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1.
Molluscan in vitro technology allows the study of the differentiation of isolated cells undergoing experimental manipulations. We have used the immunofluorescence technique and laser scanning microscopy to investigate the organization of muscle proteins (actin, myosin, paramyosin, and twitchin) and the localization of neurotransmitters (serotonin and FMRFamide) in cultured mussel larval cells. Differentiation into muscle and neuron-like cells occurs during the cultivation of mussel cells from premyogenic and prenervous larval stages. Muscle proteins are colocalized in contractile cells through all stages of cultivation. The cultivation of mussel cells on various substrates and the application of integrin receptor blockers suggest that an integrin-dependent mechanism is involved in cell adhesion and differentiation. Dissociated mussel cells aggregate and become self-organized in culture. After 20 days of cultivation, they form colonies in which serotonin- and FMRFamide-immunoreactive cells are located centrally, whereas muscle cells form a contractile network at the periphery. The pattern of thick and thin filaments in cultivated mussel cells changes according to the scenario of muscle arrangement in vivo: initially, a striated pattern of muscle filaments forms but is then replaced by a smooth muscle pattern with a diffuse distribution of muscle proteins, typical of muscles of adult molluscs. Myogenesis in molluscs thus seems to be a highly dynamic and potentially variable process. Such a “flexible” developmental program can be regarded as a prerequisite for the evolution of the wide variety of striated and smooth muscles in larval and adult molluscs.  相似文献   

2.
The development of contractile apparatus was subjected to comparative analysis during ontogenesis of the mussel Mytilus trossulus. Indirect immunofluorescence with the polyclonal antibody against mussel twitchin, a protein of thick filaments, and florescent phalloidin as a marker of filamentous cell actin were used to monitor changes in the developing muscle system at different larval stages. The first definitive muscle structures were found at the late trochophore stage (36 h after fertilization) and starting from the midveliger stage (96 h), striated muscles, which are never present in adult mussels, were distinctly seen. The striated muscle periodicity was 1.25 microm in both mussle larvae and adult scallop. The contractile activities of veliger and adult muscles were measured using an electronic signal-processing videosystem. This work is the first complex study of morphological, biochemical, and physiological characteristics of the muscle system in the larvae and adult mollusks.  相似文献   

3.
The development of contractile apparatus was subjected to comparative analysis during ontogenesis of the mussel Mytilus trossulus. Indirect immunofluorescence with the polyclonal antibody against mussel twitchin, a protein of thick filaments, and fluorescent phalloidin as a marker of filamentous cell actin were used to monitor changes in the developing muscle system at different larval stages. The first definitive muscle structures were found at the late trochophore stage (36 h after fertilization) and starting from the midveliger stage (96h), striated muscles, which are never present in adult mussels, were distinctly seen. The striated muscle periodicity was 1.25 μm in both mussel larvae and adult scallop. The contractile activities of veliger and adult muscles were measured using an electronic signal-processing video workstation. This work is the first complex study of morphological, biochemical, and physiological characteristics of the muscle system in the larvae and adult molluscs.  相似文献   

4.
The main finding of the present study is the discovery of the possibility of a morphofunctional myogenic differentiation of larval mussel cells in vitro. The shape and extensive cytoskeletal network of the cultured contracting cells mimic largely those of smooth muscle cells in vivo. However, the behavior and protein composition of these cells are not completely identical with those of smooth muscle cells. Contracting mussel cells in vitro, as well as differentiated smooth muscles, demonstrate both phasic and tonic contractions. The paramyosin to myosin ratio in the cultured mussel cells is far less than that in the muscles of veliger larvae and adult mussels. We have found the protein carpets with various adhesive characteristics determine different development pathways. Myogenic differentiation is only observed in spreading cells. Non-spreading adherent cells plated on collagen carpet show high synthetic activity but the commitment of contractile phenotype is inhibited. Our results confirm that the myogenic program established in early embryogenesis of molluscs can be realized during the cultivation of cells from premyogenic larval stages.  相似文献   

