Holy Tolloido: Tolloid cleaves SOG/Chordin to free DPP/BMPs
Authors: Mary C Mullins
Journal: Trends in Genetics (1998)

The molecular mechanism by which a group of zygotically acting genes identified almost 15 years ago[1Nüsslein-Volhard C. et al.Roux's Arch. Dev. Biol. 1984; 193: 267-282Crossref PubMed Scopus (854) Google Scholar]establishes dorsal cell fates in Drosophila is just being elucidated. A similar mechanism has been found in the establishment of the dorsal–ventral axis in the vertebrate embryo. The key genes in these processes are TGF-β molecules, Decapentaplegic (DPP) in flies and bone morphogenetic proteins (BMPs) in vertebrates. Conservation of genes in the DPP/BMP pathway in vertebrates and invertebrates strongly indicates that the basic mechanism establishing dorsal–ventral pattern formation has been conserved throughout evolution. Although both sets of genes establish non-neural ectodermal derivatives, the axes from which these cell types originate have opposite designations to each other: ventral in vertebrates is dorsal in invertebrates, and vice versa[2Nübler-Jung K. Arendt D. Roux's Arch. Dev. Biol. 1994; 203: 357-366Crossref Scopus (72) Google Scholar]. Here I discuss the mechanism recently identified by which the metalloprotease Tolloid in flies, frogs and zebrafish functions in a double inhibition mechanism to establish the graded activity of DPP/BMPs along the dorsal–ventral axis. To place the role of tolloid/Xolloid into context, I first review data on the function of other genes in the pathway. Of the seven zygotic genes with ventralized phenotypes in Drosophila, dpp is thought to be the key gene. dpp mutants display the strongest ventralized phenotype: a complete loss of the dorsal laterally derived epidermis and the dorsal-most amnioserosa, and a concomitant expansion in the ventral neurogenic ectoderm[3Ferguson E. Curr. Opin. Genet. Dev. 1996; 6: 424-431Crossref PubMed Scopus (96) Google Scholar](see fate map in Fig. 1). A phenotypic series of dpp alleles, along with dpp gene and mRNA dosage experiments, indicate that DPP acts as a morphogen to specify different positional values in the dorsal half of the Drosophila embryo[3Ferguson E. Curr. Opin. Genet. Dev. 1996; 6: 424-431Crossref PubMed Scopus (96) Google Scholar]. In situ hybridization analysis, however, shows that dpp mRNA levels are uniform throughout the dorsal half of the embryo[4Rusch J. Levine M. Curr. Opin. Genet. Dev. 1996; 6: 416-423Crossref PubMed Scopus (124) Google Scholar]. Hence, the hypothesized gradient of DPP activity cannot be generated by differing amounts of dpp mRNA; a post-transcriptional mechanism must establish the DPP activity gradient. In vertebrates, BMP2 and BMP4 are the orthologues of dpp, and have similar properties to DPP. BMP4 ventralizes embryos when overexpressed in dorsal regions, while a dominant-negative form of this protein, or antisense expression, dorsalizes the frog embryo[5Graff J.M. Cell. 1997; 89: 171-174Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar]. In zebrafish the swirl mutant, which exhibits the strongest phenotype among the zebrafish dorsalized mutants[6Mullins M.C. et al.Development. 1996; 123: 81-93Crossref PubMed Google Scholar, 7Nguyen, V.H. et al. Dev. Biol. (in press)Google Scholar], has been identified as a mutation in the Bmp2b gene[7Nguyen, V.H. et al. Dev. Biol. (in press)Google Scholar, 8Kishimoto Y. et al.Development. 1997; 124: 4457-4466Crossref PubMed Google Scholar]. The phenotypic series of zebrafish dorsalized mutants[6Mullins M.C. et al.Development. 1996; 123: 81-93Crossref PubMed Google Scholar, 7Nguyen, V.H. et al. Dev. Biol. (in press)Google Scholar], and the dose-dependent specification of different tissues by BMP4 (Refs [9Dosch R. et al.Development. 1997; 124: 2325-2334Crossref PubMed Google Scholar, 10Wilson P.A. et al.Development. 