VAMP2 Marks Quiescent Satellite Cells and Myotubes,but not Activated Myoblasts

We examined the expression and intracellular localization of vesicle-associated membrane protein 2 (VAMP2) during the differentiation of skeletal muscle cells by immunofluorescence microscopy. In isolated single myofibers, VAMP2 was expressed in quiescent satellite cells, downregulated in proliferating myoblastic cells, and re-expressed with differentiation. In the myoblastic cell line C2C12, VAMP2 was expressed at a low level in the proliferating stage, and then increased after differentiation into myotubes. Based on these results, we propose that VAMP2 can be used as a molecular marker for both quiescent satellite cells and myotubes, but not for proliferating myoblasts. We also found the partial colocalization of VAMP2 with transferrin- or Rab11-labeled vesicles in myotubes, suggesting a role of VAMP2 in the trafficking of recycling endosomes.     

Contrasting roles for MyoD in organizing myogenic promoter structures during embryonic skeletal muscle development

Background : Among the complexities of skeletal muscle differentiation is a temporal distinction in the onset of expression of different lineage‐specific genes. The lineage‐determining factor MyoD is bound to myogenic genes at the onset of differentiation whether gene activation is immediate or delayed. How temporal regulation of differentiation‐specific genes is established remains unclear. Results: Using embryonic tissue, we addressed the molecular differences in the organization of the myogenin and muscle creatine kinase (MCK) gene promoters by examining regulatory factor binding as a function of both time and spatial organization during somitogenesis. At the myogenin promoter, binding of the homeodomain factor Pbx1 coincided with H3 hyperacetylation and was followed by binding of co‐activators that modulate chromatin structure. MyoD and myogenin binding occurred subsequently, demonstrating that Pbx1 facilitates chromatin remodeling and modification before myogenic regulatory factor binding. At the same time, the MCK promoter was bound by HDAC2 and MyoD, and activating histone marks were largely absent. The association of HDAC2 and MyoD was confirmed by co‐immunoprecipitation, proximity ligation assay (PLA), and sequential ChIP. Conclusions: MyoD differentially promotes activated and repressed chromatin structures at myogenic genes early after the onset of skeletal muscle differentiation in the developing mouse embryo. Developmental Dynamics 244:43–55, 2015. © 2014 Wiley Periodicals, Inc.     

Distribution of synaptobrevin/VAMP 1 and 2 in rat brain

The synaptobrevin/vesicle-associated membrane protein (VAMP) family of proteins, which are essential for neurotransmitter release, are the vesicle donor soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor (SNARE) proteins first described in synaptic vesicles at nerve terminals.

Two synaptobrevin/VAMP isoforms are involved in calcium-dependent synaptic vesicle exocytosis, synaptobrevin/VAMP 1 and synaptobrevin/VAMP 2. However, the functional significance of these two highly homologous isoforms remains to be elucidated.

Here, we used immunohistochemical, immunofluorescence and confocal microscope techniques to localize the two synaptobrevin/VAMP isoforms in rat brain areas, particularly in nerve terminals. Our results show that the two isoforms are present in the rat central nervous system and that their expression overlaps in some areas. However, a distinct distribution pattern was detected. Synaptobrevin/VAMP 2 is the most abundant isoform in the rat brain and is widely distributed. Although synaptobrevin/VAMP 1 is less abundant, it is the main isoform in particular brain areas (e.g. zona incerta at the subthalamus or nerve terminals surrounding thalamic neurons). The colocalization of synaptophysin with synaptobrevin/VAMP 1 demonstrates the presence of this isoform in subsets of nerve terminals. These results indicate that each synaptic vesicle donor SNARE protein isoform could have a specialized role in the neurosecretory process.     

Loss of VAMP5 in mice results in duplication of the ureter and insufficient expansion of the lung

Background : Vesicle‐associated membrane protein 5 (VAMP5) is a member of the SNARE protein family, which regulates the docking and fusion of membrane vesicles within cells. Previously, we reported ubiquitous expression of VAMP5 proteins in various organs except the brain and small intestine. However, the precise roles of VAMP5 in each organ remain unclear. To explore the roles of VAMP5 in vivo, we generated VAMP5 knockout (KO) mice. Results : VAMP5 KO mice showed low birth rate and low body weight. KO embryos grew normally in the uterus, and tended to die around birth. Anatomical analysis revealed that viable KO mice often exhibited duplication of the ureter, and dead KO mice showed insufficient expansion of the lung. VAMP5 was localized in the epithelial cells of the ureter and terminal bronchiole. Conclusions : VAMP5 KO mice showed a low birth rate and abnormalities of the urinary and respiratory systems. VAMP5 KO mice died around birth, possibly due to defects in vesicoureteral flow and breathing. The results presented could provide a basis for future studies to understand the roles of VAMP5 protein. Developmental Dynamics 247:754–762, 2018. © 2018 Wiley Periodicals, Inc.     

