Summary Pax 3 regulates morphogenetic cell behavior in vitro coincident with activation of a PCP / non-canonical Wnt-signaling cascade

Pax3, a member of the paired-family of transcription factors, is required for a number of fundamental processes during vertebrate development (Dahl et al., 1997). In mice, mutations to Pax3 result in the splotch ( sp) phenotype which is characterized by a number of early developmental defects (Epstein et al., 1991). These defects, among others, include impaired neural tube closure, absence of limb musculature and the impaired development of several structures derived from the neural crest. In humans, mutations to Pax3give rise to the pigmentation, hearing and facio-skeletal anomalies observed in Waardenburg syndrome (Baldwin et al., 1992; Tassabehji et al., 1992). The mechanisms by which Pax3 and other Pax genes regulate morphological development are poorly understood, however, as the cellular and molecular processes that these genes regulate remain ill defined. Tissue morphogenesis is characterized by the coordinated effect of several different cell behaviors including changes to cell shape, position, adhesion, proliferation and death (Atchley and Hall, 1991; Gumbiner, 1996). Patterning of the vertebrate neural tube, for example, involves a coordinated set of cell behaviors known as convergent extension, which results in the simultaneous narrowing of the tissue in one dimension and its lengthening in a perpendicular dimension (Colas and Schoenwolf, 2001). Characterization of Drosophila and vertebrate morphogenetic mutants has revealed the common use of conserved signaling cascades such as the c-Jun-Nterminal kinase/stress activated protein kinase (JNK/SAPK) pathway during morphogenesis (Noselli, 1998; Noselli and Agnes, 1999; Sokol, 2000). JNK signaling pathways have been specifically implicated in processes such as dorsal closure, thorax closure and in planar tissue/cell polarity (Martin-Blanco et al., 2000; Noselli, 1998; Zeitlinger and Bohmann, 1999). Additionally, a signaling cascade involving a Wnt receptor, Frizzled, and a downstream effector, Dishevelled, is implicated in the regulation of morphogenesis in Drosophilaand is also coupled to JNK activation in tissue polarization (Sokol, 2000). This Frizzled-mediated signaling cascade, which regulates planar cell polarity (PCP) in Drosophila, appears homologous to a Wnt-signaling cascade recently shown to regulate convergent extension movements in vertebrates (Heisenberg et al., 2000; Wallingford et al., 2000). Numerous studies have suggested a role for Pax genes in the regulation of Wnt signaling (Dahl et al., 1997; Uusitalo et al., 1999). One such study has revealed that the combined activity of Pax3 and Pax7 is required for normal Wnt4 expression in the neural tube during murine development (Mansouri and Gruss, 1998). Analogous to the consequences of mutant Pax genes, mutations to Wntand to JNK genes in mice also result in morphological defects in specific tissues. For example, as for Pax3mutant mice, mutations to distinct Wnt genes result in neural tube patterning defects (Uusitalo et al., 1999). Thus, the basis of these and other data, we hypothesized that Pax proteins regulate one or more of the aforementioned 531