1997; 124: 3177-3184Crossref PubMed Google Scholar, 11Neave B. et al.Mech. Dev. 1997; 62: 183-195Crossref PubMed Scopus (148) Google Scholar]) suggest the action of a morphogen, possibly BMP2 and/or BMP4, in establishing the vertebrate dorsal–ventral axis. In Drosophila, genetic evidence suggests that Short Gastrulation (SOG) represses the activity of DPP. sog is expressed ventrally, but functions in both ventral and dorsal regions as shown by the sog mutant phenotype: a partial loss of the dorsal-most amnioserosa, and a reduction in ventral cuticular structures, associated with an expansion in the dorsal laterally derived epidermis[3Ferguson E. Curr. Opin. Genet. Dev. 1996; 6: 424-431Crossref PubMed Scopus (96) Google Scholar, 12Zusman S.B. et al.Dev. Biol. 1988; 129: 417-427Crossref PubMed Scopus (63) Google Scholar]. dpp is epistatic to sog in dpp sog double-mutant embryos[13Holley S.A. Ferguson E.L. BioEssays. 1997; 19: 281-284Crossref PubMed Scopus (81) Google Scholar], and sog loss-of-function mutations suppress a weak dpp ventralized phenotype[14Wharton K.A. et al.Development. 1993; 117: 807-822PubMed Google Scholar, 15François V. et al.Genes Dev. 1994; 8: 2602-2616Crossref PubMed Scopus (270) Google Scholar]. These results suggest that sog acts upstream of dpp in dorsal and ventral regions to inhibit DPP signalling. sog dose-dependent effects are observed in the dorsal-most cells of the embryo, suggesting a graded inhibitory action for SOG in modulating DPP activity[16Biehs B. et al.Genes Dev. 1996; 10: 2922-2934Crossref PubMed Scopus (160) Google Scholar]. In vertebrates, chordin is the orthologue of sog and functions in a similar manner[3Ferguson E. Curr. Opin. Genet. Dev. 1996; 6: 424-431Crossref PubMed Scopus (96) Google Scholar, 5Graff J.M. Cell. 1997; 89: 171-174Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar, 17Thomsen G.H. Trends Genet. 1997; 13: 209-211Abstract Full Text PDF PubMed Scopus (88) Google Scholar]. When ectopically expressed in ventral regions chordin dorsalizes the embryo and induces a secondary axis in Xenopus. This effect can be counteracted by overexpression of BMP4. As with sog and dpp, an antagonistic relationship exists between chordin and BMP4, which occurs through direct binding. Secreted Chordin binds BMP4 extracellularly with high affinity and thus prevents BMP4 from binding its receptor[5Graff J.M. Cell. 1997; 89: 171-174Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar, 17Thomsen G.H. Trends Genet. 1997; 13: 209-211Abstract Full Text PDF PubMed Scopus (88) Google Scholar]. Consistent with chordin functioning to dorsalize the embryo via antagonism of BMPs, null mutations of chordin in zebrafish (chordino mutants) produce a moderate ventralized phenotype[18Schulte-Merker S. et al.Nature. 1997; 387: 862-863Crossref PubMed Scopus (334) Google Scholar, 19Fisher S. et al.Development. 1997; 124: 1301-1311PubMed Google Scholar], and in swirl chordino double mutants swirl is epistatic to chordino[13Holley S.A. Ferguson E.L. BioEssays. 1997; 19: 281-284Crossref PubMed Scopus (81) Google Scholar]. tolloid is a positive regulator of DPP activity in Drosophila. Mutants of tolloid exhibit a moderate ventralized phenotype, in which the amnioserosa and part of the dorsal epidermis are absent, while the ventral ectoderm is enlarged[20Ferguson E.L. Anderson K.V. Development. 1992; 114: 583-597PubMed Google Scholar]. Overexpression of dpp in tolloid mutant embryos partially suppresses the tolloid mutant phenotype[20Ferguson E.L. Anderson K.V. Development. 1992; 114: 583-597PubMed Google Scholar]. Because tolloid does not regulate the transcription of dpp, tolloid probably functions upstream of, or in conjunction with, dpp to increase its activity. tolloid encodes a member of the astacin family of metalloproteases closely related to BMP1 (Ref. [21Shimell M.J. et al.Cell. 1991; 67: 469-481Abstract Full Text PDF PubMed Scopus (254) Google Scholar]). Particular tolloid alleles enhance the phenotype of weak dpp alleles, suggesting that DPP and Tolloid interact directly or within the same complex, possibly by Tolloid cleaving DPP (Refs [22Finelli A.L. et al.Development. 