Zelda Coordinates Early Gene Networks Figure 3. Zld binds to defined enhancer regions of sc/sisB, zen, and sna. RNA expression profiles from the 1–2 hr (blue) and 2–3 hr (green) datasets, Zld ChIP/input ratios (binding peaks), and hotspots [5] were viewed on the Integrated Genome Browser (IGB) [55]. All expression peaks are on the same scale (maximum value 15K). Hotspots are shown as orange rectangles; height reflects the hotspot score, which depends on the number of factors bound and the strength of their binding [5]. Zld binding peaks are shown in blue with significance scores from the Ringo algorithm (see Materials and Methods) shown above as blue rectangles. Below the Zld peaks are: TAGteam sites (purple lines; limited to CAGGTAG, TAGGTAG, CAGGTAA), cis-regulatory modules (CRMs from REDfly denoted as filled red boxes [16], from other sources, denoted as open red boxes), gene models using genome version BDGP R5/dm3 (black rectangles; red arrows denote direction of transcription). Zld binding was observed at the known enhancers of sc/sisB and zen that contain in vivo relevant TAGteam sites, and both the primary and shadow (open red box) enhancers of sna [17]. Peaks were also observed just upstream of the TSS of zen and sna, and to a region downstream of sc/sisB. RNA expression of sc/sisB and zen is mostly absent in zld2, while that of sna is less affected. bcd transcripts, which are maternally loaded, are overall similar in wild-type and zld2 embryos. doi:10.1371/journal.pgen.1002339.g003 bound regions according to binding strength from highest to lowest in ten non-overlapping windows, and then analyzed the GO terms of the genes closest to those bound regions. Several groups of early genes were enriched (Figure S3A, S3B). For example, all of the zygotic genes involved in X-chromosome counting/sex determination (sisA, sc/sisB, os/sisC, dpn, run) [2] appeared in the top 10% window (ChIP profiles shown in Figure S4; run in Figure 4), making this GO term highly enriched (Figure S3B). sc/sisB is also known to function in proneural development [19], and interestingly many genes involved in neurogenesis were enriched in highly bound regions and strongly down-regulated in zld2 (Figure S4B, Table S2), defining another battery of Zld target genes. Also strongly bound were genes involved in cellularization, such as Sry-a, nullo, and slam (Figure S5, Table S2), along with cell cycle regulators such as frs that are involved in the nc 14 lengthening, which is concurrent with cellularization [20–25]. Many of the enriched GO terms were associated with DV, AP, and terminal patterning (Figure S3A, S3B). This was expected for the DV genes expressed in the dorsal region such as zen and dpp since they are abolished in zld2 [3]. What was unexpected was strong Zld binding to genes activated by Dpp/Smads such as Ance and C15 (Figure S6), because they are downstream in the DV hierarchy and not expressed until nc 14 [26]. This suggests a feed forward loop whereby Zld directly regulates both dpp and its targets. Also unexpected was the strong binding to the ventrally expressed DV genes, which are activated by the Dl morphogen (Figure 3 and Figure 4). sna and twi are high-level Dl targets expressed in the ventral-most region, the mesoderm, while rho, brk, and sog are lower-level targets expressed in domains of increasing width in the lateral region, the neuroectoderm [27]. However, unlike the dorsally expressed genes, these genes were not significantly affected in the 1–2 hr profiling studies (Table S2). This seeming contradiction prompted us to investigate their expression patterns by in situ hybridization, which provides higher spatial and temporal resolution. Closer inspection of twi, brk, sog, and rho revealed that their expression was delayed in zld2 (Figure 4), as we noted previously for sna [3], suggesting that Zld acts in combination with Dl to ensure precise temporal activation of these genes. Like sna [3], twi expression recovered and appeared normal by nc 14 (Figure 4B), but the lateral stripes of rho, brk, and sog narrowed to about 5–6 cells wide (arrows in Figure 4D, 4F, 4H), the region of intermediate levels of Dl and where the gradient is steepest [9–11]. In addition, within the narrow domain, PLoS Genetics | www.plosgenetics.org 5 October 2011 | Volume 7 | Issue 10 | e1002339