Pax3/Pax7 mark a novel population of primitive myogenic cells during development

Skeletal muscle serves as a paradigm for the acquisition of cell fate, yet the relationship between primitive cell populations and emerging myoblasts has remained elusive. We identify a novel population of resident Pax3+/Pax7+, muscle marker-negative cells throughout development. Using mouse mutants that uncouple myogenic progression, we show that these Pax+ cells give rise to muscle progenitors. In the absence of skeletal muscle, they apoptose after down-regulation of Pax7. Furthermore, they mark the emergence of satellite cells during fetal development, and do not require Pax3 function. These findings identify critical cell populations during lineage restriction, and provide a framework for defining myogenic cell states for therapeutic studies.     

Cadherin-11 is highly expressed in rhabdomyosarcomas and during differentiation of myoblasts in vitro

Rhabdomyosarcomas bear a morphological and genetic resemblance to developing skeletal muscle. Apart from myogenic marker genes (bHLH factors, myosin, actin), cell adhesion molecules such as N-cadherin and N-CAM have been reported to be expressed both in rhabdomyosarcomas and during myogenesis. The present study demonstrates the expression of another cadherin, cadherin-11, in rhabdomyosarcomas and during differentiation of myoblasts in vitro: cadherin-11, a predominantly mesenchymal cell adhesion molecule, is highly expressed in embryonal rhabdomyosarcomas and alveolar rhabdomyosarcomas, which do not bear the Pax-3–FKHR fusion previously described. Cadherin-11 is down-regulated in normal skeletal muscle and after myotube formation in vitro. The results of this study suggest that cadherin-11 might be involved in myogenesis and that rhabdomyosarcomas may re-express or fail to down-regulate cadherin-11. Since alveolar rhabdomyosarcomas bearing the t(2;13) translocation do not express cadherin-11, it is postulated that Pax-3 and cadherin-11 might be linked and involved in the same myogenic pathway. Copyright © 1999 John Wiley & Sons, Ltd.     

VAMP2 interacts directly with the N terminus of Kv2.1 to enhance channel inactivation

Recently, we demonstrated that the Kv2.1 channel plays a role in regulated exocytosis of dense-core vesicles (DCVs) through direct interaction of its C terminus with syntaxin 1A, a plasma membrane soluble NSF attachment receptor (SNARE) component. We report here that Kv2.1 interacts with VAMP2, the vesicular SNARE partner that is also present at high concentration in neuronal plasma membrane. This is the first report of VAMP2 interaction with an ion channel. The interaction was demonstrated in brain membranes and characterized using electrophysiological and biochemical analyses in Xenopus oocytes combined with an in vitro binding analysis and protein modeling. Comparative study performed with wild-type and mutant Kv2.1, wild-type Kv1.5, and chimeric Kv1.5N/Kv2.1 channels revealed that VAMP2 enhanced the inactivation of Kv2.1, but not of Kv1.5, via direct interaction with the T1 domain of the N terminus of Kv2.1. Given the proposed role for surface VAMP2 in the regulation of the vesicle cycle and the important role for the sustained Kv2.1 current in the regulation of dendritic calcium entry during high-frequency stimulation, the interaction of VAMP2 with Kv2.1 N terminus may contribute, alongside with the interaction of syntaxin with Kv2.1 C terminus, to the activity dependence of DCV release.     

Raf kinase inhibitor protein1 is a myogenic inhibitor with conserved function in avians and mammals

Background: Raf Kinase Inhibitor Protein1 (RKIP) is a tumor suppressor that is present in several adult tissues. It functions as an inhibitor of both Raf/Mek/Erk and NF?B signaling when unphosphorylated, but following phosphorylation the ability to inhibit Raf/Mek/Erk signaling is lost and RKIP becomes an activator of G‐protein coupled receptor signaling. In neonates and adults, RKIP is known to be expressed in muscle; however, its physiological function is currently unknown. Results: In this study, we show by in situ hybridization and immunofluorescence that RKIP is also expressed in developing chick embryonic muscle, and mouse C2C12 myoblasts. Furthermore, we demonstrate that, in these systems, it functions as an inhibitor of myogenesis: increased levels of RKIP suppress myotube differentiation whereas decreasing RKIP promotes differentiation. Additionally, we show that the ability of RKIP to inhibit myogenesis is dependent upon its phosphorylation state as only the nonphosphorylated form of RKIP suppresses myogenesis. Conclusions: This study, therefore, clearly demonstrates that RKIP has conserved functions as a myogenic inhibitor in both mammalian and avian muscle. Developmental Dynamics 245:902–912, 2016. © 2016 Wiley Periodicals, Inc.     