5.
The early pupal heart of the fruit fly Drosophila melanogaster has recently been the subject of intense physiological and molecular work, yet it has not been well described, nor has it been compared with the heart of the adult fly. In the work reported here, the hearts of adults and early pupae of D. melanogaster were studied by scanning and transmission electron microscopy and by light microscopy. The hearts of adults and early pupae both consist of a tube of circular striated muscle one cell in thickness. The alary muscles, which suspend the heart, are more delicate in the adult compared to the early pupa. The pericardial cells in both early pupae and adults are connected to the heart by connective tissue radiating from the alary muscles or dorsal diaphragm. We confirm that four major changes occur in the heart during metamorphosis: 1) a conical chamber is formed de novo in the first and second abdominal segments; 2) the adult heart curves to conform to the contour of the abdomen; 3) a layer of longitudinal striated muscle appears on the ventral surface of the heart; 4) a fourth pair of ostia is added to the three already present in the early pupa; and note additionally that 5) the ostia appear as simple openings in the heart of the early pupa but are valve‐like in the adult. J. Morphol. 240:225–235, 1999. © 1999 Wiley‐Liss, Inc.  相似文献   

6.
The appearance of muscle proteins in the contractile apparatus of the mussel Mytilus trossulus was subjected to comparative analysis during ontogenesis. It was established, with the use of Western blot analysis and electrophoresis in polyacrylamid gel in the presence of sodium dodecylsulfate, that proteins of the contractile apparatus of mussel muscles express long before the formation of the first functionally active muscle system of the veliger larvae. Paramyosin is present in egg cells; twitchin, myorod, and actin appear at the stage of blastula (12 h after fertilization), and myosin appears at the trochophore stage (17 h after fertilization). The quantitative relation of muscle proteins was studied in actomyosin extracts of larvae obtained from different developmental stages. It was shown that the ratios actin/myosin and paramyosin/myosin at the veliger stage (96 h after fertilization) were found to be similar to those in the striated muscles of invertebrates.  相似文献   

7.
This study describes the anatomical and developmental aspects of muscular development from the early embryo to competent larval stage in the gastropod Ilyanassa obsoleta. Staining of F‐actin revealed differential spatial and temporal patterns of several muscles. In particular, two major muscles, the larval retractor and pedal retractor muscles originate independently and display distinct developmental patterns similar to observations in other gastropod species. Additionally, together with the larval retractor muscle, the accessory larval muscle developed in the embryo at the trochophore stage. Therefore, both these muscles develop prior to ontogenetic torsion. The pedal retractor muscle marked the most abundant growth in the mid veliger stage. Also during the middle stage, the metapodial retractor muscle and opercular retractor muscle grew concurrently with development of the foot. We show evidence that juvenile muscles, such as the buccal mass muscle and siphon muscle develop initially during the late veliger stage. Collectively, these findings substantiate that larval myogenesis involves a complex sequence of events that appear evolutionary conserved within the gastropods, and set the stage for future studies using this model species to address issues concerning the evolution and eventual fates of larval musculature in molluscs. J. Morphol., 2009. © 2009 Wiley‐Liss, Inc.  相似文献   

8.
The expression of MLCK- and PEVK-domains of twitchin, as well as the unique N-terminal domain of myorod in early development of the mussel Mytilus trossulus has been studied. The MLCK-domain of twitchin and the unique N-terminal domain of myorod appear at the early stages of development, whereas the PEVK-domain of twitchin is present only in muscles of adult mussel. The sizes of genes of the N-terminal domain of myorod, obtained at the blastula stage and from the adult animal are similar, but the proteins have significant differences in the amino acid sequences. Consequently, myorod and twitchin appear at early stages of larval mussels before the formation of “adult” muscles capable of catch contraction, and at these stages both proteins are isoforms, which differ from the isoforms of adult animals. It is possible that the MLCK-domain in the “larval” isoform of twitchin is necessary for regulating the formation of the contractile apparatus of molluscan smooth muscles, while the PEVK-domain is important for the regulation of the catch state in muscles of adult animals.  相似文献   

