Bone morphogenetic proteins and noggin: inhibiting and inducing fungiform taste papilla development

Fungiform papillae are epithelial specializations that develop in a linear pattern on the anterior mammalian tongue and differentiate to eventually contain taste buds. Little is known about morphogenetic and pattern regulation of these crucial taste organs. We used embryonic rat tongue, organ cultures to test roles for bone morphogenetic proteins, BMP2, 4 and 7, and antagonists noggin and follistatin, in development of papillae from a stage before morphological initiation (E13) or from a stage after the pre-papilla placodes have formed (E14). BMPs and noggin proteins become progressively restricted to papilla locations during tongue development. In E13 cultures, exogenous BMPs or noggin induce increased numbers of fungiform papillae, in a concentration-dependent manner, compared to standard tongue cultures; BMPs, but not noggin, lead to a decreased tongue size at this stage. In E14 cultures, however, exogenous BMP2, 4 or 7 each inhibits papilla formation so that there is a decrease in papilla number. Noggin substantially increases number of papillae in E14 cultures. Using beads for a highly localized protein delivery, papillae are inhibited in the surround of BMP-soaked beads and induced in large clusters around noggin-soaked beads. Follistatin, presented in culture medium or by bead, does not alter papilla formation or number. In all fungiform papillae that form under various culture conditions, the molecular marker, sonic hedgehog, is within each papilla. However, the BMP inhibitory effect on papillae is not prevented by disrupting sonic hedgehog signaling through addition of cyclopamine to cultures. BMPs and noggin alter cell proliferation in tongue epithelium in opposite ways, demonstrated with Ki67 immunostaining. We propose that the BMPs and noggin, colocalized within papilla placodes and the fungiform papillae per se, have opposing inhibitory and activating or inducing roles in papilla development in linear patterns. We present a model for these effects.     

Enhanced Osteogenesis of Adipose Derived Stem Cells with Noggin Suppression and Delivery of BMP-2

Bone morphogenetic proteins (BMPs) are believed to be the most potent osteoinductive factors. However, BMPs are highly pleiotropic molecules and their supra-physiological high dose requirement leads to adverse side effects and inefficient bone formation. Thus, there is a need to develop alternative osteoinductive growth factor strategies that can effectively complement BMP activity. In this study, we intrinsically stimulated BMP signaling in adipose derived stem cells (ASCs) by downregulating noggin, a potent BMP antagonist, using an RNAi strategy. ASCs transduced with noggin shRNA significantly enhanced osteogenic differentiation of cells. The potency of endogenous BMPs was subsequently enhanced by stimulating ASCs with exogenous BMPs at a significantly reduced dose. The level of mineralization in noggin shRNA treated ASCs when treated with BMP-2 was comparable to that of control shRNA treated cell treated with 10-fold more BMP-2. The complementary strategy of noggin suppression + BMP-2 to enhance osteogenesis was further confirmed in 3D in vitro environments using scaffolds consisting of chitosan (CH), chondroitin sulfate (CS), and apatite layer on their surfaces designed to slowly release BMP-2. This finding supports the novel therapeutic potential of this complementary strategy in bone regeneration.     

Noggin and chordin have distinct activities in promoting lineage commitment of mouse embryonic stem (ES) cells

To examine the role of secreted signaling molecules and neurogenic genes in early development, we have developed a culture system for the controlled differentiation of mouse embryonic stem (ES) cells. In the current investigation, two of the earliest identified BMP antagonists/neural-inducing factors, noggin and chordin, were expressed in pluripotent mouse ES cells. Neurons were present as early as 24 h following transfection of ES cells with a pCS2/noggin expression plasmid, with differentiation peaking at 72 h. With neuronal differentiation, stem cell marker genes were down-regulated and neural determination genes expressed. Coculture experiments and exposure to noggin-conditioned medium produced similar neuronal differentiation of control ES cells, while addition of BMP-4 to noggin expressants strikingly inhibited neuronal differentiation. Transfection of ES cells with a pCS2/chordin expression vector or exposure to chordin-conditioned medium produced a more complex pattern of differentiation; ES cells formed neurons, mesenchymal cells as well as N-CAM-positive, nestin-positive neuroepithelial progenitors. These data suggest that, consistent with their different expression fields, noggin and chordin may play distinct roles in patterning the early mouse embryo.     

