Secreted protein Noggin4 participates in the formation of forebrain structures in <Emphasis Type="Italic">Xenopus laevis</Emphasis> by inhibiting the Wnt/beta-catenin signaling pathway

Noggin proteins are important regulators of the early development of the vertebrate neural system. Previously, it has been traditionally thought that vertebrates have only one noggin gene (Noggin1), whose main function is the inhibition of BMP signaling pathway during the formation of dorsoventral polarity in embryos. Then other proteins of this family were discovered, and the studies of Noggin2 protein showed that noggin proteins also participate in the modulation of Nodal/Activin and Wnt/beta-catenin signaling pathways in the early development of amphibian head structures. The purpose of this study is to investigate the properties of another noggin protein, Noggin4. We proved that Noggin4 plays an important role in the formation of head structure in clawed frog, since it inhibits the activity of Wnt/beta-catenin signaling pathway. At the same time, unlike Noggin1 and Noggin2, Noggin4 does not inhibit the activity of TGF-beta signaling pathways (BMP and Nodal/Activin).     

Noggin Expression in the Adult Retina Suggests a Conserved Role during Vertebrate Evolution

Vertebrates share common mechanisms in the control of development and in the maintenance of neural and retinal function. The secreted factor Noggin, a BMP inhibitor, plays a crucial role in neural induction during embryonic development. Moreover, we have shown its involvement in retinal differentiation of pluripotent cells. Here we show Noggin expression in the adult retina in three vertebrate species. Four Noggin genes are present in zebrafish (Danio rerio; ZbNog1, 2, 3, 5), three in frog (Xenopus laevis; XenNog1, 2 and 4), and one in mouse (Mus musculus; mNog). Quantitative RT-PCR experiments show the presence of ZbNog3 and ZbNog5 mRNAs, but not ZbNog1 and ZbNog2, in the adult zebrafish retina. All three genes are expressed in the frog retina, and mNog in the mouse. Immunohistochemistry data show that Noggin proteins are predominantly localized in the Golgi apparatus of photoreceptors and in the fibers of the outer plexiform layer. Lower expression levels are also found in inner plexiform layer fibers, in ganglion cells, in the ciliary marginal zone, and in retinal pigmented epithelium. Our results show that Noggin has a specific cellular and sub-cellular expression in the adult vertebrate retina, which is conserved during evolution. In addition to its established role during embryonic development, we postulate that Noggin also exerts a functional role in the adult retina.     

The secreted protein Noggin4 is an activator of the Wnt/PCP-signaling pathway

The protein Noggin4 of the African clawed frog Xenopus laevis has been shown to act as a modulator of the “noncanonical” Wnt/PCP-signaling pathway that plays an important role in the regulation of cell motility. Induction of disturbances in the expression of Noggin4 led to the activation of Wnt/PCP-pathway and the related anomalies of early embryonic development. The Noggin4 protein can bind the Wnt11 protein that normally contributes to the activation of the Wnt/PCP-pathway and of enhancing the activator effect of this protein in luciferase assays. Thus, Noggin4 can be used as a tool for specific experimental regulation of the activity of the Wnt/PCP pathway.     

Use of the luciferase reporter constructs for investigation of the capacity of Noggin2 protein to inhibit cell signaling pathways in Xenopus laevis embryos

Noggin (Noggin1) protein inhibits Smad1-dependent TGF-?? (BMP) signaling cascades by extracellular binding of BMP proteins. Recently, we identified two previously unknown members of the Noggin family, Noggin2 and Noggin4 proteins. In this work, using luciferase reporter constructs, it was shown that Noggin2 is able to inhibit in addition to the BMP-signaling cascade, the Activin/Nodal and Wnt signaling pathways in Xenopus laevis early embryos. The inhibition efficiency of Noggin2 is comparable to well-known extracellular inhibitors Cerberus and Follistatin. In addition to revealing novel properties of Noggin2, this work demonstrates that luciferase reporter constructs are a convenient tool for studying the regulation of molecular signaling cascades in the model of Xenopus laevis embryos.     

Evidence for a role of Smad6 in chick cardiac development.