9.
The mussel cells from premyogenic larval stages are capable of differentiation into smooth muscle cells in vitro. However, the behavior and protein composition of these cells are not completely identical to those of smooth muscle cells of adult mussels. In this study we compared some properties of mussel muscle cells forming from cells of trochophore (premyogenic larval stage) in vitro with those of muscle cells of veliger and adult mussel. We found a substantial difference between the contractile apparatus protein composition of veliger muscle and cultivated cells. Myorod, one of the molecular markers of the phenotype of mollusc smooth muscle cells (Shelud'ko et al., 1999, Comp Biochem Physiol 122:277-285), is not a constituent of the contractile apparatus of veliger muscle. At the same time the protein composition of contractile apparatus in cultivated cells was similar to that of adult Mytilus muscles. There were only few quantitative differences between them. The contractile activity of cultivated cells was changing in time. The kinetic parameters of first spontaneous contractions were similar to those of phasic contractions, while their period was close to that of tonic contractions. After 50-55 hrs cultivation the cells produced both phasic and tonic contractions, but the character of contractile activity of cultivated cells was regulated after six days of cultivation only. However, there were no muscle cells in vitro, whose contractile activity was similar to that of veliger muscle cells. So, we concluded that properties of muscle cells forming from premyogenic larval mussel cells in culture are similar to those of muscle cells of the adult mussel, but not of veliger.  相似文献   

10.
Abstract. A light and electron immunohistochemical study was carried out on the body wall muscles of the chaetognath Sagitta friderici for the presence of a variety of contractile proteins (myosin, paramyosin, actin), regulatory proteins (tropomyosin, troponin), and structural proteins (α‐actinin, desmin, vimentin). The primary muscle (~80% of body wall volume) showed the characteristic structure of transversely striated muscles, and was comparable to that of insect asynchronous flight muscles. In addition, the body wall had a secondary muscle with a peculiar structure, displaying two sarcomere types (S1 and S2), which alternated along the myofibrils. S1 sarcomeres were similar to those in the slow striated fibers of many invertebrates. In contrast, S2 sarcomeres did not show a regular sarcomeric pattern, but instead exhibited parallel arrays of 2 filament types. The thickest filaments (~10–15 nm) were arranged to form lamellar structures, surrounded by the thinnest filaments (~6 nm). Immunoreactions to desmin and vimentin were negative in both muscle types. The primary muscle exhibited the classical distribution of muscle proteins: actin, tropomyosin, and troponin were detected along the thin filaments, whereas myosin and paramyosin were localized along the thick filaments; immunolabeling of α‐actinin was found at Z‐bands. Immunoreactions in the S1 sarcomeres of the secondary muscle were very similar to those found in the primary muscle. Interestingly, the S2 sarcomeres of this muscle were labeled with actin and tropomyosin antibodies, and presented no immunore‐actions to both myosin and paramyosin. α‐Actinin in the secondary muscle was only detected at the Z‐lines that separate S1 from S2. These findings suggest that S2 are not true sarcomeres. Although they contain actin and tropomyosin in their thinnest filaments, their thickest filaments do not show myosin or paramyosin, as the striated muscle thick myofilaments do. These peculiar S2 thick filaments might be an uncommon type of intermediate filament, which were labeled neither with desmin or vimentin antibodies.  相似文献   

