The unanticipated involvement of several intraflagellar transport proteins in the mammalian Hedgehog (Hh) pathway has hinted at a functional connection between cilia and Hh signal transduction. Here we show that mammalian Smoothened (Smo), a seven-transmembrane protein essential for Hh signalling, is expressed on the primary cilium. This ciliary expression is regulated by Hh pathway activity; Sonic hedgehog or activating mutations in Smo promote ciliary localization, whereas the Smo antagonist cyclopamine inhibits ciliary localization. The translocation of Smo to primary cilia depends upon a conserved hydrophobic and basic residue sequence homologous to a domain previously shown to be required for the ciliary localization of seven-transmembrane proteins in Caenorhabditis elegans. Mutation of this domain not only prevents ciliary localization but also eliminates Smo activity both in cultured cells and in zebrafish embryos. Thus, Hh-dependent translocation to cilia is essential for Smo activity, suggesting that Smo acts at the primary cilium.

Understanding the genetic architecture of evolutionary change remains a long-standing goal in biology. In vertebrates, skeletal evolution has contributed greatly to adaptation in body form and function in response to changing ecological variables like diet and predation. Here we use genome-wide linkage mapping in threespine stickleback fish to investigate the genetic architecture of evolved changes in many armor and trophic traits. We identify .100 quantitative trait loci (QTL) controlling the pattern of serially repeating skeletal elements, including gill rakers, teeth, branchial bones, jaws, median fin spines, and vertebrae. We use this large collection of QTL to address long-standing questions about the anatomical specificity, genetic dominance, and genomic clustering of loci controlling skeletal differences in evolving populations. We find that most QTL (76%) that influence serially repeating skeletal elements have anatomically regional effects. In addition, most QTL (71%) have at least partially additive effects, regardless of whether the QTL controls evolved loss or gain of skeletal elements. Finally, many QTL with high LOD scores cluster on chromosomes 4, 20, and 21. These results identify a modular system that can control highly specific aspects of skeletal form. Because of the general additivity and genomic clustering of major QTL, concerted changes in both protective armor and trophic traits may occur when sticklebacks inherit either marine or freshwater alleles at linked or possible "supergene" regions of the stickleback genome. Further study of these regions will help identify the molecular basis of both modular and coordinated changes in the vertebrate skeleton. U NDERSTANDING the quantitative genetic architecture underlying evolutionary change in nature remains a major goal in genetics. The past two decades have seen a rapid increase in experimental data from various model systems, generating vigorous debate over the relative importance of coding vs. regulatory alleles, the prevalence of pleiotropy, and the role of large-effect mutations during adaptation to new environments (Stern and Orgogozo 2008;Streisfeld and Rausher 2011;Rockman 2012).One particularly interesting genetic architecture found in several natural systems is close linkage of loci controlling multiple, often coadaptive, phenotypes. Such trait clusters, sometimes called "supergenes," have been observed in primroses (Darwin 1877;Mather 1950;Li et al. 2011), butterflies (Clarke et al. 1968Mallet 1989;Joron et al. 2006), snails (Murray and Clarke 1976), and fish (Winge 1927;Protas et al. 2008;Roberts et al. 2009;Tripathi et al. 2009). Trait clusters could result from recombination suppression (Noor et al. 2001), for example through chromosomal inversions (Lowry and Willis 2010;Joron et al. 2011;Fishman et al. 2013). Alternatively, trait clusters could result from tightly linked loci or pleiotropic effects of individual genes (Mallet 1989;Studer and Doebley 2011 Cis-regulatory changes may predominate during morphological evoluti...

We describe a method for the highly efficient and precise targeted modification of gene trap loci in mouse embryonic stem cells (ESCs). Through the Floxin method, gene trap mutations are reverted and new DNA sequences inserted using Cre recombinase and a shuttle vector, pFloxin. Floxin technology is applicable to the existing collection of 24,149 compatible gene trap cell lines, which should enable the high-throughput modification of many genes in mouse ESCs.