Biochemical characterization of the Drosophila dpp protein, a member of the transforming growth factor beta family of growth factors.

Abstract

The decapentaplegic (dpp) gene of Drosophila melanogaster Is required for pattern formation in the embryo and for viability of the epithelial cells in the imaginal disks. The dpp protein product predicted from the DNA sequence is similar to members of a family of growth factors that includes transforming growth factor 0 (TGF-1). We have produced polyclonal antibodies to a recombinant dpp protein made in bacteria and used a metallothionein promoter to express a dpp cDNA in Drosophila S2 cells. Similar to other proteins in the TGF-4 family, the dpp protein produced by the Drosophila cells was proteolytically cleaved, and both portions of the protein were secreted from the cells. The amino-terminal 47-kilodalton (kDa) peptide was found in the medium and in the proteins adhering to the plastic petri dish. The carboxy-terminal peptide, the region with sequence similarity to the active ligand portion of TGF-4, was found extracellularly as a 30-kDa homodimer. Most of the 30-kDa homodimer was in the S2 cell protein adsorbed onto the surface of the plastic dish. The dpp protein could be released into solution by increased salt concentration and nonionic detergent. Under these conditions, the amino-terminal and carboxy-terminal portions of dpp were not associated in a stable complex.Analysis of mutant alleles in the Drosophila decapentaplegic (dpp) gene indicates that the dpp gene product is required for the proper development of the embryonic dorsal hypoderm (18), for viability of larvae (42), and for cell viability of the epithelial cells in the imaginal disks (5, 45). Molecular isolation of the dpp DNA and mapping of mutant lesions onto the molecular map indicates that the gene spans over 50 kilobases (kb) of DNA (R. D. St. Johnston, F. M. Hoffmann, R. K. Blackman, D. Segal, R. Grimaila, R. W. Padgett, H. A. Irick, and W. M. Gelbart, Genes Dev., in press). The two protein-coding exons are located near the center of this region, and their expression is driven from at least five promoters distributed across 20 kb of 5' DNA. dpp expression in the larval imaginal disks requires cis-regulatory elements distributed across 25 kb of DNA 3' to the coding exons.The two exons that are common to all of the transcriptional units contain an open reading frame whose predicted protein product is 588 amino acids in length; the carboxyterminal 100 amino acids have sequence similarity to proteins in the transforming growth factor P (TGF-P) superfamily, which at this time includes five TGF-1s, Mullerian inhibiting substance (MIS), inhibins, the Xenopus protein Vg-1, the mouse protein Vgr-1, and three human bone morphogens (BMPs) (7,10,24,25,35,47,50,52). Sequence similarities to this 100-amino-acid region are highest between dpp and human bone morphogenetic proteins (75%) or murine Vgr-1 protein (77%) and range between 23 and 57% for the other members of the TGF-1 family (25,52); the conserved amino acids include all seven cysteine residues in dpp. All of the vertebrate proteins examined to date are secreted proteins in which the carboxy-...

Morphogen transport

“…Similar to the hindered diffusion model of FGF transport discussed above, transient binding of Dpp to HSPGs might slow the diffusion of Dpp (Panganiban et al, 1990a;Fujise et al, 2003;Belenkaya et al, 2004;Han et al, 2004;Takei et al, 2004;Akiyama et al, 2008;Dejima et al, 2011). Reduced levels of Dpp are observed in HSPG-deficient tissues (Fujise et al, 2003;Belenkaya et al, 2004;Han et al, 2004;Takei et al, 2004;Akiyama et al, 2008), and increased levels of HSPGs in the source (Müller et al, 2012) using a two-dimensional geometry similar to the geometry described by Yu et al (Yu et al, 2009).…”

“…Dpp is a secreted TGFβ superfamily member and a homolog of vertebrate bone morphogenetic proteins (BMPs) (Panganiban et al, 1990a;Panganiban et al, 1990b;Lecuit et al, 1996;Nellen et al, 1996;Lecuit and Cohen, 1998;Entchev et al, 2000;Teleman and Cohen, 2000;Gibson et al, 2002;Belenkaya et al, 2004;Kruse et al, 2004;Kicheva et al, 2007;Schwank et al, 2011;Zhou et al, 2012). A Dpp gradient forms from a localized source and patterns the wing imaginal disc [an epithelial wing precursor tissue that is initially flat and later develops a slight curvature and multiple folds (Sui et al, 2012)] over several days during the larval stages of Drosophila development (Entchev et al, 2000;Teleman and Cohen, 2000).…”

Early patterning of the spider embryo: a cluster of mesenchymal cells at the cumulus produces Dpp signals received by germ disc epithelial cells

“…The isolated cDNA clone encodes a polypeptide of 370 amino acids with a putative signal sequence and a putative proteolytic cleavage site (Panganiban et al, 1990) between 255 and 256 amino acid residues. The C-terminal 102 amino acid residues of the deduced protein shows 65% and 74% identity with the corresponding region of Drosophila Dpp and zebrafish BMP2, respectively, and lower percentages of identity with those of Drosophila 60A (50% identity) and Screw (42% identity) (Fig. Y. Akiyama-Oda and H. Oda …”

Dominant negative mutants of transforming growth factor-beta 1 inhibit the secretion of different transforming growth factor-beta isoforms.

“…The latter band, however, is usually barely detectable following autoradiography, presumably due to the very sticky nature of the mature factor and the resulting interaction with cells, matrix, and cell culture substrate (19a). This stickiness was also apparent with the dpp gene product, the BMP-2/4 homolog in Drosophila melanogaster (29). The efficiency of vgr-1 secretion is thus best evaluated by measuring the level of secretion of the vgr-1 precursor segment, which unfortunately cannot be carried out by using antibody-specific detection.…”