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First published online August 18, 2003
doi: 10.1242/10.1242/dev.00684

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Physical properties of Tld, Sog, Tsg and Dpp protein interactions are predicted to help create a sharp boundary in Bmp signals during dorsoventral patterning of the Drosophila embryo

Department of Genetics, Cell Biology and Development, Howard Hughes Medical Institute, University of Minnesota, Minneapolis, MN 55455, USA

View larger version (87K): [in a new window]   Fig. 1. Tsg is epistatic to Tld. Embryos were hybridized with antisense digoxigenin-labeled rhomboid probes. Mutant embryos were identified by lack of hybridization to lacZ transcripts produced from the FM7, ftz-lacZ and TM3, ftz-lacZ balancer chromosomes. Embryos are positioned with their dorsal side upwards and anterior towards the left. (A) Wild-type embryo, (B) tsg mutant embryo, (C) tld mutant embryo and (D) tsg, tld mutant embryo. The expression of rho on the dorsal side is broader and weaker in tsg mutants or tsg, tld double mutant embryos, than that in the wild type. In the tld mutant embryo, rho was expressed in its normal ventral domain, but not on the dorsal side, except for a thin anterior stripe.

View larger version (96K): [in a new window]   Fig. 2. Wing phenotypes produced by ectopic expression of different combinations of Sog, Tsg and Tld. (A) Wing from a fly expressing the A9-Gal4 driver alone. (B) Wing from a A9-Gal4>UAS-atld (activated Tld) fly. There is some loss of margin bristles but all veins form normally. (C) Wing from a A9-Gal4>UAS-sog, tsg fly. Wings are smaller and most veins are missing. (D) Wing from a A9-Gal4>UAS-sog, tsg, atld fly. Note that wing size is close to normal and the formation of most veins is recovered.

View larger version (10K): [in a new window]   Fig. 3. Sog cleavage by Tld is modulated by Tsg. (A) The indicated combinations of proteins were incubated for 4 hours at 25°C, and Sog fragments were analyzed using an anti-Sog-CR1 antibody or anti-myc antibody. Tld requires Dpp to cleave Sog. In this experiment, the Dpp concentration was 10-9 M. The molecular weight of each fragment was calculated relative to markers as: 110 kDa for a, 80 kDa for b, 65 kDa for c, 110 kDa for d, 65 kDa for e, 50 kDa for f and 33 kDa for g. (B) Sog is cleaved in at least five sites. The site marked with an asterisk is cleaved during secretion and was identified to be between R79-H80 by protein sequencing. This removes the type II transmembrane domain. The sites I, II, III and IV were cleaved by Tld in a Dpp-dependent fashion. Processing at the C-terminal site IV to produce fragment g was greatly enhanced in the presence of Tsg.

View larger version (60K): [in a new window]   Fig. 5. Sog is cleaved by Tld in a Dpp-dose dependent fashion. (A) Sog, Tld and the indicated amount of Dpp were incubated for 4 hours at 25°C and Sog fragments were analyzed by western blotting using anti-Sog-CR1 or anti-Myc antibody. Processed fragments were detected at 3x10-10 M of Dpp. (B) Sog, Tld, Tsg and the indicated amount of Dpp were incubated for 4 hours at 25°C, and Sog fragments were analyzed as above. Tsg was used at the same concentration as Dpp. Cleavage fragments were observed at 10-10 M of Dpp. Arrows a-g show the same molecular weight fragments of Sog as shown in Fig. 3.

View larger version (90K): [in a new window]   Fig. 4. Characterization of in vitro cell-based Dpp signaling assay. (A) Dose dependency of Dpp in the signaling assay was investigated. S2 cells expressing Flag-Mad proteins were incubated with the indicated amounts of Dpp protein for 3 hours at 25°C, and the cell extracts were blotted and analyzed by western blotting using anti-phosphoMad or anti-Flag antibody. Dpp activity was detected at as low as 10-10 M Dpp, and at 10-9 M Dpp, the signals were saturated. (B) Dpp signals through Tkv as a type I receptor and Punt as a type II receptor in S2 cells. S2 cells were transfected with Flag-Mad for control (C), or Flag-Mad and dsRNA of tkv, sax, or punt (R). Three days after transfection, cells were collected split into two fractions, one of which was used for a phosphoMad signaling assay and the other was used for RT-PCR.

View larger version (57K): [in a new window]   Fig. 6. Time course assay of Sog processing. (A) Sog, Tld and Dpp (3x10-10 M) were incubated at 25°C for the indicated time, and Sog fragments were analyzed on western blots using anti-Sog-CR1 or anti-Myc antibodies. The processed fragments were barely seen at 240 minutes. (B) Sog, Tld, Dpp (3x10-10 M), and Tsg (3x10-10 M) were incubated in the same conditions as above. Processed fragments were detected with as little as 15 minutes of incubation. Arrows a-g indicate the same fragments of Sog shown in Fig. 3.

View larger version (60K): [in a new window]   Fig. 7. Tld reverses the Sog and Tsg synergistic inhibition of Dpp signaling in vitro. (A) The indicated combination of proteins, including 10-9 M of Dpp, were pre-incubated for 6 hours and further incubated with S2 cells expressing Flag-Mad for 3 hours. The cell extracts were analyzed by anti-phosphoMad antibody for Dpp signals (upper panel) and by anti-Flag antibody for total Mad protein expression (lower panel). Dpp signaling was strongly blocked by Sog and Tsg, but this inhibition was reversed by the co-incubation with Tld. (B) Dpp signal (10-10 M Dpp, 1/10 the amount used in A) was blocked by the same concentration of Sog as used in A, and this inhibition was partially reversed by Tld (lane 3). (C) The indicated combination of proteins was incubated as in A, and the collected supernatants were applied to a 4-12% NuPAGE gel and processed fragments were detected by western blotting using anti-Sog-CR1 and anti-Myc antibodies. Full-length Sog is barely detectable in Dpp/Sog/Tsg/Tld. Cleaved C-terminal fragments were detected by anti-Myc antibody, but with the exception of fragment (a) N terminal fragments are not detected by anti-Sog-CR1 antibody, suggesting that, in the presence of cells, these fragments are unstable (compare with Fig. 3A). (D) The supernatants of the indicated protein combinations from the experiment shown in B were analyzed for processing. Sog is partially cleaved by the incubation with Tld, and Dpp signals were partially restored. In all panels, arrows labeled a-g correspond to the fragments illustrated in Fig. 3B.

View larger version (42K): [in a new window]   Fig. 8. Tld activity mirrors Dpp concentration helping to create a sharp boundary in signal reception. The graphs illustrate proposed temporal changes within the dorsal half of the embryo in the extracellular concentrations of Dpp and Sog as development proceeds from early to late cellular blastoderm. The green circles below the black line represent cells receiving Dpp signal. The darker green indicates a higher level of cumulative signal received over the entire time span. Yellow and orange represent cells receiving low level of Dpp signal because of early blockage in Dpp signaling by Sog diffusion. Red circles represent Sog-expressing cells. The embryos at the right show P-Mad accumulation with time. Embryos were stained using PMad primary antibody at 1/1000 dilution and anti-rabbit HRP at 1/200 dilution. The embryos were developed for 20 minutes.

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