Arginylation Regulates Neural Crest Migration Figure 1. Wnt1-Ate1 mice have perinatal lethality. (A) A Wnt1-Ate1 pup that died at birth from breathing defects as shown in the Video S1. In such pups labored breathing leads to bloating (left) and prominent air accumulation in the stomach (right). (B) Wnt1-Ate1 pups that survive after birth (indicated by arrows) shown at postnatal day 5 (P5) are smaller than their wild-type littermates and are significantly less active at day 9 (Video S2), suggesting growth and behavioral retardation. (C) Wnt1-Ate1 (KO) mice that survive to adulthood are smaller than control (WT, top) and have short snouts and abnormally shaped skulls (bottom). doi:10.1371/journal.pgen.1000878.g001 dependent morphogenesis are often accompanied by the defects in palate and other craniofacial structures that originate from the neural crest cell lineage [33–36]. Palate defects in particular are known to correlate with breathing problems. It is hypothesized that shortened or cleft palates affect the separation between the digestive and respiratory tract, causing the inhaled air to be directed to the stomach rather than the lungs and resulting in air accumulation in the stomach and lethality similar to that seen in Wnt1-Ate1 mice at P0 [35,37,38]. To test whether Wnt1-Ate1 mice have defects in the palate and/or surrounding structures, we performed postmortem analysis of the pups that died at P0 with breathing abnormalities, by removing the lower jaw and visually analyzing the throat and the roof of the oral cavity. Several defects were observed during this analysis. First, the majority of the analyzed mutant pups had an abnormally large entrance to the nasopharynx and a reduced area normally covered by the soft palate (denoted by two shorter perpendicular arrows and one longer arrow in Figure 2A, respectively). In control animals (Figure 2A, left) the entrance to the nasopharynx was tightly shut and resembled a vertical slit surrounded by a small area of soft tissue. In the mutant animals (Figure 2A, right), the nasopharynx entrance appeared partially opened, assuming a triangular shape with a larger surrounding area, suggesting that this structure was prevented from closing either because of being structurally defective, or because the musculature that controls it did not operate properly. Closer observations (Figure 2B) showed that while in control mice (left) the entrance to the nasopharynx was partially closed by the soft palate, in the mutants (right) the soft palate in this area was either short (not shown) or missing (Figure 2B), leaving a gap that would be expected to permit the air to travel unrestrictedly throughout the passages connected to the oral cavity in this area. To confirm that the defects we see are indeed palate defects, we fixed the newborn Wnt1-Ate1 pups and their control littermates and sectioned them sagittally through the middle, in the area where the palate appears as the horizontal line separating the oral and the nasal cavity. Consistent with our morphological observations, palates in all the conditional knockout pups analyzed at P0 appeared short, unable to reach the area of the throat where the separation between the trachea and the esophagus occurs (Figure 2C). The average distance between the end of the palate and the epiglottis in the mutant mice was approximately 10 times larger than that in control (Figure 2D). Thus, Wnt1-Ate1 knockout mice, similar to other mouse models with neural crest migration defects, have severe malformations of the soft palate that likely cause the breathing problems and misdirected air flow resulting in early postnatal lethality (Figure 1A and Video S1). Since Wnt family genes, in addition to the neural crest, have been implicated in the functioning of other organs, including lungs [39,40], we tested whether, in addition to the palate, other defects in Wnt1-Ate1 mice may contribute to the breathing problems and/or early lethality. Histological examination of the sagittal PLoS Genetics | www.plosgenetics.org 3 March 2010 | Volume 6 | Issue 3 | e1000878