Bone morphogenetic proteins (BMPs) are morphogenetic signaling molecules essential for embryonic patterning. To obtain molecular insight into the influence of BMPs on morphogenesis, we searched for new genes directly activated by BMP signaling. In vitro cultured mouse embryonic stem (ES) cells were used, cultivated in chemically defined growth medium (CDM). CDM-cultured ES cells responded very selectively to stimulation by various mesoderm inducers (BMP2/4, activin A, and basic fibroblast growth factor). BMP2/4 rapidly induced transcript levels of the homeobox genes Msx-1 and Msx-2 and the proto-oncogene JunB, whereas c-jun transcripts displayed delayed albeit prolonged increase. Using differential display cDNA cloning, six direct BMP target genes were identified. These include Id3, which showed strong mRNA induction, and the moderately induced One important stage in the development of the vertebrate embryo is reached when the three germ layers are formed and the body plan gets established during the process of gastrulation (1, 2). Our understanding of the molecular mechanism mediating gastrulation is still insufficient, although over the past years many secreted growth factor-like molecules have been identified to play important roles in these early embryonic events (3). Among these signaling molecules are several members of the BMP 1 family. BMP-related growth factors belong to the TGF-␤-superfamily and have been identified in a wide variety of organisms, ranging from insects to mammals (4). BMPs have originally been isolated for their ability to induce ectopic bone formation when injected under the skin or into the muscle of rodents (5, 6), but meanwhile many of the BMPs have been implicated in a variety of other developmental interactions, including very early embryonic inductive events. For example in Drosophila embryonic dorsal-ventral patterning is partly accomplished through the action of DPP (decapentaplegic) (7,8), which is the BMP member most closely related to mammalian BMP2 and BMP4. At the functional level these factors can substitute for one another in vertebrate and Drosophila embryos. Human BMP4 is able to rescue the dorsalventral pattern defects of dpp null mutants (9), whereas Drosophila DPP protein can induce ectopic bone in mice (10). The striking evolutionary conservation of BMP2, BMP4, and DPP suggested that these molecules play crucial roles in early vertebrate development, as is confirmed meanwhile by many studies. In Xenopus laevis BMP4 ventralizes early mesoderm (11-13) and promotes the differentiation of epidermis from ectoderm (14). Blocking BMP2/4 receptor activity in the ventral part of the embryo eliminates blood formation and dorsalizes the mesoderm (15, 16), whereas in contrast overexpression of BMP4 RNA leads to an increased expression of ventral genes and inhibits the formation of anterior structures (11,12,17). Use of a dominant-negative BMP2/4 receptor to block BMP signals in the ectoderm (18), or disaggregation of ectodermal cells (19), causes differentiation into neural ti...

The cDNAs encoding the human bone morphogenetic proteins BMP-2 and BMP-4 in an eukaryotic expression vector were permanently transferred into the murine mesenchymal progenitor cell line C3H10T1/2. Originally, these cells are known to differentiate into myotubes, adipocytes, and chondrocytes upon the addition of azacytidine. Permanent transfection of genes encoding human BMP-2 and BMP-4 induces differentiation into the osteogenic lineage. The osteogenic differentiation potential of C3H10T1/2 cells is substantiated by histochemical and genetic analyses of marker genes typical or specific for osteogenesis, including the parathyroid hormone receptor, alkaline phosphatase, osteopontin, osteonectin, and osteocalcin. In addition to osteoblast formation, development into adipocytes and chondrocytes is also observed, suggesting that BMP-2 and BMP-4 induce differentiation into three mesenchymal lineages.

M-cadherin is a classical calcium-dependent cell adhesion molecule that is highly expressed in developing skeletal muscle, satellite cells, and cerebellum. Based on its expression pattern and observations in cell culture, it has been postulated that M-cadherin may be important for the fusion of myoblasts to form myotubes, the correct localization and function of satellite cells during muscle regeneration, and the specialized architecture of adhering junctions in granule cells of cerebellar glomeruli. In order to investigate the potential roles of M-cadherin in vivo, we generated a null mutation in mice. Mutant mice were viable and fertile and showed no gross developmental defects. In particular, the skeletal musculature appeared essentially normal. Moreover, muscle lesions induced by necrosis were efficiently repaired in mutant mice, suggesting that satellite cells are present, can be activated, and are able to form new myofibers. This was also confirmed by normal growth and fusion potential of mutant satellite cells cultured in vitro. In the cerebellum of M-cadherin-lacking mutants, typical contactus adherens junctions were present and similar in size and numbers to the equivalent junctions in wild-type animals. However, the adhesion plaques in the cerebellum of these mutants appeared to contain elevated levels of N-cadherin compared to wild-type animals. Taken together, these observations suggest that M-cadherin in the mouse serves no absolutely required function during muscle development and regeneration and is not essential for the formation of specialized cell contacts in the cerebellum. It seems that N-cadherin or other cadherins can largely compensate for the lack of M-cadherin.

ABSTRACT

Parathyroid hormone (PTH)-mediated gene activation was assessed in the osteoblast-like rat cell line ROS17/2.8 with two PTH fragments harboring distinct activating domains: PTH-(1-34) and PTH-(28-48). The PTH response of genes expressed immediate early in the cell cycle or in the osteoblast developmental sequence was investigated. In addition, subtractive cloning was used to identify genes in ROS17/2.8 cells that are activated by the two PTH domains. PTH-(1-34) immediately increased the transcript levels of c-fos and c-jun at a considerably higher rate than PTH-(28-48). A significant immediate PTH effect on osteoblastic marker genes could not be detected, with the exception of elevated ornithine decarboxylase transcript levels. However, continuous application of PTH-(1-34) increased transcript levels of the osteoblast-specific osteocalcin gene and reduced those of other osteoblastic marker genes including alkaline phosphatase and the PTH/PTH-related peptide receptor. By subtractive cloning, nine cDNAs were isolated corresponding to mRNAs directly up-regulated by PTH-(1-34) or PTH-(28-48). Among these were a cyclic phosphodiesterase, a (cytosine 5)-methyltransferase, an 80-kDa protein kinase C substrate, junB, and a novel GC-binding protein. Three cDNAs are unknown at present. Interestingly, in all cases, the efficiency of gene activation by PTH-(28-48) was substantially lower in comparison with PTH-(1-34). PTH-mediated protein kinase C signaling in ROS17/2.8 cells may therefore constitute a minor pathway in comparison with the dominant cAMP/protein kinase A cascade.

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