Bone morphogenetic proteins (BMPs), members of the transforming growth factor-beta (TGF-beta) superfamily, are obligatory growth factors for early embryogenesis and heart formation. SMAD proteins transduce signals of the TGF-beta superfamily. We isolated chicken Smad6 (cSmad6), a member of inhibitory SMADs, and found its expression to be remarkably restricted to the developing heart, eyes, and limbs. cSmad6 expression was detected in the cardiogenic region of stage 5 embryos and overlapped Nkx2-5 and bmp-2, -4, and -7 expression. Throughout development, cSmad6 was expressed strongly in the heart, primarily in the myocardium, endocardium, and endocardial cushion tissue. Myocardial expression of cSmad6 was stronger in the forming septum, where highly localized expression of bmp-2 and -4 was also observed. Ectopically applied BMP-2 protein induced the expression of cSmad6, a putative negative regulator of BMP-signaling pathway, in anterior medial mesoendoderm of stage 4-5 embryos. In addition, blocking of BMP signaling using Noggin downregulated cSmad6 in cardiogenic tissue. cSmad1, one of the positive mediators of BMP signaling, was also expressed in cardiogenic region, but was not BMP-2 inducible. Our data suggest that cSmad6 has a role in orchestrating BMP-mediated cardiac development. We propose the possible mechanism of action of cSmad6 as modulating BMP signal by keeping a balance between constitutively expressed pathway-specific cSmad1 and ligand-induced inhibitory cSmad6 in the developing heart.     

Modulation of BMP signaling by Noggin is required for the maintenance of palatal epithelial integrity during palatogenesis

BMP signaling plays many important roles during organ development, including palatogenesis. Loss of BMP signaling leads to cleft palate formation. During development, BMP activities are finely tuned by a number of modulators at the extracellular and intracellular levels. Among the extracellular BMP antagonists is Noggin, which preferentialy binds to BMP2, BMP4 and BMP7, all of which are expressed in the developing palatal shelves. Here we use targeted Noggin mutant mice as a model for gain of BMP signaling function to investigate the role of BMP signaling in palate development. We find prominent Noggin expression in the palatal epithelium along the anterior-posterior axis during early palate development. Loss of Noggin function leads to overactive BMP signaling, particularly in the palatal epithelium. This results in disregulation of cell proliferation, excessive cell death, and changes in gene expression, leading to formation of complete palatal cleft. The excessive cell death in the epithelium disrupts the palatal epithelium integrity, which in turn leads to an abnormal palate-mandible fusion and prevents palatal shelf elevation. This phenotype is recapitulated by ectopic expression of a constitutively active form of BMPR-IA but not BMPR-IB in the epithelium of the developing palate; this suggests a role for BMPR-IA in mediating overactive BMP signaling in the absence of Noggin. Together with the evidence that overexpression of Noggin in the palatal epithelium does not cause a cleft palate defect, we conclude from our results that Noggin mediated modulation of BMP signaling is essential for palatal epithelium integrity and for normal palate development.     

Identification of genes associated with regenerative success of <Emphasis Type="Italic">Xenopus laevis</Emphasis>hindlimbs

Background  

Epimorphic regeneration is the process by which complete regeneration of a complex structure such as a limb occurs through production of a proliferating blastema. This type of regeneration is rare among vertebrates but does occur in the African clawed frog Xenopus laevis, traditionally a model organism for the study of early development. Xenopus tadpoles can regenerate their tails, limb buds and the lens of the eye, although the ability of the latter two organs to regenerate diminishes with advancing developmental stage. Using a heat shock inducible transgene that remains silent unless activated, we have established a stable line of transgenic Xenopus (strain N1) in which the BMP inhibitor Noggin can be over-expressed at any time during development. Activation of this transgene blocks regeneration of the tail and limb of Xenopus tadpoles.     

