Muscle tissue histogenesis of the lymph hearts of chick embryos

By means of electron microscope autoradiography, the ultrastructure of muscle fibers, and the capacity if muscle of cell nuclei of 3H-thymidine (3H-T) incorporating of were studed in developing lymph hearts of 0-13 day old chick embryos, rather active sarcomerogenesis developing lymph hearts of 9-13 day old chick embryos, a rather active sarcomerogenesis being observed. Filament of intermediate size microtubules, Golgi complexes, centrioles, and numbers free ribosomes and polysomes were observed in the sarcoplasm. The sarcoplasmic reticulum channels were not numerous, their terminal cisterns often formed "subsarcolemmal cisternae". Between muscle fibers, cell junctions of fasciae adherentes type were observed. Two hours after 3H-T administrations, only mononuclear cells without myofilaments were labeled. If fixation was made 70 hours after 3H-T administration, then the label was found in addition on muscle fiber nuclei. These data evidence that the lymph heart muscle tissue histogenesis undergoes the same patterns of development as does the somatic muscle tissue.     

An early post‐traumatic reaction of lymph‐heart striated muscle fibers in adult frog Rana temporaria during the first postoperative week: An electron microscopic and autoradiographic study

According to the current opinion, lymph‐heart striated muscle represents a specialized type of skeletal muscles in frogs. Here, we studied muscle fibers in mechanically damaged lymph hearts during the first postoperative week using electron‐microscopic autoradiography. We present evidence that both, the satellite cells and pre‐existing muscle fibers bordering the site of injury, contribute directly to the lymph‐heart muscle regeneration. Several muscle fibers located in the vicinity of the damaged area displayed features of nuclear and sarcoplasmic activation. We also observed ultrastructural changes indicating activation of a few satellite cells, namely decondensation of chromatin, enlargement of nuclei and nucleoli, appearance of free ribosomes and rough endoplasmic reticulum tubules in the cytoplasm. Electron‐microscopic autoradiography showed that 4 h after single ³H‐thymidine administration on the seventh day after injury not only the activated satellite cells, but also some nuclei of myofibers bordering the injured zone are labeled. We showed that both, the myonuclei of fibers displaying the signs of degenerative/reparative processes in the sarcoplasm and the myonuclei of the fibers enriched with highly organized myofibrils, can re‐enter into the S‐phase. Our results indicate that the nuclei of lymph‐heart myofibers can reactivate DNA synthesis during regenerative myogenesis, unlike the situation in regenerating frog skeletal muscle where myogenic cells do not synthesize DNA at the onset of myofibrillogenesis. J. Morphol. 276:1525–1534, 2015. © 2015 Wiley Periodicals, Inc.     

Mast cells of lymph hearts during ontogenesis of frogs Rana temporaria

Electron microscopic observations of the lymph hearts of tadpoles and yearling frogs of Rana temporaria showed that mast cells (MCs) were present not only between muscle fibers (population of resident MCs), but in the cavities of lymph heart (population of circulating MCs), too. There were some differences in the ultrastructure of the resident MCs at each studied stage of larval development. The first recognizable MCs were revealed in the lymph hearts at premetamorphosis (stages 39-41). MCs presented as mononuclear relatively small and slightly elongated cells with a few immature secretory granules and numerous free ribosomes, polysomes and short cisternae of rough endoplasmic reticulum (RER) in the cytoplasm. Chromatin of their nuclei was poorly condensed; the Golgi apparatus was moderately developed. At pro-metamorphosis (stages 44-45), we revealed MCs at different levels of their differentiation. Some MCs demonstrated an active process of granulogenesis in their cytoplasm. Among densely packed cytoplasmic organelles, immature secretory granules were closely associated with cisternae of RER and free ribosomes. Other MCs appeared as more differentiated cells. They were characterized by a predominantly heterochromatic nuclei and cytoplasm filled with polymorphic and heterogeneous granules. MCs also showed a reduction in the number of free ribosomes and cisternae of RER in the cytoplasm. On the contrary, the Golgi apparatus was well developed. Stacks of Golgi cisternae, detaching vacuoles, and progranules occupied the perinuclear region. The majority of the outlines above ultrastructural features of differentiated MCs were typical for MCs of yearling frogs. At metamorphic climax (stages 52-53), MCs often tightly contacted with macrophages. We did not reveal apoptotic MCs. However, some MCs exhibited morphological features typical for programmed necrosis-like death, which was characterized by mitochondria swelling, dilatation of cisternae of RER and nuclear envelope, plasma membrane rupture and subsequent loss of intracellular contents. Electron microscopical immunocytochemistry revealed the localization of atrial natriuretic peptide (ANP), substance S (SP) and heat shock protein (Hsp70) in the secretory granules of the resident and circulating MCs at different stages of tadpole development and in yearling frogs.     