11.
SUMMARY Myogenesis of two representatives of Platyhelminthes, Stylostomum sanjuania and Pseudoceros canadensis, was followed from egg deposition until well‐differentiated free‐swimming larval stages, using F‐actin staining and confocal laserscanning microscopy. Zonulae adhaerentes are the only structures to stain before 50% of development between egg deposition and hatching in S. sanjuania, and before 67% of development in P. canadenis. Subsequently, irregular fibers appear in the embryo, followed by a helicoid muscle close to the apical pole. Three longitudinal muscle pairs form, of which the dorsal pair remains more pronounced than the others. Gradually, new muscles form by branching or from double‐stranded muscle zones adjacent to existing muscles. This results in an elaborate muscular bodywall that consists of a single helicoid muscle as well as multiple circular and longitudinal muscles. Diverse retractor muscles insert at the sphincter muscles around the stomodeum. The overall arrangement and formation mode of the larval musculature appears very similar in both species, although only P. canadensis has a primary circular muscle posterior to the helicoid muscle. Muscle formation in the apical region of the embryo precedes that at the abapical pole and the primary longitudinal muscles form slightly later than the primary circular muscles. Myogenesis and larval myoanatomy appears highly conserved among polyclad flatworms, but differs significantly from that of other trochozoan clades. Our data suggest that the larval muscular ground pattern of polyclad larvae comprises a bodywall consisting of a helicoid muscle, circular and longitudinal muscles, several retractor muscles, and sphincter muscles around the stomodeum.  相似文献   

12.
We used confocal microscopy and immunohistochemistry to characterize the morphology of the subcutaneous neurons and the innervation of the slime glands and striated muscles in the hagfish Myxine glutinosa. A rich plexus of 5HT‐, ChAT‐ and TH‐positive neurons is described in the capsule of the slime glands. These neurons, like those of the subcutaneous plexus, receive pericellular terminations from the axons of central cells. Capsular neurons receive innervation from 5HT‐positive and nNOS‐positive nerve fibres. Other nerve endings belonging to two separate nerve populations are identified in the striated muscles. They contain TH and nNOS immunoreactivity. Due to the lack of any topographical labelling, the cell origin and the projections of the neurons into the cranial and spinal nerves are unknown. This study provides anatomical evidence of multiple (5HT and nNOS) peripheral innervation of the neurons. However, it does not provide information about the function of these neurons in the hagfish. We suggest that hagfish neurons have a phylogenetic relationship with the spinal group of the dorsal cells of lampreys and the supramedullary cells of teleosts.  相似文献   

13.
Objectives: Four adult non‐human primates Papio ursinus were used to study induction of bone formation by recombinant human transforming growth factor‐β2 (hTGF‐β2) together with muscle‐derived stem cells. Materials and methods: The hTGF‐β2 was implanted in rectus abdominis muscles and in calvarial defects with and without addition of morcellized fragments of striated muscle, harvested from the rectus abdominis or temporalis muscles. Expression of osteogenic markers including osteogenic protein‐1, bone morphogenetic protein‐3 and type IV collagen mRNAs from generated specimens was examined by Northern blot analysis. Results: Heterotopic intramuscular implantation of 5 and 25 μg hTGF‐β2 combined with 100 mg of insoluble collagenous bone matrix yielded large corticalized mineralized ossicles by day 30 with remodelling and induction of haematopoietic marrow by day 90. Addition of morcellized rectus abdominis muscle to calvarial implants enhanced induction of bone formation significantly by day 90. Conclusions: In Papio ursinus, in marked contrast to rodents and lagomorphs, hTGF‐β2 induced large corticalized and vascularized ossicles by day 30 after implantation into the rectus abdominis muscle. This striated muscle contains responding stem cells that enhance the bone induction cascade of hTGF‐β2. Induction of bone formation by hTGF‐β2 in the non‐human primate Papio ursinus may occur as a result of expression of bone morphogenetic proteins on heterotopic implantation of hTGF‐β2; the bone induction cascade initiated by mammalian TGF‐β proteins in Papio ursinus needs to be re‐evaluated for novel molecular therapeutics for induction of bone formation in clinical contexts.  相似文献   