Paraxis is required for somite morphogenesis and differentiation in Xenopus laevis

Background : In most vertebrates, the segmentation of the paraxial mesoderm involves the formation of metameric units called somites through a mesenchymal‐epithelial transition. However, this process is different in Xenopus laevis because it does not form an epithelial somite. Xenopus somitogenesis is characterized by a complex cells rearrangement that requires the coordinated regulation of cell shape, adhesion, and motility. The molecular mechanisms that control these cell behaviors underlying somite formation are little known. Although the Paraxis has been implicated in the epithelialization of somite in chick and mouse, its role in Xenopus somite morphogenesis has not been determined. Results : Using a morpholino and hormone‐inducible construction approaches, we showed that both gain and loss of function of paraxis affect somite elongation, rotation and alignment, causing a severe disorganization of somitic tissue. We further found that depletion or overexpression of paraxis in the somite led to the downregulation or upregulation, respectively, of cell adhesion expression markers. Finally, we demonstrated that paraxis is necessary for the proper expression of myotomal and sclerotomal differentiation markers. Conclusions : Our results demonstrate that paraxis regulates the cell rearrangements that take place during the somitogenesis of Xenopus by regulating cell adhesion. Furthermore, paraxis is also required for somite differentiation. Developmental Dynamics 244:973–987, 2015. © 2015 Wiley Periodicals, Inc.     

MyoD and Myogenin expression during myogenic phases in brown trout: a precocious onset of mosaic hyperplasia is a prerequisite for fast somatic growth.

Muscle cell recruitment (hyperplasia) during myogenesis in the vertebrate embryo is known to occur in three consecutive phases. In teleost fish (including zebrafish), however, information on myogenic precursor cell activation is largely fragmentary, and comprehensive characterization of the myogenic phases has only been fully undertaken in a single slow-growing cyprinid species by examination of MEF2D expression. Here, we use molecular techniques to provide a comprehensive characterization of MyoD and Myogenin expression during myogenic cell activation in embryos and larvae of brown trout, a fast-growing salmonid with exceptionally large embryos. Results confirm the three-phase pattern, but also demonstrate that the second and third phases begin simultaneously and progress vigorously, which is different from the previously described consecutive activation of these phases. Furthermore, we suggest that Pax7 is expressed in myogenic progenitor cells that account for second- and third-phase myogenesis. These findings are discussed in relation to teleost myotome development and to teleost growth strategies.     

Pax3 regulation of FGF signaling affects the progression of embryonic progenitor cells into the myogenic program

Pax3/7-dependent stem cells play an essential role in skeletal muscle development. We now show that Fgfr4 lies genetically downstream from Pax3 and is a direct target. In chromatin immunoprecipitation (ChIP)-on-chip experiments, Pax3 binds to a sequence 3' of the Fgfr4 gene that directs Pax3-dependent expression at sites of myogenesis in transgenic mouse embryos. The activity of this regulatory element is also partially dependent on E-boxes, targets of the myogenic regulatory factors, which are expressed as progenitor cells enter the myogenic program. Other FGF signaling components, notably Sprouty1, are also regulated by Pax3. In vivo manipulation of Sprouty expression reveals that FGF signaling affects the balance between Pax-positive progenitor cells and committed myoblasts. These results provide new insight into the Pax-initiated regulatory network that modulates stem cell maintenance versus tissue differentiation.     

Involvement of Wnt4 signaling during myogenic proliferation and differentiation of skeletal muscle.

The direct effects of Wnt4 on myogenic proliferation and differentiation of skeletal muscle precursors are examined. Wnt4 cDNA was misexpressed in the presumptive limb fields on the right side of stage 16 chick embryos. Muscle development was evaluated at stage 37 with hematoxylin-eosin staining and immunohistochemical staining for fast and slow types of the myosin heavy chain (MyHC). Overexpression of Wnt4 resulted in up-regulation of Pax7 and MyoD1 expression. The muscle mass showed a significant increase compared with that of the control limb. The area for fast MyHC-expressing cells showed a significant increase, whereas a slight decrease was observed for slow MyHC-expressing cells. Wnt4 acted as a stimulator during myogenic proliferation and differentiation, especially, for fast-type muscle in C2C12 cells. The present results are identical to those of myostatin knockout, suggesting that Wnt4 is acting against myostatin as an antagonizing signal for myostatin.     

