In vivo evidence that BMP signaling is necessary for apoptosis in the mouse limb

To determine the role of Bone morphogenetic protein (BMP) signaling in murine limb development in vivo, the keratin 14 promoter was used to drive expression of the BMP antagonist Noggin in transgenic mice. Phosphorylation and nuclear translocation of Smad1/5 were dramatically reduced in limbs of the transgenic animals, confirming the inhibition of BMP signaling. These mice developed extensive limb soft tissue syndactyly and postaxial polydactyly. Apoptosis in the developing limb necrotic zones was reduced with incomplete regression of the interdigital tissue. The postaxial extra digit is also consistent with a role for BMPs in regulating apoptosis. Furthermore, there was persistent expression of Fgf8, suggesting a delay in the regression of the AER. However, Msx1 and Msx2 expression was unchanged in these transgenic mice, implying that induction of these genes is not essential for mediating BMP-induced interdigital apoptosis in mice. These abnormalities were rescued by coexpressing BMP4 under the same promoter in double transgenic mice, suggesting that the limb abnormalities are a direct effect of inhibiting BMP signaling.     

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.     

A constitutively active form of neurokinin 1 receptor and neurokinin 1 receptor-mediated apoptosis in glioblastomas

Previous studies have shown that neurokinin 1 receptor (NK1R) occurs naturally in human glioblastomas and its stimulation causes cell proliferation. In the present study we show that stimulation of NK1R in human U373 glioblastoma cells by substance P increases Akt phosphorylation by 2.5-fold, with an EC₅₀ of 57 nM. Blockade of NK1R lowers basal phosphorylation of Akt, indicating the presence of a constitutively active form of NK1R; similar results are seen in U251 MG and DBTRG-05 glioblastoma cells. Linkage of NK1R to Akt implicates NK1R in apoptosis of glioblastoma cells. Indeed, treatment of serum-starved U373 cells with substance P reduces apoptosis by 53 ± 1% ( p  <   0.05), and treatment with NK1R antagonist L-733,060 increases apoptosis by 64 ± 16% ( p  <   0.01). Further, the blockade of NK1R in human glioblastoma cells with L-733,060 causes cleavage of Caspase-3 and proteolysis of poly (ADP-ribose) polymerase. Experiments designed to elucidate the mechanism of NK1R-mediated Akt phosphorylation revealed total involvement of non-receptor tyrosine kinase Src and phosphatidyl-3-kinase, a partial involvement of epidermal growth factor receptor, and no involvement of mitogen-activated protein/extracellular signal-related kinase. Taken together, the results of the present study indicate a key role for NK1R in glioblastoma apoptosis.     

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.     

Specific induction of cranial placode cells from Xenopus ectoderm by modulating the levels of BMP,Wnt, and FGF signaling

The neural–epidermal boundary tissues include the neural crest and preplacodal ectoderm (PPE) as primordial constituents. The PPE region is essential for the development of various sensory and endocrine organs, such as the anterior lobe of the pituitary, olfactory epithelium, lens, trigeminal ganglion, and otic vesicles. During gastrulation, a neural region is induced in ectodermal cells that interacts with mesendodermal tissue and responds to several secreted factors. Among them, inhibition of bone morphogenetic protein (BMP) in the presumptive neuroectoderm is essential for the induction of neural regions, and formation of a Wnt and fibroblast growth factor (FGF) signaling gradient along the midline determines anterior–posterior patterning. In this study, we attempted to specifically induce PPE cells from undifferentiated Xenopus cells by regulating BMP, Wnt, and FGF signaling. We showed that the proper level of BMP inhibition with an injection of truncated BMP receptor or treatment with a chemical antagonist triggered the expression of PPE genes. In addition, by varying the amount of injected chordin, we optimized specific expression of the PPE genes. PPE gene expression is increased by adding an appropriate dose of an FGF receptor antagonist. Furthermore, co‐injection with either wnt8 or the Wnt inhibitor dkk‐1 altered the expression levels of several region‐specific genes according to the injected dose. We specifically induced PPE cell differentiation in animal cap cells from early‐stage Xenopus embryos by modulating BMP, Wnt, and FGF signaling. This is not the first research on placode induction, but our simple method could potentially be applied to mammalian stem cell systems. genesis 53:652–659, 2015. © 2015 Wiley Periodicals, Inc.     

