(GG) An internal stack of the same embryo as in F (the corresponding internal stack for the wild-type embryo in I is shown in KK). genes using the UAS-Gal4 system. We used a salivary-gland-specificfork head-Gal4line to restrict expression to the salivary glands, in combination with reporters of cell shape and the cytoskeleton. We identified a number of genes known to affect salivary gland formation, confirming the effectiveness of the screen. In addition, we found many genes not implicated previously in this Losmapimod (GW856553X) process, some having known functions in other tissues. We report the initial characterization of a subset of genes, includingchickadee,rhomboid1,egalitarian,bitesize, andcapricious, through comparison of gain- and loss-of-function phenotypes. DURING development and organogenesis most tissues arise from layers of epithelial cells that reorganize through complex morphogenetic movements. Many adult organs consist of tubular arrangements of epithelial sheets, and these tubules form during development through a process called tubulogenesis. There are a number of ways to generate tubules (Lubarskyand Krasnow2003). One important process is the direct conversion of epithelial sheets into tubules through wrapping (Colasand Schoenwolf2001) or budding (Hoganand Kolodziej2002). Cells undergoing tubulogenesis change their shapes drastically from a cuboidal or columnar epithelial shape to a wedge shape or conical shape and then back to a more columnar epithelial shape once positioned inside the tube. Cell shape is determined by the intracellular cytoskeleton, primarily actin Losmapimod (GW856553X) and microtubules. The cytoskeleton is closely coupled to cellcell adhesion Losmapimod (GW856553X) as well as to adhesion to the extracellular matrix. We are interested in understanding how the cytoskeleton and thus cell shape is regulated and coordinated during tubulogenesis. We chose to perform a gain-of-function screen rather than a mutagenesis-based loss-of-function screen as phenotypes observed in the latter might be subtle and thus missed or phenotypes in a given tissue might be obscured by disruption of other tissues and many genes might also have redundant functions. In contrast, the gain-of-function/overexpression approach allows a particular Losmapimod (GW856553X) tissue and gene to be targeted, and many such screens have been successfully conducted in the past (for examples, seeRrthet al.1998;Molnaret al.2006;Bejaranoet al.2008). The screen presented here uses the formation of the salivary glands in the Drosophila embryo as an assay system. The screen is based on a collection of transposable elements (EP elements) generated byRrthet al.(1998)that contain UAS sites that respond to the yeast transcription factor Gal4 that is followed by a promoter directing expression, when activated, of genes located downstream 3 of the EP insertion site. If combined through crosses with a tissue-specific source of Gal4 (Hendersonand Andrew2000;Zhouet al.2001), overexpression (and in some cases antisense expression) of a downstream gene will be activated only in the target tissue, which in our case are the embryonic salivary glands in the Drosophila embryo. Salivary gland formation in Drosophila is probably the simplest form of tubulogenesis (Lubarskyand Krasnow2003). A patch of 200 cells in the ventral epidermis of the embryo within parasegment 2 becomes specialized to form a salivary gland primordium, the placode, with 100 cells on either side of the embryo. This fate determination occurs through a combination of the activities of the homeotic genessex combs reduced(scr),extradenticle(exd), andhomothorax(hth) and dorsal signaling bydecapentaplegic(dpp) (Panzeret al.1992;Hendersonet al.1999;Hendersonand Andrew2000). Withoutscr,exd, andhthfunction, no salivary glands form. Different subpopulations of cells are found in the invaginated gland, such as the secretory cells and the common and individual duct cells. Their distinction depends on EGF signaling from the ventral midline (Kuoet al.1996;Habermanet al.2003). Once the cells have become specialized at stage 10 of embryogenesis, no further cell division occurs within the primordium, and no cells are lost through apoptosis (Campos-Ortegaand Hartenstein1985;Bateand MartinezArias1993;Myatand Andrew2000a). Invagination initiates in the dorsal posterior corner of the primordium, with all future secretory cells invaginating in a precise order, Sema3b followed by invagination of the duct cells and formation of the ducts (Myatand Andrew2000b). A key gene essential for the invagination isfork head(fkh). Fkh is a winged-helix transcription factor, and in its absence all of the cells fated to form the glands remain on the surface of the embryo as they fail to undergo apical constriction (Myatand Andrew2000a). Once inside the embryo, the glands have to navigate their way through the surrounding tissues, including the visceral mesoderm and central nervous system, to reach their extended final position parallel to the midline and anteriorposterior axis. They are guided by cues from the surrounding tissues (Kolesnikovand Beckendorf2005;Harrisand Beckendorf2007;Harriset al.2007). Also, after initially invaginating in a posteriordorsal direction, the glands turn and further extend into the embryo in a direction parallel to the anteriorposterior embryonic axis in a process dependent on integrins and downstream signals (Bradleyet al.2003;Vininget al.2005). A few factors that impinge on the cytoskeleton and cell shape during salivary gland morphogenesis have previously been identified. The actin cytoskeleton is modified through proteins such as.