Neural cell adhesion molecule is expressed by smooth muscle cells during the development of the rat vascular system

Summary The presence and distribution of neural cell adhesion molecule (N-CAM) was examined by light and electron microscopical immunocytochemistry in the descending thoracic aorta, the superior mesenteric artery and mesenteric arteries from fetal and adult rats (embryonic day 15 to post-natal day 90). In embryonic and early post-natal rats, N-CAM immunoreactivity was localized in perivascular nerves, in the smooth muscle cell plasma membrane and basal lamina. In nerves, N-CAM-immunoreactive sites were found associated with both the axon and Schwann cell membranes. N-CAM immunoreactivity was also found associated with the surface of adventitial fibroblast-like cells and with collagen fibrils, in regions where these fibrils were in contact with smooth muscle cells. In mature vessels N-CAM immunoreactivity was found to be restricted to the perivascular innervation and the surface of fibroblast-like cells. These observations indicate that N-CAM is expressed transiently in rat vascular tissues during development and is localized not only on the surface of smooth muscle cells but also in association with extracellular matrix components.     

Expression of myogenic regulatory factors in chicken embryos during somite and limb development

The expression of the myogenic regulatory factors (MRFs), Myf5, MyoD, myogenin (Mgn) and MRF4 have been analysed during the development of chicken embryo somites and limbs. In somites, Myf5 is expressed first in somites and paraxial mesoderm at HH stage 9 followed by MyoD at HH stage 12, and Mgn and MRF4 at HH stage 14. In older somites, Myf5 and MyoD are also expressed in the ventrally extending myotome prior to Mgn and MRF4 expression. In limb muscles a similar temporal sequence is observed with Myf5 expression detected first in forelimbs at HH stage 22, MyoD at HH stage 23, Mgn at HH stage 24 and MRF4 at HH stage 30. This report describes the precise time of onset of expression of each MRF in somites and limbs during chicken embryo development, and provides a detailed comparative timeline of MRF expression in different embryonic muscle groups.     

Myotonic dystrophy protein kinase is expressed in embryonic myocytes and is required for myotube formation

Myotonic dystrophy (DM1) is a multi-systemic disease caused by a triplet nucleotide repeat expansion in the 3' untranslated region of the gene coding for myotonic dystrophy protein kinase (DMPK). The primary pathophysiology of DM1 is thought to result from RNA transport and processing defects. The function of DMPK in development or any potential role in DM1 remains unknown. Here we report a novel role for DMPK in myogenesis. We have discovered a specific expression pattern of DMPK in mouse and chick embryonic development. DMPK is expressed in postmitotic cardiac and skeletal myocytes and developmental signaling centers. During cardiac myocyte maturation, DMPK migrates from perinuclear to cellular membrane localization. Manipulating DMPK levels in cultured cardiac and skeletal myocytes has revealed a key role for DMPK in myocyte differentiation. Overexpression of DMPK induces cell rounding and apoptosis in myocytes. In addition, DMPK is necessary for myogenin expression in differentiating C2C12 myoblasts.     

平行结构支架材料对大鼠成肌细胞α-肌动蛋白的影响

目的：根据大鼠成肌细胞骨骼肌α-肌动蛋白mRNA的表达变化，初步探讨借助支架材料的有序排列构建具有理想方向性组织工程化骨骼肌的可行性。方法：将医用可吸收缝合线平行折叠并制备成类圆柱体状，与大鼠成肌细胞L6体外复合培养。以18s RNA为内参照，运用半定量RT-PCR检测大鼠成肌细胞骨骼肌α-肌动蛋白mRNA在体外培养早期的变化。结果：随着体外培养时间的延长，大鼠骨骼肌α-肌动蛋白的mRNA表达水平逐渐升高，并于体外培养第4d，维持在相对稳定的水平。结论：大鼠成肌细胞与具有平行结构支架材料体外复合培养早期，骨骼肌α-肌动蛋白的基因表达与骨骼肌再生有一定程度的吻合。     

Why is cyclooxygenase-2 expressed in neuroendocrine cells of the human alimentary tract?

During immunohistochemical studies of cyclooxygenase-2 (COX-2) on human coion cancers, many strong COX-2-immunoreactive cells scattered in the bottom of the crypt of normal coion mucosa were unexpectedly noticed. COX-2-immunoreactive cells were morphologically very similar to neuroendocrine cells and were confirmed immunohistochemically to be positive for chromogranin A or serotonin. Moreover, COX-2-immunoreactive neuroendocrine cells were observed not only in the large intestine, but also in the stomach, duodenum and small intestine. The role of COX-2 expression in neuroendocrine cells of the human alimentary tract was speculated.