1994; 120: 861-870PubMed Google Scholar, 23Childs S.R. O'Connor M.B. Dev. Biol. 1994; 162: 209-220Crossref PubMed Scopus (62) Google Scholar]). This notion is strengthened by the fact that BMP1 was originally isolated in a protein complex with BMP2 and BMP3, and also TGF-β molecules[24Wozney J.M. et al.Science. 1988; 242: 1528-1534Crossref PubMed Scopus (3367) Google Scholar]. However, the recent identification of the procollagen protease as BMP1 (Ref. [25Kessler E. et al.Science. 1996; 271: 360-362Crossref PubMed Scopus (458) Google Scholar]) and the similarity between SOG and procollagen[15François V. et al.Genes Dev. 1994; 8: 2602-2616Crossref PubMed Scopus (270) Google Scholar], in addition to the observation that Chordin binds BMP4 directly, suggest that tolloid acts upstream of dpp to cleave SOG proteolytically, thus releasing DPP so that it can bind its receptor[26Holley S.A. et al.Cell. 1996; 86: 607-617Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar]. Three recent papers show that this is the probable mechanism by which Tolloid acts in frogs[27Piccolo S. et al.Cell. 1997; 91: 407-416Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar], flies[28Marqués G. et al.Cell. 1997; 91: 417-426Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar]and fish[29Blader P. et al.Science. 1997; 278: 1937-1940Crossref PubMed Scopus (169) Google Scholar]. Piccolo et al.[27Piccolo S. et al.Cell. 1997; 91: 407-416Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar]and Blader et al.[29Blader P. et al.Science. 1997; 278: 1937-1940Crossref PubMed Scopus (169) Google Scholar]show that overexpression of Xolloid/tolloid (X/tolloid)moderately ventralizes frog and fish embryos. The alterations in gene expression and morphological defects are remarkably similar to zebrafish chordino mutant embryos, the phenotype expected if Tolloid specifically represses Chordin activity. Both groups also generated dominant-negative forms of X/Tolloid, which have the expected opposite effect of dorsalizing the embryo when overexpressed. Epistasis was examined to determine where in the BMP pathway tolloid acts. A dominant-negative form of the BMP type I receptor, when overexpressed, blocks signalling by BMP4 and dorsalizes the embryo[5Graff J.M. Cell. 1997; 89: 171-174Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar, 17Thomsen G.H. Trends Genet. 1997; 13: 209-211Abstract Full Text PDF PubMed Scopus (88) Google Scholar]. Injection of the ventralizing frog or fish X/tolloid mRNA, together with the dominant-negative BMP receptor, results in embryos with the dorsalized dominant-negative BMP receptor phenotype[27Piccolo S. et al.Cell. 1997; 91: 407-416Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar, 29Blader P. et al.Science. 1997; 278: 1937-1940Crossref PubMed Scopus (169) Google Scholar]. Thus, X/tolloid acts upstream of the BMP receptor. Ectopic expression of chordin, or two additional secreted factors that function in the organizer and inhibit BMP function (noggin and follistatin) dorsalize the embryo and induce secondary axes in Xenopus[5Graff J.M. Cell. 1997; 89: 171-174Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar, 17Thomsen G.H. Trends Genet. 1997; 13: 209-211Abstract Full Text PDF PubMed Scopus (88) Google Scholar]. Co-injection of X/tolloid with noggin[27Piccolo S. et al.Cell. 1997; 91: 407-416Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar, 28Marqués G. et al.Cell. 1997; 91: 417-426Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar, 29Blader P. et al.Science. 1997; 278: 1937-1940Crossref PubMed Scopus (169) Google Scholar]or follistatin[27Piccolo S. et al.Cell. 1997; 91: 407-416Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar]results in identical dorsalization phenotypes. In striking contrast, embryos co-injected with X/tolloid and chordin do not form secondary axes and can be ventralized, indicating that X/tolloid acts upstream of chordin to inhibit Chordin from blocking BMP signalling[27Piccolo S. et al.Cell. 1997; 91: 407-416Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar, 28Marqués G. et al.Cell. 1997; 91: 417-426Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar, 29Blader P. et al.Science. 