mice were identified by coat color. Five chimeras (2 each for1B3 and 1A4, 1 for 1H2) were mated with CD-1 females to test for germline transmission. All of them were demonstrated as Arginylation Regulates Neural Crest Migration germline chimeras. The neo cassette was removed by mating the chimeras with ROSA26FLP1 (Jax stock#: 003946) homozygous females. To obtain Wnt1-Ate1 mice, Ate1-floxed mice were crossed with the mouse strain Tg(Wnt1-GAL4)11Rth Tg(Wnt1-cre)11Rth/J (The Jackson Laboratory) expressing Cre recombinase under the neural crest-inducing Wnt1 promoter. The following primers were used for genotyping of the final mouse strain (see Figure S1B for a typical genotyping gel): for Ate1-floxed allele, Ate1gtLoxF (59-TG- CCTCCAGCATTGGATGAA-39) and Ate1gtLoxR (59-CCATG- GGTCTCCAATTTGCA-39); For ROSA locus and Wnt trans- gene, primers recommended by the Jackson Laboratory were used as described on their web site for each corresponding strain. Ate1 antibody Rat monoclonal antibodies against a mixture of full-length bacterially expressed Ate1-1 and Ate1-2 were custom produced by Absea (http://www.absea-antibody.com). Reactivity with individ- ual Ate1 isoforms was verified by Western blots against purified bacterially expressed Ate1, isoforms 1–4. Clone 6F11 that interacts equally with all four Ate1 isoforms was used for section staining shown in Figure S3 and Figure S4. Anatomical and histological analysis For gross anatomical examination, mice at different postnatal stages were euthanized, dissected and observed for possible organ and tissue abnormalities, and individual organs were collected and compared between the mutants and the matching controls. For obtaining the images of the roof of the mouth and nasopharynx entrance shown in Figure 2 newborn mice were fixed in 4% paraformaldehyde in PBS and stored at 4uC. Fixed samples were washed in PBS and lower jaws were removed, and then the palate and the entrance of nasopharynx were observed under a dissection microscope. For histological analysis, mice and isolated mouse organs including X-gal stained embryos were fixed in 4% paraformalde- hyde in PBS, paraffin embedded, and sectioned. For observation and analysis of general organ morphology sections were stained with hematoxylin and eosin. For immunohistochemistry shown in Figure S3 and Figure S4, paraffin-embedded sections were deparaffinized with xylene, re-hydrated with sequential metha- nol:PBS series (95:5, 70:30, 50:50 and 30:70), washed with PBS, blocked with PBS supplemented with 0.1% Triton X-100 and BSA, and treated with anti-Ate1 (described above, 1:50), anti-beta- III tubulin (R&D Systems, MAB1195, 1:100), anti-phospho histone H3 (Santa Cruz Biotechnology, sc-8656, 1:500), anti- cleaved caspase 3 (Cell Signaling Technology, #9661, 1:100) or anti-talin (Santa Cruz Biotechnology, sc-7534, 1:500) antibodies in PBS supplemented with 0.1% Triton X-100. After washing with 0.1% Triton X-100 in PBS, samples were treated with fluorescent dye-conjugated secondary antibodies, washed with PBS and mounted in Aqua Poly/Mount (Polysciences, Inc., 18606). Samples were observed under a fluorescent microscope. Whole-mount skeletal staining Whole-mount skeletons of newborn and adult mice were stained with alizarin red S and alcian blue 8GS as described in [41]. Whole-mount immunostaining for enteric nerves Fixation and blocking in guts excised from embryos at E16.5 was performed as described in [49]. After blocking, guts were incubated with anti beta-III tubulin antibody for 2 days at 1:100 dilution in 5% BSA in TST buffer (20 mM Tris-HCl (pH 8.0), PLoS Genetics | www.plosgenetics.org 12 March 2010 | Volume 6 | Issue 3 | e1000878