Utilizing both in situ hybridization and quantitative mRNA analysis, we investigated the changes in the gene network state caused by the removal of one or both of the early acting enhancers. brk5’ deletion generally phenocopied the gene mutant, including expansion of the BMP ligand decapentaplegic (dpp) as well as inducing variability in amnioserosa tissue cell number suggesting a loss of canalization. In contrast, brk3’ deletion presented unique phenotypes including dorsal expansion of several ventrally expressed genes and a decrease in amnioserosa cell number. Similarly, deletions were made for two enhancers associated with the gene short-gastrulation (sog), sog.int and sog.dist, demonstrating that they also exhibit distinct patterning phenotypes and affect canalization. In summary, this study shows that similar gene expression driven by coacting enhancers can support distinct, and sometimes complementary, functions within gene regulatory networks and, moreover, that phenotypes associated with individual enhancer deletion mutants can provide insight into new gene functions.Developmental genes are often regulated by multiple enhancers exhibiting similar spatiotemporal outputs, which are generally considered redundantly acting though few have been studied functionally. Using CRISPR-Cas9, we created deletions of two enhancers,  brinker (brk) and short-gastrulation (sog) from the genome of D. melanogaster fruit fly using CRISPR-Cas9 genome editing. Surprisingly, opposite phenotypes relating to some target genes are associated with the enhancer deletions. Deletion of one enhancer generally exhibits phenotypes in early embryo patterning similar to respective gene mutants; whereas, in contrast, deletion of the other presents unique phenotypes including change in cell number for a particular tissue in the embryo, the amnioserosa. In summary, this study shows that coacting enhancers driving similar expression outputs can support distinct, and sometimes complementary, functions to differentially impact the development of embryos and that the individual mutation of these enhancers can provide insight into new gene functions.Genes expressed during development are often regulated by multiple cis-regulatory sequences, non-coding DNA, also known as enhancer sequences. Many instances have been found where two or more distinct enhancer sequences support similar spatiotemporal outputs relating to a single gene. These enhancers have generally been considered redundantly acting, or overlapping in activity, though few have been studied functionally. We created deletions of coacting enhancer pairs associated with the genes  Drosophila embryos, brinker (brk) and short gastrulation (sog), which are each associated with two enhancers that control their expression in the early embryo  when both enhancers should be active (yw), brkΔ5’, brkΔ3’, and brkΔ5’Δ3’. First, as proof of principle, total counts of brk mRNA were measured and, as expected, the brkΔ5’ data is similar to wildtype (as the brk3’ enhancer is predominantly acting at late stage 5), whereas counts are decreased for the brkΔ3’ and brkΔ5’Δ3’ datasets  