Department of Biology, New Mexico State University, Las Cruces, New Mexico, USA.
PLoS One. 2012;7(8):e44583. doi: 10.1371/journal.pone.0044583. Epub 2012 Aug 31.
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.
组织特异性转录因子被认为与信号通路合作，以促进模式组织特化，部分通过共同调节转录。果蝇 Pax6 同源物 Eyeless 与 Hedgehog、Decapentaplegic 和 Notch 信号通路形成一个复杂的、尚未完全理解的调控网络，以控制眼睛特异性基因表达。我们报告了一种组合方法，包括 mRNAseq 和微阵列分析，以鉴定 Eyeless 和 Hedgehog、Decapentaplegic 或 Notch 共同调节的靶标。多项分析表明，与以前只涉及 Eyeless 的研究相比，Eyeless 和 Hedgehog、Decapentaplegic 或 Notch 共同过表达的转录组提供了更完整的眼睛发育图景：（1）主成分分析和双向层次聚类显示，Eyeless+信号因子的转录组比单独过表达 Eyeless 时更接近眼睛控制转录组；（2）与单独过表达 Eyeless 相比，至少有 3 倍以上的基因上调；（3）基于基因本体分析，与单独过表达 Eyeless 相比，上调的基因具有更多样的功能。通过利用 RNA 干扰的二次筛选，我们表明预测基因 CG4721 在眼睛发育中起作用。CG4721 编码一种 Neprilysin 家族金属蛋白酶，对 Eyeless+Notch 的反应高度上调，证实了我们方法的有效性。鉴于果蝇和脊椎动物眼睛发育之间的相似性，作为 Ey+信号因子的潜在靶标鉴定出的大量新基因将为我们理解果蝇和人类眼睛发育提供新的见解。
