Decapentaplegic

 The decapentaplegic (dpp) gene plays a key role in patterning both embryonic and adult structures in the fruitfly Drosophila. The name of the locus underscores the multiple requirements for dpp activity during development and refers to the wide range of defects caused by mutations in the gene (decapentaplegic = 15 defects) (1). dpp is one of the few haplo-insufficient loci in Drosophila; consequently, even a 50% reduction in dpp activity leads to embryonic lethality. Less severe alleles of dpp are viable but result in adults that have defects in structures derived from one or more imaginal discs. The dpp gene encodes a protein that is homologous to secreted growth factors of the transforming growth factor-b (TGF-b) superfamily. Ligands that belong to this group of signaling molecules have been identified in animals across the phylogenetic spectrum and are involved in cellular and developmental processes ranging from regulation of the cell cycle and the extracellular matrix, to establishment of embryonic pattern and aging. Genetic and molecular studies of dpp and the genes involved in dpp signal transduction have proven to be of broad general interest, because the basic mechanisms involved in generating and receiving TGF-b signals are evolutionarily conserved.

1. Molecular Features

The dpp gene spans ~55 kb of DNA, the bulk of which consists of a large array of cis-regulatory elements (2). dpp messenger RNA is expressed in a complex spatial and temporal pattern that reflects the multiple roles the ligand plays during development. Three major cis-acting domains regulate transcription of the gene: an upstream dpp-shv region required for expression in the embryonic gut and pupal wing, a central dpp-Hin region that directs embryonic expression, and a downstream dpp-disk region that regulates transcription in imaginal discs (2). These dispersed regulatory elements contribute to the genetic complexity of the locus and mediate dpp transcription in response to inputs from a variety of signaling pathways. For example, dpp expression in the blastoderm stage embryo is regulated by Dorsal, a transcription factor homologous to mammalian NFkB. Later in embryogenesis, dpp transcription in the dorsal-most cells of the germband is controlled by genes acting in the Jun kinase signaling pathway, while expression of dpp in the midgut is dependent on multiple factors, including homeobox DNA-binding proteins, the growth factor Wingless, and Dpp itself. During imaginal disc development, localized expression of dpp in the wing and leg discs is regulated by the hedgehog and wingless genes.

 The dpp locus gives rise to three major and two minor transcripts through the use of alternative promoters that are utilized at different stages of development (2). While the mRNAs have unique 5′-untranslated sequences, they share common second and third exons that contain an open reading frame coding for a single 588-amino-acid-residue protein. Dpp is most closely related to the vertebrate bone morphogenetic proteins BMP-2 and BMP-4, and it shares ~75% identity with these proteins in the carboxy-terminal region that constitutes the mature ligand domain. While the vertebrate BMPs were first identified by their ability to induce ectopic bone, they are now known to have important roles in embryonic development in a number of organisms, including frogs, mice, and humans. Dpp and BMP-4 can substitute for one another functionally, because a human BMP-4 transgene can rescue patterning defects in a Drosophila embryo lacking Dpp (3). Conversely, the fly protein can induce the formation of ectopic bone when injected subcutaneously into rats. Like other ligands belonging to the TGF-b superfamily, Dpp is processed and secreted as a disulfide-linked dimer of ~30kDa.

 2. Dpp Signal Transduction

 Dpp acts as a secreted ligand to influence the developmental fate of cells that receive the signal. Genes involved in Dpp signal transduction have been primarily identified using two strategies. Some genes have been recovered in genetic screens to isolate enhancers of weak dpp alleles, while others have been isolated in low stringency hybridization screens, based on their homology to TGF-b signaling components identified in other organisms.

