No abstract

Both decapentaplegic (dpp) protein and 60A protein have been implicated in pattern formation during Drosophila melanogaster embryogenesis. Within the C-terminal domain, dpp and 60A are similar to human bone morphogenetic protein 2 (75% identity) and human osteogenic protein 1 (70% identity), respectively. Both recombinant human bone morphogenetic protein 2 and recombinant human osteogenic protein 1 have been shown to induce bone formation in vivo and to restore large diaphyseal segmental defects in various animal models. We examined whether the Drosophila proteins, dpp and 60A, have the capacity to induce bone formation in mammals by using the rat subcutaneous bone induction model. Highly purified recombinant dpp and 60A induced the formation of cartilage, bone, and bone marrow in mammals, as determined by histological observations and by measurements of the specific activity of alkaline phosphatase and calcium content of the implants, thereby demonstrating that related proteins from phylogenetically distant species are capable of inducing bone formation in mammals when placed in sites where progenitor cells are available.Embryonic bone development begins with migration of mesenchymal cells to a predetermined site where they either condense, proliferate, and differentiate directly into boneforming cells or pass through an intermediate cartilage stage before they are replaced with bone. In adult life, bone has a remarkable potential to repair itself upon fracture through a process that recapitulates embryonic bone development. Urist (1) and Reddi and Huggins (2) have shown that the cellular events involved in embryonic bone development are reproduced in predictable intervals in subcutaneous implants of demineralized bone matrix in rats. By employing a reconstitution assay in the rat subcutaneous bone induction model (3, 4) and molecular cloning approaches, several osteogenic proteins (OPs), also called bone morphogenetic proteins [BMPs; BMP-2 through BMP-6, OP-1 (also called BMP-7), and OP-2] have been identified (5-8). The predicted amino acid sequences of these proteins indicate that they are all members of the transforming growth factor f8 (TGF-f3) superfamily, sharing a high degree of homology within the C-terminal seven-cysteine domain (9).The TGF-,p superfamily members are signaling molecules thought to be responsible for specific morphogenic events during development (9, 10). For example, increasing concentrations of Xenopus activins can cause animal cap cells to differentiate into various cell types (11) while BMP-4 (closely related to BMP-2) can instruct a ventral posterior positional cell fate on developing mesoderm in the Xenopus blastula (12, 13). In the mouse, localized expression of BMPs has been reported in skin, heart, nervous system, and developing limbs (14). A recent study demonstrates that mutation of BMP-5The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 so...

We have analyzed a new limb mutant in the chicken that we nameoligozeugodactyly (ozd). The limbs of this mutant have a longitudinal postaxial defect, lacking the posterior element in the zeugopod(ulna/fibula) and all digits except digit 1 in the leg. Classical recombination experiments show that the limb mesoderm is the defective tissue layer in ozd limb buds. Molecular analysis revealed that theozd limbs develop in the absence of Shh expression, while all other organs express Shh and develop normally. NeitherPtc1 nor Gli1 are detectable in mutant limb buds. However,Bmp2 and dHAND are expressed in the posterior wing and leg bud mesoderm, although at lower levels than in normal embryos. Activation ofHoxd11-13 occurs normally in ozd limbs but progressively declines with time. Phase III of expression is more affected than phase II,and expression is more severely affected in the more 5′ genes. Interestingly, re-expression of Hoxd13 occurs at late stages in the distal mesoderm of ozd leg buds, correlating with formation of digit 1. Fgf8 and Fgf4 expression are initiated normally in the mutant AER but their expression is progressively downregulated in the anterior AER. Recombinant Shh protein or ZPA grafts restore normal pattern toozd limbs; however, retinoic acid fails to induce Shh in ozdlimb mesoderm. We conclude that Shh function is required for limb development distal to the elbow/knee joints, similar to the Shh-/-mouse. Accordingly we classify the limb skeletal elements as Shh dependent or independent, with the ulna/fibula and digits other than digit 1 in the leg being Shh dependent. Finally we propose that the ozd mutation is most likely a defect in a regulatory element that controls limb-specific expression of Shh.

A variety of genetic evidence suggests that a gradient of Decapentaplegic (Dpp) activity determines distinct cell fates in the dorsal region of the Drosophila embryo, and that this gradient may be generated indirectly by an inverse gradient of the BMP antagonist Short gastrulation (Sog). It has been proposed that Sog diffuses dorsally from the lateral neuroectoderm where it is produced, and is cleaved and degraded dorsally by the metalloprotease Tolloid (Tld). Here we show directly that Sog is distributed in a graded fashion in dorsal cells and that Tld degradation limits the levels of Sog dorsally. In addition, we find that Dynamin-dependent retrieval of Sog acts in parallel with degradation by Tld as a dorsal sink for active Sog.

Cotton (Gossypium hirsutum L.) cotyledon tissues have been efficiently transformed and plants have been regenerated. Cotyledon pieces from 12-day-old aseptically germinated seedlings were inoculated with Agrobacterium tumefaciens strains containing avirulent Ti (tumor-inducing) plasmids with a chimeric gene encoding kanamycin resistance. After three days cocultivation, the cotyledon pieces were placed on a callus initiation medium containing kanamycin for selection. High frequencies of transformed kanamycin-resistant calli were produced, more than 80% of which were induced to form somatic embryos. Somatic embryos were germinated, and plants were regenerated and transferred to soil. Transformation was confirmed by opine production, kanamycin resistance, immunoassay, and DNA blot hybridization. This process for producing transgenic cotton plants facilitates transfer of genes of economic importance to cotton.