A marked reduction in the expression ofSox6levels was observed in those cells transduced with theGsc-K3Rlentivirus when compared to cells infected with theWt-Gsc(Fig. required for optimal transcriptional activation of Gsc. Our results identify the first physiological pathway regulated by Wwp2in vivoas well as identify a unique non-proteolytic mechanism through which the Wwp2 controls craniofacial development. Ubiquitin is an important post-translational modifier with profound effects on various aspects of protein biology through altering turnover rate, subcellular localization and/or the activity of target proteins1,2. Ubiquitin is usually conjugated to target proteins through an enzymatic process mediated by E3 ubiquitin ligases, like Wwp2. As a member of the Nedd4-family of E3 ligases, Wwp2 contains a N-terminal C2 domain name, a WW domain name, and a C-terminal catalytic HECT domain name3. The HECT domain name of Wwp2 contains an active cysteine residue that mediates ubiquitin transfer from E2 enzymes to itself and then to target proteins. Wwp2 can facilitate the conjugation of ubiquitin to a number of proteinsin vitro46. However, thein vivofunction of Wwp2 is currently unknown. This is generally true for most members of mammalian Nedd4 family of E3 ligases since several studies have exhibited key functions for these proteins in regulating specific signaling pathwaysin vitroyet there is a dearth ofin vivodata to indicate that these ligases regulate specific physiological processes. Therefore, the ability to demonstrate a role for Wwp2in vivowould aid in emphasizing the biological relevance of the Nedd4 family of E3 ubiquitin ligases. To address what physiological Triisopropylsilane processes Wwp2 regulates, we generated mice deficient in Wwp2. We utilized gene-trap technology to generateWwp2-null mice using embryonic stem cells that contained the bacterial -galactosidase (LacZ) gene inserted in intron 34 of theWwp2locus (Fig. 1a). The insertion of LacZ at this location results in complete ablation ofWwp2transcript and protein levels in mice homozygous for the gene-trapped allele (Wwp2Gt/Gt) (Fig. 1bc). Analysis of litters given birth to to male and female mice heterozygous for the Wwp2 gene trap allele (Wwp2Gt/+) revealed that matureWwp2Gt/Gtmice are runted when compared to age and sex-matched wild-type (WT) controls (Fig. 1de).Wwp2Gt/Gtmice display abnormal cranial facial development that is characterized by the presence of a domed skull and a shortened snout (Fig. 1f). While we observed 100% penetrance in the development of craniofacial abnormalities inWwp2Gt/Gtmice, the severity of the phenotype was variable. A subset ofWwp2Gt/Gtmice developed a more profound craniofacial malformation characterized by a misaligned jaw and a twisted snout (Fig. 1g). Misalignment of the jaw in theWwp2Gt/Gtmice resulted in the chronic overgrowth of the mandibular incisors (Fig. 1g). To further quantitate the cranial facial abnormalities, we utilized quantitative computed tomography (-QCT) to scan the skulls of theWwp2Gt/Gtand WT mice. The nasal bones ofWwp2Gt/Gtmice were decreased by 20% in length and were twisted by 10 to 25 in those mice with the more severe asymmetric nasal bone phenotype (Fig. 1hI). == Physique 1. Craniofacial patterning defects are present EPLG6 inWwp2GT/GTmice. == a)Schematic depicting the position of B-gal insertion into the Wwp2 locus.b)Analysis of Wwp2 transcript levels by qPCR in WT mice (WT),Wwp2GT/+(GT/+) andWwp2GT/GT(GT/GT) mice. Values represent means s.d. (n=6 for each genotype)c)Immunoprecipitation-Western blot analysis depicting the absence of Wwp2 protein in theWwp2GT/GTmice.d)Photograph of four-week aged male WT andWwp2GT/GTmice.e)Body weight of male and female WT andWwp2GT/GTat four-weeks of age. Values represent means s.d. (n=6 for each genotype)f)Presence of shortened snout inWwp2GT/GTmice as shown grossly (left) as well as by alizarin red staining of WT andWwp2GT/GTskulls (right).g)Photograph depicting misaligned jaw and overgrown of mandibular incisor ofWwp2GT/GT(GT/GT) mice.h)CT analysis of skulls from WT mice as well asWwp2GT/GTmice depicting the shortened (middle) or twisted nasal bone (right) in theWwp2GT/GTmice.i)Quantitative analysis of the distances between the various landmarks in the skulls of four-week aged Wt andWwp2GT/GTmice. Values represent means Triisopropylsilane s.d. (n=8 for each genotype) (*p<0.001). Uncropped images of blots are shown inSupplementary Fig.S7. TheWwp2gene-trap allele allows LacZ to be expressed from the endogenousWwp2locus (Fig. 1a). Thus LacZ can be used as a surrogate marker to trackWwp2expressionin vivo. Staining of whole-mount Triisopropylsilane skulls isolated from WT andWwp2Gt/Gtneonatal mice revealed LacZ to be present in the nasofrontal region of the skull consistent with the type of craniofacial abnormalities observed inWwp2Gt/Gtmice (Fig.2a). Additional immunostaining analysis of WT andWwp2Gt/Gtskulls using an anti-X-gal antibody revealed specific LacZ staining patterns in regions of the skull that also positive for Safranin O, a stain used to detect cartilage proteoglycans (Fig. 2bc). Serial histological sections of Wt skull were analyzed byin situhybridization for the expression ofWwp2and immunostaining for Sox9.Wwp2could be detected in the cartilaginous regions of the skull at various stages of development that were also positive for Sox9 demonstrating.
