The mammalian Tolloid-like 1 gene, Tll1, is necessary for normal septation and positioning of the heart.
Mammalian Tolloid-like 1 (mTLL-1) is an astacin-like metalloprotease, highly similar in domain structure to the morphogenetically important proteases bone morphogenetic protein-1 (BMP-1) and Drosophila Tolloid. To investigate possible roles for mTLL-1 in mammalian development, we have used gene targeting in ES cells to produce mice with a disrupted allele for the corresponding gene, Tll1. Homozygous mutants were embryonic lethal, with death at mid-gestation from cardiac failure and a unique constellation of developmental defects that were apparently confined solely to the heart. Constant features were incomplete formation of the muscular interventricular septum and an abnormal and novel positioning of the heart and aorta. Consistent with roles in cardiac development, Tll1 expression was specific to precardiac tissue and endocardium in 7.5 and 8.5 days p.c. embryos, respectively. Tll1 expression was also high in the developing interventricular septum, where expression of the BMP-1 gene, Bmp1, was not observed. Cardiac structures that were not affected in Tll1-/- embryos either showed no Tll1 expression (atrio-ventricular cushions) or showed overlapping expression of Tll1 and Bmp1 (aortico-pulmonary septum), suggesting that products of the Bmp1 gene may be capable of functionally substituting for mTLL-1 at sites in which they are co-expressed. Together, the various data show that mTLL-1 plays multiple roles in formation of the mammalian heart and is essential for formation of the interventricular septum. (+info)
Bone morphogenetic protein 1 regulates dorsal-ventral patterning in early Xenopus embryos by degrading chordin, a BMP4 antagonist.
Bone morphogenetic protein 1 (BMP1) is a metalloprotease that ventralises dorsal mesoderm when overexpressed in early Xenopus embryos. Here we show that Xenopus BMP1 blocks the dorsalising activity of chordin, but not noggin or DeltaxBMPR, when coexpressed in the ventral marginal zone and degrades chordin protein in vitro. We also show that a dominant-negative mutation for XBMP1 (dnBMP1) dorsalises ventral mesoderm in vivo, and blocks degradation of chordin by both XBMP1 and Xolloid, a closely related Xenopus metalloprotease, in vitro. dnBMP1 does not dorsalise ventral mesoderm in UV-irradiated embryos, demonstrating that this activity is dependent upon a functional organiser--the natural source of chordin in Xenopus gastrulae. Our results suggest that XBMP1 may regulate the availability of chordin during vertebrate embryogenesis. (+info)
Mammalian BMP-1/Tolloid-related metalloproteinases, including novel family member mammalian Tolloid-like 2, have differential enzymatic activities and distributions of expression relevant to patterning and skeletogenesis.
Vertebrate bone morphogenetic protein 1 (BMP-1) and Drosophila Tolloid (TLD) are prototypes of a family of metalloproteases with important roles in various developmental events. BMP-1 affects morphogenesis, at least partly, via biosynthetic processing of fibrillar collagens, while TLD affects dorsal-ventral patterning by releasing TGFbeta-like ligands from latent complexes with the secreted protein Short Gastrulation (SOG). Here, in a screen for additional mammalian members of this family of developmental proteases, we identify novel family member mammalian Tolloid-like 2 (mTLL-2) and compare enzymatic activities and expression domains of all four known mammalian BMP-1/TLD-like proteases [BMP-1, mammalian Tolloid (mTLD), mammalian Tolloid-like 1 (mTLL-1), and mTLL-2]. Despite high sequence similarities, distinct differences are shown in ability to process fibrillar collagen precursors and to cleave Chordin, the vertebrate orthologue of SOG. As previously demonstrated for BMP-1 and mTLD, mTLL-1 is shown to specifically process procollagen C-propeptides at the physiologically relevant site, while mTLL-2 is shown to lack this activity. BMP-1 and mTLL-1 are shown to cleave Chordin, at sites similar to procollagen C-propeptide cleavage sites, and to counteract dorsalizing effects of Chordin upon overexpression in Xenopus embryos. Proteases mTLD and mTLL-2 do not cleave Chordin. Differences in enzymatic activities and expression domains of the four proteases suggest BMP-1 as the major Chordin antagonist in early mammalian embryogenesis and in pre- and postnatal skeletogenesis. (+info)
Expression of chick BMP-1/Tolloid during patterning of the neural tube and somites.
The expression pattern described here is that of the chick BMP-1/Tolloid family of secreted metalloproteinases during early stages of development. BMP-1/Tolloid transcripts are expressed in the blastoderm, at gastrulation stages and as the neural plate forms and neural tube folds, BMP-1/Tolloid is found at the neural plate/ectodermal transition. Expression is maintained in the premigratory neural crest, and transiently in the migrating cephalic neural crest cells. BMP-1/Tolloid is also expressed in the caudal, but not in the anterior notochord, and in the ventral neural tube at the time of dorso-ventral patterning. Further sites of BMP-1/Tolloid expression are the lateral plate mesoderm and the dermotome and the myotome of the somites. (+info)
Bone morphogenetic protein 1 is an extracellular processing enzyme of the laminin 5 gamma 2 chain.