14.
Summary The presence and distribution pattern of paramyosin have been examined in different invertebrate muscle cell types by means of Western blot analysis and electron microscopy immunogold labelling. the muscles studied were: transversely striated muscle with continuous Z lines (flight muscle fromDrosophila melanogaster), transversely striated muscle with discontinuous Z lines (heart muscle from the snailHelix aspersa), obliquely striated body wall muscle from the earthwormEisenia foetida, and smooth muscles (retractor muscle from the snail and pseudoheart outer muscular layer from the earthworm). Paramyosin-like immunoreactivity was localized in thick filaments of all muscles studied. Immunogold particle density was similar along the whole thick filament length in insect flight muscle but it predominated in filament tips of fusiform thick filaments in both snail heart and earthworm body wall musculature when these filaments were observed in longitudinal sections. In obliquely sectioned thick filaments, immunolabelling was more abundant at the sites where filaments disappeared from the section. These results agree with the notion that paramyosin extended along the whole filament length, but that it can only be immunolabelled when it is not covered by myosin. In all muscles examined, immunolabelling density was lower in cross-sectioned myofilaments than in longitudinally sectioned myofilaments. This suggests that paramyosin does not form a continuous filament. The results of a semiquantitative analysis of paramyosin-like immunoreactivity indicated that it was more abundant in striated than in smooth muscles, and that, within striated muscles, transversely striated muscles contain more paramyosin than obliquely striated muscles.  相似文献   

15.
Myogenesis is currently investigated in a number of invertebrate taxa using combined techniques, including fluorescence labeling, confocal microscopy, and 3D imaging, in order to understand anatomical and functional issues and to contribute to evolutionary questions. Although developmental studies on the gross morphology of bivalves have been extensively pursued, organogenesis including muscle development has been scarcely investigated so far. The present study describes in detail myogenesis in the scallop Nodipecten nodosus (Linnaeus, 1758) during larval and postmetamorphic stages by means of light, electron, and confocal microscopy. The veliger muscle system consists of an anterior adductor muscle, as well as four branched pairs of striated velum retractors and two pairs of striated ventral larval retractors. The pediveliger stage exhibits a considerably elaborated musculature comprising the velum retractors, the future adult foot retractor, mantle (pallial) muscles, and the anterior and posterior adductors, both composed of smooth and striated portions. During metamorphosis, all larval retractors together with the anterior adductor degenerate, resulting in the adult monomyarian condition, whereby the posterior adductor retains both myofiber types. Three muscle groups, i.e., the posterior adductor, foot retractor, and pallial muscles, have their origin prior to metamorphosis and are subsequently remodeled. Our data suggest a dimyarian condition (i.e., the presence of an anterior and a posterior adductor in the adult) as the basal condition for pectinids. Comparative analysis of myogenesis across Bivalvia strongly argues for ontogenetic and evolutionary independence of larval retractors from the adult musculature, as well as a complex set of larval retractor muscles in the last common bivalve ancestor.  相似文献   

16.
Integrins play a key role in the intermediation and coordination between cells and extracellular matrix components. In this study, we first determined the presence of the β integrin‐like protein and its presumptive ligand, fibronectin‐like protein, during development and in some adult tissues of the bivalve mollusc Mytilus trossulus. We found that β integrin‐like protein expression correlated with the development and differentiation of the digestive system in larvae. Besides the presence of β integrin‐like protein in the digestive epithelial larval cells, this protein was detected in the hemocytes and some adult tissues of M. trossulus. The fibronectin‐like protein was detected firstly at the blastula stage and later, the FN‐LP‐immunoreactive cells were scattered in the trochophore larvae. The fibronectin‐like protein was not expressed in the β integrin‐positive cells of either the veliger stage larvae or the adult mussel tissues and the primary hemocyte cell culture. Despite the β integrin‐ and fibronectin‐like proteins being expressed in different cell types of mussel larvae, we do not exclude the possibility of direct interaction between these two proteins during M. trossulus development or in adult tissues.  相似文献   

17.
The expression of MLCK- and PEVK-domains of twitchin, as well as the unique N-terminal domain of myorod in early development of the mussel Mytilus trossulus has been studied. The MLCK-domain of twitchin and the unique N-terminal domain of myorod appear at the early stages of development, whereas the PEVK-domain of twitchin is present only in muscles of adult mussel. The sizes of genes of the N-terminal domain of myorod, obtained at the blastula stage and from the adult animal are similar, but the proteins have significant differences in the amino acid sequences. Consequently, myorod and twitchin appear at early stages of larval mussels before the formation of "adult" muscles capable of catch contraction, and at these stages both proteins are isoforms, which differ from the isoforms of adult animals. It is possible that the MLCK-domain in the "larval" isoform of twitchin is necessary for regulating the formation of the contractile apparatus of molluscan smooth muscles, while the PEVK-domain is important for the regulation of the catch state in muscles of adult animals.  相似文献   

