Figure 7. Targets of key patterning factors are more likely to be expressed if bound by Zld. (A) Bar graph showing the number of bound target genes of DV [35] and AP [13,14] transcription factors (red) that are also bound by Zld (blue) divided into two groups: targets that are expressed (+) or not expressed (2) in blastoderm embryos (from polII ChIP-chip data of [14]). The analysis was restricted to genes within 2 kb of bound regions. (B–C) Heat maps showing the fraction of target genes that two factors have in common, including all target genes (B) or blastoderm-expressed targets (C). The number in each box represents the fraction of genes bound by a factor denoted in the row that are also bound by the factor denoted in the column. For example, 59% of all DTS targets are also bound by Zld (B). This number increases to 76% for those DTS targets that are expressed at 2– 4 hrs (C). doi:10.1371/journal.pgen.1002339.g007 both of these ideas. Zld protein accumulates to high levels by one hour of development (Figure 1B, 1D), which coincides with the onset of zygotic genome activation [6]. Within a two-hour period, the embryo cellularizes, determines X-chromosome dosage, patterns its body plan, and gets ready for gastrulation. By virtue of a single factor these processes are coordinately activated. Zelda Coordinates Early Gene Networks One can predict that increasing Zld levels in early embryos would advance timing of activation. Our initial attempts to increase Zld protein levels by adding copies of Zld rescue constructs did not yield higher Zld protein levels (data not shown), indicating that Zld levels may be tightly regulated. However, ten Bosch et al. [2] showed that doubling the number of TAGteam sites in the zen enhancer led to precocious expression, supporting the idea that Zld acts to time zygotic gene activation. In the absence of Zld, all direct targets are either: 1) not expressed, 2) delayed but recover, or 3) delayed but do not recover fully. For example, genes involved in sex determination, cellulariza- tion, dorsal patterning, and proneural development are strongly down-regulated in zld2 and never recover (Table S2; Figures S4, S5, S6). In contrast, genes involved in AP and ventral patterning were not significantly down-regulated, and how they recovered depended on how they responded to other factors. The high-level Dl targets sna and twi recovered by nc 14, but the lower-level targets sog, brk, and rho did not recover their normal patterns in zld2; instead they were expressed sporadically in a narrow domain with great variability among embryos (Figure 4). It appears that intermediate levels of Dl are no longer sufficient for robust and faithful target- gene expression, and lower levels cannot activate them at all; thus, the Dl gradient cannot be interpreted without Zld. These effects are likely due to the lack of direct Zld input, as mutation of the TAGteam sites in the sog primary enhancer caused a similar narrowing of the reporter expression domain [38]. Indirect effects of delayed twi expression may also contribute, since mutation of Twi binding sites in the rho enhancer also resulted in a narrower domain [39]. These observations suggest that Zld not only acts as a timer for Dl target-gene activation, but also potentiates Dl morphogenetic activity over a broad range in the neuroectoderm in order to establish multiple threshold responses. Along the AP axis, Zld may function in a similar way with Bcd. In zld2, the hb border shifts anteriorly (Figure 6), indicating that in regions of low-level Bcd, Zld enhances the sensitivity of target genes to morphogen concentra- tions. These results imply that Zld may promote transcription by acting synergistically with the patterning morphogens. It is important to note that the observed delay in expression does not necessarily mean the gene is activated later, but that without the synergy factor, there are not enough detectable transcripts at the time when assayed. Sporadic expression may reflect a similar situation. Timing within networks Beyond the role of Zld in timing transcriptional initiation is a more elaborate timing mechanism, exemplified by the sequential appearance of the gap genes. How does Zld achieve differential activation of target genes within a network? A simple model would suggest that the activation of Zld target genes correlates with the strength of Zld binding to their regulatory elements. We noticed that the earlier activated genes in the segmentation network had higher binding scores than those activated later. gt, tll, and all of the primary pair-rule genes, which are abundantly expressed by nc 10, had higher binding scores (Figure 5, Figure 6) than kni and Kr, which become abundant later in nc 11 and nc 12, respectively. Later-acting genes such as secondary pair-rule genes, segment polarity genes, and the homeotic genes were bound, but had lower binding scores (Figure S7 and data not shown). Such a mechanism where timing of activation is dependent on strength of binding was shown for the Pha-4 transcription factor in C. elegans pharyngeal development. Pha-4 regulates a wide array of genes expressed at different stages, and the onset of target-gene expression depends on the affinity of Pha-4 binding sites in the regulatory regions of those target genes [40,41]. An intriguing possibility for the early Drosophila embryo is that as Zld levels rise in the first hour of PLoS Genetics | www.plosgenetics.org 10 October 2011 | Volume 7 | Issue 10 | e1002339