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.

Affolter, The Decapentaplegic morphogen gradient: from pattern formation to growth regulation. 2007, Pubmed

Affolter, The Decapentaplegic morphogen gradient: from pattern formation to growth regulation. 2007, Pubmed
Akiyama, Dally regulates Dpp morphogen gradient formation by stabilizing Dpp on the cell surface. 2008, Pubmed
Bangi, Dpp and Gbb exhibit different effective ranges in the establishment of the BMP activity gradient critical for Drosophila wing patterning. 2006, Pubmed
Basler, Compartment boundaries and the control of Drosophila limb pattern by hedgehog protein. 1994, Pubmed
Belenkaya, Drosophila Dpp morphogen movement is independent of dynamin-mediated endocytosis but regulated by the glypican members of heparan sulfate proteoglycans. 2004, Pubmed
Birsoy, XPACE4 is a localized pro-protein convertase required for mesoderm induction and the cleavage of specific TGFbeta proteins in Xenopus development. 2005, Pubmed , Xenbase
Creemers, Modulation of furin-mediated proprotein processing activity by site-directed mutagenesis. 1993, Pubmed
Cui, BMP-4 is proteolytically activated by furin and/or PC6 during vertebrate embryonic development. 1998, Pubmed , Xenbase
Cui, The activity and signaling range of mature BMP-4 is regulated by sequential cleavage at two sites within the prodomain of the precursor. 2001, Pubmed , Xenbase
De Bie, Processing specificity and biosynthesis of the Drosophila melanogaster convertases dfurin1, dfurin1-CRR, dfurin1-X, and dfurin2. 1995, Pubmed
Degnin, Cleavages within the prodomain direct intracellular trafficking and degradation of mature bone morphogenetic protein-4. 2004, Pubmed , Xenbase
Eldar, Self-enhanced ligand degradation underlies robustness of morphogen gradients. 2003, Pubmed
Entchev, Gradient formation of the TGF-beta homolog Dpp. 2000, Pubmed
Gibson, Lumenal transmission of decapentaplegic in Drosophila imaginal discs. 2002, Pubmed
Goldman, Mutation of an upstream cleavage site in the BMP4 prodomain leads to tissue-specific loss of activity. 2006, Pubmed , Xenbase
Horton, Gene splicing by overlap extension: tailor-made genes using the polymerase chain reaction. 1990, Pubmed
Immerglück, Induction across germ layers in Drosophila mediated by a genetic cascade. 1990, Pubmed
Kao, The entire mesodermal mantle behaves as Spemann's organizer in dorsoanterior enhanced Xenopus laevis embryos. 1988, Pubmed , Xenbase
Kelley, A concentration-dependent endocytic trap and sink mechanism converts Bmper from an activator to an inhibitor of Bmp signaling. 2009, Pubmed
Kicheva, The Decapentaplegic morphogen gradient: a precise definition. 2008, Pubmed
Künnapuu, The Drosophila DPP signal is produced by cleavage of its proprotein at evolutionary diversified furin-recognition sites. 2009, Pubmed
Lecuit, Dpp receptor levels contribute to shaping the Dpp morphogen gradient in the Drosophila wing imaginal disc. 1998, Pubmed
Lee, Mosaic analysis with a repressible cell marker (MARCM) for Drosophila neural development. 2001, Pubmed
Molloy, Intracellular trafficking and activation of the furin proprotein convertase: localization to the TGN and recycling from the cell surface. 1994, Pubmed
Nakayama, Regulation of BMP/Dpp signaling during embryonic development. 2000, Pubmed , Xenbase
Nellen, Direct and long-range action of a DPP morphogen gradient. 1996, Pubmed
Nelsen, Site-specific cleavage of BMP4 by furin, PC6, and PC7. 2009, Pubmed , Xenbase
O'Connor, Shaping BMP morphogen gradients in the Drosophila embryo and pupal wing. 2006, Pubmed
Ohkawara, Action range of BMP is defined by its N-terminal basic amino acid core. 2002, Pubmed , Xenbase
Pallavi, Egfr/Ras pathway mediates interactions between peripodial and disc proper cells in Drosophila wing discs. 2003, Pubmed
Panganiban, A Drosophila growth factor homolog, decapentaplegic, regulates homeotic gene expression within and across germ layers during midgut morphogenesis. 1990, Pubmed
Reuter, Homeotic genes regulate the spatial expression of putative growth factors in the visceral mesoderm of Drosophila embryos. 1990, Pubmed
Roebroek, Cloning and functional expression of Dfurin2, a subtilisin-like proprotein processing enzyme of Drosophila melanogaster with multiple repeats of a cysteine motif. 1992, Pubmed
Roebroek, cDNA sequence of a Drosophila melanogaster gene, Dfur1, encoding a protein structurally related to the subtilisin-like proprotein processing enzyme furin. 1991, Pubmed
Sampath, Drosophila transforming growth factor beta superfamily proteins induce endochondral bone formation in mammals. 1993, Pubmed
Shimmi, Facilitated transport of a Dpp/Scw heterodimer by Sog/Tsg leads to robust patterning of the Drosophila blastoderm embryo. 2005, Pubmed
Sopory, Regulation of bone morphogenetic protein-4 activity by sequence elements within the prodomain. 2006, Pubmed , Xenbase
Staehling-Hampton, Ectopic decapentaplegic in the Drosophila midgut alters the expression of five homeotic genes, dpp, and wingless, causing specific morphological defects. 1994, Pubmed
St Johnston, Decapentaplegic transcripts are localized along the dorsal-ventral axis of the Drosophila embryo. 1987, Pubmed
Tanimoto, Hedgehog creates a gradient of DPP activity in Drosophila wing imaginal discs. 2000, Pubmed
Teleman, Dpp gradient formation in the Drosophila wing imaginal disc. 2000, Pubmed
Thomas, Furin at the cutting edge: from protein traffic to embryogenesis and disease. 2002, Pubmed
Vilmos, crossveinless defines a new family of Twisted-gastrulation-like modulators of bone morphogenetic protein signalling. 2005, Pubmed
Wehrli, arrow encodes an LDL-receptor-related protein essential for Wingless signalling. 2000, Pubmed
Zecca, Direct and long-range action of a wingless morphogen gradient. 1996, Pubmed