Expression patterns of bone morphogenetic proteins (Bmps) in the developing mouse tooth suggest roles in morphogenesis and cell differentiation

Bone morphogenetic proteins (BMP) are secretory signal molecules which have a variety of regulatory functions during morphogenesis and cell differentiation. Teeth are typical examples of vertebrate organs in which development is controlled by sequential and reciprocal signaling between the epithelium and mesenchyme. In addition, tooth development is characterized by formation of mineralized tissues: the bone-like dentin and cementum as well as epithelially derived enamel. We have performed a comparative in situ hybridization analysis of the expression of six different Bmps (Bmp-2 to Bmp-7) starting from initiation of tooth development to completion of crown morphogenesis when dentine and enamel matrices are being deposited. Bmps-2, -4, and -7 were frequently codistributed and showed marked associations with epithelial-mesenchymal interactions. Their expression shifted between the epithelium and mesenchyme starting from the stage of tooth initiation. They were subsequently expressed in the enamel knot, the putative signaling center regulating tooth shape. Their expression domains prior to and during the differentiation of the dentine-forming odontoblasts and enamel-forming ameloblasts was in line with functions in regulation of cell differentiation and/or secretory activities of the cells. The expression of Bmp-3 was confined to mesenchymal cells, in particular to the dental follicle cells which give rise to the cementoblasts, forming the hard tissue covering the roots of teeth. Bmp-5 was expressed only in the epithelial ameloblasts. It was upregulated as the cells started to polarize and intense expression continued in the secretory ameloblasts. Bmp-6 was expressed only weakly in the dental mesenchyme during bud and cap stages. Our results are in line with regulatory functions of Bmps at all stages of tooth morphogenesis. Bmps-2, -4, and -7 are conceivably parts of signaling networks regulating tooth initiation and shape development. They as well as Bmp-5 may be involved in the induction and formation of dentine and enamel, and Bmp-3 in the development of cementum. The remarkable overlaps in the expression domains of different Bmp genes may implicate functional redundancy and/or formation of active heterodimers between different BMPs.     

A study of patients with Nelson''s syndrome

Tooth development is regulated by a reciprocal series of epithelial-mesenchymal interactions. Bmp4 has been identified as a candidate signalling molecule in these interactions, initially as an epithelial signal and then later at the bud stage as a mesenchymal signal (Vainio et al. [1993] Cell 75:45-58). A target gene for Bmp4 signalling is the homeobox gene Msx-1, identified by the ability of recombinant Bmp4 protein to induce expression in mesenchyme. There is, however, no evidence that Bmp4 is the endogenous inducer of Msx-1 expression. Msx-1 and Bmp-4 show dynamic, interactive patterns of expression in oral epithelium and ectomesenchyme during the early stages of tooth development. In this study, we compare the temporal and spatial expression of these two genes to determine whether the changing expression patterns of these genes are consistent with interactions between the two molecules. We show that changes in Bmp-4 expression precede changes in Msx-1 expression. At embryonic day (E)10.5-E11.0, expression patterns are consistent with BMP4 from the epithelium, inducing or maintaining Msx-1 in underlying mesenchyme. At E11.5, Bmp-4 expression shifts from epithelium to mesenchyme and is rapidly followed by localised up-regulation of Msx-1 expression at the sites of Bmp-4 expression. Using cultured explants of developing mandibles, we confirm that exogenous BMP4 is capable of replacing the endogenous source in epithelium and inducing Msx-1 gene expression in mesenchyme. By using noggin, a BMP inhibitor, we show that endogenous Msx-1 expression can be inhibited at E10.5 and E11.5, providing the first evidence that endogenous Bmp-4 from the epithelium is responsible for regulating the early spatial expression of Msx-1. We also show that the mesenchymal shift in Bmp-4 is responsible for up-regulating Msx-1 specifically at the sites of future tooth formation. Thus, we establish that a reciprocal series of interactions act to restrict expression of both genes to future sites of tooth formation, creating a positive feedback loop that maintains expression of both genes in tooth mesenchymal cells.     