1997; 278: 1937-1940Crossref PubMed Scopus (169) Google Scholar]. Although Chordin, Noggin and Follistatin all function by inhibiting and binding BMPs (Refs [17Thomsen G.H. Trends Genet. 1997; 13: 209-211Abstract Full Text PDF PubMed Scopus (88) Google Scholar, 30Fainsod A. et al.Mech. Dev. 1997; 63: 39-50Crossref PubMed Scopus (309) Google Scholar]), X/Tolloid specifically inactivates only one of these factors—Chordin. The epistasis between sog and tolloid in flies suggests that these genes have a relationship that is similar to their vertebrate counterparts. Double-mutant tolloid sog embryos exhibit the sog mutant phenotype[28Marqués G. et al.Cell. 1997; 91: 417-426Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar]. Thus in the absence of sog, no apparent phenotype or function is revealed for tolloid, indicating that tolloid functions upstream of sog to repress SOG activity. The analysis of epistasis suggests that the X/Tolloid protease acts by cleaving Chordin/SOG and thus prevents it from inactivating BMPs/DPP. All three groups provide biochemical evidence for this mechanism. The incubation of medium from a cell-culture system expressing the secreted vertebrate or fly X/Tolloid protein with Chordin or SOG results in cleavage of Chordin[27Piccolo S. et al.Cell. 1997; 91: 407-416Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar, 29Blader P. et al.Science. 1997; 278: 1937-1940Crossref PubMed Scopus (169) Google Scholar]and SOG (Ref. [28Marqués G. et al.Cell. 1997; 91: 417-426Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar]). Unlike the vertebrate Tolloid, however, SOG proteolysis is greatly enhanced by the presence of DPP (Ref. [28Marqués G. et al.Cell. 1997; 91: 417-426Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar]), presumably in a DPP–SOG complex. Cleavage requires the X/Tolloid protease domain, because a point mutation predicted to inactivate the protease does not cleave Chordin[27Piccolo S. et al.Cell. 1997; 91: 407-416Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar]or SOG (Ref. [28Marqués G. et al.Cell. 1997; 91: 417-426Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar]). Piccolo et al.[27Piccolo S. et al.Cell. 1997; 91: 407-416Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar]further demonstrated that the Xolloid-digested Chordin no longer possesses dorsalizing activity. Medium containing Chordin induces muscle in Xenopus tissue explants and inhibits the ventralizing action of BMPs, while medium containing Chordin/Xolloid does not. Moreover, the BMP released from Xolloid-digested Chordin–BMP complexes was biologically active to ventralize animal cap cells[27Piccolo S. et al.Cell. 1997; 91: 407-416Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar]. These data reveal a double inhibition mechanism by which a gradient of DPP and, probably, BMP activity is generated along the dorsal–ventral axis of invertebrate and vertebrate embryos to pattern the axis (Fig. 2). Chordin and X/Tolloid both act by inhibitory mechanisms. Chordin and, probably, SOG bind BMPs/DPP, abrogating receptor–ligand interaction and downstream signalling. The X/Tolloid protease acts positively to increase DPP/BMP activity by inhibiting Chordin/SOG action through direct cleavage of Chordin/SOG. All of these proteins are secreted, and at least DPP and SOG can diffuse to opposite sides of the embryo. DPP/BMPs and Tolloid are expressed in one half of the embryo, whereas SOG/Chordin is expressed in the opposite half (see Fig. 1). SOG/Chordin inhibits DPP/BMP activity in the region where SOG/Chordin is expressed, thus allowing neural tissue development. In addition SOG and, presumably, Chordin diffuse to the opposite pole where SOG reduces DPP/BMP activity in a graded manner. If the sole function of Tolloid is to digest SOG protein, then the tolloid mutant phenotype indicates that SOG diffuses to the dorsal-most part of the embryo. Uniform dorsal expression of the Tolloid protease[28Marqués G. et al.Cell. 1997; 91: 417-426Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar]results in degradation in dorsal regions of SOG/Chordin in SOG–DPP