According to the current paradigm for BMP signaling, the ligand binds a heteromeric complex of two structurally related transmembrane serine-threonine kinases, called the type I and type II receptors (4, 5). Formation of the ligand–receptor complex allows the type II kinase to phosphorylate and activate the type I receptor. A type II receptor, Punt, as well as two type I receptors, Thick veins (Tkv) and Saxophone (Sax), have been implicated in Dpp signaling. Although the role of Punt and Tkv as receptors for Dpp is well established, it appears that Sax may primarily mediate the response to other BMP-related ligands in Drosophila. Activation of Tkv results in the direct phosphorylation of a cytoplasmic protein encoded by mothers against dpp (mad). This modification triggers Mad to form a complex with Medea (a structurally related protein), and it enables their translocation from the cytoplasm into the nucleus. Mad and Medea contain DNA-binding domains and are thought to regulate the expression of downstream target genes in association with other transcription factors (4, 6). The Mad family of proteins is evolutionarily conserved. The human homologue of Medea (DPC4) has been identified as a tumor- suppressor gene, a result that appears logical in light of the known antiproliferative effects of TGF-b.

3. Biological Role of Dpp

Dpp plays an important role in the specification of cell fate and morphogenesis in a number of tissues (1). Mutations in dpp that interfere with production of an active ligand affect all developmental events regulated by the gene, while mutations that disrupt specific enhancer elements affect dpp function in a tissue- or stage-specific manner. Among the processes that require dpp signaling are: oogenesis; establishment of dorsal–ventral pattern during embryogenesis; morphogenetic movements of dorsal closure; subdivision of the mesoderm along the dorsal–ventral axis; specification of the visceral mesoderm and endoderm in the embryonic gut; development of the heart, gastric cecae, salivary glands, and the trachea; and growth and patterning of imaginal discs.

The ability of Dpp to trigger distinct responses in a single field of cells has generated a great deal of interest in understanding the mechanisms underlying dpp function. Dpp has been shown to specify cell fate in a concentration-dependent manner; that is cells respond to different thresholds of Dpp by following distinct pathways of differentiation. A critical issue that arises is how gradients of dpp activity are established, and how such gradients are interpreted. Based on recent studies, two alternative mechanisms have evolved: one is based on diffusion of Dpp from its site of synthesis to generate a protein concentration gradient, and the second involves an inhibitor that diffuses into the domain where dpp is expressed and interferes with Dpp signaling in a graded manner. This results in a gradient of ligand activity, rather than concentration. Both types of gradients are discussed further below.

 Adult structures in the fruitfly arise from imaginal discs, small groups of epithelial cells that are set aside during embryogenesis. These discs grow and are patterned during the larval and pupal stages in response to different signals. In the wing imaginal disc, Dpp acts as a long-range morphogen to specify cell fate along the anterior–posterior axis. dpp is expressed in a narrow domain of cells at the anterior–posterior compartment boundary, from where it diffuses to generate a gradient of protein in the surrounding tissue. Cells up to 20 cell diameters away respond to different threshold concentrations of Dpp by activating the transcription of target genes like spalt and optomotor blind (7, 8). Expression of spalt occurs close to the source of dpp protein, while optomotor blind expression overlaps with, and extends further than, spalt expression. The nested domains of Dpp target gene expression further subdivide the wing disc into distinct regions that are specified by the combination of genes expressed.

During early development, a gradient of Dpp signaling is required to establish cell fates within the dorsal half of the embryo. Peak levels of Dpp signaling specify the dorsal-most amnioserosa tissue, while lower levels are required to specify the dorsal ectoderm (9, 10). Reduction in the level of Dpp signaling results in progressive loss of dorsal structures, while increasing concentrations of Dpp can induce dorsal cell fates. Since dpp mRNA is expressed uniformly in all dorsal cells, it is generally believed that Dpp activity, rather than its concentration, is graded. A number of extracellular proteins are involved in generating a gradient of Dpp signaling in the embryo. A second BMP ligand, Screw, acts synergistically with Dpp to enhance signaling in the dorsal-most cells. The activity of these ligands is antagonized by a secreted factor, Short gastrulation (Sog), that can prevent ligand binding to the receptor. The inhibitor Sog is expressed in ventral cells and diffuses dorsally to generate a ligand gradient of the opposite polarity. In addition, a metalloproteinase, Tolloid (Tld), promotes signaling in dorsal cells by cleaving Sog and releasing the ligand. Thus modulation of ligand activity at multiple levels contributes to establishment of a gradient of BMP signaling in the embryo (11-14).