Cyr61, product of a growth factor-inducible immediate-early gene, regulates chondrogenesis in mouse limb bud mesenchymal cells

Chondrogenesis during embryonic skeletal development involves the condensation of mesenchymal cells followed by their differentiation into chondrocytes. We describe herein a previously unrecognized regulator of mammalian chondrogenesis encoded by a murine growth factor-inducible immediate-early gene, cyr61. The Cyr61 protein is a secreted, heparin-binding protein (379 amino acids with 38 conserved cysteines) that promotes cell adhesion, migration, and proliferation. The expression pattern of the cyr61 gene during embryogenesis is tissue specific and temporally regulated. Most notably, cyr61 is transiently expressed in mesenchymal cells of both mesodermal and neuroectodermal origins undergoing chondrogenesis, suggesting that Cyr61 may play a role in the development of the embryonic skeleton. In this communication, we demonstrate that the Cyr61 protein promotes chondrogenesis in micromass cultures of limb bud mesenchymal cells in vitro and is likely to play a similar role in vivo based on the following observations: (1) Cyr61 is present in the embryonic limb mesenchyme during chondrogenesis in vivo and in vitro; (2) purified recombinant Cyr61 protein added exogenously to micromass cultures promotes chondrogenesis as judged by precocious expression of type II collagen, increased [35S]sulfate incorporation, and larger Alcian blue-staining cartilage nodules; (3) Cyr61 enhances cell-cell aggregation, an initial step in chondrogenesis, and promotes chondrogenic differentiation in cultures plated at subthreshold cell densities that are otherwise unable to support differentiation; and (4) neutralization of the endogenous Cyr61 with specific antibodies inhibits chondrogenesis. Taken together, these results identify Cyr61 as a novel player in chondrogenesis that contributes to the development of the mammalian embryonic skeleton.     

Odontogenic epithelium induces similar molecular responses in chick and mouse mandibular mesenchyme

Previous observations have shown that, during the initiation phase of odontogenesis, signals from mouse odontogenic epithelium can elicit teeth in non-odontogenic but neural crest-derived mesenchyme isolated from ectopic sites including chick mandibular mesenchyme. In the present study the formation of ectopic tooth buds and dental mesenchyme in chick mandibular mesenchyme was examined using heterospecific recombinations between E11 mouse odontogenic epithelium and stage 23 chick lateral mandibular mesenchyme. Both morphological criteria and chick-specific probes for Msx-1, Msx-2, and Bmp-4 mRNAs were used as markers for early dental mesenchyme. Our results demonstrated that interactions of mouse odontogenic epithelium with chick mandibular mesenchyme induce early changes in the chick mandibular mesenchyme including the appearance of a translucent zone, cell proliferation, and induction of expression of Msx-1, Msx-2, and Bmp-4, which have been shown to be associated with the formation of dental mesenchyme. In addition, tooth bud-like structures that resemble E13 tooth buds in vivo both morphologically and in their patterns of gene expression formed after 6 days in the heterospecific recombinations. The tooth bud-like structures consist of invaginated mouse mandibular epithelium and condensed chick mandibular mesenchyme expressing high levels of Msx-1 and Bmp-4, but undetectable levels of Msx-2. Unlike the induction of Msx-1, Msx-2, and Bmp-4 in the underlying mesenchyme, which is specific for signals derived from odontogenic epithelium, the induction of a translucent zone and cellular proliferation in the underlying mesenchyme may be related to the growth-promoting potential of embryonic epithelia and not be specific to signals derived from the odontogenic epithelium. Similar to mouse odontogenic epithelium, agarose beads soaked in recombinant BMP-4 induced a translucent zone, cellular proliferation, and expression of Msx-1, Msx-2, and Bmp-4 in chick mandibular mesenchyme after 24 hours. These observations together showed that avian mandibular mesenchyme has odontogenic potential that is expressed upon interactions with inductive signals from mouse odontogenic epithelium. Similar to odontogenesis in vivo, formation of dental mesenchyme in chick mandibular mesenchyme is mediated by the activation of Msx-1, Msx-2, and Bmp-4.     

Capture and expansion of bone marrow-derived mesenchymal progenitor cells with a transforming growth factor-beta1-von Willebrand's factor fusion protein for retrovirus-mediated delivery of coagulation factor IX