HyBMP5-8b, a BMP5-8 orthologue, acts during axial patterning and tentacle formation in hydra

Developmental gradients play a central role in axial patterning in hydra. As part of the effort towards elucidating the molecular basis of these gradients as well as investigating the evolution of the mechanisms underlying axial patterning, genes encoding signaling molecules are under investigation. We report the isolation and characterization of HyBMP5-8b, a BMP5-8 orthologue, from hydra. Processes governing axial patterning are continuously active in adult hydra. Expression patterns of HyBMP5-8b in normal animals and during bud formation, hydra's asexual form of reproduction, were examined. These patterns, coupled with changes in patterns of expression in manipulated tissues during head regeneration, foot regeneration as well as under conditions that alter the positional value gradient indicate that the gene is active in two different processes. The gene plays a role in tentacle formation and in patterning the lower end of the body axis.     

Opposing Nodal/Vg1 and BMP signals mediate axial patterning in embryos of the basal chordate amphioxus

The basal chordate amphioxus resembles vertebrates in having a dorsal, hollow nerve cord, a notochord and somites. However, it lacks extensive gene duplications, and its embryos are small and gastrulate by simple invagination. Here we demonstrate that Nodal/Vg1 signaling acts from early cleavage through the gastrula stage to specify and maintain dorsal/anterior development while, starting at the early gastrula stage, BMP signaling promotes ventral/posterior identity. Knockdown and gain-of-function experiments show that these pathways act in opposition to one another. Signaling by these pathways is modulated by dorsally and/or anteriorly expressed genes including Chordin, Cerberus, and Blimp1. Overexpression and/or reporter assays in Xenopus demonstrate that the functions of these proteins are conserved between amphioxus and vertebrates. Thus, a fundamental genetic mechanism for axial patterning involving opposing Nodal and BMP signaling is present in amphioxus and probably also in the common ancestor of amphioxus and vertebrates or even earlier in deuterostome evolution.     

Regulation of cardiomyocyte apoptosis in ischemic reperfused mouse heart by glutathione peroxidase

Apoptosis, a genetically controlled programmed cell death, has been found to play a role in ischemic reperfusion injury in several animal species including rats and rabbits. To examine whether this is also true for other animals, an isolated perfused mouse heart was subjected to 30 min of ischemia followed by 2 h of reperfusion. Experiments were terminated before ischemia (baseline), after ischemia, and at 30, 60, 90 and 120 min of reperfusion. At the end of each experiment, hearts were processed for the evaluation of apoptosis and DNA laddering. The in situ end labeling (ISEL) technique was used to detect apoptotic cardiomyocyte nuclei while DNA laddering was evaluated by subjecting the DNA obtained from the cardiomyocytes to 1.8% agarose gel electrophoresis followed by photographing under UV illumination. The results of our study revealed that apoptotic cells appear only after 60 min of reperfusion as demonstrated by the intense fluorescence of the immunostained genomic DNA when observed under fluorescence microscopy. None of the ischemic hearts showed any evidence of apoptosis. These results were corroborated with the findings of DNA fragmentation showing increased ladders of DNA bands in the same reperfused hearts representing integer multiples of the internucleosomal DNA length (about 180 bp). Since our previous studies showed a role of glutathione peroxidase (GSHPx) in apoptotic cell death, we performed identical experiments using isolated hearts from GSHPx-l knockout mice and transgenic mice overexpressing GSHPx-l. GSHPx-l knockout mice showed evidence of apoptotic cell death even after 30 min of reperfusion. Significant number of apoptotic cells were found in the cardiomyocytes as compared to non-transgenic control animals. To the contrary, very few apoptotic cells were found in the hearts of the transgenic mice overexpressing GSHPx-l. Hearts of GSHPx-l knockout mice were more susceptible to ischemia/reperfusion injury while transgenic mice overexpressing GSHPx- 1 were less susceptible to ischemia reperfusion injury compared to non-transgenic control animals. The results of this study clearly demonstrate a role of GSHPx in ischemia/reperfusion-induced apoptosis in mouse heart.     

Matrix GLA protein is a developmental regulator of chondrocyte mineralization and, when constitutively expressed, blocks endochondral and intramembranous ossification in the limb

Matrix GLA protein (MGP), a gamma-carboxyglutamic acid (GLA)-rich, vitamin K-dependent and apatite-binding protein, is a regulator of hypertrophic cartilage mineralization during development. However, MGP is produced by both hypertrophic and immature chondrocytes, suggesting that MGP's role in mineralization is cell stage-dependent, and that MGP may have other roles in immature cells. It is also unclear whether MGP regulates the quantity of mineral or mineral nature and quality as well. To address these issues, we determined the effects of manipulations of MGP synthesis and expression in (a) immature and hypertrophic chondrocyte cultures and (b) the chick limb bud in vivo. The two chondrocyte cultures displayed comparable levels of MGP gene expression. Yet, treatment with warfarin, a gamma-carboxylase inhibitor and vitamin K antagonist, triggered mineralization in hypertrophic but not immature cultures. Warfarin effects on mineralization were highly selective, were accompanied by no appreciable changes in MGP expression, alkaline phosphatase activity, or cell number, and were counteracted by vitamin K cotreatment. Scanning electron microscopy, x-ray microanalysis, and Fourier-transform infrared spectroscopy revealed that mineral forming in control and warfarin-treated hypertrophic cell cultures was similar and represented stoichiometric apatite. Virally driven MGP overexpression in cultured chondrocytes greatly decreased mineralization. Surprisingly, MGP overexpression in the developing limb not only inhibited cartilage mineralization, but also delayed chondrocyte maturation and blocked endochondral ossification and formation of a diaphyseal intramembranous bone collar. The results show that MGP is a powerful but developmentally regulated inhibitor of cartilage mineralization, controls mineral quantity but not type, and appears to have a previously unsuspected role in regulating chondrocyte maturation and ossification processes.     

