Ciliogenesis defects in embryos lacking inturned or fuzzy function are associated with failure of planar cell polarity and Hedgehog signaling Tae Joo Park 1 , Saori L Haigo 2 & John B Wallingford 1 The vertebrate planar cell polarity (PCP) pathway has previously been found to control polarized cell behaviors rather than cell fate. We report here that disruption of Xenopus laevis orthologs of the Drosophila melanogaster PCP effectors inturned (in) or fuzzy (fy) affected not only PCP-dependent convergent extension but also elicited embryonic phenotypes consistent with defective Hedgehog signaling. These defects in Hedgehog signaling resulted from a broad requirement for Inturned and Fuzzy in ciliogenesis. We show that these proteins govern apical actin assembly and thus control the orientation, but not assembly, of ciliary microtubules. Finally, accumulation of Dishevelled and Inturned near the basal apparatus of cilia suggests that these proteins function in a common pathway with core PCP components to regulate ciliogenesis. Together, these data highlight the interrelationships between cell polarity, cellular morphogenesis, signal transduction and cell fate specification. The planar cell polarity (PCP) cascade is a conserved signaling pathway that governs the polarization of cells within the plane of a cell sheet. In vertebrates, PCP signals control cell polarity during convergent extension, a patterned cell rearrangement that results in the lengthening and narrowing of a tissue. Such tissue reorganization is of principal importance during embryonic morphogenesis. In particular, convergent extension is essential for vertebrate neural tube closure 1 . In D. melanogaster , a wide array of PCP pathway components have been identified 2,3 , and orthologs of the designated ‘core PCP’ genes 2,3 , including Dishevelled (Dvl), Flamingo (Celsr1) and Strabismus (Vangl2), have each been shown to control convergent extension during vertebrate neural tube closure 1,2 . In D. melanogaster , the ankyrin-repeat protein Diego is another crucial component of the PCP signaling apparatus 4,5 . Recent studies have shown that a related vertebrate protein, Inversin, is essential for convergent extension and, like Diego, interacts with PCP signaling components such as Vangl2 (ref. 6). Curiously, Inversin is a micro- tubule-associated protein essential for cilia morphogenesis 7 , and another ciliary protein, Bardet-Biedl Syndrome protein 4 (BBS4), is also implicated in planar polarity and convergent extension 8 . These data raise the possibility that ciliogenesis may be important for PCP signaling. Cilia have also recently been suggested to be critical to the functioning of other signaling pathways such as the Hedgehog cascade 9–12 . Although PCP signaling is known to affect the polarity of stereocilia in the vertebrate ear 13 , the possibility that the PCP signaling cascade may be more broadly involved in ciliogenesis has not yet been examined. Although ciliary proteins influence Hedgehog and PCP signal transduction in vertebrates 6,8–12 , this does not seem to be the case in D. melanogaster (for example, see ref. 14). In fact, the extent to which PCP signaling mechanisms and functions differ between flies and vertebrates is not known, in part because many D. melanogaster PCP components have yet to be examined in any vertebrate. Two such examples are the D. melanogaster ‘PCP effector’ proteins encoded by the inturned (in) and fuzzy (fy) genes 15,16 . These proteins have little conserved domain structure to suggest their function 15,16 , and in genetic studies, they behave quite differently from the ‘core PCP’ genes 3,17 . For example, they function epistatically downstream of dishevelled; mutation of effector genes disrupts cell polarity without affecting the polarized accumulation of core PCP proteins 3,17 . Here, we report the first analysis of Inturned and Fuzzy function in a vertebrate animal (Xenopus laevis). We find that, although these proteins do influence convergent extension, they also are broadly required for Hedgehog signaling. Rather than implicating Inturned and Fuzzy in Hedgehog signaling directly, our data suggest that the Hedgehog signaling defects are a secondary phenotype stemming from a failure of ciliogenesis in these embryos. In fact, we show that Inturned and Fuzzy control the assembly of an apical actin network that is essential for the normal orientation of ciliary microtubules. Finally, we report the accumulation of Dishevelled and Inturned protein at the apical surface of ciliated cells, suggesting that these proteins regulate ciliogenesis through a conserved PCP signaling pathway. These data demonstrate an important role for Inturned and Fuzzy in the organization of cell polarity and cellular morphology Received 14 October 2005; accepted 23 January 2006; published online 19 February 2006; doi:10.1038/ng1753 1 Department of Molecular Cell and Developmental Biology & Institute for Cellular and Molecular Biology, University of Texas, Austin, Texas 78712, USA. 2 Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California 94720, USA. Correspondence should be addressed to J.W.B. (wallingford@mail.utexas.edu). NATURE GENETICS VOLUME 38 [ NUMBER 3 [ MARCH 2006 303 ARTICLES © 2006 Nature Publishing Group http://www.nature.com/naturegenetics