Sopory S , Kwon S , Wehrli M , Christian JL .

R01 HD42598 NICHD NIH HHS , R01GM67029 NIGMS NIH HHS , R01 HD037976-09 NICHD NIH HHS , R01 HD042598-05 NICHD NIH HHS , R01 HD037976 NICHD NIH HHS , R01 HD042598 NICHD NIH HHS , R01 GM067029 NIGMS NIH HHS

	Fig. 1. Model for regulation of BMP4 signaling by sequential cleavage. (A) When proBMP4 is sequentially cleaved at the S1 and then the S2 sites, the mature ligand is released from the cleaved prodomain and is relatively stable, enabling it to signal to distal cells. (B) When proBMP4 is cleaved at the S1 site alone, the mature ligand remains non-covalently associated with the prodomain and this complex is targeted for degradation in the lysosome (blue shape) either directly within the biosynthetic pathway of synthesizing cells (upper panel) or via endocytic targeting in signal receiving cells (lower panel). This lowers the rate of ligand production at the source and/or enhances the rate of ligand degradation in receiving cells. As a result, BMP4 generated by cleavage at the S1 site alone signals is present at lower steady state levels.
	Fig. 2. Dpp is cleaved at two sites within the prodomain. (A) Alignment of sequences flanking the cleavage site(s) of BMP4 and BMP2 from human, chick and Xenopus (Xen), and DPP from Drosophila (fly). (B) Schematic illustration of cleavage sites in wild type and cleavage variant forms of proDpp. Shaded bar represents the prodomain, white bar the region between the two cleavage sites and black bar the mature ligand domain. (C) Dpp precursors were expressed in S2 cells and Western blots of cell media were probed with antibodies specific for the HA-tag in the prodomain or the myc-tag in the mature domain. Bands corresponding to uncleaved precursor, prodomain following cleavage at the S1 site, prodomain following cleavage at the S2 site, and mature ligand are indicated schematically to the right of the gel. The same bands were observed in Western blots of cell lysates. (D) Radiolabeled native or cleavage mutant forms of proDpp were incubated with recombinant Dfurin or Hfurin for the times indicated. All results were reproduced in at least three independent experiments.
	Fig. 3. Effect of optimal and minimal cleavage motifs on production of mature Dpp in Xenopus embryos. RNA (2 ng) encoding Dpp precursor proteins was injected into two-cell Xenopus embryos. Levels of precursor and cleavage products were examined by Western blot of embryonic extracts (lysate) or blastocoele fluid (BF) collected at the blastula stage from uninjected (UI) embryos or Dpp-expressing embryos. Bands corresponding to uncleaved precursor, prodomain fragments and mature ligand are indicated to the right of the gel. Results were reproduced in three independent experiments.
	Fig. 4. Cleavage of the S2-site is required for normal wing development. Normal leg (A), wing (B) and eye (C) formation in wild type (wt) flies is contrasted with that in dppd8/dppd10 mutant flies that have truncated legs (D) tiny winglets (E) and ventrally receding eyes (F). Expression of UAS-DppWT-myc under the control of dpp-Gal4 fully rescued leg (G) and eye (I), and partially rescued wing development (H) in all dppd8/dppd10 mutants. UAS-Dpp-mycS2KK did not rescue the truncated legs (J) or missing wings (K) in any flies but partially rescued ventral eye development in almost all flies examined (L). Double arrows (C, F, I and L) indicate the distance from the ventral eye margin to the bristles in wild type, mutant and rescued animals. Arrows (A, G) indicate the claws whereas arrowheads (D, J) highlight distal truncation with loss of tarsal segments. Co, cox; fe, femur; ti, tibia; ta, tarsus with segments I–V.
	Fig. 5. Cleavage of Dpp at the S2 site is required for activation of pMAD in the wing disc. Wing discs with clones of cells (marked by GFP) expressing proDppWT (A–A″) or proDppS2KK (B-B″) were immunostained to detect pMAD. Endogenous pMAD staining in the absence of ectopic Dpp is also shown (C′, C″). Representative clones outside of the endogenous dpp expression domain are outlined. Arrows denote induction of pMAD outside of clones.