Vizualizing the morphogen adsorption gradient in the <Emphasis Type="Italic">Xenopus laevis</Emphasis> embryo using fluorescently labeled heparin-binding motif of BMP4 morphogen

Spatial distribution of heparan sulfates in the embryonic extracellular matrix at midgastrula stage has been demonstrated in the Xenopus laevis embryo model. Towards this end, fluorescently labeled fusion protein EGFP-hbmBMP4 made up by green fluorescent protein (EGFP) and heparin-binding motif of Bone Morphogenetic Protein 4 (BMP4) was produced in the E. coli expression system. Xenopus laevis embryos at midgastrula stage (stage 11) were fixed and cut along the anteroposterior axis and then incubated with EGFP-hbmBMP4. The fluorescently labeled samples were analyzed in fluorescence microscope. The spatial distribution of fluorescence intensity reflecting BMP4 adsorption on the embryonic extracellular matrix proved to be similar to the corresponding distribution pattern for the Noggin1 heparin-binding motif obtained previously. The highest intensity zone was detected around the dorsal blastopore lip; another high intensity zone, although slightly less prominent, was observed in the ventral blastopore lip region. Since on one hand, heparin-binding sites significantly differ in their organization in BMP4 and Noggin1 proteins and, on the other hand, spatial adsorption distribution patterns for these proteins coincide in the embryo, it appears that all secreted morphogens containing a heparin-binding site share a single adsorption gradient in the embryonic extracellular space.     

Effects of bone morphogenic protein 4 (BMP4) and its inhibitor,Noggin, on <Emphasis Type="Italic">in vitro</Emphasis>maturation and culture of bovine preimplantation embryos

Background  

BMP4 is a member of the transforming growth factor beta (TGFbeta) superfamily and Noggin is a potent BMP inhibitor that exerts its function by binding to BMPs preventing interactions with its receptors. The aim of this work was to investigate the role of BMP4 and Noggin, on oocytes in vitro maturation (m experiments) and embryos in vitro development (c experiments) of bovine.     

Noggin and BMP4 co-modulate adult hippocampal neurogenesis in the APPswe/PS1ΔE9 transgenic mouse model of Alzheimer’s disease

In addition to the subventricular zone, the dentate gyrus of the hippocampus is one of the few brain regions in which neurogenesis continues into adulthood. Perturbation of neurogenesis can alter hippocampal function, and previous studies have shown that neurogenesis is dysregulated in Alzheimer disease (AD) brain. Bone morphogenetic protein-4 (BMP4) and its antagonist Noggin have been shown to play important roles both in embryonic development and in the adult nervous system, and may regulate hippocampal neurogenesis. Previous data indicated that increased expression of BMP4 mRNA within the dentate gyrus might contribute to decreased hippocampal cell proliferation in the APP_swe/PS1_ΔE9 mouse AD model. However, it is not known whether the BMP antagonist Noggin contributes to the regulation of neurogenesis. We therefore studied the relative expression levels and localization of BMP4 and its antagonist Noggin in the dentate gyrus and whether these correlated with changes in neurogenesis in 6-12 mo old APP_swe/PS1_ΔE9 transgenic mice. Bromodeoxyuridine (BrdU) was used to label proliferative cells. We report that decreased neurogenesis in the APP/PS1 transgenic mice was accompanied by increased expression of BMP4 and decreased expression of Noggin at both the mRNA and protein levels; statistical analysis showed that the number of proliferative cells at different ages correlated positively with Noggin expression and negatively with BMP4 expression. Intraventricular administration of a chimeric Noggin/Fc protein was used to block the action of endogenous BMP4; this resulted in a significant increase in the number of BrdU-labeled cells in dentate gyrus subgranular zone and hilus in APP/PS1 mice. These results suggest that BMP4 and Noggin co-modulate neurogenesis.     

Oxidative stress promotes autophagic cell death in human neuroblastoma cells with ectopic transfer of mitochondrial PPP2R2B (Bβ2)

Background

Bone morphogenetic proteins (BMPs) are pleiotropic members of the TGF-beta superfamily which regulate many biological processes during development and adult tissue homeostasis and are implicated in the pathogenesis of a number of human diseases. Their involvement in both normal and aberrant physiology creates a need for rapid, sensitive and methodologically simple assays to evaluate their activity from a variety of biological samples. Previously alkaline phosphatase based assays, ELISA and luciferase based bioassays have been developed to evaluate either individual or total BMP activity. In this paper, we describe a highly sensitive, rapid and specific cell based assay for the simultaneous quantification of total and isoform specific BMP activity from biological samples.