Dynamics of BMP signaling in limb bud mesenchyme and polydactyly

Mutations in the Bone Morphogenetic Protein (BMP) pathway are associated with a range of defects in skeletal formation. Genetic analysis of BMP signaling requirements is complicated by the presence of three partially redundant BMPs that are required for multiple stages of limb development. We generated an inducible allele of a BMP inhibitor, Gremlin, which reduces BMP signaling. We show that BMPs act in a dose and time dependent manner in which early reduction of BMPs result in digit loss, while inhibiting overall BMP signaling between E10.5 and E11.5 allows polydactylous digit formation. During this period, inhibiting BMPs extends the duration of FGF signaling. Sox9 is initially expressed in normal digit ray domains but at reduced levels that correlate with the reduction in BMP signaling. The persistence of elevated FGF signaling likely promotes cell proliferation and survival, inhibiting the activation of Sox9 and secondarily, inhibiting the differentiation of Sox9-expressing chondrocytes. Our results provide new insights into the timing and clarify the mechanisms underlying BMP signaling during digit morphogenesis.     

Expression pattern of the homeotic gene Bapx1 during early chick gastrointestinal tract development

Regulation of the Bone Morphogenetic Protein (BMP) signaling pathway is essential for the normal development of vertebrate gastrointestinal (GI) tract, but also for the differentiation of the digestive mesenchymal layer into smooth muscles and submucosal layer. Different studies demonstrated that Bapx1 (for bagpipe homeobox homolog 1) negatively regulates the BMP pathway, but its precise expression pattern during the development and the differentiation of the GI tract mesenchyme actually remains to be examined. Here, we present the spatio-temporal expression profile of Bapx1 in the chick GI tract. We show that Bapx1 is first expressed in the undifferentiated mesenchyme of the gizzard and the colon. After the differentiation of the digestive mesenchyme, we found Bapx1 strongly expressed in the gizzard smooth muscle and in the submucosa layer of the colon. This expression pattern provides new insights into the roles of Bapx1 during the regionalization of the GI tract and the differentiation of the digestive mesenchyme of the colon and the stomach.     

Expression pattern of the homeotic gene Bapx1 during early chick gastrointestinal tract development

Regulation of the Bone Morphogenetic Protein (BMP) signaling pathway is essential for the normal development of vertebrate gastrointestinal (GI) tract, but also for the differentiation of the digestive mesenchymal layer into smooth muscles and submucosal layer. Different studies demonstrated that Bapx1 (for bagpipe homeobox homolog 1) negatively regulates the BMP pathway, but its precise expression pattern during the development and the differentiation of the GI tract mesenchyme actually remains to be examined. Here, we present the spatio-temporal expression profile of Bapx1 in the chick GI tract. We show that Bapx1 is first expressed in the undifferentiated mesenchyme of the gizzard and the colon. After the differentiation of the digestive mesenchyme, we found Bapx1 strongly expressed in the gizzard smooth muscle and in the submucosa layer of the colon. This expression pattern provides new insights into the roles of Bapx1 during the regionalization of the GI tract and the differentiation of the digestive mesenchyme of the colon and the stomach.     

BMP2, BMP4, and their receptors are expressed in the differentiating muscle tissues of mouse embryonic tongue

To investigate the role of bone morphogenetic proteins (BMPs) in the differentiation process of skeletal muscle, we analyzed the in vivo expression of BMP2 and BMP4, of BMP receptors (BMPR) IA, IB, and II, and of activin receptors (ActR) IA, II, and IIB in mouse tongue muscle between embryonic day 11 (E11) and E17. The mRNA expression levels for BMP2 were 5-fold to 11-fold greater than those for BMP4 between E13 and E17 (P < 0.05-0.01). Expression of the BMP2, BMPRIB, ActRIA, ActRII, and ActRIIB proteins was first observed at E13. Expression of BMP2 and BMPRIB was detected in the whole area of the differentiating muscle tissues identified by immunostaining for fast myosin heavy chain (fMHC), but that of ActRIA, ActRII, and ActRIIB was detected only in the peripheral area of the differentiating muscle tissues. In the E15 tongue, all of the BMPs, BMPRs, and ActRs studied herein were expressed in the whole area of the differentiating muscle tissues identified by immunostaining for fMHC. These results suggest that BMPs play a role in the differentiation of tongue muscle tissues at E15 but have little or no effect at E13. This study was supported in part by grants-in-aid for funding scientific research (no. 16591871 to A.Y.) from the Bio-ventures and High-Technology Research Center of the Ministry of Education, Culture, Sports, Science, and Technology of Japan and by the Science Research Promotion Fund from the Promotion and Mutual Aid Corporation for Private Schools of Japan.     

