Two-domain expression patterns of tll and hkb. (a) Locally activated Torso signaling induces tll and hkb through relief of Cic-mediated repression. (b) and (c) Spatial patterns of tll (b) and hkb (c) mRNAs in wild-type embryos. (d) and (e) Expression patterns of tll (d) and hkb (e) in embryos that lack Bcd (bcd). (f) and (g) Expression patterns of tll (f) and hkb (g) in embryos that lack Trunk (trk), which is a ligand for Torso.
Dose-dependent effects of Bcd and Cic on tll expression. (a) The Bcd activation gradient works against the inverse transcriptional repression gradient of Cic in activating many of its targets. (b) Quantification of the boundaries of anterior tll expression. (c) and (d) Quantified boundaries of the anterior tll in embryos with different levels of Bcd (c) and Cic (d). Averaged boundaries are shown with error bars indicating standard error of the mean (s.e.m). The numbers of embryos used in the analysis are N = 23 (bcd), N = 95 (bcd/+), N = 49 (wt), N = 42 (bcd[4x]), N = 65 (cic), N = 56 (cic/+), and N = 32 (cic[4x]). (e) These results are consistent with incoherent feedforward mechanism, where Bcd and Torso activate both tll and an anterior repressor of tll.
Anterior repression of tll depends on Hkb and Dl. (a) Quantification of the boundary of anterior hkb. (b) and (c) Quantified boundaries of the anterior hkb in embryos with different levels of Bcd (b) and Cic (c). (d) Averaged boundaries are shown with error bars indicating s.e.m. The numbers of embryos used in the analysis are N = 101 (bcd), N = 64 (bcd/+), N = 52 (wt), N = 67 (bcd[4x]), N = 39 (cic), N = 103 (cic/ +), and N = 41 (cic[4x]). (e) Dorsal view of an embryo stained with Hkb protein (green) and tll transcript (red). The pattern of Hkb closely matches with the repression domain of tll. (e) Pattern of tll in Hkb mutant embryo. (f) Lateral view of an embryo stained for Dl protein and tll mRNA. (g) tll mRNA pattern in an embryo that lacks Dl signaling and Hkb protein.
Kni is an additional repressor of anterior tll. (a)–(d) Expression pattern of kni in wild-type embryos (a), embryos that lack Bcd (b), Dl signaling (c), or Cic (d). (e) Expression pattern of kni in embryos with one copy of bcd. (f) A model for the regulation of anterior-ventral stripe pattern of kni by Bcd, Dl, and Torso signaling. (g) Lateral view of an embryo co-stained for kni (green) and tll (red) mRNAs. (h) tll expression pattern in an embryo that lacks Kni and Hkb.
Cic-dependent control of posterior domains of tll and hkb. (a)–(c) Simultaneous detection of Cic protein (a) and tll (b) and hkb (c) mRNAs in a single embryo using FISH. (d) Quantification of nuclear Cic in the posterior half of the embryo. The boundaries of tll and hkb are indicated by red and green arrows, respectively. (e) and (f) Normalized spatial gradient of tll (e) and hkb (f) mRNAs. (g) Bar graph of average intensities of Cic at the boundaries of tll (red) and hkb (green). Each bar represents an average of 52 data with error bars indicating s.e.m. (h) Changes in the boundaries of tll and hkb in embryos with different copies of cic gene (see Sec. II for details on the quantification). Posterior pole is denoted as 0% embryo length, and error bars are s.e.m. The number of embryos in each background are N = 65 (cic), N = 56 (cic/+), N = 49 (wt), and N = 32 (cic[4x]) for tll and N = 39 (cic), N = 103 (cic/+), N = 52 (wt), and N = 41 (cic[4x]) for hkb.
Effect of perturbing the Cic gradient. (a) Simultaneous staining of Cic in wild-type and cic embryos reveal very low level of Cic this mutant. (b) Quantified nuclear Cic in the posterior half of the wild-type embryo (red, N = 21) and embryos from cic flies (black, N = 14). Error-bars are s.e.m. (c) Assuming that Cic is downregulated completely at the posterior pole of cic embryos, we obtained normalized Cic gradient in wild-type embryos (red, N = 21), which show that Cic is degraded to approximately 20% of the maximum at the posterior pole. Similar approach was used to determine normalized gradient from cic heterozygous embryos (green, N = 33) and embryos with two extra copies of cic (blue, N = 25). Error bars are s.e.m. (d) Nuclear gradient of Cic in the posterior half of wild-type (red, N = 30) and embryos with two extra copies of cic (blue, N = 32). Filled yellow and green arrows denote the boundaries of tll and hkb, respectively, in wild-type embryos while empty arrows represent the two gene expression boundaries in the mutant embryos.
A model for the regulation of tll and hkb. (a) A regulatory network of anterior tll and hkb by the three maternal signals. (b) A regulatory network of posterior tll and hkb by locally activated Torso. (c)–(e) Spatial distributions of Bcd (c), Cic (d), and nuclear Dl (e) gradients that provide spatially distributed inputs to the models shown in (a) and (b). (f) Bcd, Cic, and nuclear Dl gradients superimposed on the same embryo.
Sequence-specific analysis of tll enhancers. (a) Expression patterns of tll enhancers in vivo; fragments are named in accordance to the original publications. 46,51 Fragments K2 and P3 reproduce the posterior pattern, fragment AD reproduces the anterior pattern and the fragment P2 reproduces both, the anterior and the posterior patterns. (b) In vivo binding patterns of five transcriptional regulators in the tll locus. 52,53 (c) Results of sequence analysis: statistical significance of binding site density in the tll locus. 41 Peaks (see the encircled numbers) correspond to significant clusters of binding sites for transcriptional regulators Bcd, Dl, Zld and Cic. (d) Alignment of DNA fragments corresponding to the identified clusters from seven species of Drosophila, binding site matches to regulatory motifs are boxed. Shaded areas mark regions highly conserved in evolution. (E) Binding motif logos for binding motifs of the transcriptional regulators used in the sequence analysis. 35,54–56
Modeling of tll and hkb expression patterns in the wild-type and mutant embryos. The model successfully predicts pattern of tll and hkb in multiple mutants corresponding to changes in the levels and spatial distribution of inductive signals and their downstream targets. The spatial distribution of inductive signals is shown as a superposition of theBcd, Cic, and nuclear Dl gradients. Predicted tll and hkb patterns are presented in the 2nd and 3rd columns, and the corresponding experimental patterns are shown in the last two columns.
Values of the model constants.
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