Results

A C2C12 cell line stably transfected with a reporter plasmid consisting of the BMP response element (BRE) from the Id1 promoter fused to a luciferase reporter gene was generated. Exposure of this cell line to human recombinant BMP2, BMP4, BMP6, BMP7, BMP9 and BMP10 induced the expression of luciferase which was quantified using a luminometer. This assay was specific for BMP activity as the other TGF-β superfamily members TGF-β 1, Nodal and Mullerian Inhibiting Substance (MIS) did not induce the reporter. Pretreatment of samples with isoform specific BMP blocking antibodies coupled with isoform specific titration analysis allowed the simultaneous identification and quantification of BMP4, BMP6 and BMP9 in serum samples.

Conclusion

The assay is rapid (<48 hours) and can be used to simultaneously measure isoform specific and total BMP activity in complex solutions.     

BMP-signaling regulates the generation of hair-cells

Bone morphogenetic proteins (BMPs) are diffusible molecules involved in a variety of cellular interactions during development. Bmp4 expression accompanies the development of the ear sensory organs during patterning and specification of sensory cell fates, yet there is no understanding of the role of BMP4 in this process. The present work was aimed at exploring the effects of BMP-signaling on the development of hair-cells. For this purpose, we studied gene expression, cell proliferation and cell death in isolated chick otic vesicles that were grown in vitro in the presence of recombinant BMP4 or the BMP-inhibitor Noggin. Cath1 was used as a marker for hair-cell specification. BMP4 reduced the number of Cath1-cells and, conversely, Noggin increased the size of the sensory patches and the number of Cath1-positive cells. The effect of BMP4 was irreversible and occurred before hair-cell specification. Lfng and Fgf10 were expressed in the prosensory domain before Cath1, and their expression was expanded by Noggin. At these stages, modifications of BMP activity did not respecify non-sensory epithelium of the otic vesicle. The expression of Bmp4 at sensory patches was suppressed by BMP4 and induced by Noggin suggesting an autoregulatory loop. Analysis of BrdU incorporation during 6 and 18 h indicated that the effects of BMP4 were due to its ability to reduce the number of actively proliferating progenitors and inhibit cell fate specification. BMP4 induced cell death within the prosensory domain of the otic vesicle, along with the expression of Msx1, but not Msx2. On the contrary, BMP-inhibition with Noggin favored hair-cell specification without changes in the overall cell proliferation. We propose that about the stage of terminal division, the balance between BMP and BMP-inhibitory signals regulates survival and specification of hair-cell precursors, the final number of sensory hair-cells being limited by excess levels of BMPs. The final size of sensory patches would hence depend on the balance between BMP4 and opposing signals.     

Mitochondrial localization of Smad5 in a human chondrogenic cell line

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-beta (TGF-beta) superfamily and regulate the formation of cartilage and bone tissues as well as other key events during development. TGF-beta superfamily signaling is mediated intracellularly by Smad proteins, some of which can translocate into the cell nucleus and influence gene expression. Although much progress has been made in understanding how TGF-beta superfamily signaling regulates expression of target genes, little formal proof has been presented regarding the intracellular distribution of the Smad proteins before their entry into the nucleus. In the literature, non-nuclear Smad proteins are generally referred to as cytoplasmic. Using confocal microscopy, we here show for the first time that immunofluorescent labeling of Smad5, one of the Smad proteins associated with BMP signaling, colocalizes with the mitochondrion-specific probe MitoTracker, demonstrating a mitochondrial distribution of Smad5 in non-stimulated chondroprogenitor cells.     

