Conditional Switches for Extracellular Matrix Patterning inDrosophila melanogaster

Abstract

An F 1 mutagenesis strategy was developed to identify conditional mutations affecting extracellular matrix (ECM) patterning. Tubulogenesis requires coordinated movement of epithelial cells and deposition of a multilayered ECM. In the Drosophila ovary, an epithelium of follicle cells creates the eggshells, including the paired tubular dorsal appendages (DAs) that act as breathing tubes for the embryo. A P-element mutagenesis strategy allowed for conditional overexpression of hundreds of genes in follicle cells. Conditional phenotypes were scored at the level of individual mutant (F 1 ) female flies. ECM pattern regulators were readily identified including MAPK signaling gene ets domain lacking (fused DAs), Wnt pathway genes frizzled 3 and osa (long DAs), Hh pathway gene debra (branched DAs), and transcription factor genes sima/HIF-1a, ush, lilli, Tfb1, broad, and foxo. In moving cells the ½Ca 21 /calcineurin pathway can regulate adhesion to ECM while adherens junctions link cells together. Accordingly, thin eggshell and DA phenotypes were identified for the calcineurin regulator calreticulin and the adherens junction component arc. Finally a tubulogenesis defect phenotype was identified for the gene pterodactyl, homologous to the mammalian serine/threonine receptorassociated protein (STRAP) that integrates the TGF-b and PI3K/AKT signaling pathways. Because phenotypes can be scored in each mutant fly before and after gene induction, this F 1 conditional mutagenesis strategy should allow for increased scale in screens for mutations affecting repeated (reiterated) events in adult animals, including gametogenesis, movement, behavior, and learning.

TGF-beta-dependent and -independent roles of STRAP in cancer

“…As previously mentioned, disruption of STRAP expression causes branching morphogenesis defects in Drosophila as well as defects in neural tube closure in mice (3, 4). These observed defects may point to a failure in cellular migration as coordinated migration is required for both developmental processes.…”

“…It has been determined that STRAP is a conserved 38 kDa protein that localizes to both the cytoplasm and nucleus of cells (1). Mutation of the Drosophila STRAP homologue, pterodactyl, has been implicated in tubulogenesis and branching morphogenesis defects (3). Furthermore, STRAP knockout in mice is embryonic lethal due to defects in neural tube closure, somitogenesis, and organogenesis (4).…”

Adult-specific over-expression of the Drosophila genes magu and hebe increases life span and modulates late-age female fecundity

“…Because PdL gene over-expression mutations are both conditional (DOX-dependent) and dominant (only one copy of the gene needs be mutated), it means that new mutations do not need to be made homozygous, and phenotypes can be assayed in each mutant (F1) female (Khokhar et al 2008). In the mutagenesis strategy employed here each mutant F1 female bearing a new PdL insertion was assayed longitudinally for fecundity phenotype before and after induction of gene over-expression by DOX.…”

“…With the conditional systems, the control and experimental (gene over-expressing) groups have identical genetic backgrounds, and therefore the effects of the transgene can be identified without confounding effects of genetic background (Sun and Tower 1999; Tower 2000; Curtis et al 2007; Giannakou et al 2008; Min et al 2008). Placing a conditional (DOX-dependent) promoter facing outward from a P element transposon (the “ PdL ” P element) allows for the efficient screening of Drosophila genes for over-expression phenotypes, including effects on life span (Landis et al 2001; Landis et al 2003) and oogenesis (Khokhar et al 2008). In particular, the dominant and conditional nature of the PdL gene-over-expression mutations allows screening for effects on reproduction at the level of the individual (F1) mutant female fly.…”

Composition of the Survival Motor Neuron (SMN) complex in Drosophila melanogaster

“…STRAP binds to the SMN complex via an interaction with Gem7 (OTTER et al 2007). In Drosophila, mutations in the STRAP orthologue, Wmd/CG3957, cause defects in development of the adult wing (KHOKHAR et al 2008), suggesting that this protein may not be essential for basal assembly of spliceosomal snRNPs. Gem6-7-8 forms a subcomplex that is tethered to SMN via interaction with Gem8 (CARISSIMI et al 2006;OTTER et al 2007).…”