Fgf-4 expression during gastrulation, myogenesis, limb and tooth development in the mouse.

Fgf-4, initially isolated as a transforming gene from human tumors, is a member of the Fibroblast Growth Factor (FGF) family. It has previously been shown by northern blot hybridization analysis to be expressed in teratocarcinoma and embryonic stem cells, suggesting that it plays a role in embryonic development. We have carried out an RNA in situ hybridization analysis of Fgf-4 expression in the developing mouse embryo, from fertilization through the 14th day of gestation (E14.5). Our results show that Fgf-4 RNA is first detected at the late blastocyst stage in cells that give rise to all of the embryonic lineages (inner cell mass cells). During the early stages of gastrulation, expression becomes restricted to the primitive streak where mesoderm and definitive endoderm are formed. Expression continues in the distal (rostral) two-thirds of the streak through approx. E10, and then is detected in the tail bud, which replaces the streak as the primary source of mesoderm. Additional sites of expression are found after the three primary germ layers are established and organogenesis begins. Fgf-4 RNA is detected transiently in the branchial arch units, the somitic myotome, the apical ectodermal ridge of the developing limb bud and the tooth bud, suggesting that the gene has multiple roles during embryogenesis. These results are compared with the expression patterns of other FGF genes. Taken together, the data suggest that individual members of the gene family are expressed sequentially in developmental pathways such as mesoderm formation and myogenesis, and play a role in specific epithelial-mesenchymal interactions.     

Myostatin inhibits myoblast differentiation by down-regulating MyoD expression

Myostatin, a negative regulator of myogenesis, is shown to function by controlling the proliferation of myoblasts. In this study we show that myostatin is an inhibitor of myoblast differentiation and that this inhibition is mediated through Smad 3. In vitro, increasing concentrations of recombinant mature myostatin reversibly blocked the myogenic differentiation of myoblasts, cultured in low serum media. Western and Northern blot analysis indicated that addition of myostatin to the low serum culture media repressed the levels of MyoD, Myf5, myogenin, and p21 leading to the inhibition of myogenic differentiation. The transient transfection of C(2)C(12) myoblasts with MyoD expressing constructs did not rescue myostatin-inhibited myogenic differentiation. Myostatin signaling specifically induced Smad 3 phosphorylation and increased Smad 3.MyoD association, suggesting that Smad 3 may mediate the myostatin signal by interfering with MyoD activity and expression. Consistent with this, the expression of dominant-negative Smad3 rescued the activity of a MyoD promoter-reporter in C(2)C(12) myoblasts treated with myostatin. Taken together, these results suggest that myostatin inhibits MyoD activity and expression via Smad 3 resulting in the failure of the myoblasts to differentiate into myotubes. Thus we propose that myostatin plays a critical role in myogenic differentiation and that the muscular hyperplasia and hypertrophy seen in animals that lack functional myostatin is because of deregulated proliferation and differentiation of myoblasts.     

Ectopic expression of Fgf-4 in chimeric mouse embryos induces the expression of early markers of limb development in the lateral ridge

The biological consequences of constitutive fibroblast growth factor-4 (fgf-4) expression were investigated in chimeric embryos prepared between wild-type host embryos and murine ES cells transfected with a construct in which expression of the murine fgf-4 gene was directed by the phosphoglycerate kinase (PGK-1) promoter. The embryos exhibit abnormalities of the limbs and the anterior central nervous system (CNS). The limb phenotype comprised the induction of growth along the lateral ridge between the definitive fore and hind limbs resembling the early phases of limb development. The CNS defects comprised a complete absence, or marked reduction in forebrain and midbrain structures and rudimentary or absent eye development. Constitutive expression of fgf-4 was also accompanied by ectopic expression of the sonic hedgehog (shh) and msx-1 genes in the lateral ridge. These findings indicate that FGF exhibits multiple activities in early development which include the ability to induce the expression of early markers of limb development in the lateral ridge. © 1996 Wiley-Liss, Inc.     

Compartmentalization of the somite and myogenesis in chick embryos are influenced by wnt expression

Muscles of the body and bones of the axial skeleton derive from specialized regions of somites. Somite development is influenced by adjacent structures. In particular, the dorsal neural tube and the overlying ectoderm have been shown to be necessary for the induction of myogenic precursor cells in the dermomyotome. Members of the Wnt family of signaling molecules, which are expressed in the dorsal neural tube and the ectoderm, are postulated to be responsible for this process. It is shown here that ectopically implanted Wnt-1-, -3a-, and -4-expressing cells alter the process of somite compartmentalization in vivo. An enlarged dorsal compartment results from the implantation of Wnt-expressing cells ventrally between the neural tube/notochord and epithelial somites, at the expense of the ventral compartment, the sclerotome. Thus, ectopic Wnt expression is able to override the influence of ventralizing signals arising from notochord and floor plate. This shift of the border between the two compartments was identified by an increase in the domain of Pax-3 expression and a complete loss of Pax-1 expression in somites close to the ectopic Wnt signal. The expanded expression of MyoD and desmin provides evidence that it is the myotome which increases as a result of Wnt signaling. Paraxis expression is also drastically amplified after implantation of Wnt-expressing cells indicating that Wnts are involved in the formation and maintenance of somite epithelium and suggesting that Paraxis is activated through Wnt signaling pathways. Taken together these results suggest that ectopic Wnts disturb the normal balance of signaling molecules within the somite, resulting in an enhanced recruitment of somitic cells into the myogenic lineage.     

