The genomic response to 20-hydroxyecdysone at the onset of Drosophila metamorphosis

Abstract

Background: The steroid hormone 20-hydroxyecdysone (20E) triggers the major developmental transitions in Drosophila, including molting and metamorphosis, and provides a model system for defining the developmental and molecular mechanisms of steroid signaling. 20E acts via a heterodimer of two nuclear receptors, the ecdysone receptor (EcR) and Ultraspiracle, to directly regulate target gene transcription.

Results: Here we identify the genomic transcriptional response to 20E as well as those genes that are dependent on EcR for their proper regulation. We show that genes regulated by 20E, and dependent on EcR, account for many transcripts that are significantly up- or downregulated at puparium formation. We provide evidence that 20E and EcR participate in the regulation of genes involved in metabolism, stress, and immunity at the onset of metamorphosis. We also present an initial characterization of a 20E primary-response regulatory gene identified in this study, brain tumor (brat), showing that brat mutations lead to defects during metamorphosis and changes in the expression of key 20E-regulated genes.

Conclusion: This study provides a genome-wide basis for understanding how 20E and its receptor control metamorphosis, as well as a foundation for functional genomic analysis of key regulatory genes in the 20E signaling pathway during insect development.

Figure 1

Validation of the temporal patterns of 20E-regulated gene expression as determined by microarray analysis. (a) Northern blot hybridizations adapted with permission from published data [27,28]. Arrow indicates E74A isoform. (b) Cluster analysis of microarray data derived from RNA samples isolated from staged wild-type animals. The colors for each time point represent the change in the level of expression relative to the average expression levels across all time points for that gene, with dark blue indicating the lowest level of expression and red indicating the highest level, as depicted on the bottom. The numbers at the top indicate hours relative to pupariation, with green bars representing the peaks of 20E titer.

Figure 2

Microarray results for EcRi and 20E organ cultures experiments. (a) Graphic depiction of the number of genes upregulated (blue) or downregulated (yellow) in EcRi late third instar larvae or prepupae at the times indicated. (b) Graphic depiction of the number of 20E-regulated genes or 20E primary-response genes that are either upregulated (blue) or downregulated (yellow) in third instar larval organ culture. (c) Venn diagram depicting the overlap between all EcR-regulated genes and the combined 20E-regulated genes and 20E primary-response genes. (d) Cluster diagram depicting the temporal expression pattern of the 479 genes in the 20E-final set, divided into those genes that are upregulated by 20E (above) or downregulated by 20E (below). Times are shown in hours relative to puparium formation, and colors are as described in Figure 1b.

Figure 3

Temporal expression patterns of EcR-dependent genes that are regulated by (a) starvation, (b) stress, or (c) infection. Upregulated (up) and downregulated (down) genes are labeled, hours are relative to puparium formation, which is marked by a black line, and colors are as described in Figure 1b.

Figure 4

Validation of seven 20E primary-response regulatory genes. (a) Northern blot analysis of RNA samples isolated from organ cultures treated with either 20E alone, 20E plus cycloheximide (20E+Cyc), or cycloheximide (Cyc) alone, for 0, 2, or 6 hours. (b) Temporal expression patterns of depicted genes with hours shown relative to puparium formation. Green bars represent the peak 20E titers. Colors in the cluster analysis are as described in Figure 1b. Hybridization to detect E74A and rp49 mRNAs was included as a control.

Figure 5

Mutations in brat lead to defects in genetic and biological responses to 20E. (a) Control w¹¹¹⁸ pharate adult. (b-d) Representative brat^k06028 mutant animals. (e) Northern blot analysis of w¹¹¹⁸ and brat^k06028 mutants staged in hours relative to pupariation. Blots were probed to detect brat mRNA and transcripts from seven different 20E-regulated genes. Hybridization to detect rp49 mRNA was included as a control for loading and transfer.