Mesenchymal stem cells give rise to the progenitors of many differentiated phenotypes, including osteocytes, chrondocytes, myocytes, adipocytes, fibroblasts, and marrow stromal cells, which are capable of self-renewal and undergo expansion in the presence of transforming growth factor-beta1 (TGF-beta1). The present study was designed to test the concept that mesenchymal progenitor cells could be selected and expanded by virtue of their intrinsic physiologic responses to TGF-beta1. Human bone marrow aspirates were initially cultured, under low serum conditions, in collagen pads or gels impregnated with a genetically engineered TGF-beta1 fusion protein bearing an auxiliary von Willebrand's factor-derived collagen-binding domain (TGF-beta1-vWF). Histologic examination of TGF-beta1-vWF-supplemented collagen pads from 8-day cultures revealed the selective survival of a population of mononuclear blastoid cells. The TGF-beta-responsive cells were expanded to form stromal/fibroblastic colonies by serum reconstitution, and further to form osteogenic colonies upon supplementation with osteoinductive factors. In comparative studies, both marrow-derived progenitor cells and mature stromal cells were transduced with a retroviral vector bearing a human factor IX construct. Both the transduced progenitor cells and mature stromal cells expressed the factor IX transgene at levels comparable to those reported for human fibroblasts. Transplantation of murine progenitor cells bearing the human factor IX vector into syngeneic B6CBA mice resulted in detectable circulating levels of the human factor IX antigen. Taken together, these data demonstrate a novel physiologic approach for the selection of mesenchymal precursor cells followed by mitotic expansion, transduction, and transplantation of these progenitor cells with retroviral vectors bearing therapeutic genes.     

Cloning of mouse diastrophic dysplasia sulfate transporter gene induced during osteoblast differentiation by bone morphogenetic protein-2

Although intensive studies have been directed at understanding osteoblastic differentiation, the molecular mechanisms are still unclear. In this study, we describe a cDNA that encodes a sulfate transporter that was cloned as a gene induced in osteoblast precursor cells in association with osteoblastic differentiation. Based on the fact that bone morphogenetic protein-2 (BMP-2) induces osteoblastic phenotypes in immature mouse fibroblastic C3H10T1/2 cells, we performed a subtraction hybridization between BMP-2-treated and untreated cells, and have isolated one clone (designated as st-ob for sulfate transporter in osteoblast) induced by BMP-2 that is constantly expressed in osteoblastic cells. The deduced amino acid sequence and proposed structure of st-ob are mostly identical to those of the human diastrophic dysplasia sulfate transporter gene product (DTDST). St-ob mRNA was abundantly expressed in the thymus, testis, calvaria and osteoblastic MC3T3-E1 cells, whereas its expression was faint in C3H10T1/2 cells. Expression of st-ob in C3H10T1/2 cells was increased by transforming growth factor-beta1 (TGF-beta1), retinoic acid and dexamethasone as well as BMP-2. Furthermore, BMP-2 increased sulfate incorporation in C3H10T1/2 cells about twice as high as the baseline level. Osteoblasts actively take up sulfate to synthesize proteoglycans, which are one of the major components of the extracellular matrix of bone and cartilage. The present study demonstrates that st-ob induced during osteoblastic differentiation is an important phenotype of osteoblasts for characterizing their function.     

The human oncoprotein MDM2 arrests the cell cycle: elimination of its cell-cycle-inhibitory function induces tumorigenesis

The human oncoprotein MDM2 (hMDM2) overexpresses in various human tumors. If amplified, the mdm2 gene can enhance the tumorigenic potential of murine cells. Here, we present evidence to show that the full-length human or mouse MDM2 expressed from their respective cDNA can inhibit the G0/G1-S phase transition of NIH 3T3 and normal human diploid cells. The protein harbors more than one cell-cycle-inhibitory domain that does not overlap with the p53-interaction domain. Deletion mutants of hMDM2 that lack the cell-cycle-inhibitory domains can be stably expressed in NIH 3T3 cells, enhancing their tumorigenic potential. The tumorigenic domain of hMDM2 overlaps with the p53-interaction domain. Some tumor-derived cells, such as Saos-2, H1299 or U-2OS, are relatively insensitive to the growth-inhibitory effects of hMDM2. These observations suggest that hMDM2 overexpression in response to oncogenic stimuli would induce growth arrest in normal cells. Elimination or inactivation of the hMDM2-induced G0/G1 arrest may contribute to one of the steps of tumorigenesis.     

Identification in the chicken of GRL1 and GRL2: two granule proteins expressed on the surface of activated leukocytes

We report the production of two monoclonal antibodies reacting, respectively, with a 92-kDa protein (GRL1) and a 40- to 65-kDa membrane glycoprotein (GRL2), both present in chicken thrombocyte and myelocyte granules. We examined the expression of GRL1 and GRL2 during the development of the hematopoietic system: GRL1 is restricted to thrombocytes and myelocytes, whereas GRL2 is present in thrombocytes, myelocytes, myeloid progenitors, and a subpopulation of erythroid progenitors. In the lymphoid lineages, neither GRL1 nor GRL2 is expressed during thymus and bursa ontogeny or on resting peripheral blood lymphocytes. However, CD3+ T lymphoblasts obtained by mitogenic stimulation of GRL2-negative quiescent T lymphocytes are stained on their surface by anti-GRL2 Mab. In vitro stimulation of thrombocytes and granulocytes with their specific secretagogues results in the expression of GRL1 and in the overexpression of GRL2 on the cell surface. These observations are consistent with the following two conclusions: the presence on the cell surface of GRL1 epitope is a marker of thrombocyte and myelocyte activation; GRL2 epitope is present on the granule membrane of leukocytes, including T cells. In that respect, GRL2 appears to share certain features with leukocyte activation antigens recently described in human.     