BMP signalling permits population expansion by preventing premature myogenic differentiation in muscle satellite cells

Satellite cells are the resident stem cells of adult skeletal muscle, supplying myonuclei for homoeostasis, hypertrophy and repair. In this study, we have examined the role of bone morphogenetic protein (BMP) signalling in regulating satellite cell function. Activated satellite cells expressed BMP receptor type 1A (BMPR-1A/Alk-3) and contained phosphorylated Smad proteins, indicating that BMP signalling is operating during proliferation. Indeed, exogenous BMP4 stimulated satellite cell division and inhibited myogenic differentiation. Conversely, interfering with the interactions between BMPs and their receptors by the addition of either the BMP antagonist Noggin or soluble BMPR-1A fragments, induced precocious differentiation. Similarly, blockade of BMP signalling by siRNA-mediated knockdown of BMPR-1A, disruption of the intracellular pathway by either Smad5 or Smad4 knockdown or inhibition of Smad1/5/8 phosphorylation with Dorsomorphin, also caused premature myogenic differentiation. BMP signalling acted to inhibit the upregulation of genes associated with differentiation, in part, through regulating Id1. As satellite cells differentiated, Noggin levels increased to antagonise BMP signalling, since Noggin knockdown enhanced proliferation and impeded myoblast fusion into large multinucleated myotubes. Finally, interference of normal BMP signalling after muscle damage in vivo perturbed the regenerative process, and resulted in smaller regenerated myofibres. In conclusion, BMP signalling operates during routine satellite cell function to help coordinate the balance between proliferation and differentiation, before Noggin is activated to antagonise BMPs and facilitate terminal differentiation.     

Expression pattern of the expanded noggin gene family in the planarian Schmidtea mediterranea

Noggin genes are mainly known as inhibitors of the Bone Morphogenetic Protein (BMP) signalling pathway. Noggin genes play an important role in various developmental processes such as axis formation and neural differentiation. In vertebrates, inhibition of the BMP pathway is usually carried out together with other inhibitory molecules: chordin and follistatin. Recently, it has been shown in planarians that the BMP pathway has a conserved function in the maintenance and re-establishment of the dorsoventral axis during homeostasis and regeneration. In an attempt to further characterize the BMP pathway in this model we have undertaken an in silico search of noggin genes in the genome of Schmidtea mediterranea. In contrast to other systems in which between one and four noggin genes have been reported, ten genes containing a noggin domain are present in S. mediterranea. These genes have been classified into two groups: noggin genes (two genes) and noggin-like genes (eight genes). Noggin-like genes are characterized by the presence of an insertion of 50–60 amino acids in the middle of the noggin domain. Here, we report the characterization of this expanded family of noggin genes in planarians as well as their expression patterns in both intact and regenerating animals. In situ hybridizations show that planarian noggin genes are expressed in a variety of cell types located in different regions of the planarian body.     

BMP antagonism by Noggin is required in presumptive notochord cells for mammalian foregut morphogenesis

Esophageal atresia with tracheoesophageal fistula (EA/TEF) is a serious human birth defect, in which the esophagus ends before reaching the stomach, and is aberrantly connected with the trachea. Several mouse models of EA/TEF have recently demonstrated that proper dorsal/ventral (D/V) patterning of the primitive anterior foregut endoderm is essential for correct compartmentalization of the trachea and esophagus. Here we elucidate the pathogenic mechanisms underlying the EA/TEF that occurs in mice lacking the BMP antagonist Noggin, which display correct dorsal/ventral patterning. To clarify the mechanism of this malformation, we use spatiotemporal manipulation of Noggin and BMP receptor 1A conditional alleles during foregut development. Surprisingly, we find that the expression of Noggin in the compartmentalizing endoderm is not required to generate distinct tracheal and esophageal tubes. Instead, we show that Noggin and BMP signaling attenuation are required in the early notochord to correctly resolve notochord cells from the dorsal foregut endoderm, which in turn, appears to be a prerequisite for foregut compartmentalization. Collectively, our findings support an emerging model for a mechanism underlying EA/TEF in which impaired notochord resolution from the early endoderm causes the foregut to be hypo-cellular just prior to the critical period of compartmentalization. Our further characterizations suggest that Noggin may regulate a cell rearrangement process that involves reciprocal E-cadherin and Zeb1 expression in the resolving notochord cells.