Clonal analysis of vertebrate myogenesis. I. Early developmental events in the chick limb

Early developmental events occurring in the prospective muscle tissue region of chick embryo leg buds have been subjected to an in vitro clonal analysis. Colony-forming cells are present at stage 20 (72 hr incubation), but none of the colonies exhibit morphological signs of muscle differentiation. After an additional 8 hr of incubation (stage 21), approximately 10% of the colony-forming cells have acquired the capacity to form multinucleated cells in vitro, and the percentage of clonable myoblasts increases to a level of approximately 60% during the next 3 days of incubation. Clonal analysis of myoblast populations within regions of the developing limb have indicated that, between stages 21 and 27, the dorsal and ventral segments of the myogenic region contain appreciably more clonable muscle cells than the anterior and posterior segments. In addition, during stages 21 and 22 there is a 3-fold difference in muscle-colony-forming cells between the proximal and distal halves of the dorsal-ventral segments, as well as between the proximal and distal halves of the anterior-posterior segments. Thus at least two temporal and regional gradients—proximal to distal and medial to lateral—of clonable myoblast content can be delineated within the developing chick limb. In addition to changes in the proportions of muscle-colony-forming cells, the extent of multinuclearity within individual muscle colonies increases with the developmental age of the embryo from which the clonable myoblasts are derived. The progressive changes in the relative proportions of muscle-colony-forming cells and in clonal morphology are discussed in terms of their possible cell lineage implications.     

Rapamycin inhibits the invasive ability of thyroid cancer cells by down-regulating the expression of VEGF-C in vitro

The aim of this study was to observe the effects of rapamycin on proliferation, apoptosis and invasion of SW579 in vitro. The proliferation and apoptosis of SW579 cells were detected by methyl thiazolyl tetrazolium and flow cytometry. Transwell assay was used to observe the changes of invasive ability of SW579 cells after being treated with rapamycin. The effects of rapamycin on the expression of mammalian target of rapamycin (mTOR) signalling and vascular endothelial growth factor C (VEGF-C) were observed by Western blot. The inhibition and apoptosis rates increased obviously when the concentration of rapamycin was 20?nm. When the rapamycin concentration was 10?nm, the invasive ability of SW579 cells changed significantly than when it was 5?nm. Our data showed that when the concentrations of rapamycin were over 20?nm, the expression of mTOR and p70S6K decreased significantly, and the expression of PTEN increased notably. There were no remarkable variations observed when we detected the expression of Akt. We found the expression of VEGF-C was high in SW579 cells and decreased slightly when the cells were treated with 5?nm rapamycin. When the concentration of rapamycin was over 5?nm, significant changes were observed. Rapamycin could inhibit the proliferation and induce the apoptosis of human thyroid cancer cells in vitro by mTOR inhibition. No obvious changes observed in the expression of AKT indicated that there might be a feedback loop effect by the mTOR inhibition induced by rapamycin. Rapamycin could inhibit the invasive ability of SW579 cells by down-regulating the expression of VEGF-C. Copyright ? 2012 John Wiley & Sons, Ltd.     

Myostatin通过Smad3下调MyoD的表达来抑制骨骼肌卫星细胞的分化

Myostatin基因,是肌肉生长的负调控因子,通过下调MyoD的表达抑制骨骼肌细胞的分化,但具体机制目前尚未完全清楚。本研究以体外培养的猪骨骼肌卫星细胞为实验材料,利用RNAi 技术,以Smad3为靶基因进行干扰研究,研究干扰前后猪骨骼肌卫星细胞增殖情况的变化以及MyoD、Myostatin基因的表达规律,进一步阐述三个基因间的调控关系。结果表明,Myostatin通过下调MyoD的表达,抑制骨骼肌卫星细胞的分化,但这种抑制作用是受Smad3调节的。     

Oroxylin A inhibits ATRA-induced IL-6 expression involved in retinoic acid syndrome by down-regulating CHOP

Production of IL-6 constituted the major cause of death in the ATRA trial called retinoic acid syndrome (RAS). LAP and LIP are active and inactive isoforms of C/EBPβ, respectively. Inactive LIP dimerized with LAP to eliminate its activity. Following treatment with ATRA, CHOP expression was increased and dimerized with LIP more preferentially than LAP to rescue function of LAP. Oroxylin A has been reported to activate CHOP, a key mediator of unfolded protein response (UPR) pathway, and resulted in apoptosis. Interestingly, we found that low concentration of oroxylin A (≦ 40 μM) showed no apoptosis effect on NB4 and HL-60 cells and decreased the CHOP protein level via promoting its degradation. MG132 was utilized to conform the effect of oroxylin A on degrading CHOP. Our results showed that oroxylin A decreased the level of IL-6 secretion of NB4 cells with or without ATRA treatment while the effect was eliminated by C/EBPβ siRNA. We conclude that oroxylin A possessed abilities of inhibiting the ATRA-induced IL-6 production via modulation of LAP/LIP/CHOP in leukemia cell lines, which could providing a therapeutic strategy for RAS.     

