Pannier and its co-repressor U-shaped participate in the same regulatory network during eye-

Our observation and previous reports of a dual regulatory role of Pnr during eye-antennal disc development may be mediated by the presence of a co-factor that modulates its regulatory role. In the developing wing imaginal disc, it has been shown that Pnr acquires a repressing mode of regulation upon heterodimerization with its co-factor U-shaped (Ush) (Fossett et al., 2001; Haenlin et al., 1997; Sorrentino et al., 2007). It has previously been stated that Ush is not expressed in the eye-antennal disc (Fossett et al., 2001; Maurel-Zaffran and Treisman, 2000). However, in our RNA-seq data we found the transcript of ush being expressed

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during eye-antennal disc development (Supplementary Figure 14A). Therefore, we hypothesized that Ush may act as a co-factor in this tissue.

A role of Ush as co-factor of Pnr requires both proteins to be present in the same cells of the eye-antennal disc. Since pnr expression in the eye-antennal disc has only been studied based on Gal4 driver lines, we first characterized the localization of Pnr using a newly generated antibody. We found that the protein is located, as previously reported, in the large nuclei of the dorsal peripodial epithelium (Figure 19B). Additionally, Pnr was detected in a few cell rows in the disc proper, most probably in a subset of the cuboidal margin cells (Supplementary Figure 15B-B’’). In later stages, the Pnr staining was less intense in the future ocellar complex region (Supplementary Figure 15AA’’). Lineage tracing experiments showed that descendants of pnr-positive cells extend further ventrally in the peripodial epithelium (Supplementary Figure 15C-C’’’). Additionally, we observed descendants of pnr-positive cells in the dorsal disc margin as well as in the disc proper (Supplementary Figure 15D-D’’’). Using a newly generated antibody against Ush, we confirmed the presence of the Ush protein during eye-antennal disc development in D. melanogaster (Figure 19B’). As shown for Pnr, the Ush protein is localized in the nuclei of the peripodial epithelium in the dorsal part of the eye-antennal disc, spanning the antennal, the ocellar and parts of the future head cuticle regions (Figure 19B’). We also observed Ush expression in potential adjacent cuboidal margin cells (Supplementary Figure 16).

Therefore, Ush and Pnr expression largely overlaps in the dorsal region of the eye-antennal disc (see also Supplementary Figure 14B), suggesting that they could indeed interact in the developing head. Please note that Ush is not only expressed during eye-antennal disc development, but also necessary for proper head development. Knockdown of ush in the dorsal developing eye-antennal disc consistently led to the loss of posterior vertical bristles (pVT – (Chyb and Gompel, 2013)), and irregularities at the border of orbital cuticle and dorsal frons (Supplementary Figure 14C), while the upregulation of ush affected the overall head shape and loss or gain of the pVT and adjacent bristles (Supplementary Figure 14C’). The effect on bristle patterns is consistent with the reported role of Ush in bristle formation on the thorax (Cubadda et al., 1997; Haenlin et al., 1997).

The co-expression of Pnr and Ush suggested that both genes may interact genetically.

To test this, we assessed the effect of gain- and loss of function of both genes on each other using the binary GAL4-UAS system in combination with Immunohistology. Since we aimed at

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modulating the expression of both genes within the endogenous domains, we used GAL4 driver lines, which drive expression in different dorsal regions of the developing eye-antennal disc.

VT042374 activity was reminiscent of the pnr expression domain in the peripodial epithelium and in marginal cells except for a small region in the presumptive ocelli domain (Figure 19A).

Regulatory elements of this line overlap with two open chromatin ATAC-seq peaks in an intronic region of the pnr locus (Supplementary Figure 17), suggesting that indeed partial endogenous pnr expression is reported. Additionally, we used the oc-GAL4 driver line that drove expression in the ocellar complex region that was not covered by the VT042374 line (Supplementary Figure 18 A-A’’).

Knock-down of pnr in the eye-antennal disc using VT042374 led to depletion of both, Pnr protein and Ush protein (Figure 19C’’-C’’’). This finding showed on the one hand that the pnr knock-down worked efficiently and suggests on the other hand that Pnr is necessary for the expression of ush. Note that this result could also be observed using the oc-GAL4 driver line, where even though the discs show great deformation after pnr knockdown, the Ush protein was clearly detected only in a smaller region (Supplementary Figure 18D’). Pnr was upregulated upon overexpression using the VT042374 driver (Figure 19C) and the oc driver (Supplementary Figure 18C). While the effect on Ush was not obvious after pnr overexpression using the VT042374 driver (Figure 19C’), slight upregulation was observed when the oc driver was used (Supplementary Figure 18C’).

The knockdown of ush using the VT042374 driver line resulted in a complete loss of Ush protein in the expected region (Figure 19D’’’), confirming that the knock-down worked efficiently. Conversely, we observed upregulation of Pnr expression in the region where RNAi against ush was driven (Figure 19D’’), suggesting that the presence of Ush results in pnr repression. Overexpression of ush using the VT042374 driver line resulted in a reduction of Pnr expression (Figure 19D). To confirm this observation, we made use of a previously reported double antenna phenotype upon loss of Pnr function (Oros et al., 2010), that we also found after pnr RNAi (Figure 19F). Intriguingly, overexpression of ush using a stronger pnr driver line (pnr-GAL4, (Fossett et al., 2001; Heitzler et al., 1996)) resulted in the same double antenna phenotype (Figure 19F’), supporting the observation that Ush is involved in repression of pnr expression.

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In summary, we could show that Ush and Pnr are spatially co-expressed during eye-antennal disc development. Our gain- and loss of function experiments showed that Ush is necessary for proper head development. Furthermore, we found evidence for genetic interactions between Ush and Pnr during eye-antennal disc development (Figure 19G), implying that both participate in the same regulatory network.

4.3.7. Overexpression of pannier phenocopies aspects of the differences observed between