Significance of bone morphogenetic protein-4 function in the initial myofibrillogenesis of chick cardiogenesis

The heart is the first organ to form and function during vertebrate embryogenesis. Using a secreted protein, noggin, which specifically antagonizes bone morphogenetic protein (BMP)-2 and -4, we examined the role played by BMP during the initial myofibrillogenesis in chick cultured precardiac mesoendoderm (mesoderm + endoderm; ME). Conditioned medium from COS7 cells transfected with Xenopus noggin cDNA inhibited the expression of sarcomeric proteins (such as sarcomeric alpha-actinin, Z-line titin, and sarcomeric myosin), and so myofibrillogenesis was perturbed in cultured stage 4 precardiac ME; however, it did not inhibit the expression of smooth muscle alpha-actin (the first isoform of alpha-actin expressed during cardiogenesis). In cultured stage 5 precardiac ME, noggin did not inhibit either the formation of I-Z-I components or the expression of sarcomeric myosin, but it did inhibit the formation of A-bands. Although BMP4 was required to induce expressions of sarcomeric alpha-actinin, titin, and sarcomeric myosin in cultured stage 6 posterolateral mesoderm (noncardiogenic mesoderm), smooth muscle alpha-actin was expressed without the addition of BMP4. Interestingly, in cultured stage 6 posterolateral mesoderm, BMP2 induced the expressions of sarcomeric alpha-actinin and titin, but not of sarcomeric myosin. These results suggest that (1) BMP4 function lies upstream of the initial formation of I-Z-I components and A-bands separately in a stage-dependent manner, and (2) at least two signaling pathways are involved in the initial cardiac myofibrillogenesis: one is an unknown pathway responsible for the expression of smooth muscle alpha-actin; the other is BMP signaling, which is involved in the expression of sarcomeric alpha-actinin, titin, and sarcomeric myosin.     

Essential role of BMPs in FGF-induced secondary lens fiber differentiation

It is widely accepted that vitreous humor-derived FGFs are required for the differentiation of anterior lens epithelial cells into crystallin-rich fibers. We show that BMP2, 4, and 7 can induce the expression of markers of fiber differentiation in primary lens cell cultures to an extent equivalent to FGF or medium conditioned by intact vitreous bodies (VBCM). Abolishing BMP2/4/7 signaling with noggin inhibited VBCM from upregulating fiber marker expression. Remarkably, noggin and anti-BMP antibodies also prevented purified FGF (but not unrelated stimuli) from upregulating the same fiber-specific proteins. This effect is attributable to inhibition of BMPs produced by the lens cells themselves. Although BMP signaling is required for FGF to enhance fiber differentiation, the converse is not true. Expression of noggin in the lenses of transgenic mice resulted in a postnatal block of epithelial-to-secondary fiber differentiation, with extension of the epithelial monolayer to the posterior pole of the organ. These results reveal the central importance of BMP in secondary fiber formation and show that although FGF may be necessary for this process, it is not sufficient. Differentiation of fiber cells, and thus proper vision, is dependent on cross-talk between the FGF and BMP signaling pathways.     

Noggin expression in a mesodermal pluripotent cell line C1 and its regulation by BMP

Osteoblasts and chondrocytes are derived from mesodermal stem cells and their differentiation is under the control of coordinated interaction among signaling molecules. Noggin is one of the signaling molecules which bind to and inactivate BMPs to induce neural tissues and dorsal mesoderm in Xenopus. However, its expression and regulation in mammalian cells has not been known. In this study, we investigated expression of noggin in murine pluripotent mesodermal cell line, C1. Noggin expression was very low in these C1 cells before they were induced to differentiate. When C1 cells were induced to differentiate into chondrocytes in aggregate cultures in the presence of dexamethasone(dex), noggin expression was significantly increased. In a sharp contrast, when the C1 cells were induced to differentiate into osteoblastic cells by the treatment with beta glycerophosphate (betaGP) and ascorbic acid (AA), noggin mRNA expression remained to be barely detectable. Noggin expression was also observed in the developing cartilage of vertebrae in 15.5 dpc mouse embryos. The noggin mRNA level in C1 cells in monolayer cultures was enhanced significantly by the treatment with BMP4/7 in a dose-dependent manner with a maximal effect at 100 ng/ml. The BMP4/7 effect on noggin expression was time dependent starting within 12 h and peaked at 24 h. These results indicate that noggin is expressed in the pluripotent mesodermal cell line C1 and that its expression is regulated by BMP.     