and level (NanoString) of expression allowed us to more fully investigate the changes in gene regulation in these mutant embryos  or antagonistic effects (i.e. opposite phenotypes) on target gene expression outputs. NanoString allows us to detect mRNA abundance, which is complementary to the spatial data provided by stainings. For example, dization , and levdization . During s mutant . The brkgenotype . However embryos .brk encodes a transcription factor that acts as a transcriptional repressor to limit BMP signaling and support patterning in the early embryo and at other stages of development including in the wing disc .All flies were reared at 23°C on standard fly media. ftz-lacZ , sogY506ftz-lacZ , brk 3’-vep-lacZ , sog.intvep-lacZ , sog.disvep-lacZ , and Hishttp://flycrispr.molbio.wisc.edu/tools) was used to identify the PAM sequences with no predicted off target hits. The gRNA plasmids were then injected into either y2 cho2 v1 P{nos-phiC31\int.NLS}X; attP2 (III) (NIG-Fly #TBX-0003) or y1 v1 P{nos-phiC31\int.NLS}X; attP40 (II) (NIG-Fly #TBX-0002). Stable gRNA transgenic lines were created and then crossed to a Cas9 expressing line . Individuals from the next generation were screened by PCR for the deletions. Alternatively, for the sogΔint and sogΔdist, a homologous recombination cassette (HRC) was created by adding 1kb homology arms to the plasmid pDsRed-attP . Lines were screened for RFP in the eyes and the RFP cassette was subsequently removed in the same way as for the single mutants. All lines represent isogenic backgrounds differing only in the gRNA line used to create the specific deletion.For CRISPR-Cas9 deletions within the genome, gRNA constructs were created by modifying the pCFD4 plasmid  to targsog, aos, zen, sna, and rho were transcribed from cDNA subcloned into pGEM-T vector or full-length cDNA as for brk . For fluorescent in situ hybridization (FISH), probes were detected using Sheep anti-digoxigenin (Life Technologies PA185378), Mouse anti-Bio (Invitrogen 03–3700), and Rabbit anti-FITC (Invitrogen A889). Fluorescently labelled secondary antibodies were all from ThermoFisher (used at 1:400). Enzymatic detection was performed using digoxigenin labelled probes followed by detection with Anti-Digoxigenin-AP antibody  and standard AP staining using NBT/BCIP. Other antibodies used in this study were Hnt , pSMAD1/5 , and dpErk .Embryos were fixed and stained following standard protocols. Antisense RNA probes labeled with digoxigenin, biotin, or FITC-UTP were used to detect reporter or in vivo gene expression as described previously . Probes brkΔ5’ and the brkΔ5’Δ3’), there were no obvious defects in cellularization seen in any of the mutants. Using nuclear shape and size combined with percent cellularization to characterize developmental stage was judged to be an adequate method to synchronously stage embryos that is independent of the genotype.Specific staging of embryo images is indicated in the associated figure legend. For early and late stage 5, the shape and length of the nucleus as well as the membrane front of the cellularizing cells was used to define either early