Effects of scandium chloride on osteogenic and adipogenic differentiation of mesenchymal stem cells

Mesenchymal stem cells(MSCs)are multi-potent cells that are able to differentiate and mature into various types of cells under a certain microenvironment for cell therapy and tissue regeneration.Scandium(Sc),an important rare earth element,recently has been intensively investigated in biomedical fields as well as industrial engineering,and chloride channels have been proven to be able to affect osteogenic differentiation.Thus,it is significant to investigate effects of ScCl₃on cell activities of MSCs.In this paper,rat bone MSCs(rBMSCs)were co-cultured with various concentrations of ScCl₃(1×10^-8,1×10^-6,and 1×10^-4mol/L)to evaluate their influence on cell proliferation as well as osteogenic and adipogenic differentiation in vitro.The results indicate that ScCl₃promotes the proliferation of rBMSCs initially,which is yet reduced upon ion accumulation.We used immunofluorescence staining,quantitative real time polymerase chain reactions,and assays measuring alkaline phosphatase activity,mineralized deposits,and intracytoplasmic lipids to reveal that rBMSCs treated with ScCl₃at concentrations of 1×10^-8-1×10^-6mol/L can enhance levels of osteogenic differentiation in a dosedependent manner and reduce adipogenic differentiation to a certain degree through Wnt/β-catenin signaling pathway.These results indicate that appropriate concentrations of ScCl₃can improve osteogenic differentiation in the lineage commitment of rBMSCs,and thus,promote bone remodeling.This study implies that ScCl₃ possesses great potentials in the treatment of bone diseases and would provide new strategy of designing composites by SiCl3 doping for biomedical applications in the future.     

C2H2-171: a novel human cDNA representing a developmentally regulated POZ domain/zinc finger protein preferentially expressed in brain

We describe a novel human zinc finger cNDA. C2H2-171. This cDNA represents an mRNA which encodes a protein of 484 amino acids and a calculated molecular weight of 54 kD. Four zinc finger-like domains are found in the C-terminal end of the protein. At the N-terminus, C2H2-171 contains a POZ/tramtrack-like domain similar to that found in the tumor associated zinc finger proteins LAZ-3/BCL-6 and PLZ-F, as well as in non-zinc finger proteins. C2H2-171 RNA is preferentially expressed in the brain, and increases during the course of murine development, with maximal expression in the adult. C2H2-171 RNA is differentially expressed in brain regions, with the highest level of expression in the cerebellum. C2H2-171 RNA was expressed at high levels in primary cerebellar granule cell neurons compared to astrocytes. The gene encoding C2H2-171 is highly conserved in vertebrates, and maps to the terminus of human chromosome 1 (1q44-ter). This chromosomal location is associated with a number of cytogenetic aberrations including those involving brain developmental anomalies and tumorigenesis. These data suggest that C2H2-171 may play an important role in vertebrate brain development and function.     

Effects of La~（3＋） on osteogenic and adipogenic differentiation of primary mouse bone marrow stromal cells

In order to elucidate the action of La3+ on bone metabolism,effects of La3+ on the osteogenic and adipogenic differentiation of pri-mary mouse bone marrow stromal cells(BMSCs) were studied by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide(MTT) test,alkaline phosphatase(ALP) activity measurement,mineralized function,oil red O stain and measurement.The results showed that La3+ pro-moted the proliferation of BMSCs except at 1×10-10 and 1×10-6 mol/L.The effect of La3+ on the osteogenic differentiation depended on con-centrations at the 7th day,but the osteogenic differentiation was inhibited at any concentration at the 14th day.La3+ promoted the formation of mineralized matrix nodules except at 1×10-8 and 1×10-5 mol/L.La3+ inhibited adipogenic differentiation except at 1×10-10 and 1×10-7 mol/L at the 10th day,and inhibited adipogenic differentiation except at 1×10-9 mol/L at the 16th day.These findings suggested that La3+ might have protective effect on bone at appropriate dose and time.This would be valuable for better understanding the mechanism of the effect of La3+ on bone metabolism.