18.
To date only few comparative approaches tried to reconstruct the ontogeny of the musculature in invertebrates. This may be due to the difficulties involved in reconstructing three dimensionally arranged muscle systems by means of classical histological techniques combined with light or transmission electron microscopy. Within the scope of the present study we investigated the myogenesis of premetamorphic, metamorphic, and juvenile developmental stages of the anaspidean opisthobranch Aplysia californica using fluorescence F‐actin‐labeling in conjunction with modern confocal laser scanning microscopy. We categorized muscles with respect to their differentiation and degeneration and found three true larval muscles that differentiate during the embryonic and veliger phase and degenerate during or slightly after metamorphosis. These are the larval retractor, the accessory larval retractor, and the metapodial retractor muscle. While the pedal retractor muscle, some transversal mantle fibers and major portions of the cephalopedal musculature are continued and elaborated during juvenile and adult life, the buccal musculature and the anterior retractor muscle constitute juvenile/adult muscles which differentiate during or after metamorphosis. The metapodial retractor muscle has never been reported for any other gastropod taxon. Our findings indicate that the late veliger larva of A. californica shares some common traits with veligers of other gastropods, such as a larval retractor muscle. However, the postmetamorphic stages exhibit only few congruencies with other gastropod taxa investigated to date, which is probably due to common larval but different adult life styles within gastropods. Accordingly, this study provides further evidence for morphological plasticity in gastropod myogenesis and stresses the importance of ontogenetic approaches to understand adult conditions and life history patterns. J. Morphol., 2008. © 2007 Wiley‐Liss, Inc.  相似文献   

19.
 Whole-mount technique using fluorescent-labelled phalloidin for actin staining and confocal laser scanning microscopy as well as semi-thin serial sectioning, scanning and transmission electron microscopy were applied to investigate the ontogeny of the various muscular systems during larval development in the limpets Patella vulgata L. and P. caerulea L. In contrast to earlier studies, which described a single or two larval shell muscles, the pretorsional trochophore-like larva shows no less than four different muscle systems, namely the asymmetrical main head/foot larval retractor muscle, an accessory larval retractor with distinct insertion area, a circular prototroch/velar system, and a plexus-like pedal muscle system. In both Patella species only posttorsional larvae are able to retract into the shell and to close the aperture by means of the operculum. Shortly after torsion the two adult shell muscles originate independently in lateral positions, starting with two fine muscle fibres which insert at the operculum and laterally at the shell. During late larval development the main larval retractor and the accessory larval retractor become reduced and the velar muscle system is shed. In contrast, the paired adult shell muscles and the pedal muscle plexus increase in volume, and a new mantle musculature, the tentacular muscle system, and the buccal musculature arise. Because the adult shell muscles are entirely independent from the various larval muscular systems, several current hypotheses on the ontogeny and phylogeny of the early gastropod muscle system have to be reconsidered. Received: 23 June 1998 / Accepted: 25 November 1998  相似文献   

20.
The arrangement and structure of sinus hair muscles in the snout of the shrew, Sorex unguiculatus, were studied by electron microscopy and serial section light microscopy. Both striated and smooth muscles are directly associated with sinus hair follicles. The striated muscle fibers originate from the base of a follicle and insert onto the superficial portion of adjoining caudally positioned follicles. Some fibers insert into the corium instead of inserting into a follicle. The fibers show a fine structure typical of red fibers. Smooth muscle cells form a network with elastic fibers beneath the corium. Some cells are directly attached to the capsule of the sinus, thus forming a type of M. arrector pili. Striated muscle fibers that appear to end in the corium are connected with the smooth muscle network through the elastic fibers which appear to function as the tendon of these two types of muscle cell.  相似文献   

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