Embryonic development requires a complex series of relative cellular movements and shape changes that are generally referred to as morphogenesis. 1995; Ashery-Padan et al., 2000; Smith et al., 2009), (Wawersik et al., 1999) or the Fgf receptor adaptor and mutants is definitely variable (Wawersik et al., 1999; Gotoh et al., 2004), either germline or presumptive lens conditional mutation of gives a very consistent failure of lens placode invagination (Grindley et MLN8054 tyrosianse inhibitor al., 1995; Ashery-Padan et al., 2000; Smith et al., 2009) that contrasts, for example, with lens placode conditional deletion of embryo, as well as with the neuroepithelial cells that contribute to the hinge-point region during neural tube closure (Lecuit and Lenne, 2007). The traveling pressure behind AC is the activation of apical non-muscle myosins (Dawes-Hoang et al., 2005), which induce contraction of actin-myosin complexes and reduce the apical area. Analysis of gastrulation offers identified that non-muscle myosin activation during AC is definitely induced by Rho kinase (Rok), which is definitely triggered and apically concentrated by RhoGEF2 and Rho1 (Barrett et al., 1997; Hacker and Perrimon, 1998; Nikolaidou and Barrett, 2004; Dawes-Hoang et al., 2005; Kolsch et al., 2007). In vertebrate morphogenesis, the requirement for non-muscle myosin-driven AC has been shown (Haigo et al., 2003; Hildebrand, 2005; Lee and Harland, 2007); however, the mechanisms that regulate its activity MLN8054 tyrosianse inhibitor are less well defined than in protein Enabled (Ena), is definitely involved in many examples of actin dynamics, including the assembly of actin filament networks (Krause et al., 2003). In mutants, the build up of apical actin is definitely MLN8054 tyrosianse inhibitor lost in mutants (Baum and Perrimon, 2001; Gates et al., 2007; Gates et al., 2009). Xena, the homolog of Ena, is definitely apically enriched in the neural plate and is required for neuroepithelial AC, neural tube closure and apical actin enrichment (Roffers-Agarwal et al., 2008). Similarly, gene-targeted mice with different allelic mixtures of its three Ena homologs, Mena (Enah Mouse Genome Informatics), Vasp and Evl, also have neural tube closure problems and mislocalization of actin in the neuroepithelium (Menzies et al., 2004; Kwiatkowski et al., 2007). These data are consistent with a role for Mena/Vasp proteins in facilitating MLN8054 tyrosianse inhibitor the localized apical assembly of actin filaments in epithelia undergoing AC. Interestingly, the Mena/Vasp proteins, via their EVH1 website, bind to proteins that contain the proline-rich consensus found in many actin-associated cytoskeletal proteins (Hildebrand and Soriano, 1999; Ball et al., 2002). Currently, it is not known how Ena/Vasp proteins are apically localized during vertebrate AC. In the present study, we utilized a mutant gene-trapped mouse collection having a disruption in [the allele (Hildebrand and Soriano, 1999)] to determine whether it is required for lens pit cell AC. Cell shape analysis revealed the cells ARHGAP26 within the lens pit of these mutants fail to fully apically constrict, resulting in smaller and misshapen lens pits. Mutant lens pits also displayed a redistribution of F-actin, similar to what is definitely observed with Shroom3 and Ena/Vasp loss-of-function mutations (Hildebrand and Soriano, 1999; Haigo et al., 2003; Menzies et al., 2004; Roffers-Agarwal et al., 2008). Because of the similarity in phenotypes and the potential for Shroom3 and Ena/Vasp proteins to interact, the function of the proline-rich EVH1-binding domain was examined. We showed that this domain is essential for Shroom3-mediated AC and apical Vasp recruitment. It was also identified that Shroom3 manifestation in the prospective lens is dependent on Pax6, therefore defining a link between inductive signaling and the morphogenesis machinery. Collectively, these data provide novel insights into mechanisms of epithelial morphogenesis and, more specifically, of lens pit invagination. MATERIALS AND METHODS Animal maintenance and use Mice were housed in a specific pathogen-free vivarium in accordance with institutional guidelines. A gestational age of 0.5 days was defined as the day of detection of a vaginal plug [embryonic day (E) 0.5]. At specific gestational age groups, fetuses were eliminated by hysterectomy after the dams had been anesthetized with isoflurane. mice [in full, experiments Ectopic lenses were induced by injecting mRNA into the animal pole of half the blastomeres of two- or.