Pannier regulates genes that are differentially expressed between D. melanogaster and D

Pnr is an interesting candidate transcription factor that may be involved in the development of differences in dorsal head morphology as well as eye size observed between D. melanogaster and D. mauritiana for the following reasons: 1. Our global clustering and motif enrichment analyses suggest that Pnr regulates many DEGs between both species. 2. pnr itself is differentially expressed between D. melanogaster and D. mauritiana. 3. Pnr is known to be expressed in the dorsal portion of the eye-antennal disc (Maurel-Zaffran and Treisman, 2000;

and see below) and it determines the dorsal-ventral axis of the retinal field in the early L2 discs (Maurel-Zaffran and Treisman, 2000; Singh et al., 2005; Singh and Choi, 2003). Additionally, later during eye-antennal disc development, Pnr influences the ratio of retinal and head cuticle fate in the dorsal disc by repressing retinal determination genes (Oros et al., 2010). Therefore, we sought to validate and refine our global differential expression data focusing on Pnr.

First, we asked at what stage of eye-antennal disc development pnr was differentially expressed between species. Based on our transcriptomic dataset we found significantly higher expression in D. mauritiana at 120h AEL (Figure 18A). This trend was further confirmed by real-time qPCR (Supplementary Figure 11).

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Figure 18. A. Expression dynamics of the pnr transcript at the three developmental stages in D. melanogaster (red) and D. mauritiana (blue). B. Network reconstruction of known interactions upstream and downstream targets of Pnr (DroID (Yu et al., 2008)) that overlap with our Pnr target gene list. Cyan nodes represent target genes that are differentially expressed between D. melanogaster and D. mauritiana in at least one of the three studied developmental stages. Grey nodes represent predicted targets of Pnr based on our target gene list but are not differentially expressed. Black edges describe potential upstream regulators of Pnr based on DroID. Red arrows point towards Pnr target genes that are annotated as being ‘activated’ by Pnr in DroID, whereas blue edges point to genes where the interaction between Pnr and the gene is annotated as ‘repressing’. Grey edges describe interactions that are annotated as direct TF-gene interactions in DroID. C. Hierarchical clustering of read counts of predicted Pnr target genes (based on our target gene list) which were found to be differentially expressed in at least one developmental stage. The cyan line in each cluster represents pnr expression, which itself is a member of Cluster 6. Left side of each cluster: Expression dynamics of genes in D. melanogaster (OreR), Right side of each cluster: Expression dynamics of genes in D. mauritiana (TAM16).

Next, we wanted to define a list of putative direct Pnr target genes. This was crucial since the database used to infer motif enrichment was based on ChIP-Chip and ChIP-seq

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experiments that were not conducted in Drosophila eye-antennal discs (Herrmann et al., 2012;

Imrichová et al., 2015). To obtain tissue and stage specific putative target genes, we assessed accessible chromatin regions by generating an ATAC-seq dataset for D. melanogaster eye-antennal discs covering the same three time points used for the transcriptomic dataset (Figure 17C). We found 14,511 unique peaks across all three timepoints. In the open chromatin regions, we revealed 1,335 Pnr-specific GATA motifs associated with 1108 genes expressed in our RNA-seq dataset (see Materials and Methods for details), suggesting that they were active during eye and head development. A cross validation of the putative Pnr target genes using the i-cisTarget tool confirmed an enrichment for Pnr, Nej, pMad and Mef2 binding sites (Supplementary Figure 12A). The identification of putative pMad target genes among Pnr targets may recapitulate the previous observation that both proteins interact physically during larval development (Kim et al., 2017). The putative Pnr target genes were highly enriched in GO terms like signal transduction, development, growth and cell cycle progression as well as in very specific terms such as compound eye development (Supplementary Figure 12B), recapitulating known functions of Pnr during eye-antennal disc development.

We further assessed the reliability of our target gene identification by searching for known target genes of Pnr. Among the putative target genes, we found Angiotensin-converting enzyme (Ance) (Supplementary Table 19), which is regulated by Pannier and pMad during Drosophila larval development (Kim et al., 2017). pnr itself is autoregulated in the wing imaginal disc (Fromental-Ramain et al., 2010, 2008). Accordingly, we found pnr as target gene as well (Figure 18C, Supplementary Table 19). We did not find wg as putative target gene, which is consistent with the study of Pereira and collegues, who suggested that Pnr does not activate wg expression in the peripodial membrane (Pereira et al., 2006). Conserved GATA motifs, that were though shown to be not responsive to Pnr bining (Pereira et al., 2006) lie indeed between significantly called peaks of a highly accessible intergenic region between the wg and wg6 loci (Supplementary Figure 13). Overall, we were able to obtain a high confidence Pnr target gene list.

Our initial cluster analysis suggested that Pnr may regulate many genes that are differentially expressed between D. melanogaster and D. mauritiana. We could confirm this observation because 67.8 % (751 of the 1,108) of the expressed target genes showed expression differences between D. melanogaster and D. mauritiana in at least one stage.

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In summary, we could show that pnr expression was significantly higher in D. mauritiana at 120h AEL and we identified a list of high confidence Pnr target genes which are mainly involved in signalling and developmental processes, cell cycle progression and growth. Most of the Pnr target genes were differentially expressed between D. melanogaster and D. mauritiana.