Academic journal article Genetics

A Targeted Gain-of-Function Screen Identifies Genes Affecting Salivary Gland Morphogenesis/Tubulogenesis in Drosophila

Academic journal article Genetics

A Targeted Gain-of-Function Screen Identifies Genes Affecting Salivary Gland Morphogenesis/Tubulogenesis in Drosophila

Article excerpt

ABSTRACT

During development individual cells in tissues undergo complex cell-shape changes to drive the morphogenetic movements required to form tissues. Cell shape is determined by the cytoskeleton and cell-shape changes critically depend on a tight spatial and temporal control of cytoskeletal behavior. We have used the formation of the salivary glands in the Drosophila embryo, a process of tubulogenesis, as an assay for identifying factors that impinge on cell shape and the cytoskeleton. To this end we have performed a gain-of-function screen in the salivary glands, using a collection of fly lines carrying EP-element insertions that allow the overexpression of downstream-located genes using the UAS-Gal4 system. We used a salivary-gland-specific fork head-Gal4 line 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 process, some having known functions in other tissues. We report the initial characterization of a subset of genes, including chickadee, rhomboid1, egalitarian, bitesize, and capricious, 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 (Lubarsky and Krasnow 2003). One important process is the direct conversion of epithelial sheets into tubules through wrapping (Colas and Schoenwolf 2001) or budding (Hogan and Kolodziej 2002). 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 and microtubules. The cytoskeleton is closely coupled to cell-cell adhesion 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 tissue and gene to be targeted, and many such screens have been successfully conducted in the past (for examples, see Rørth et al. 1998;Molnar et al. 2006; Bejarano et 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 by Rørth et 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 39 of the EP insertion site. If combined through crosses with a tissuespecific source of Gal4 (Henderson and Andrew 2000; Zhou et 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 (Lubarsky and Krasnow 2003). 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. …

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