Details

Autor(en) / Beteiligte

• Molinié, Benoit

Titel

Investigation of Dpp Target Genes on a Genome-Wide Scale in Early Drosophila Embryos

Ort / Verlag

ProQuest Dissertations & Theses

Erscheinungsjahr

2009

Quelle

ProQuest Dissertations & Theses A&I

Beschreibungen/Notizen

• During early Drosophila development, a gradient of the TGF-β signalling molecule subdivides the embryonic dorsal ectoderm into amnioserosa in response to peak signalling, and dorsal epidermis in response to lower levels of signalling. Currently, only a limited number of Decapentaplegic (Dp7) target genes have been identified, and the molecular mechanisms by which these tissues differentiate are unknown. To identify additional Dp7 target genes, I designed an approach based on the use of transgenic Drosophila lines that induce ectopic Dp7 activity in the embryo in a series of microarray experiments. First, the expression array protocols were developed by comparing transcription levels of genes expressed in gastrulation defective (gd7) embryos (showing no peak level of dp7 expression) and in gd7;St2-sog embryos (even stripe number 2 (St2); Short gastrulation [Sog]) (showing ectopic peak level of dp7 expression). In a second microarray experiment, the levels of gene expression in both St2-dpp embryos that ectopically express Dp7 as well as in wild type embryos were compared, thus allowing me to generate an initial list of potential novel Dp7 target genes. A second novel list of potential Dp7 target genes was generated based on the comparison of the Mata-Gal4;UAS-tkvQD line, which artificially mimics dp7 ubiquitous over-expression, to Mata-Gal4;+/+. A master list of potential Dp7 target genes was finally constructed based on the common elements identified from the first two lists. Known Dpp target genes such as u-shape (ush), hindsight (hnt, also known as pebbled, peb, but referred to here as hnt), pannier (pnr), Related to angiotensin converting enzyme (race) were identified, as well as novel genes such as CG32299, CG16869, CG3218, and CG31902. The changes in expression levels as observed on microarrays were subsequently confirmed on Drosophila embryos using RNA in situ hybridisation, and precisely quantified by quantitative real time PCR (qRT-PCR). Three novel potential Dp7 target genes dFollistatin (Fs), Roughest (rst), and Sema-5c (Sema-5c) were investigated in detail to confirm their eligibility as Dp7 target genes. These genes were chosen based on the similarity of their expression patterns to those of known Dp7 target genes, and their involvement in the TGF-β pathway regulation in mammalian cell lines. The results obtained from the microarray analyses led to the creation of a reliable novel list of potential Dp7 target genes. Follistatin function was characterized using gain/loss-of-function mutant Drosophila lines and by generating St2-Fs transgenic lines ectopically expressing Fs. These analyses were proven to be consistent with previous reports showing that mammalian Fs is a Dp7 pathway down-regulator. The function of Rst and Sema-5c in the Dp7 pathway were tested using rst-/-, and sema5c-/- knock-out lines showing Sema-5c as a potential Dp7 pathway down-regulator and Rst as a Dp7 pathway modulator. Finally, chromatin immunoprecipitation was carried out on cellular extracts obtained from embryos over-expressing Dp7 and the Dp7 cytoplasmic mediators R-Smad and co-Smad Mad and Medea in order to be hybridised in the future to Affymetrix-GeneChip Drosophila Tiling 2.0R arrays.