or late stage 5. Early stage 5 (nuclear cycle (nc)14 A and B) included all embryos in nc14  that arThe following primers were used to generate riboprobes:dppi-f ccagaactagaaaaccggaagcdppi-t7-r gaaatTAATACGACTCACTATAgggCGCCTGTGCTAAAGACCCTGstumps-f TGGCCCAGAACATCGTCAGTTTstumps-r-t7 gaaatTAATACGACTCACTATAgggATGAGACTTCACCTGCTCCTGGATtld B-f ATGTGGATGAGTGTTCAATtld B-r-T7 gaaatTAATACGACTCACTATAgggTCCCTTCGCTGGACCTCTCATRace-f ATGAGACTGTTTCTGCTAGCCCTGCRace-T7-r GAAAATTAATACGACTCACTATAGGGACGCAAGCAGAAGGCACAGATAnetA–f ATGATCCGTGGAATCTTGCTCCTGCnetA-T7-r AATTTAATACGACTCACTATAGGGCTTTGCACTCATTGGCTTCCTTGGCpnr-f ATCTCAAACCCTCGCTCAGCpnr-T7-r aagtaatacgactcactatagggagaCGAGGTGGCCATCAGTTTGGsog ex1-f TCAGGTTCAGTCGCTCTTGAsog ex1-T7-r AATTTAATACGACTCACTATAGGGGTGTCGGACTCCTCGAACATdpp and rho was measured using ImageJ. First, an ellipse was fit to the outside of the embryo image, and the perimeter of this ellipse was measured; then, an arc was manually drawn along this ellipse matching the extent of fluorescent signal for each gene measured. The length of the arc was measured, and a ratio of arc length to full circumference was determined. The width of zen at stage 6 was measured using chopped sections that were co-stained with DAPI to mark each of the nuclei. The cells expressing zen were counted manually. The amnioserosa cell number was counted using stage 10–13 stage embryos stained with Hnt antibody. Nuclei on the lateral side of the embryo only were counted manually in ImageJ by planing through the z stacks and using the Cell Counter plug-in. Box plots were created using BoxPlotR (http://shiny.chemgrid.org/boxplotr/).Embryos were sectioned along the anterior-posterior axis manually using a razor blade, and cylindrical mid-embryo sections were mounted on the cut side allowing for imaging of the axial plane. Percentage of embryo circumference for χ2 [The width of the pMad gradient was measured as described  with theχ2 , we usedχ2 . The scastumps were made in ImageJ. Z-stack projections were created using the sum slices function and then the heat map was applied using the LUT function.Heatmap representation of images for pMad and Grids were drawn on standard 10 cm apple juice collection plates, dividing each into 160 quadrants. One embryo was transferred from a two hour collection into each of the quadrants of the grid. The plates were aged at 25°C in a humidified chamber for 30 hours, and the number of hatched and unhatched embryos was manually counted. This was repeated for a minimum of three times on different days for each genotype.brk mutant lines and nuclear morphology was observed on a Zeiss LSM800 microscope. Embryos were collected at nc14C for each genotype. This specific stage was determined to be when the nuclei were elongated but not yet disorganized, and the membrane front  fully encapsulates the nuclei [WT, brkΔ5’Δ3’) or 5  individual embryos were analyzed for each genotype.NanoString was run as described . H2A-RFPe nuclei . Once exbrkΔ5’ and brkΔ3’ were verified by crossing deletions made with different gRNA lines in trans to check for offsite effects. For brkΔ5’Δ3’, two independent mutant lines