In this and all other subsequent figures lateral views of embryos are shown with anterior to the left and dorsal up, unless otherwise noted. (A,C) Stills from movies (n = 3 for each) of the two indicated sog_Distal MS2-yellow reporter variants sog_Distal (A) or sogD_ΔOpa (C) in which five predicted Opa-binding sites were mutated as shown (H) and transcription detected in vivo via MS2-MCP-GFP imaging (Koromila and Stathopoulos, 2019) at three representative timepoints: nc13, nc14B, and nc14C. Blue dots indicate presence of GFP+ signal, representing nascent transcripts labeled by the MS2-MCP system; thresholding was applied and remaining signals identified by the Imaris Bitplane software, for visualization purposes only. Nuclei were labeled by Nup-RFP (Lucas et al., 2013). Scale bar represents 50 μm. (B) Plots of number of active nuclei, defined by counting dots (x-axis) versus relative DV axis embryo-width (EW) position (y-axis), analyzed from representative stills from movies of three embryos at nc14C. (D, E) Anti-Opa (D) and anti-Zld (E) antibody staining of early wild-type embryos at the indicated stages. (F) Integrative Genomics Viewer (IGV) genome browser track of the sog locus showing Zld and Opa ChIP-seq data for embryos at two timepoints: nc13-14 and nc14 late for Zld (GSM763061 and GSM763061, respectively; Harrison et al., 2011) and 3 hr and 4 hr for Opa. Zld nc13-14, Zld nc14 late and Opa 3 hr ChIP-seq samples are of overlapping timepoints, whereas Opa 4 hr ChIP-seq sample is later. Gray shading marks the region of sog_Distal enhancer location. (G) JASPAR consensus binding site for Opa based on mammalian Zic proteins identified by bacterial one-hybrid (Sen et al., 2010; Noyes et al., 2008). (H) Location of 5 sequences within the 650 bp sog_Distal enhancer region that match the Jaspar Opa consensus binding site allowing 1 bp mismatch. Mutated Opa sites introduced to eliminate binding are shown in blue, creating sogD_ΔOpa (C; see Materials and methods). Bases in bold (7 bp) indicate matches to the Opa de novo motifs identified by ChIP-seq analysis (see J). For sake of comparison to consensus sequence, reverse complement sequence is shown for a subset. (I) Consensus binding site for Mus musculus Zic3/Opa homolog identified using ChIP-seq (Lim et al., 2010). (J,K) Sequence logo representations of the most significant and abundant motifs, likely consensus binding sites, identified by HOMER de novo motif analysis in the Opa 3 hr and Opa 4 hr (J), or Zld nc13-14 and Zld nc14 late (K) ChIP-seq datasets defined (Central motif enrichment p-values 1e-566, 1e-354, 1e-3283, and 1e-2173, respectively). Grey-shaded box indicates the shared region between Opa motifs.