Activation of retinoblastoma protein in mammary gland leads to ductal growth suppression, precocious differentiation, and adenocarcinoma.

The retinoblastoma (Rb) tumor suppressor controls cellular proliferation, survival, and differentiation and is functionally inactivated by mutations or hyperphosphorylation in most human cancers. Although activation of endogenous Rb is thought to provide an effective approach to suppress cell proliferation, long-term inhibition of apoptosis by active Rb may have detrimental consequences in vivo. To directly test these paradigms, we targeted phosphorylation-resistant constitutively active Rb alleles, Rb Delta Ks, to the mouse mammary gland. Pubescent transgenic females displayed reduced ductal elongation and cell proliferation at the endbuds. Post-puberty transgenic mice exhibited precocious cellular differentiation and beta-casein expression and extended survival of the mammary epithelium with a moderate but specific effect on the expression of E2F1, IGF1R alpha, and phospho-protein kinase B/AKT. Remarkably, approximately 30% Rb Delta K transgenic females developed focal hyperplastic nodules, and approximately 7% exhibited full-blown mammary adenocarcinomas within 15 mo. Expression of the Rb Delta K transgene in these mammary tumors was reduced greatly. Our results suggest that transient activation of Rb induces cancer by extending cell survival and that the dual effects of Rb on cell proliferation and apoptosis impose an inherent caveat to the use of the Rb pathway for long-term cancer therapy.     

Twist plays an essential role in FGF and SHH signal transduction during mouse limb development

Loss of Twist gene function arrests the growth of the limb bud shortly after its formation. In the Twist(-/-) forelimb bud, Fgf10 expression is reduced, Fgf4 is not expressed, and the domain of Fgf8 and Fgfr2 expression is altered. This is accompanied by disruption of the expression of genes (Shh, Gli1, Gli2, Gli3, and Ptch) associated with SHH signalling in the limb bud mesenchyme, the down-regulation of Bmp4 in the apical ectoderm, the absence of Alx3, Alx4, Pax1, and Pax3 activity in the mesenchyme, and a reduced potency of the limb bud tissues to differentiate into osteogenic and myogenic tissues. Development of the hindlimb buds in Twist(-/-) embryos is also retarded. The overall activity of genes involved in SHH signalling is reduced.Fgf4 and Fgf8 expression is lost or reduced in the apical ectoderm, but other genes (Fgf10, Fgfr2) involved with FGF signalling are expressed in normal patterns. Twist(+/-);Gli3(+/XtJ) mice display more severe polydactyly than that seen in either Twist(+/-) or Gli3(+/XtJ) mice, suggesting that there is genetic interaction between Twist and Gli3 activity. Twist activity is therefore essential for the growth and differentiation of the limb bud tissues as well as regulation of tissue patterning via the modulation of SHH and FGF signal transduction.     

Dual requirement of ectodermal Smad4 during AER formation and termination of feedback signaling in mouse limb buds

BMP signaling is pivotal for normal limb bud development in vertebrate embryos and genetic analysis of receptors and ligands in the mouse revealed their requirement in both mesenchymal and ectodermal limb bud compartments. In this study, we genetically assessed the potential essential functions of SMAD4, a mediator of canonical BMP/TGFß signal transduction, in the mouse limb bud ectoderm. Msx2‐Cre was used to conditionally inactivate Smad4 in the ectoderm of fore‐ and hindlimb buds. In hindlimb buds, the Smad4 inactivation disrupts the establishment and signaling by the apical ectodermal ridge (AER) from early limb bud stages onwards, which results in severe hypoplasia and/or aplasia of zeugo‐ and autopodal skeletal elements. In contrast, the developmentally later inactivation of Smad4 in forelimb buds does not alter AER formation and signaling, but prolongs epithelial‐mesenchymal feedback signaling in advanced limb buds. The late termination of SHH and AER‐FGF signaling delays distal progression of digit ray formation and inhibits interdigit apoptosis. In summary, our genetic analysis reveals the temporally and functionally distinct dual requirement of ectodermal Smad4 during initiation and termination of AER signaling. genesis 51:660–666. © 2013 Wiley Periodicals, Inc.     