Development alters the expression of calcium currents in chick limb motoneurons

Calcium current types expressed in vertebrate spinal motoneurons have not been previously resolved. We have resolved three types in chick limb motoneurons identified by retrograde labeling and report that dramatic changes in their expression take place during development in vivo. T-, N-, and L-type calcium currents were distinguished on the basis of kinetics, voltage dependence, and unique pharmacological sensitivities. Developmental changes were characterized by studying motoneurons isolated from embryos at three stages spanning neuromuscular system development. T currents were dominant at the earliest stage. Motoneurons from embryos 2 days older showed much reduced T currents and much increased N and L currents. We suggest that mature motoneurons will be dominated by N- and L-type calcium currents and that T current may serve developmental roles.     

CRP2 transcript expression pattern in embryonic chick limb

We are using the model of the developing mouse embryo to elucidate the pattern of arginase expression in mammalian cells in normal animals and in arginase I (AI) deficiency during development by digoxigenin-labeled RNA in situ hybridization. Our goal is to understand the regulation of these isozymes, with the expectation that this knowledge will help patients suffering from AI deficiency. We found that AI mRNA was widely and strongly expressed in the normal developing mouse embryo; in contrast, a relatively strong AII mRNA signal was found only in the intestine. In the AI knockout mouse embryo, no AII overexpression was found. These results indicated that arginases are needed in mouse embryonic development and AI is the principal form required. The strong AI expression in the peripheral nervous system suggests that the pathogenesis of the neurological retardation in AI deficiency may be conditioned by AI deficiency in the nervous system during embryonic development.     

Blockade of L-type voltage-gated Ca channel inhibits ischemia-induced neurogenesis by down-regulating iNOS expression in adult mouse

Neurogenesis in the adult mammalian hippocampus may contribute to repairing the brain after injury. The signals that regulate neurogenesis in the dentate gyrus following ischemic stroke insult are not well known. We have previously reported that inducible nitric oxide synthase (iNOS) expression is necessary for ischemia-stimulated neurogenesis in the adult dentate gyrus. Here, we show that mice subjected to 90 min of middle cerebral artery occlusion (MCAO) significantly increased the number of new neurons and up-regulated iNOS expression in the dentate gyrus. Blockade of the L-type voltage-gated Ca(2+) channel (L-VGCC) prevented neurogenesis in the dentate gyrus and subventricular zone (SVZ), and down-regulated iNOS expression in the dentate gyrus after cerebral ischemia. This study suggests that Ca(2+) influx through L-VGCC is involved in ischemia-induced neurogenesis by up-regulating iNOS expression.     

Proteoglycan synthesis by primary chick skeletal muscle during in vitro myogenesis

The proteoglycans synthesized by primary chick skeletal muscle during in vitro myogenesis were compared with those of muscle-specific fibroblasts. Cultures of skeletal muscle cells and muscle fibroblasts were separately labeled using [35S] sulfate as a precursor. The proteoglycans of the cell layer and medium were separately extracted and isolated by ion-exchange chromatography on DEAE-Sephacel followed by gel filtration chromatography on Sepharose CL-2B. Two cell layer-associated proteoglycans synthesized both by skeletal muscle cells and muscle fibroblasts were identified. The first, a high molecular weight proteoglycan, eluted from Sepharose CL-2B with a Kav of 0.07 and contained exclusively chondroitin sulfate chains with an average molecular weight greater than 50,000. The second, a relatively smaller proteoglycan, eluted from Sepharose CL-2B with a Kav of 0.61 and contained primarily heparan sulfate chains with an average molecular weight of 16,000. Two labeled proteoglycans were also found in the medium of both skeletal muscle and muscle fibroblasts. A high molecular weight proteoglycan was found with virtually identical properties to that of the high molecular weight chondroitin sulfate proteoglycan of the cell layer. A second, smaller proteoglycan had a similar monomer size (Kav of 0.63) to the cell layer heparan sulfate proteoglycan, but differed from it in that this molecule contained primarily chondroitin sulfate chains with an average molecular weight of 32,000. Studies on the distribution of these proteoglycans in muscle cells during in vitro myogenesis demonstrated that a parallel increase in the relative amounts of the smaller proteoglycans occurred in both the cell layer and medium compared to the large chondroitin sulfate proteoglycan in each compartment. In contrast, muscle-derived fibroblasts displayed a constant ratio of the small proteoglycans of the cell layer and medium fractions, compared to the larger chondroitin sulfate proteoglycan of the respective fraction as a function of cell density. Our results support the concept that proteoglycan synthesis is under developmental regulation during skeletal myogenesis.