Figure S4 Ate1 deletion in Wnt1-Ate1 mice affects some but not all of the peripheral nervous system. Midbrain (m) and hindbrain (h) area of the control (WT) and Wnt1-Ate1 (KO) E12.5 embryo double-stained for the neuronal marker beta-III tubulin and Ate1. Ate1 is prominently missing from the midbrain and some peripheral nervous system structures (arrowheads). Found at: doi:10.1371/journal.pgen.1000878.s004 (3.87 MB TIF) Figure S5 Enteric neurons are positioned normally in Wnt1- Ate1 mice. Mid-areas of whole mount E16.5 guts stained for beta- III tubulin. Found at: doi:10.1371/journal.pgen.1000878.s005 (2.44 MB TIF) Figure S6 Impaired cell migration in Wnt1-Ate1-R26R neural crest explants. X-gal staining of control (left four panels) and Wnt1-Ate1 (right four panels) explants derived from E8.5 embryos show that while LacZ-expressing cells in control actively emigrate from the explant and reach the periphery of the expanding cell mass, cells in the mutant stay closer to the explant and do not appear to venture out on their own. 106 images on the periphery show higher magnifications of the regions boxed in the 26 images of the corresponding explants in the center, illustrating the cell emigration from the explant mass in control (left) and absence of such emigration in the knockout (right). 10 control and 6 mutant explants were analyzed. Found at: doi:10.1371/journal.pgen.1000878.s006 (4.60 MB TIF) Figure S7 Ate1 knockout in Wnt1-Ate1 embryos does not result in increased rates of apoptosis. Left, lower magnification image of a control DNaseI-treated embryo stained with TUNEL. Boxed regions outline the areas of the head (1), pharyngeal arches (2), and back (3), shown magnified in the right-hand panels for TUNEL- stained wild-type, Wnt1-Ate1, negative, and positive control embryos as marked. Levels of TUNEL staining in Wnt1-Ate1 and wild-type embryos are similar to those in the negative control and do not show any prominent differences from each other. 4 wild-type and 2 Wnt1-Ate1 embryos were analyzed. Found at: doi:10.1371/journal.pgen.1000878.s007 (1.82 MB TIF) Figure S8 Ate1 knockout in Wnt1-Ate1 embryos does not result in increased rates of apoptosis. Cross sections of wild-type (top) and Wnt1-Ate1 (bottom) embryos at E9.5 stained with an antibody to cleaved caspase 3. The area shown includes neural tube with adjacent population of migratory neural crest cells (see Figure S13C for X-gal staining of a similar section). Levels of cleaved caspase 3 staining in Wnt1-Ate1 and wild-type embryos do not show any prominent differences from each other and from the negative control with secondary antibody only. Found at: doi:10.1371/journal.pgen.1000878.s008 (1.22 MB TIF) Figure S9 Ate1 knockout does not affect neural crest cell proliferation rates. X-gal stained E9.5 wild-type (top) and Wnt1- Ate1 (bottom) embryos were sectioned and immunostained for cell proliferation marker phospho-histone H3. No differences in staining were observed between wild-type and Wnt1-Ate1 in X- gal stained tissues. Found at: doi:10.1371/journal.pgen.1000878.s009 (4.03 MB TIF) Figure S10 Ate1 knockout does not affect neural crest cell proliferation rates. Neural crest explants from E8.5 and E9.5 Ate1 knockout embryos labeled with BrdU after 2 days in culture. Levels of BrdU staining are similar in wild-type and knockout explants, suggesting no differences in proliferation rates of the neural crest cells. Found at: doi:10.1371/journal.pgen.1000878.s010 (3.57 MB TIF) Figure S11 Ate1 knockout results in reduced focal adhesions. Left panels, fluorescence staining of the edge of the cell monolayer Arginylation Regulates Neural Crest Migration moving into the wound with anti-paxillin (top), anti-talin (middle), and anti-focal adhesion kinase (FAK, bottom) to visualize focal adhesions. Knockout cells show a dramatic reduction in focal adhesion area and number. Bar, 20 mm. Middle panels, Western Blotting comparison of the focal adhesion protein levels in wild- type and Ate1 knockout cells. Loading was adjusted by weight of the packed cell pellets and verified by loading control–actin for paxillin, and tubulin for talin and FAK, as shown in the image. Right panels, quantification of the number of paxillin focal adhesions per mm of the wound edge and of the area of talin focal adhesions per cell leading edge shows that the number of prominent focal adhesions in wild-type exceeds that in the knockout by over 5-fold. Error bars for paxillin represent SEM for the measurements in 21 and 18 different images in WT and KO, respectively. Error bars for talin represent SEM for the measurements of 15 WT and 18 KO cells. Found at: doi:10.1371/journal.pgen.1000878.s011 (2.18 MB TIF) Figure S12 Ate1 knockout results in reduced focal adhesions. Left panels, fluorescence staining of single cells with anti-talin (top) and anti-paxillin (bottom) to visualize focal adhesions. Knockout cells show a dramatic reduction in focal adhesion area and number. Right panels, quantification of the number of focal adhesion area per cell shows that the number of prominent focal adhesions in wild-type exceeds that in the knockout by several fold. Quantifications shown represent measurements of 15 WT and 15 KO cells (paxillin) and 21 WT and 25 KO cells (talin). Found at: doi:10.1371/journal.pgen.1000878.s012 (2.07 MB TIF) Figure S13 Ate1 knockout results in reduced focal adhesions. Cross sections of wild-type (A) and Wnt1-Ate1 (B) embryos at E9.5 stained with antibody to talin. C, a similar area from an X-gal stained embryo showing the location of the migratory neural crest cell population used for the quantification of talin levels shown in D. Error bars represent SEM for measurements of 6 regions taken from each of the 5 wild-type and 4 Wnt1-Ate1 sections (30 wild- type and 24 Wnt1-Ate1 measurements), p-value,0.01. Found at: doi:10.1371/journal.pgen.1000878.s013 (2.98 MB TIF) Figure S14 Images of X-gal-stained E9.5 wild-type (left) and Wnt1-Ate1 (right) embryos used for obtaining higher magnifica- tion views shown in Figure 4 of the main text. Embryos were staged by somite count, indicated for each embryo on the top right next to the head. For some embryos (marked with X and asterisks) somite counts were not performed. These embryos were staged by comparison with their littermates as follows: *–littermates (shown on right) had 25 and 28 somites; **–littermate (shown on left) had 21 somites; ***–littermates had 22–26 somites; X–four embryos from the same litter lined up on the bottom for comparison. Found at: doi:10.1371/journal.pgen.1000878.s014 (4.29 MB TIF) Figure S15 Images of Sox10-stained E9.5 and E10.5 wild-type and Wnt1-Ate1 embryos used for obtaining higher magnification views shown in Figure 4 of the main text. Found at: doi:10.1371/journal.pgen.1000878.s015 (4.99 MB TIF) Video S1 Newborn Wnt1-Ate1 pup (left bottom) exhibits shallow, rapid breathing, eventually resulting in the accumulation of air in the abdominal cavity and death (see Figure 1 in the main text). In contrast its littermate control (right top) breathes regularly and exhibits no visible abnormalities. Found at: doi:10.1371/journal.pgen.1000878.s016 (4.61 MB AVI) Video S2 Four littermate mice at P9 show different behavior depending on the genotype. While control mice (two larger pups) move actively, Wnt1-Ate1 mice (two smaller pups, one on right and one hidden under the other pups at the beginning of the PLoS Genetics | www.plosgenetics.org 14 March 2010 | Volume 6 | Issue 3 | e1000878