Epithelial cells maintained in culture medium containing low calcium proteolytically process laminin 5 (alpha3beta3gamma2) within the alpha3 and gamma2 chains (). Experiments were designed to identify the enzyme(s) responsible for the laminin 5 processing and the sites of proteolytic cleavage. To characterize the nature of laminin 5 processing, we determined the N-terminal amino acid sequences of the proteolytic fragments produced by the processing events. The results indicate that the first alpha3 chain cleavage (200-l65 kDa alpha3) occurs within subdomain G4 of the G domain. The second cleavage (l65-l45 kDa alpha3) occurs within the lIla domain, 11 residues N-terminal to the start of domain II. The gamma chain is cleaved within the second epidermal growth factor-like repeat of domain Ill. The sequence cleaved within the gamma2 chain matches the consensus sequence for the cleavage of type I, II, and III procollagens by bone morphogenetic protein-1 (BMP-1), also known as type I procollagen C-proteinase (). Recombinant BMP-1 cleaves gamma2 in vitro, both within intact laminin 5 and at the predicted site of a recombinant gamma2 short arm. alpha3 is also cleaved by BMP-1 in vitro, but the cleavage site is yet to be determined. These results show the laminin alpha3 and gamma2 chains to be substrates for BMP-1 in vitro. We speculate that gamma2 cleavage is required for formation of the laminin 5-6 complex and that this complex is directly involved in assembly of the interhemidesmosomal basement membrane. This further suggests that BMP-1 activity facilitates basement membrane assembly, but not hemidesmosome assembly, in the laminin 5-rich dermal-epidermal junction basement membrane in vivo. (+info)
Profiling of genes which are differentially expressed in mouse liver in response to adenoviral vectors and delivered genes.
The effects of transgene delivery by adenoviral vectors were studied by probing a 588 gene, mouse cDNA array with mRNA derived from infected liver. The liver tissues were obtained from naive mice and mice infected with replication-deficient adenovirus, adenovirus expressing transforming growth factor beta1 (TGFbeta1), and adenovirus expressing connective tissue growth factor (CTGF). Expression of 98 genes was detected in the array analysis. The increased expression of the transcripts for Stat1, gamma interferon-induced monokine (MIG) and interferon regulatory factor 1 (IRF1) clearly demonstrated the immune response induced by infection with a first generation, replication-incompetent adenovirus. In vivo expression of TGFbeta1 led to a down-regulation of genes involved in the immune response. The increased expression of u-PAR1, laminin receptor and BMP-1 confirms the importance of CTGF and TGFbeta1 in angiogenesis, and tissue repair. Expression of the serine protease inhibitors, Spi 2.4 and Spi 2, is also increased in response to AdTGFbeta1 and AdCTGF. (+info)
Is chordin a long-range- or short-range-acting factor? Roles for BMP1-related metalloproteases in chordin and BMP4 autofeedback loop regulation.
Diffusible morphogen models have been used widely to explain regional specification of tissues and body axes during animal development. The three-signal model for patterning the dorsal-ventral axis of the amphibian embryo proposes, in part, that a factor(s) secreted from Spemann's organizer is responsible for converting lateral marginal zone into more dorsal cell fates. We examine the possibility that chordin, a secreted inhibitor of bone morphogenetic protein (BMP) signaling and candidate "dorsalizing signal," is a long-range-acting factor. We show that chordin can, when overexpressed, act directly over distances of at least 450 microm in the early Xenopus embryo to create a gradient of BMP signaling. However, since lower levels of chordin can still induce secondary axes and these amounts of chordin act only locally to inhibit a BMP target gene, we suggest that chordin likely acts as a short-range signal in vivo. Furthermore, BMP1, a secreted metalloprotease that cleaves chordin protein in vitro, inhibits chordin's axis-inducing effects, suggesting that BMP1 functions to negatively regulate chordin's action in vivo. A dominant-negative mutant BMP1 blocks the in vitro cleavage of chordin protein by wild-type BMP1 and induces secondary axes when injected ventrally. We argue that BMP1 and Xolloid are probably functionally redundant metalloproteases and may have two roles in the early Xenopus embryo. One role may be to inhibit the action of low-level chordin protein expressed throughout the entire embryo and a possible second role may be to inhibit activation of a juxtacrine cell relay, thereby confining chordin's action to the organizer region preventing chordin from functioning as a long-range-acting factor. (+info)
Bone morphogenetic protein-1 processes probiglycan.
Bone morphogenetic protein-1 (BMP-1) is a metalloprotease that plays important roles in regulating the deposition of fibrous extracellular matrix in vertebrates, including provision of the procollagen C-proteinase activity that processes the major fibrillar collagens I-III. Biglycan, a small leucine-rich proteoglycan, is a nonfibrillar extracellular matrix component with functions that include the positive regulation of bone formation. Biglycan is synthesized as a precursor with an NH(2)-terminal propeptide that is cleaved to yield the mature form found in vertebrate tissues. Here, we show that BMP-1 cleaves probiglycan at a single site, removing the propeptide and producing a biglycan molecule with an NH(2) terminus identical to that of the mature form found in tissues. BMP-1-related proteases mammalian Tolloid and mammalian Tolloid-like 1 (mTLL-1) are shown to have low but detectable levels of probiglycan-cleaving activity. Comparison shows that wild type mouse embryo fibroblasts (MEFs) produce only fully processed biglycan, whereas MEFs derived from embryos homozygous null for the Bmp1 gene, which encodes both BMP-1 and mammalian Tolloid, produce predominantly unprocessed probiglycan, and MEFs homozygous null for both the Bmp1 gene and the mTLL-1 gene Tll1 produce only unprocessed probiglycan. Thus, all detectable probiglycan-processing activity in MEFs is accounted for by the products of these two genes. (+info)