Dpp and Gbb exhibit different effective ranges in the establishment of the BMP activity gradient critical for Drosophila wing patterning

Morphogen gradients ensure the specification of different cell fates by dividing initially unpatterned cellular fields into distinct domains of gene expression. It is becoming clear that such gradients are not always simple concentration gradients of a single morphogen; however, the underlying mechanism of generating an activity gradient is poorly understood. Our data indicate that the relative contributions of two BMP ligands, Gbb and Dpp, to patterning the wing imaginal disc along its A/P axis, change as a function of distance from the ligand source. Gbb acts over a long distance to establish BMP target gene boundaries and a variety of cell fates throughout the wing disc, while Dpp functions at a shorter range. On its own, Dpp is not sufficient to mediate the low-threshold responses at the end points of the activity gradient, a function that Gbb fulfills. Given that both ligands signal through the Tkv type I receptor to activate the same downstream effector, Mad, the difference in their effective ranges must reflect an inherent difference in the ligands themselves, influencing how they interact with other molecules. The existence of related ligands with different functional ranges may represent a conserved mechanism used in different species to generate robust long range activity gradients.     

Over-expression of fibroblast growth factor-2 causes defective bone mineralization and osteopenia in transgenic mice

Over-expression of human FGF-2 cDNA linked to the phosphoglycerate kinase promoter in transgenic (TgFGF2) mice resulted in a dwarf mouse with premature closure of the growth plate and shortening of bone length. This study was designed to further characterize bone structure and remodeling in these mice. Bones of 1-6 month-old wild (NTg) and TgFGF2 mice were studied. FGF-2 protein levels were higher in bones of TgFGF2 mice. Bone mineral density was significantly decreased as early as 1 month in femurs from TgFGF2 mice compared with NTg mice. Micro-CT of trabecular bone of the distal femurs from 6-month-old TgFGF2 mice revealed significant reduction in trabecular bone volume, trabecular number (Tb.N), and increased trabecular separation (Tb.Sp). Osteoblast surface/bone surface, double-labeled surface, mineral apposition rate, and bone formation rates were all significantly reduced in TgFGF2 mice. There were fewer TRAP positive osteoclasts in calvaria from TgFGF2 mice. Quantitative histomorphometry showed that total bone area was similar in both genotypes, however percent osteoclast surface, and osteoclast number/bone surface were significantly reduced in TgFGF2 mice. Increased replication of TgFGF2 calvarial osteoblasts was observed and primary cultures of bone marrow stromal cells from TgFGF2 expressed markers of mature osteoblasts but formed fewer mineralized nodules. The data presented indicate that non-targeted over-expression of FGF-2 protein resulted in decreased endochondral and intramembranous bone formation. These results are consistent with FGF-2 functioning as a negative regulator of postnatal bone growth and remodeling in this animal model.     

基因"慢"的克隆及其在小鼠组织中的表达

Formin蛋白家族由结构相关的蛋白组成,它们都有两个formin同源功能区（formin homology domains）,FH1和FH2。这些基因上的多种变异均导致动物四肢畸形,提示这些基因在肢体发育中起重要作用。我们自小鼠肢体基因库中分离出了一个新基因：“慢”,该基因含有FH1和FH2。我们检测了该基因在小鼠胚胎及成体的表达情况,并对可能的功能意义进行了讨论。     

Effects of cytosine arabinoside on in vivo and in vitro mouse limb development

Summary When pregnant mice were exposed to 40 mg per kg of cytosine arabinoside (ara-C) on days 10 to 12 of gestation, adactylous limbs with large, distally located blisters were found when the fetuses were examined on day 18. Embryonic limbs exposed transplacentally under identical conditions and explanted to culture exhibited the same morphological abnormality as did limbs exposed directly in culture to 0.1 to 1 μg per ml of ara-C. Two noncytotoxic analogues of ara-C, uridine arabinoside (ara-U) and hypoxanthine arabinoside (ara-HX), had no influence on morphological differentiation of limbs in vitro. Ara-C alone caused a dose-related decrease in uptake of³H-thymidine and³⁵SO₄ in cultured limb buds. Production of this morphologically distinct malformation in vitro will allow detailed biochemical investigations on the effect of ara-C limb ectodermal-mesenchymal interactions. Supported by NIH Postdoctoral Fellowship No. CM02660. Supported by NIEHS Predoctoral Fellowship No. ES00127. Supported by NIEHS Toxicology Training Grant No. ES00127.