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First published online 23 February 2005
doi: 10.1242/dev.01722

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Peak levels of BMP in the Drosophila embryo control target genes by a feed-forward mechanism

Department of Biology, New York University, 100 Washington Square East, New York, NY 10003, USA

View larger version (55K): [in a new window]   Fig. 1. Race expression depends on high levels of dpp and zen activity. Dorsal views of stage 5/6 embryos with anterior towards the left. Embryos were hybridized with Race (A-E) or zen (F) probes. (A) Wild type. Race transcripts are present in a five- to six-cell wide dorsal stripe in the main body region and in two broader patches in the region that will form head structures. (B) Race is absent in dpphr4 homozygotes. (C) Race is expressed weakly in a broad domain in the main body region of sogSY06 hemizygous embryos. The head domains are relatively strong and also broader. (D) Race expression is normal in dpphr4 heterozygotes. (E,F) Race transcripts are absent from the main body region, but appear normally in the head region of dpphr4/+; zenw36/+ embryos (E), although zen expression is normal (F). The expression in the head domains appears to be less sensitive to a drop in dpp and zen activities.

View larger version (83K): [in a new window]   Fig. 2. The purpose of the BMP gradient peak is to set up the zen domain. All views of embryos are dorsal, except K (lateral). Embryos are of the genotypes wild type 2X dpp (A-C), 4X dpp (D-F), 4X dpp, zenw36 (G-I) and UAS-zen driven by maternal-Gal4 (J-L; see Materials and methods), and were stained with anti-phospho Mad (PMad) antibodies (A,D,G,J), or hybridized with zen (B,E,H,K) or Race (C,F,I,L) RNA probes. Increasing the dose of dpp to four copies broadens the domain of peak level PMad to 10-12 cells (D; see inset for higher magnification view of one or two rows of cells, turned sideways). The domain of zen (E) and Race (F) broaden likewise. In zen mutant embryos with 4X dpp, the zen domain broadens (H), but that of Race does not (I), presumably because Zen proteins are absent (Rushlow et al., 1987). When zen is ubiquitously expressed throughout the embryo (K), the Race domain broadens to encompass the lower-level PMad domain (L), indicating that lower levels of PMad can activate Race if Zen is present. Ectopic Race expression is also observed in the posterior region (L, arrow) where PMad is present.

View larger version (68K): [in a new window]   Fig. 3. The binding of both Mad and Zen to the Race enhancer is required for proper Race expression. (A) DNAse I footprinting analysis of Mad and Zen GST fusion proteins bound to a 255 bp fragment that includes the proximal region of the Race 533 bp enhancer (349-533) and 70 nucleotides from the Bluescript vector. The fragment is end-labeled at the vector end. Lane 1, chemical degradation of the probe on G+As; lanes 2 and 6, DNAse I digestion of the DNA probe. Increasing amounts of Mad (500 ng, 1500 ng and 4500 ng in lanes 3-5 respectively) and Zen (20 ng, 60 ng and 200 ng in lanes 7-9 respectively) were incubated with the fragment prior to DNAse I digestion. The region protected by Mad is depicted as a blue rectangle, the hatched half denoting weaker protection. The regions protected by Zen are shown as red ovals. The nucleotide sequence of the protected regions are shown below the gel with the overlap between the Zen and Mad footprints shown in purple. Putative core binding sites are underlined for Zen (Han et al., 1989) and boxed for Mad (boxes a,b,c,f,g) (Kim et al., 1996) and Medea (boxes d,e) (Xu et al., 1998; Pyrowolakis et al., 2004). The boxes are also marked on the G+A sequence. (B) Schematic representation of the full-length Race 533 bp enhancer fused to a lacZ reporter gene, and a transgenic embryo carrying this construct in situ hybridized with lacZ probes. lacZ expression is identical to the Race pattern. The ring of staining in the head region is an artifact of the vector. (C) Embryo carrying a deletion of the Mad-binding region (nucleotides 432-497). lacZ expression is severely reduced. (D) Embryo carrying mutations in the ATTA core sites of the Zen-binding sites (underlined, see Materials and methods). lacZ expression is absent.

