Analysis of the Transcriptomes Downstream of Eyeless and the Hedgehog, Decapentaplegic and Notch Signaling Pathways in Drosophila melanogaster

Abstract

Tissue-specific transcription factors are thought to cooperate with signaling pathways to promote patterned tissue specification, in part by co-regulating transcription. The Drosophila melanogaster Pax6 homolog Eyeless forms a complex, incompletely understood regulatory network with the Hedgehog, Decapentaplegic and Notch signaling pathways to control eye-specific gene expression. We report a combinatorial approach, including mRNAseq and microarray analyses, to identify targets co-regulated by Eyeless and Hedgehog, Decapentaplegic or Notch. Multiple analyses suggest that the transcriptomes resulting from co-misexpression of Eyeless+signaling factors provide a more complete picture of eye development compared to previous efforts involving Eyeless alone: (1) Principal components analysis and two-way hierarchical clustering revealed that the Eyeless+signaling factor transcriptomes are closer to the eye control transcriptome than when Eyeless is misexpressed alone; (2) more genes are upregulated at least three-fold in response to Eyeless+signaling factors compared to Eyeless alone; (3) based on gene ontology analysis, the genes upregulated in response to Eyeless+signaling factors had a greater diversity of functions compared to Eyeless alone. Through a secondary screen that utilized RNA interference, we show that the predicted gene CG4721 has a role in eye development. CG4721 encodes a neprilysin family metalloprotease that is highly up-regulated in response to Eyeless+Notch, confirming the validity of our approach. Given the similarity between D. melanogaster and vertebrate eye development, the large number of novel genes identified as potential targets of Ey+signaling factors will provide novel insights to our understanding of eye development in D. melanogaster and humans.

Pax6 Interactions with Chromatin and Identification of Its Novel Direct Target Genes in Lens and Forebrain

“…Considering the central role of Pax6 in eye formation, it has been estimated that Drosophila ey ultimately controls the expression of several thousand genes [40], [41], [42]. However, the proportion of directly or indirectly regulated genes is not known, as the majority of Pax6-direct targets in different tissues and at specific developmental stages remain to be determined [26], [43], [44].…”

Temporal regulation of Dpp signaling output in the Drosophila wing

“…To alter output based on Dpp concentration, for example, pMad-binding sites differ in both affinity (Wharton et al, 2004) and spacing (Lin et al, 2006). In addition, pMad-mediated transcription can be affected by the proximity of other transcription-factor binding sites, which allows selector genes or other signaling pathways to affect the functional output of Dpp signaling (Liang et al, 2012;Nfonsam et al, 2012).…”

The design of experiments for the transcriptome studies by high-throughput sequencing methods

“…По этим причинам планирование глубины секвенирования транскриптома в каждом конкретном эксперименте должно учитывать не только расчетную, но и по возможности ранее определенную протяжен-ность транскриптома у исследуемого вида. Например, протяженность транскриптома дрозофилы ненамного меньше транскриптома у млекопитающих (Nfonsam et al, 2012). В то же время у злаковых средняя протяжен-ность транскриптома в два -три раза больше, чем у мыши (Krasileva et al, 2013), поэтому для этих видов растений целесообразно установить глубину секвенирования не менее чем в 4-5 × 10 9 п. н.…”