 Similar antagonistic interactions involving homologous proteins are involved in patterning the dorsal–ventral axis in vertebrate embryos. In Xenopus, BMP-4 promotes ventral development, while a homologue of Sog (Chordin) promotes dorsal cell fates. Recently an amphibian homologue of Tld ) Xolloid) has been shown to cleave Chordin (12). These and other studies suggest that the dorsal–ventral axes in Drosophila and vertebrates are specified by a similar mechanism, although they are inverted relative to one another. The extensive parallels between TGF-b/BMP signaling in vertebrates and invertebrates allow one to extend the insights gained from studying Dpp signaling in Drosophila to other organisms, including humans.

References

1. F. A. Spencer, F. M. Hoffman, and W. M. Gelbart (1982) Decapentaplegic: a gene complex affecting morphogenesis in Drosophila melanogaster. Cell 28, 451–461. 

2. R. D. St. Johnston, F. M. Hoffman, R. K. Blackman, D. Segal, R. Grimaila, R. W. Padgett, H. A. Irick, and W. M. Gelbart (1990) The molecular organization of the decapentaplegic gene in Drosophila melanogaster. Genes Dev. 4, 1114–1127. 

3. R. W. Padgett, J. M. Wozney, and W. M. Gelbart (1993) Human BMP sequences can confer normal dorsal–ventral patterning in the Drosophila embryo. Proc. Natl. Acad. Sci. USA 90, 2905-2909.

4.  C.-H. Heldin, K. Miyazono, and P. ten Dijke (1997) TGF- signalling from cell membrane to nucleus through SMAD proteins. Nature 390, 465–471. 

5. J. Massague (1996) TGF- signaling: receptors, transducers, and Mad proteins. Cell 85, 947–950.

6. K. Arora, H. Dai, S. G. Kazuko, J. Jamal, M. B. O''Connor, A. Letsou, and R. Warrior (1995( The Drosophila schnurri gene acts in the Dpp/TGF- signaling pathway and encodes a transcription factor homologous to the human MBP family. Cell 81, 781–790. 

7. T. Lecuit, W. J. Brook, M. Ng, M. Calleja, H. Sun, and S. M. Cohen (1996) Two distinct mechanisms for long-range patterning by Decapentaplegic in the Drosophila wing. Nature 381, 1387-1393.

8. D. Nellen, R. Burke, G. Struhl, and K. Basler (1996) Direct and long-range action of a DPP morphogen gradient. Cell 85, 357–368. 

9. E. L. Ferguson and K. V. Anderson (1992) Decapentaplegic acts as a morphogen to organize dorsal–ventral pattern in the Drosophila embryo. Cell 71, 451–461. 

10. K. A. Wharton, R. P. Ray, and W. M. Gelbart (1993) An activity gradient of decapentaplegic is necessary for the specification of dorsal pattern elements in the Drosophila embryo. Development 117, 807–22. 

11. G. Marqués, M. Musacchio, M. J. Shimell, K. Wunnenberg-Stapleton, K. W. Cho, and M. B. O''Connor (1997) Production of a DPP activity gradient in the early Drosophila embryo through the opposing actions of the SOG and TLD proteins. Cell 91, 417–426. 

12. S. Piccolo, E. Agius, B. Lu, S. Goodman, L. Dale, and E. M. De Robertis (1997) Cleavage of Chordin by Xolloid metalloprotease suggests a role for proteolytic processing in the regulation of Spemann organizer activity. Cell 91, 407–416. 

13. J. L. Neul and E. L. Ferguson (1998) Spatially restricted activation of the SAX receptor by SCW modulates DPP/TKV signaling in Drosophila dorsal-ventral patterning. Cell 95, 483–494. 

14. M. Nguyen, S. Park, G. Marqués, and K. Arora (1998) Interpretation of a BMP activity gradient in Drosophila embryos depends on synergistic signaling by two type  I receptors, SAX and TKV. Cell 95, 495–506.