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.

The ever-increasing speed and resolution of modern microscopes make the storage and post-processing of images challenging and prevent thorough statistical analyses in developmental biology. Here, instead of deploying massive storage and computing power, we exploit the spherical geometry of zebrafish embryos by computing a radial maximum intensity projection in real time with a 240-fold reduction in data rate. In our four-lens selective plane illumination microscope (SPIM) setup the development of multiple embryos is recorded in parallel and a map of all labelled cells is obtained for each embryo in <10 s. In these panoramic projections, cell segmentation and flow analysis reveal characteristic migration patterns and global tissue remodelling in the early endoderm. Merging data from many samples uncover stereotypic patterns that are fundamental to endoderm development in every embryo. We demonstrate that processing and compressing raw image data in real time is not only efficient but indispensable for image-based systems biology.

casanova (cas) mutant zebrafish embryos lack endoderm and develop cardia bifida. In a substractive screen for Nodal-responsive genes, we isolated an HMG box-containing gene, 10J3, which is expressed in the endoderm. The cas phenotype is rescued by overexpression of 10J3 and can be mimicked by 10J3-directed morpholinos. Furthermore, we identified a mutation within 10J3 coding sequence that cosegregates with the cas phenotype, clearly demonstrating that cas is encoded by 10J3. Epistasis experiments are consistent with an instructive role for cas in endoderm formation downstream of Nodal signals and upstream of sox17. In the absence of cas activity, endoderm progenitors differentiate into mesodermal derivatives. Thus, cas is an HMG box-containing gene involved in the fate decision between endoderm and mesoderm that acts downstream of Nodal signals. The endoderm germ layer generates the structures of the digestive and respiratory tracts. In addition, endoderm is crucial in the organization and/or induction of neighboring tissues, such as the head and the heart (Grapin-Botton and Melton 2000). In the zebrafish, endoderm derives from cells positioned at the blastoderm margin of the late blastula (Warga and Nusslein-Volhard 1999). Although endoderm and mesoderm progenitors partially overlap, most mesoderm progenitors come from positions relatively far away from the very margin at this stage.The molecular pathway leading to endoderm formation is only partially understood. Specification of endoderm requires Nodal signaling (Kimelman and Griffin 2000). Zebrafish mutants lacking the Nodal-related factors Squint (Sqt) and Cyclops (Cyc) fail to form endoderm (Feldman et al. 1998;Sampath et al. 1998). Similarly, endoderm does not form in embryos defective in both maternal and zygotic components of one-eyed pinhead (MZoep), which encodes an EGF-CFC protein required for cells to respond to Nodal signals (Schier et al. 1997;Strähle et al. 1997;Zhang et al. 1998;Gritsman et al. 1999). In zebrafish, Nodals induce endoderm presumably via activation of the type I TGF␤ receptor TARAM-A (Tar; Renucci et al. 1996;Peyrieras et al. 1998), the mix-like homeobox transcription factor MIXER (bonnie and clyde, bon; Kikuchi et al. 2000), and the zinc-finger transcription factor GATA5 (faust; Reiter et al. 1999Reiter et al. , 2001). Both transcription factors require a third gene, casanova (cas), to efficiently induce the endoderm-specific sox17 gene (Alexander and Stainier 1999) and to allow marginal cells to achieve the proper endodermal program. At gastrula stages, cas mutant embryos express sox17 neither in endoderm precursors nor in the forerunner cells, a small group of noninvoluting mesendodermal cells at the dorsal margin (Melby et al. 1996). At later stages, cas mutants lack a gut tube and develop a heart condition known as cardia bifida. cas activity is required cell-autonomously for endoderm development and endodermal expression of foxA2 . Thus, cas acts within endoderm precursors, downstream of the Nodal signals Cyc and Sqt and the ...