made using the same gRNA pair was crossed in the same manner. No differences were seen between the single mutant embryos and the trans-heterozygous embryos in the expression of brk, dpp, Race, or zen so all analyses were done using a single mutant line for each deletion.Individual deletion lines for brk or sog gene expression, specifically. For brk analysis, males of the genotype YW, brkΔ5’, brkΔ3’, or brkΔ5’Δ3’ were crossed to females of genotype brkM68/FM7 ftz-lacZ. To identify trans-heterozygous embryos  and assay for changes in amnioserosa, embryos were stained with a combination of antibodies: anti-βGal to detect the balancer, anti-Sxl to identify female embryos, and anti-Hnt to detect amnioserosa. Similar trends were seen as for homozygous enhancer mutants :3’grna-f attttaacttgctatttctagctctaaaacCGCCTCGGCCGGCGTCGCTGCgacgttaaattgaaaataggtc3’grna-r attttaacttgctatttctagctctaaaacCGGGGTGGAAAAGCGACCCGCgacgttaaattgaaaataggtc5’grna-f tatataggaaagatatccgggtgaacttcGTACCTGTTCGATCCTTCATgttttagagctagaaatagcaag5’grna-r attttaacttgctatttctagctctaaaacTTGGGCTTTCGTTGCACAACgacgttaaattgaaaataggtcSog.int grna-f tatataggaaagatatccgggtgaacttcGTCAAAATCTTTAGTTAAAGgttttagagctagaaatagcaagSog.int grna-r: attttaacttgctatttctagctctaaaacTAGATCCCGGGATTTGTGCCgacgttaaattgaaaataggtcSog.distgrna-f:tatataggaaagatatccgggtgaacttcGATGATAGGTGGACTCTTGATgttttagagctagaaatagcaagSog.distgrna-r:attttaacttgctatttctagctctaaaacAGCGAATACGTGGAATTCCTCgacgttaaattgaaaataggtcSet 2 (second independent generation of deletions used to test for 2nd site mutations):3’grna2-f tatataggaaagatatccgggtgaacttcGTTCCAAAACTTTAATCTGTTgttttagagctagaaatagcaag3’grna2-r attttaacttgctatttctagctctaaaac GCAATCATTCTTCAATTCATCgacgttaaattgaaaataggtc5’grna2-f tatataggaaagatatccgggtgaacttcGACCCGATGAAGGATCGAACgttttagagctagaaatagcaag5’grna2-r attttaacttgctatttctagctctaaaacGCTTTCGTTGCACAACTTTAgacgttaaattgaaaataggtcHDR templates:Sog.int LA-DSR-f gtacgtgaattccgaaactcgcgtgtgttatctaSog.int LA-DSR-r ctagcggcggccgctaactaaagattttgactagtSog.int RA-DSR-f gtacgtggcgcgccatcccgggatttgtgccSog.int RA-DSR-r ctagcgctgcaggcggcagacagttgaataaaSog.dist LA-DSR-f gtacgtcccgggtccatccccacaccatttatSog.dist LA-DSR-r ctagcggcggccgcgaatacgtggaattcctttcgSog.dist RA-DSR-f gtacgtactagtaagagtccacctatcatcccagtSog.dist RA-DSR-r ctagcgggcgcgccagatgcgccagaagtacgCRISPR-Cas9 Deletions:Deleted base pairs capitalized. Indel/added sequence shown in bold italics.brkΔ5’:tggtattaaaactgaaaatcaatctaaaaatcaaccattgataacattttattgaatcaaaccaaaagccaaattgattcctgaatccaaaagacccgatgaaggatcgaacaggtactacgatgatattggtcgGAAAATACCTGCGCATCCTGGTGGTTTATGGTGCGGCCGTAAATGCAAGCCAAGTTCTTTACGGCTTCTCTGGCACAAACCCTAAATGTGGATTACGCTAATATTGCCCCCCCTAATAAAAACGGTCGTTGTCCAGGGCCGAATATTGCGTCTGATTGGTTTTTCCCACGATTACAATTAGCCGGACGGACACAAACTGACCTGAGCTGACCCGCAAAAAGACACGGTTGTCCGGCAGTCGGAACTGAAGGAAACTAAAGGAAACTGAGGGCAGGTCAGCGCTATGGATTGTGCACTAAGTTGCTTAATCCGACGGGAAATCCAAAACACAACCCGAGCCCGATCCTTCGCTCCTTCGATTTAAGCCAAAGTTAGAGGCACAGGCACACATGTGTGTTTGGTTTGAACGGGAAAGCCCCATTTTAAAGCTGGCCAACCAACGGCAACACATGTTCATGTTAGGACCGATACAGGTTGACATTCCCTGGAAGGATGCACCTCTGGGAGATTCCCACAACCGGCAGCAGGTCATGTCCAACCGATCGTTGCGGGAGCCACTTGTCCCGAAAAAATCCAAAGAAACTATCAAGTGGCGTTTAGGGAAACTCAAAACTTTCCAACCACACCATATCTTTCTAACGCCACACAATAAACTGGTATGATCACTGTTAAGATCAAAATGGGCTAAATAAAGCGATCATGAATCATTTACTATAAACTGAAGAGTTTTCTATTCTTTAATATAAAAGAAAAGATGAGTAACACCCTACAAAAATTTTAGGACTACAGATCTACCCACCGATGATGATCAACCCTTCCCAAAAAAAAGTCACAGCTTGTATTTCGTAAAGATTTCAGATTTCTTAAGACACGATCACCAACTGATCTATGGACTTCTTAAACCATTGGGCTTTCGTTGCACAACTTTAGGGATTTGTTTTTTTTTTTTTTTTTTTGATTCCACATCCGATTTCTTCTTCGAAGCACTTCGATCTTTTCTCacatcagtttgatcgacaatcgacgatcgatggctgtgtgaaaacaagtttcgtaactatgtaaattaacctccabrkΔ3’:GGAGAGCAGAGAGCAAGCAGGAGCCAAGAGGCAAAGCCAAAAGAAATGCGTTTCTTTTATTTTTGACGCGTCTTAGATTCTTCCTCTGCCGCTTCTTCTCTCGTTTTTTTCTTTCACTTTTTTGTTTTTTTTTTTTTTGTTTTTGATTTCTTGGAATCGCATCTACTTTGAACCTTCCACGTTCCGACGGAAGACTCAGAGACTCGACTGCATTGTATATTTGAATTTCATTTCAATTTAAATACGATTTTCTATGAAAATGCACGCACCACAAATACGTGCAATCGTTATCCAAACCATCTACCCCTCCCCTCCTCCCCTTTATCACCATGCTCTTTGCCTCCGCCGTCCTCTGCTTTTTTTTTGTTTTTTACCCCATACATGTACATAATACGATCCAAGTTTCATGAAAAAAACCGGAGAAACAAATTGATCATGTTTTGAATGCATTTCTGTTTGATTTCAATTTCGATTTGTGTACTCCATAAATGCGAAAGAATCTTGGATTGGAGTTGGGGGGATAAAATGGATGGATATATGGGGTGGGGTAAATCAGAATTATACGGCATATGCGGATCTAATCTAATAATATACAATATTACTGAGTCGCATACCCTAGAAATCGCACATTGATTAGCACAACCTGTCCAAATTATCCGAGCAAAATCCCCCCAAATCACGCAACACTCTAAACTGATCCGAAAGATCCTAACAGTTGAAAATCCACCTAGTCGAGCTAAAACCAAATTGTCAGAGGATTGCCTTGAATGGCCCACTTAAGCCAGGCGTGCGGCTGAAAGGAGCCGAGGATTCCTTGCCGAATCCGAATCGGAATCCTGTGCCAAGGACAGCATGCAACGCACCGATGCGAGTGCCGAATGACCTTTGGGAAAATACGTGTTGATCGACATCCATCGGCAATTGTTTGAGATCTTCGGAAATCTCTCACCTTTCGAATGGGAAATTCCTTAGGATCATCGAGCTAGCTGGTCAGAGAAGGCCGTTCATAAGCATGCAAATAAAGATTTCACACCTCGGCCAGCGCACTAGGGTGCATGGTGCAGCATAGGGCTCTCAGCCAAAGGACACGAAACGAAATGCATGGAGATCGGGACACCTGGGAAATACCCAACCTGCCCGAAACATATGGACGCACAACGCACAGGAGGGATCTCTCGCTCTGAATGGAAAGCAGAAACTCTCTATAGGCACTAGCTATCCATCCAAGTGGTTCCCGAAAAAAAAAACCCTCACAATATAATTCGTAAATCCCAGCCGCAGGTATTTAGTTCCGCCTTTGTCCATCTTTGGAATTCCCAATGCTATTATTGCACCTAAGCCACTGGAGTGGGAGGTTTTGCAGTGTGTGTGCGCGTTGCAAGTTGCTGCCGGTGAGCAACACATCGAATTCGTCCCATCTCTGTCTGGCCGCGACTCGTCATCTCTCTCTCTCTCTCTCTCTCTCTGATCTCTCTGTCCCGCTCTGGTTCGTGCTCTTGCTGTCGCCGGCGCCGCAGGTCAACAGCGATGTGTGGCATTTTCTAACAGTTGCTGCTGCTGTCGCAGGTTGCAGGTTGCATGTTTTGTTGCAGTTGCTGCAGCTTATATGACGCTTACATTTGTTATATGTATCTGCCACTGGGGGTTTTCCGTCAATATTGCAGGTTGCATAGCTTATGCTGCAGCTTCTATTGATGACTAATTTATTGTTGCTTGCTTGCTTGAGCAAGTTGCTTTAGGTTTACATTAAAACGGCACATTTCTTGGCATCAGTTGCACTCTCTGTTGGTATAATTGTTGCTGTTTCTAGTATCAAGTGTACTAATGTTGCAGGTGCTGCGCATGCATATGTTGCAAGTCTGTTGTTTATTGTAATGTACCCATAAAACTGCATATATTTATACATAGGTGTTTAGATTTCTACTATTATCGCTGAAAACTTGTTTTTCTATTCAATCTTGCTCATAAAATGGTTCAAATATATATAGATTTGCTGCACTCCCGACTTAATTCTTCCAAAATGGCTGTTAAATTTCAAACTATTGCCCATGTTGTTTTCGTTTAAATCGCTTATGGTTTTTCGGATGTTGTTTTTCGTTCAATCGCTGGCTGATTTTCTTGCTCTCCCGAAAATCCACACGCACATTTTCATTGTCGACAAATTTTCAGACGGCAGGACAAAGAATTTTTCATTCGTTTGTTTTCCACGCACTCGGTCTGAATGGCCAAAAAGGAAAATGTGACGAGCTAGAGGGAAATAGGGCACATTTTTGGGGGCAAGCGAGATGACCAAAAAAGACCTGCGATCCGTTCCGATCCGCTCCGGGTCGCTTTTCCACCCCgggggcattgttttcagaccgagagtatatagtagaatctcctattatatcttctattcctatttgtatttgtatttgtacgagacgggcagaaagaacgagaggaatggagatagagggbrk3’ in