Community effect triggers terminal differentiation of myogenic cells derived from muscle satellite cells by quenching Smad signaling

A high concentration of bone morphogenetic proteins (BMPs) stimulates myogenic progenitor cells to undergo heterotopic osteogenic differentiation. However, the physiological role of the Smad signaling pathway during terminal muscle differentiation has not been resolved. We report here that Smad1/5/8 was phosphorylated and activated in undifferentiated growing mouse myogenic progenitor Ric10 cells without exposure to any exogenous BMPs. The amount of phosphorylated Smad1/5/8 was severely reduced during precocious myogenic differentiation under the high cell density culture condition even in growth medium supplemented with a high concentration of serum. Inhibition of the Smad signaling pathway by dorsomorphin, an inhibitor of Smad activation, or noggin, a specific antagonist of BMP, induced precocious terminal differentiation of myogenic progenitor cells in a cell density-dependent fashion even in growth medium. In addition, Smad1/5/8 was transiently activated in proliferating myogenic progenitor cells during muscle regeneration in rats. The present results indicate that the Smad signaling pathway is involved in a critical switch between growth and differentiation of myogenic progenitor cells both in vitro and in vivo. Furthermore, precocious cell density-dependent myogenic differentiation suggests that a community effect triggers the terminal muscle differentiation of myogenic cells by quenching the Smad signaling.     

Bone morphogenetic protein signaling and the initiation of lens fiber cell differentiation

Previous studies showed that the retina produces factors that promote the differentiation of lens fiber cells, and identified members of the fibroblast growth factor (FGF) and insulin-like growth factor (IGF) families as potential fiber cell differentiation factors. A possible role for the bone morphogenetic proteins (BMPs) is suggested by the presence of BMP receptors in chicken embryo lenses. We have now observed that phosphorylated SMAD1, an indicator of signaling through BMP receptors, localizes to the nuclei of elongating lens fiber cells. Transduction of chicken embryo retinas and/or lenses with constructs expressing noggin, a secreted protein that binds BMPs and prevents their interactions with their receptors, delayed lens fiber cell elongation and increased cell death in the lens epithelium. In an in vitro explant system, in which chicken embryo or adult bovine vitreous humor stimulates chicken embryo lens epithelial cells to elongate into fiber-like cells, these effects were inhibited by noggin-containing conditioned medium, or by recombinant noggin. BMP2, 4, or 7 were able to reverse the inhibition caused by noggin. Lens cell elongation in epithelial explants was stimulated by treatment with FGF1 or FGF2, alone or in combination with BMP2, but not to the same extent as vitreous humor. These data indicate that BMPs participate in the differentiation of lens fiber cells, along with at least one additional, and still unknown factor.     

Myo/Nog cell regulation of bone morphogenetic protein signaling in the blastocyst is essential for normal morphogenesis and striated muscle lineage specification

Cells that express MyoD mRNA, the G8 antigen and the bone morphogenetic protein (BMP) inhibitor noggin (Nog) are present in the epiblast before gastrulation. Ablation of “Myo/Nog” cells in the blastocyst results in an expansion of canonical BMP signaling and prevents the expression of noggin and follistatin before and after the onset of gastrulation. Once eliminated in the epiblast, they are neither replaced nor compensated for as development progresses. Older embryos lacking Myo/Nog cells exhibit severe axial malformations. Although Wnts and Sonic hedgehog are expressed in ablated embryos, skeletal muscle progenitors expressing Pax3 are missing in the somites. Pax3+ cells do emerge adjacent to Wnt3a+ cells in vitro; however, few undergo skeletal myogenesis. Ablation of Myo/Nog cells also results in ectopically placed cardiac progenitors and cardiomyocytes in the somites. Reintroduction of Myo/Nog cells into the epiblast of ablated embryos restores normal patterns of BMP signaling, morphogenesis and skeletal myogenesis, and inhibits the expression of cardiac markers in the somites. This study demonstrates that Myo/Nog cells are essential regulators of BMP signaling in the early epiblast and are indispensable for normal morphogenesis and striated muscle lineage specification.     

Basic FGF and suppression of BMP signaling sustain undifferentiated proliferation of human ES cells

Human embryonic stem cells (hESCs) are routinely cultured on fibroblast feeder layers or in fibroblast-conditioned medium (CM). Bone morphogenetic proteins (BMPs) have previously been shown to induce hESC differentiation, in apparent contrast to mouse embryonic stem (ES) cells, in which BMP4 synergizes with leukemia inhibitory factor (LIF) to maintain self-renewal. Here we demonstrate that hESCs cultured in unconditioned medium (UM) are subjected to high levels of BMP signaling activity, which is reduced in CM. The BMP antagonist noggin synergizes with basic fibroblast growth factor (bFGF) to repress BMP signaling and sustain undifferentiated proliferation of hESCs in the absence of fibroblasts or CM. These findings suggest a basic difference in the self-renewal mechanism between mouse and human ES cells and simplify the culture of hESCs.