Zelda Coordinates Early Gene Networks Figure 6. Zld regulates timing within the gap gene network. Wild-type (wt; A, C, E, G, I) and zld2 (B, D, F, H, J) embryos were hybridized as indicated. Wild-type activation of gt (A) and tll (G) was detectable earlier than that of kni (C) and Kr (E). In zld2 embryos, all gap genes were delayed 1– 2 nc (B, D, F, H, data not shown for hb), and cross-regulatory interactions were subsequently affected. Abnormal ectopic activation was observed for kni (D) and Kr (F). All gap domains were expanded and/or shifted (B, D, F, H, J), and the gt (B) and Kr (F) domains overlap, which is not seen in wild- type (compare with A and E). (K–O) IGB views of the genomic regions indicated. Multiple Zld-bound regions surround each gap gene (O). Note that the genes activated earlier have higher binding scores (K, N). doi:10.1371/journal.pgen.1002339.g006 appear to bind Zld in vitro (Figure 2D). It is possible that the enrichment of these sites in Zld-bound regions is due to recruitment of Zld by components of complexes that directly interact with these sequences, or to opportunistic Zld interactions. Thus, at least for hotspots with the CAGGTA motif, which was discovered in the hotspots with highest complexity (bound by 12– 14 factors) [5]), it is possible that Zld binding is involved in their establishment [5]. Zld times zygotic gene activation and promotes robust expression The idea of an ‘‘initial step in the cascade of zygotic gene interactions that control development’’ was first proposed by Edgar and Schubiger [6], and the idea of a ‘‘timer’’ in early development that functions alongside the spatially restricted morphogens was proposed by ten Bosch et al. [2] and De Renzis et al. [4] for CAGGTAG sites. Our combined results on Zld extend PLoS Genetics | www.plosgenetics.org 9 October 2011 | Volume 7 | Issue 10 | e1002339