and Chordin–BMP complexes. The consequence is increasing concentrations of free DPP/BMP in a direction that is opposite to that of the SOG/presumptive Chordin gradient. A signalling gradient of DPP/BMP activity is generated, which patterns the dorsal–ventral axis (Fig. 1). A contradiction remains regarding the role played by SOG in development of the amnioserosa. sog mutant embryos lack most (but not all) fully differentiated amnioserosa[12Zusman S.B. et al.Dev. Biol. 1988; 129: 417-427Crossref PubMed Scopus (63) Google Scholar], although an early marker for the prospective amnioserosa is expanded in sog mutant embryos[15François V. et al.Genes Dev. 1994; 8: 2602-2616Crossref PubMed Scopus (270) Google Scholar, 16Biehs B. et al.Genes Dev. 1996; 10: 2922-2934Crossref PubMed Scopus (160) Google Scholar]. This poses two conundrums. First, the amnioserosa is postulated to be specified by the highest levels of DPP activity (Fig. 1), which is consistent with the expansion of an early marker, but ultimately the amnioserosa is reduced, rather than expanded, in sog mutant embryos. Loss of sog, an inhibitor of dpp, is expected to increase, not decrease, cell types specified by high DPP activity. Two models are presented to explain this contradiction. A model by Holley et al.[26Holley S.A. et al.Cell. 1996; 86: 607-617Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 28Marqués G. et al.Cell. 1997; 91: 417-426Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar]proposes that SOG actually increases DPP levels in the dorsal-most areas. SOG–DPP complexes in dorsal–lateral regions would diffuse to the dorsal-most regions, where Tolloid cleaves SOG and releases DPP. In this manner, higher DPP levels could be produced in the dorsal-most regions, which would be required for complete differentiation of the amnioserosa at a stage following the initial specification of the amnioserosa. Hence, loss of SOG would reduce DPP levels in the dorsal-most regions, resulting in an ultimate loss of differentiated amnioserosa. The second model postulates that an additional signal is required for amnioserosa differentiation and that the signal is absent in sog mutant embryos[28Marqués G. et al.Cell. 1997; 91: 417-426Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar]. Interestingly, Marqués et al.[28Marqués G. et al.Cell. 1997; 91: 417-426Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar]propose that a cleavage product of SOG or, alternatively, twisted gastrulation (which encodes a putative growth factor[31Mason E.D. et al.Genes Dev. 1994; 8: 1489-1501Crossref PubMed Scopus (123) Google Scholar]) might be this signal. In this model, twisted gastrulation function must depend on sog, in some unknown manner. Both models account for a loss of the amnioserosa, but they do not address a second unresolved issue, the presence of a small amount of amnioserosa in sog mutant embryos. The presence of some amnioserosa and dorsal–lateral ectoderm in sog mutant embryos suggests that different levels of DPP activity remain in the sog mutant. How is this generated? Perhaps screw, a TGF-β molecule, is involved. screw acts upstream of, or together with, dpp, perhaps in a more active heterodimer with DPP, to specify dorsal cell fates[32Arora K. et al.Genes Dev. 1994; 8: 2588-2601Crossref PubMed Scopus (175) Google Scholar, 33Ferguson E. Anderson K. Cell. 1992; 71: 451-461Abstract Full Text PDF PubMed Scopus (407) Google Scholar]. Further work lies ahead to unravel these puzzles and establish the precise roles played by the vertebrate genes in pattern formation of the embryo. In summary, through a powerful combination of genetics, molecular biology and biochemistry a double inhibition mechanism between X/Tolloid, a long-range acting Chordin/SOG, and BMPs/DPP, appears to generate an activity gradient of DPP/BMPs, which establishes different cell fates along the dorsal–ventral axis. I thank C. Ferguson, P. Klein, and M. Granato for discussions and helpful comments on the manuscript. My apologies to the many authors whose work was not cited due to space limitations.