RESUMO
Transcription factors (TFs) are key regulators of cell fate. The estimated 755 genes that encode DNA binding domain-containing proteins comprise â¼ 5% of all Drosophila genes. However, the majority has remained uncharacterized so far due to the lack of proper genetic tools. We generated 594 site-directed transgenic Drosophila lines that contain integrations of individual UAS-TF constructs to facilitate spatiotemporally controlled misexpression in vivo. All transgenes were expressed in the developing wing, and two-thirds induced specific phenotypic defects. In vivo knockdown of the same genes yielded a phenotype for 50%, with both methods indicating a great potential for misexpression to characterize novel functions in wing growth, patterning, and development. Thus, our UAS-TF library provides an important addition to the genetic toolbox of Drosophila research, enabling the identification of several novel wing development-related TFs. In parallel, we established the chromatin landscape of wing imaginal discs by ChIP-seq analyses of five chromatin marks and RNA Pol II. Subsequent clustering revealed six distinct chromatin states, with two clusters showing enrichment for both active and repressive marks. TFs that carry such "bivalent" chromatin are highly enriched for causing misexpression phenotypes in the wing, and analysis of existing expression data shows that these TFs tend to be differentially expressed across the wing disc. Thus, bivalently marked chromatin can be used as a marker for spatially regulated TFs that are functionally relevant in a developing tissue.
Assuntos
Padronização Corporal/genética , Drosophila melanogaster/embriologia , Discos Imaginais/embriologia , Fatores de Transcrição/genética , Asas de Animais/embriologia , Animais , Animais Geneticamente Modificados , Cromatina/genética , Cromatina/metabolismo , Metilação de DNA/genética , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Regulação da Expressão Gênica no Desenvolvimento , Histonas/genética , Fenótipo , Regiões Promotoras Genéticas/genética , Estrutura Terciária de Proteína/genética , Interferência de RNA , RNA Polimerase II/genética , RNA Interferente PequenoRESUMO
Imaginal discs, especially the wing imaginal disc, are powerful model systems to study organ development. The traditional methods to analyze wing imaginal discs depend on the laborious and time-consuming dissection of larvae. "Omics"-based approaches, such as RNA-seq, ChIP-seq, proteomics and lipidomics, offer new opportunities for the systems-level investigation of organ development. However, it is impractical to manually isolate the required starting material. This is even more problematic when experiments strive for enhanced temporal and spatial resolution. The mass isolation workflow discussed in this review, solves this problem. The semi-automated sorting of 1000 wing imaginal discs in less than 3h forms the basis of a workflow that can be connected to biochemical analyses of organ patterning and growth. In addition to the mass isolation workflow we briefly describe key "omics" technologies and their applications. The combination of mass isolation and "omics"-approaches ensures that the wing imaginal disc will continue to be a key model organ for studying developmental processes, both on the genetic, but increasingly also on the biochemical level.