View larger version (73K): [in a new window]   Fig. 4. Zen binding is enhanced in the presence of Mad. (A) Schematic representation of the 42 bp wild-type (wt-42) and mutant oligonucleotides that eliminate the core Smad-(Sm-42) or Zen-(Zm-42) binding sites, and the 128 bp DNA fragment from the Race enhancer used in electrophoretic mobility shift assays (EMSAs) showing Medea-(blue circle), Mad-(blue ovals) and Zen-(red ovals) binding sites. (B) DNA binding requires the core consensus sites. 32P-labeled wild type (lanes 1-4) and mutant oligonucleotides (Sm-42, lanes 5-8; Zm-42, lanes 9-12) were incubated with no protein (lanes 1,5,9); and 100 ng Mad (lanes 2,6,10), 100 ng Medea (lanes 3,7,11) or 10 ng Zen (lanes 4,8,12). Mad and Medea produce a single complex of bound probe, whereas Zen produces two complexes. The slower migrating Zen complex could be due to a Zen/Zen/DNA complex. Mutant Sm-42 (or Zm-42) eliminated the binding of Mad/Medea (or Zen) without affecting the binding of Zen (or Mad/Medea). (C) 32P-labeled wild-type DNA fragments were incubated with no protein (lanes 1 and 5), increasing amounts of Mad (1 ng, 2 ng, and 5 ng in lanes 2-4, respectively), increasing amounts of Zen (0.1 ng, 0.3 ng, and 1 ng in lanes 6-9, respectively) or increasing amounts of Zen in the presence of 1 ng of Mad (lanes 10-13). The amounts of Zen in lanes 10-13 were the same as in lanes 6-9. More Zen complexes are shifted in lanes 10-13 compared with lanes 6-9. In lane 13, a supershifted complex (arrow) is visible above the Zen complex (arrowhead) that may be due to the formation of a Mad/Zen/DNA complex or a Zen/Zen/DNA complex. (D) Similar experiment as in C, except that Zen-Del was used instead of wild-type Zen. The same amounts of proteins were used as in C. Enhancement of Zen binding by Mad was not observed.

View larger version (30K): [in a new window]   Fig. 5. Mad and Zen proteins interact. (A) Schematic representation of the full-length and truncated forms of Zen proteins used in the in vitro protein interaction assays. Black bars represent the homeodomain of Zen (amino acids 90-149) (Rushlow et al., 1987) and the hatched bar conatins the putative Mad-Zen interaction domain (amino acids 152-199). (B) Autoradiogram of 5% of the amounts of 35S-labeled in vitro translated Zen proteins used in each binding reaction. Lane 1, full-length Zen; lanes 2-7, truncated Zen proteins a-f. (C) Results of GST pull-down assays with full-length and truncated Zen proteins. Odd-numbered lanes were reactions using GST protein (negative controls). Even-numbered lanes were reactions using GST-MadN with the following Zen proteins: lane 2, full length; lane 4, a; lane 6, b; lane 8, c; lane 10, d; lane 12, e; lane 14, f; lane 16, Zen-Del. Results are also summarized on the right side of A. Zen-Del has background binding with GST alone. In repeated experiments, the difference between GST and GST-MadN lanes for Zen-Del was somewhat variable and consistently insignificant.

View larger version (76K): [in a new window]   Fig. 6. The Mad-Zen interaction is necessary for Race activation. Late stage 5 embryos were hybridized in situ with a Race probe (A,B) or Race and lacZ probes (to determine the zen mutant embryos; C,D). (A) Embryo expressing UAS-zen driven by the maternal Gal4 driver. Ectopic Race expression is visible in the posterior region and the dorsal stripe is broader. (B) Embryo expressing UAS-zen-Del driven by the maternal Gal4 driver. Ectopic Race transcripts are absent. (C) zenw36 mutant embryo expressing UAS-zen driven by the maternal Gal4 driver. Race expression is restored. (D) zenw36 mutant embryo expressing UAS-zen-Del driven by the maternal Gal4 driver. Race expression is absent, indicating that Zen-Del is not able to rescue the zen mutant phenotype.