brkΔ5’Δ3’:gaagcgaatgccaatgcacaaggagctaaacgagtgcgagcgagacgggcagaaagaacgagaggaatggagatagagggAGCAGGAGCCAAGAGGCAAAGCCAAAAGAAATGCGTTTCTTTTATTTTTGACGCGTCTTAGATTCTTCCTCTGCCGCTTCTTCTCTCGTTTTTTTCTTTCACTTTTTTGTTTTTTTTTTTTTTGTTTTTGATTTCTTGGAATCGCATCTACTTTGAACCTTCCACGTTCCGACGGAAGACTCAGAGACTCGACTGCATTGTATATTTGAATTTCATTTCAATTTAAATACGATTTTCTATGAAAATGCACGCACCACAAATACGTGCAATCGTTATCCAAACCATCTACCCCTCCCCTCCTCCCCTTTATCACCATGCTCTTTGCCTCCGCCGTCCTCTGCTTTTTTTTTGTTTTTTACCCCATACATGTACATAATACGATCCAAGTTTCATGAAAAAAACCGGAGAAACAAATTGATCATGTTTTGAATGCATTTCTGTTTGATTTCAATTTCGATTTGTGTACTCCATAAATGCGAAAGAATCTTGGATTGGAGTTGGGGGGATAAAATGGATGGATATATGGGGTGGGGTAAATCAGAATTATACGGCATATGCGGATCTAATCTAATAATATACAATATTACTGAGTCGCATACCCTAGAAATCGCACATTGATTAGCACAACCTGTCCAAATTATCCGAGCAAAATCCCCCCAAATCACGCAACACTCTAAACTGATCCGAAAGATCCTAACAGTTGAAAATCCACCTAGTCGAGCTAAAACCAAATTGTCAGAGGATTGCCTTGAATGGCCCACTTAAGCCAGGCGTGCGGCTGAAAGGAGCCGAGGATTCCTTGCCGAATCCGAATCGGAATCCTGTGCCAAGGACAGCATGCAACGCACCGATGCGAGTGCCGAATGACCTTTGGGAAAATACGTGTTGATCGACATCCATCGGCAATTGTTTGAGATCTTCGGAAATCTCTCACCTTTCGAATGGGAAATTCCTTAGGATCATCGAGCTAGCTGGTCAGAGAAGGCCGTTCATAAGCATGCAAATAAAGATTTCACACCTCGGCCAGCGCACTAGGGTGCATGGTGCAGCATAGGGCTCTCAGCCAAAGGACACGAAACGAAATGCATGGAGATCGGGACACCTGGGAAATACCCAACCTGCCCGAAACATATGGACGCACAACGCACAGGAGGGATCTCTCGCTCTGAATGGAAAGCAGAAACTCTCTATAGGCACTAGCTATCCATCCAAGTGGTTCCCGAAAAAAAAAACCCTCACAATATAATTCGTAAATCCCAGCCGCAGGTATTTAGTTCCGCCTTTGTCCATCTTTGGAATTCCCAATGCTATTATTGCACCTAAGCCACTGGAGTGGGAGGTTTTGCAGTGTGTGTGCGCGTTGCAAGTTGCTGCCGGTGAGCAACACATCGAATTCGTCCCATCTCTGTCTGGCCGCGACTCGTCATCTCTCTCTCTCTCTCTCTCTCTCTGATCTCTCTGTCCCGCTCTGGTTCGTGCTCTTGCTGTCGCCGGCGCCGCAGGTCAACAGCGATGTGTGGCATTTTCTAACAGTTGCTGCTGCTGTCGCAGGTTGCAGGTTGCATGTTTTGTTGCAGTTGCTGCAGCTTATATGACGCTTACATTTGTTATATGTATCTGCCACTGGGGGTTTTCCGTCAATATTGCAGGTTGCATAGCTTATGCTGCAGCTTCTATTGATGACTAATTTATTGTTGCTTGCTTGCTTGAGCAAGTTGCTTTAGGTTTACATTAAAACGGCACATTTCTTGGCATCAGTTGCACTCTCTGTTGGTATAATTGTTGCTGTTTCTAGTATCAAGTGTACTAATGTTGCAGGTGCTGCGCATGCATATGTTGCAAGTCTGTTGTTTATTGTAATGTACCCATAAAACTGCATATATTTATACATAGGTGTTTAGATTTCTACTATTATCGCTGAAAACTTGTTTTTCTATTCAATCTTGCTCATAAAATGGTTCAAATATATATAGATTTGCTGCACTCCCGACTTAATTCTTCCAAAATGGCTGTTAAATTTCAAACTATTGCCCATGTTGTTTTCGTTTAAATCGCTTATGGTTTTTCGGATGTTGTTTTTCGTTCAATCGCTGGCTGATTTTCTTGCTCTCCCGAAAATCCACACGCACATTTTCATTGTCGACAAATTTTCAGACGGCAGGACAAAGAATTTTTCATTCGTTTGTTTTCCACGCACTCGGTCTGAATGGCCAAAAAGGAAAATGTGACGAGCTAGAGGGAAATAGGGCACATTTTTGGGGGCAAGCGAGATGACCAAAAAAGACCTGCGATCCGTTCCGATCCGCTCCGGGTCGCTTTTCCACCCCggcattgttttcagaccgagagtatatagtagaatctcctattatatcttctattcctatttgtasogΔint:GCGGCCGCGGACATATGCACACCTGCGATCATAACTTCGTATAATGTATGCTATACGAAGTTATAGAAGAGCACTAGTAAAGATCTCCATGCATAAGGCGCGCCAAGGGGTATTAACTGTTGCCTGTTGCTTTCGACATTTCTACCTCCGCTGCGATTGCATAAGTTGCCAATGCCATTGCGCATACGCCGTGTCGTCTATATGGCTATATGGCTATATGGCTGTATGGTGCGGGGAAATCCCCGTAATCGCAGGTAGAATTCCAGCCGGTGCCGAGGCGGGACCTGCTCGCACCTCTAATCCCGCCAGGGTTTTCGGGACATGGGATATTCCCGACGGCACAGCATAGCACTCCGTTTTCTTTTTTTTTTTTTATTATTATTGTGTCCAGTTTTAATCCGGAAAGCGGGAATTCCCTTCCGCTCGCTGCCTGCACTGCGCTGCGCAGACGCATCGGCGTCCGTAAGCCGCTTACCAAAAAGATACGGGTATACCCAAATGGATGCCTGCCCATGTATATAGACCATTGGGTGGTATGGACCATGGACCATAAAGCGGCACCCAATTGCAATTTGTTGCAACTCACACGCTTGATTAGCTGCATTTCCTGTTTGCAAACCCCCCTCCCAATACTCCCTACACTCATATATTATATATGCATATAAATGTATGTGTATATTGCATACTATTATGTTCCCGAGTTGCAGTTTTTTTTTCCTTTTTTTTCCACTATTTCTTTCTTGGTTCGCTGCCGTTGTTGCATTTTCAATTAAAAACAGATTGCGGTTTACGCTGCAGCGACTTTGAAACGTTACTCGGCTTACACACAAAATGCAGCGCGATAGTAATGATGATTAACAGGTTATGTTAGGGCTTAGTTGATACGCCGTAGatcccgggatttgtgccggaaccggaacgaatatcgaatcaatcgcatatccttaaagttcgaactagtcaaaatctttagttasogΔdist:GGCCGCGGACATATGCACACCTGCGATCGTAGTGCCCCAACTGGGGTAACCTTTGAGTTCTCTCAGTTGGGGGCGTAGATAACTTCGTATAATGTATGCTATACGAAGTTATAGAAGAGCACTAGTGGGTAGTCCACATTCCATTCCATCGAAAGGAATTCCACGTATTCGCTGGGCGTTTCGGGGGCGGACTGGGCATATGGGCGTGGTGGGAGGGGGTGGGCGTCACGGGGCGTGGCCACACGTGAAACGGCTAATTTGTTGATGCAAATGCGGGTGTGGCCTGTTCTCGGATCGGAGTCCCCAGCACGCGCCCACAAATAAATAAATGGCTGGAGATGGAAATGGAAACCGATGACGGGGAATAGGGAATGACGGCTGGGTAGCGGGGGAGACGGGGATGGGAACTGGGGGGCTGGGGTCTTGGACCGAGATCGCAGCCAATGGCGACAATTGGCATTCACCGTCGCAATTAATCATCATCACCGTCCGATTGACAATCGGGCCACAAATAAATCAAAATCAAAGGCATAAGAAAGCGAATGCAGCGTCGGCATACAACTGCACTGAAAGAAAAATATGTATGAAGCTTCTGGTCAAGCAAACCGCAAGAAGCGCATTTAAGCTCAAGTATGCTCAAGTATTTGTTGTGTAACTTTAGATGATTCAAAATCGCTTTCTTATGTCTTTTTTGACTGCACTTGCCCTGAATTTCGTCGAGTGTAGTTCCCAATCATTGCAACAATTCATCAAGTATGCAAACACACGCGGCCGAATCAATCAATGGCGAGTAGTAGGAGTGCATGGAGGATGAGAAGGAGGAGAAGTTGGTTGAGAGGTCATCGTTGCGATTCTGCGATTCAGCAGTTCCACAGAAGGTGTCGTAATCCTGGACGCAAGGGTGCACGGACCAACTGACAGGGGCAAGTGCGTCCTGTGCCACCAGATGACGCACGATGCGGCCGGAAAAACCCAAAATCAAAAACCGAAAACCGAAAACCTGGTCAGAGTTTCCGAAAACCAAAGAGCCAACATCGAATGCGGCACAATAACCCGATTGTCTGCGAATACCCACGATGATCTAGAATCGCACGGAGAGCACTCTCACGCATCCGTGGCCATATGGGTGCGGCCAAATCGGAAATTCCCAGGACAGGTAGAATGCATTGGATATACGGGTATACGGATTGGAATTGGGATTGGGATTGGGACTAGCACCAGGTTGCAACGCCCGCCAAGAAGCCAATTTAAATAAGCAGCATAAACAAAAGCGACAGCGTTTTATGATCCCCGCTCCTTATCCTTGCACAAGGATATCGCCATGGCCACGCAGGTAGGAATAGCAGATATGGCGGCAATGATGCGCCAACCGCACTGCTTCGTCCTGGTCCTGGTCGGATGGGCTTTTCCCACGCAACCGCGACCTTATCTGCGCCCCTTTTATGAGGCTGCATCTGTTTTCGCACCTCGATGCCGTTGGCATTATAGCCACATGTGTATGGTGGGAATTTCCGATCGACCAGCCTACCTGTTCCGCTGAAACCCGGGAATCTGTCCATCCTGAGCTTCCACACACACACACACACACACACAGGTCAGTCGGCATCAATTGGCTGCCATAAACATATAACAATCAATATTGAATCCTTTATCGTAGAATTTGTTGTATATGCCCATTGCAGTCCTTCGATTAAATGATGGCTAAAATGAATAAAATGAGTTGCTAGGTTCGAAGTAGGTGAAAACTGGTCAATAAGCCATATCCTTTATGATTAGATATTCTAGTAATCTGGCATGAAATACACAAATATAATAGCGAGTGATTAGTTTTATATAAGTATTTCTATTAAACCTCTAATAATTACCGCTTTACATATAGATTTTTTTAGTTGGATGCGGGCTTTAGTACCATATAGCTTTACCTGAATCTTATCGTTTGCTTTGGCTGATCCCCCAGGATCTGCGACATAATGCCAAACACAAGCAACTTGTTCGCCTGATCTTCGCAATCCCAGCAGGTTAACTACCCGATTTTATCGAGCAACAGCAAACTCTTTGAACTGATTTGCTATATTTCATTTATGGCCGATTCCAAGTCCATGTCCATTTCCCATTTCCATGTTCGTGTCCAAGTCCGTTGTTCGTTGTCCATTTCGATGTCAGTCATTCATTTTTGTGCTCGACTGCTAATGCCTTAAAGCCAAAATCCATTTCGTCGTTTGGCCATATAATCCAACATTATACATTAAATATGGCACTTGAAAGCACCGCGCGTCTGCATGCCAATTTCAAGGCCCCTTTTCCTCCACTTCACCACCCACAGCATACCATCCCATATCCCACCGGAATTTTCCGATTTTCCCCATTTTCCCCCAACAACACCACCACCTTGCTGCGATCGCACTTGTCGCTCGCGCCGGCGCCTTCAGGGTCGTCAATTTTTTGAGCGACATTTCGAAACGGTGGCTAAAATCACAAAAGCCAACAGAAAAAAATAAAAATCAACTATGGCGAAAAAGTAATACAAATGAAATCCGAAAACAAATAAGCGATTTTGTTTGTGGGGAGAAATGATGATGATCGGCGCCAGACGGACATAAAAACGCCAAAAGAAACGGAAAGGAAGCCTGGGAAAGGCGCATTTCCACTTTGTTTATTCGGAGCTCTGAAGCTCTTTTTAATGGTTTCTTTTTTTTTTTTTTTTTGGCCAAATGGCCAATTAATGCGATTTACTTGATACGCTTTTGCATTTTGTTGTTGCTTTACGACGGCGGATATTAATTAATTGGCCCGTCTCTGGGGAGCAATTCCCATTGTGCCCATCaagagtccacctatcatcccagttcgcagtccaaacctgacaatcgattaaaattacctctccagttgcacagatcaacggggtagtccacattccattccatcgaaaggaattccacgtattcgcS1 Fig(A) Dark field images of lateral view of cuticle preps from first instar larvae. (B) Bright field images taken with 40X objective of dentical bands in the A2 abdominal segment. Ventral view with anterior to the left.