	Fig. 6. Cleavage at the S2 site is not required for ectopic expression of labial in the midgut primordium. (A–C) Stages 14–15 nontransgenic embryos (WT), or those expressing DppWT or DppS2KK precursors using the mesodermal driver 24B-Gal4 were immunostained to detect Labial expression in the midgut endoderm. (D) Quantification of labial stripe width. Values represent the mean number (+/− s.d.) of labial expressing cells in a stripe. The width of the labial stripe in WT embryos was significantly different than that in embryos expressing DppWT or DppS2KK precursors (p < 0.05) but there was no significant difference in the number of labial cells between embryos expressing DppWT or DppS2KK precursors. (E,F) dppS4 homozygous embryos expressing either proDppWT or proDppS2KK immunostained to detect labial expression in the endoderm.
	Fig. 7. The S2 site of proDpp is selectively cleaved in the wing disc, but not in the embryonic mesoderm. Western blots of proteins from pooled wing disc extracts or from embryo immunoprecipitates derived from nontransgenic flies (none) or from transgenic flies expressing proDPPWT or proDPPS2KK were probed with antibodies specific for the HA-tag in the prodomain or the myc-tag in the mature domain of each precursor protein. Bands corresponding to uncleaved precursor, prodomain fragments and mature ligand are indicated to the right of the gel. The top and middle panels on the left are short and long exposures, respectively, of the same Western blot. The upper mature ligand bands detected with the anti-myc antibody are most likely due to incomplete deglycosylation (based on predicted molecular weight of S1 cleaved mature Dpp and comparison with duplicate protein extracts that had not been deglycosylated with PNGaseF, as indicated at the top of the gel). Bands indicated by an asterisk are most likely precursor degradation products. The arrowhead indicates the faint band corresponding to S1-only cleaved prodomain generated from DPPWT in the wing disc.
	Fig. S1. Myc-epitope-tagged Dpp retains heparin binding capacity. Xenopus BMP4myc, BMP4ΔRKKmyc (in which the heparin binding domain has been deleted) or Dpp6Xmyc precursor proteins were expressed in HEK cells. Cell media was incubated with heparin-sepharose beads which were washed to remove unbound proteins and then boiled to release bound proteins. Western blots of cell media (input) or proteins that did not (unbound) or that did (bound) bind to heparin beads were probed with antibodies specific for the myc-tag in the mature domain.
	Fig. S2. Cleavage at the S2 site is required for Dpp to induce overgrowth of the wing disc. (A–C) Wing discs dissected from transgenic flies expressing proDppWT (A–A′) or proDppS2KK (B–B′) under the control of Ubx-GAL4, or from nontransgenic flies (C–C′) were immunostained to detect pMAD. Horizontal images of pMAD staining were obtained at the level of either the Disc proper (DP) (A–C) or the peripodial epithelium (PE) (A′–C′) using confocal microscopy. Arrows indicate the relative width of discs along the A–P axis.
	Fig. S3. Dpp generated from proDPPS2KK can induce localized overgrowth of the wing disc when clones abut the periphery. Autofluorescence (A,B) and GFP imaging (A′,B′) of wing discs with large clones of cells (marked by GFP) expressing proDppS2KK. Arrows indicate the borders of the expanded region of the wing disc induced by ectopic Dpp. In (B), overgrowth is also apparent in the folding pattern of the expanded portion of the disc (small arrowheads) that resembles the normal pattern (large arrowheads).
	Fig. S4. Immunoreactive Dpp is present in clones of cells expressing wild type and S2KK-mutant precursors. Wing discs with clones of cells (marked by GFP) expressing proDppWT or proDppS2KK were permeabilized and immunostained to detect myc.

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