development a ‘‘temporal’’ concentration gradient is formed such that interaction with higher affinity binding sites would occur before that with lower affinity sites, thus differentially activating target genes. A second timing mechanism is provided by the intrinsic properties of the regulatory motifs established by Zld. Our data revealed that Zld functions in several coherent feed forward loops, for example, binding both the XSE (X-chromosome signal element) genes (such as sisA) and Sxl, dpp and its targets, and twi and rho (Figure S9). Embedded in this type of motif is a mechanism of temporal control since a delay in the activation of the third gene in the loop occurs because of its dependence on accumulation of the second gene product [42,43]. For example, the activation of Sxl is 2– 3 nc later than that of the XSE genes [2]. In addition, experiments that abolished the TAGteam sites in the SxlPe enhancer caused a 3 nc delay in reporter expression, demonstrating a direct role for these sites, and hence Zld, in timing transcriptional activation [2]. Zld also functions in an incoherent feed forward loop whereby one branch of the loop has the opposite sign [43]. Zld promotes transcription of both the pair-rule and gap genes, while gap proteins repress pair-rule genes (Figure S9). The primary pair-rule gene transcripts are easily detectable by nc 10 (Figure 5), even before some of the gap genes, giving a new perspective on the canonical segmentation gene hierarchy in which the pair-rule genes are downstream of the gaps. Early strong activation of the pair-rule genes may be essential to guarantee transcriptional activation before repressor gradients overwhelm the AP axis. Zld increases expressivity of target genes We can extract clues from our results about how Zld may function on a mechanistic level. First, Zld appears in zygotic nuclei very early (Figure 1C), before Bcd and Dl, possibly binding to target genes first. Second, loss of Zld results in delayed transcriptional activation and, in many cases, weak and/or sporadic expression (Figure 4, Figure 5, Figure 6). Third, Zld binding is frequently found at early enhancers (both primary and shadow), as well as close to the TSS of genes, hinting at a role in recruitment of the transcriptional machinery. Fourth, Zld binding coincides with hotspots (Figure 3 and Figure S1), which were found to correlate with regions of nucleosome depletion [5]. Together these observations suggest that Zld increases the transcriptional activity, or expressivity, of target genes. Mechanistically, Zld binding could facilitate either the access of other factors (both activators and repressors) to DNA or the interaction of these factors with the transcriptional machinery, an idea put forth by Bradley et al. [44] after observing a correlation between the evolutionary turnover of the CAGGTAG site along with the patterning factor binding sites. An alternative mechanism to ensure robust and coordinated early embryonic expression is pol II pausing (reviewed in [45]). Many Zld target genes such as sog were shown to exhibit polymerase pausing [46]. The delayed and sporadic expression in zld2 could be explained by lack of paused pol II. It is evident from our results that Zld coordinates the onset of transcriptional activity of the early gene networks during the MZT (Figure S9). Considering that Zld is also expressed at later times in development [47], we predict that Zld will act similarly to increase expressivity of genes in networks that function, for example, in central nervous system development in mid-stage embryos and imaginal disc patterning in larval development. In these processes, similar to the MZT, a simple strategy may be used to collectively activate and temporally control batteries of genes required for establishing the proper gene circuitry. Zelda Coordinates Early Gene Networks Materials and Methods Fly strains The yw strain was used to obtain wild-type embryos, and the zld294 allele was used to obtain zld2 germline clones as previously described [3]. Antibody production and Western blotting Rabbit polyclonal antibodies against Zld were generated using the C-terminal part of Zld (amino acids 1240–1470) [3], containing a cluster of four zinc fingers fused to GST. For chromatin immunoprecipitation experiments, the antibodies were purified from the serum bleeds by antigen affinity chromatography [48] against purified recombinant-Zld protein coupled to an affinity column. For Western blotting, 50 appropriately staged Drosophila embryos were homogenized in SDS Laemmli loading buffer and briefly centrifuged. The protein concentration of the supernatant was determined (Bradford), and equal amounts of protein were loaded in each lane of a 6% SDS-PAGE gel (40 mg per lane). The blotting and transfer were performed according to standard procedures [48]. In situ hybridization and antibody staining Embryos were fixed and hybridized as previously described [3] using digoxygenin-UTP (Roche Biochemicals) labeled RNA probes synthesized from subcloned cDNA sequences or genomic intronic DNA sequences (for sog and rho). Antibody staining was performed by incubating fixed embryos with rat anti-Zld antibodies (1:200 dilution) followed by incubation with AlexaFluor 488 donkey anti- rat IgG (1:500 dilution) secondary antibodies (Invitrogen). Embryos were visualized by fluorescence microscopy using an FX-A Nikon microscope and by Nomarski optics using a Zeiss Axiophot microscope. Flourescent in situ hybridization (FISH) was performed as previously described [17] using intronic probes for sog and rho, sheep anti-DIG antibodies (Roche Biochemicals), and AlexaFluor 555 donkey anti-sheep IgG secondary antibodies (Invitrogen). Images were acquired as previously described [17] using a Leica TCS SP5 confocal microscope (406 oil immersion objective) with 102461024 resolution and approximately 250 nm/pixel. More than 15 Z-sections from nc 14 embryos were taken at 0.5 mm intervals to capture as many nascent transcripts in nuclei as possible. Confocal image processing The Z-sections containing pixel intensities higher than the median intensity of all pixels were selected for analysis. For each position in the X–Y plane, the pixel with the strongest intensity across all Z-sections was defined as the intensity value for that X–Y position. All of the Z-sections from the DAPI channel were processed by Helicon Focus (HelicoSoft) to generate clear images of nuclei, which were identified by customized Matlab scripts. Every FISH signal was assigned to the closest nucleus, only when the distance between a FISH signal and the center of a nucleus was smaller than 1.56 the radius of the nucleus. The assigned nuclei were considered as with expression and pseudo-colored. DNA binding assays Electrophoretic mobility shift assays (EMSA, or gel shift assays) were performed as previously described [3]. The following oligo- nucleotide sequences were derived from genomic DNA sequences. Each is 21 nucleotides in length and contains a TAGteam or TATCGAT site plus surrounding sequences: zen1: CACTATT- TAGGTAGACACTGT, zen2: TGGGTTTCAGGTAGGTGAA- TA, zen3: ATAAACACAGGCAGCTGGTGC, eve3: ACAATTG- CAGGTAAGTAGAGC, nullo-1: AAAGGATCAGGTACCCG- PLoS Genetics | www.plosgenetics.org 11 October 2011 | Volume 7 | Issue 10 | e1002339