(TIF)Click here for additional data file.S2 FigbrkΔ5’Δ3’) or 5  individual embryos are shown by black bars. WT shown in black, brkΔ5’ in gold, brkΔ3’ in blue and brkΔ5’Δ3’ in grey. Genes broken into groups based on expression level for ease of display.Results for 70 genes assayed by NanoString in nc14C (late stage 5) embryos see . The res(TIFF)Click here for additional data file.S3 FignetA and (B) stumps gene loci. Brk binding is significant  only in late stage 5. Flybase defined protein coding regions for each gene shown in blue under Brk ChIP-seq tracks.(A-B) Screen shots from database of Brk ChIP-seq data  showing (TIF)Click here for additional data file.S4 Figtsg and (B) vn, or immunostained with an antibody to (C) dpErk. (D) Cross section of stage 6 embryos showing zen expression (green). Magnified image shows only dorsal one-third of embryo. Representative images for each genotype, further quantified in E. (E) Box plot of width, in number of cells, expressing zen. P-values determined by Welsh’s t-test comparing brkΔ5’, brkΔ3’, and brkΔ5’Δ3’ to WT for zen were P = 0.4, P = 5.5x10-5, P = 0.06, respectively. Significance indicated in graph by *P<0.05, ***P<0.0001. (F) Percentage of embryos showing normal (blue) vs disrupted (orange) expression of Race in early stage 6 embryos. Number of embryos counted for each graph in this figure indicated under genotype.(A-C) Dorsal view of stage 6 embryos hybridized with riboprobes to (A) (TIF)Click here for additional data file.S5 Figbrk and egr. egr expression is diminished or lost in the brkΔ5’ and brkΔ5’Δ3’ embryos. (B) In situ hybridization of late stage 5 embryos, dorsal views, with riboprobes to egr. egr expression remains low in the brkΔ5’ but is expanded in the brkΔ3’ embryos. (C) FISH staining of late stage 5 embryos, lateral views, with riboprobes to Cv-2. White arrows indicate expanded Cv-2 expression in the brkΔ5’ and brkΔ5’ Δ3’ embryos. (D) Model of canalization loop acting to regulate amnioserosa cell number, reproduced from [egr, (F) Cv-2, and (G) zen loci.(A) FISH staining of early stage 5 embryos, lateral views, with riboprobes to ced from . (E-G) Sced from  showing (TIF)Click here for additional data file.S6 Figtld (green), ind and sna (both purple). All embryos are trans-heterozygous females of the genotypes indicated. Consistent with the patterns seen in the homozygous brk CRISPR mutants, tld is expanded ventrally, beyond the domain of ind expression in the trans-heterozygous embryos with brkΔ5’ and brkΔ5’ Δ3’ but not significantly in brkΔ3’. (F) Comparison of number of amnioserosa cells in homozygous brk enhancer mutants to trans-heterozygous combinations with brk- gene mutant. Homozygous mutant data is reproduced from (A-E) FISH staining of late stage 5 embryos, lateral views, with riboprobes to (TIF)Click here for additional data file.S1 DatasetExcel file containing raw counts for all graphically represented data depicted in Figs (XLSX)Click here for additional data file.