In D. melanogaster, the subdivision of dorsal ectoderm into amnioserosa (AS) and dorsal epidermis is controlled by the combined actions of two morphogens of the TGF-β family, Decapentaplegic (Dpp) and Screw (Scw).
More...In D. melanogaster, the subdivision of dorsal ectoderm into amnioserosa (AS) and dorsal epidermis is controlled by the combined actions of two morphogens of the TGF-β family, Decapentaplegic (Dpp) and Screw (Scw). They form an extracellular gradient with peak levels of Dpp/Scw signalling in the dorsal-most region of the embryo. Dpp/Scw signalling is transduced to the nucleus by a complex containing the phosphorylated Smad transcription factor, pMad and the co-Smad, Medea, which are responsible for the transcriptional activation of a number of targets in specific dorsal territories, including zerknullt (zen) gen. zen encodes a homeobox transcription factor, which is expressed for a brief period of time in the early developing embryo and is required for all aspects of AS formation, including the initial cell shape changes that drive the morphogenesis of the AS epithelium. However, the number and nature of Zen-target genes involved in AS differentiation subsequent to its specification are currently unknown. The only known target genes of Zen are the so called u-shaped genes; they encode transcription factors that control the maintenance of AS after Zen activity has faded away, so they are not informative for elucidating the specification of morphological properties on a cellular level. In this project, we attempted to identify Zen-target genes required for AS differentiation and/or maintenance.
Methods: D. melanogaster flies were grown at 22 ºC on standard cornmeal, molasses, agar, yeast medium. Staged embryos were obtained as described in Zuñiga et al. (2009). Fly strain was zen7, a null allele balanced over TM3, ftz-lacZ (Rushlow et al., 1987). zen7 homozygous embryos were selected using a genotyping procedure described by Ghanim and White (2006). In our case, both the absence of lacZ specific band together with the presence of a control band (non-coding region of gene Dtg) were indicators of homozygous lethal embryos. For RNA extractions, extracts of homozygous (zen7) and heterozygous (zen7/TM3, ftz-lacZ) embryos at stage 5 or stage 6-7 of embryogenesis , previously preserved in RNAWIZ reagent (N = 100) were pooled, then samples were carefully homogenized in a 1.5 mL Eppendorf tube on ice with the aid of RNAse-free polypropylene pellet pestle. After homogenization, RNA extraction was performed using standard protocols. RNA was quantified using Qubit® RNA HS Assay Kit (Thermo Fisher) and the integrity was assayed in a Tape station 2200 (Agilent Technologies). Samples were treated with Turbo DNA-free DNase (Ambion) to remove contaminating DNA. Two biological replicates of RNA from of homozygous and heterozygous embryos were sequenced using XXX and the sequence reads that passed quality filters were analyzed using XXX
Overall design: Two biological replicates of RNA from zen7 homozygous embryos and zen7/TM3, ftz-lacZ heterozygous embryos (reference) at stage 5 or stage 6-7 of embryonic development were sequenced using Illumina HiSeq 2000.
Less...