Muscle development in vitro following X irradiation

Myogenic cells obtained from 12-day-old embryonic chicken hind limb and breast muscle were exposed to 5000 rads of X irradiation. Although 10% of the initial cell dissociates were killed by irradiation, the remaining cells were comparable to controls in plating efficiency and light microscopic morphology. Moreover, there was no increase or loss of cells for at least 72 hr in vitro when plated at a density of 2 × 10⁶ cells/60-mm plate. It was found that muscle cell fusion after irradiation proceeded at the same rate and to the same relative extent as in control cultures. Myotubes developed normally; cross-striations were prominent by 5 to 7 days of culture and the cells maintained a well-differentiated state for periods of at least 3 weeks in vitro. In control cultures continuously labeled with 1 μCi/ml of [³H]TdR, 75% of the nuclei within myotubes were heavily labeled by 118 hr; less than 15% of the nuclei within syncytia of irradiated cultures were labeled. Quantitative microphotometry of Feulgen-stained cultures demonstrated that all nuclei within control and irradiated myotubes contained the 2C complement of DNA. Similar experiments conducted with cells released from limbs and breasts of 10-day-old embryos revealed lower absolute levels of cytoplasmic fusion in both control and irradiated samples, however, there was slightly more cell death after exposure to X rays in 10-day-old than 12-day-old material. Nevertheless, considerable cell fusion occurred in irradiated limb and breast cell cultures, consistent with the conclusion that the commitment to myogenesis of prefusion myoblasts is extremely stable even in the face of massive ionizing radiation and that neither cell division nor replication of DNA is an obligatory prerequisite for the in vitro fusion and subsequent differentiation of skeletal muscle obtained from 10- and 12-day-old chick embryos.     

SENSITIVITY OF EARLY EMBRYONIC THYMIC LYMPHOID DEVELOPMENT TO 3H-THYMIDINE OF HIGH SPECIFIC ACTIVITY

The pulse technique, using high specific activity ³H-TdR to selectively kill cells in cell cycle, was applied to the thymic anlagen of chick embryos. With optimal specific and total ³H-TdR activities and pulse times of 2–4 hr the subsequent lymphoid development in organ culture of the thymic anlagen of 10-day-old chick embryos could be almost completely inhibited. The most important effect of the ³H-TdR was on the lymphoid precursor cells of the anlagen. The thymic epithelium appeared more resistant to ³H-TdR and allowed a lymphoid development of pulsed anlagen grafted to the chorioallantoic membrane of chick embryos when new lymphoid precursor cells were provided. The lymphoid precursor cells of the thymic anlagen of 10-day-old chick embryos therefore appeared to be in cell cycle with short generation time. The thymic anlagen of 8-, 9- and 10-day-old but not 7-day-old embryos showed a lymphoid development in organ culture. They did not differ with respect to the sensitivity to hot pulses of ³H-TdR. Thus no evidence of a lag in the onset of lymphoid precursor cell proliferation during the development of the early embryonic chick thymus was noted.     

Glycosaminoglycan synthesis by the early embryonic chick heart

Glycosaminoglycans of embryonic chick hearts labeled in situ were characterized by means of labeled precursor incorporation, electrophoretic mobility, sensitivity to testicular hyaluronidase, elution characteristics from CPC-cellulose columns, and hexosamine content. During the initial period of overt cardiac muscle differentiation (approximately stage 10) chondroitin sulfates are not detectable but an undersulfated component is present. Chondroitin sulfate synthesis appears shortly after overt muscle differentiation. Hyaluronate is present both during and after overt myocardial differentiation. Although epimerization of ³H-glucosamine-derived labeled UDP-N-acetyl-d-glucosamine occurs (determined by recovery of incorporated labeled galactosamine), label does not appear in chondroitin sulfate. ³H-Glucosamine is thus a relatively specific precursor for unsulfated glycosaminoglycans, a fact that we exploited in demonstrating their distribution radioautographically. Glycosaminoglycan synthesis was also examined in hearts labeled (a) in isolated organ culture, (b) in situ but exposed directly to the medium by removal of the splanchnopleure. In both cases fully sulfated chondroitin sulfate and chondroitin are not synthesized. Hearts make only hyaluronate and undersulfated chondroitin sulfate.     

Chick endogenous lectin enhances chondrogenesis of cultured chick limb bud cells

We have studied the effect of β-d-galactoside-specific lectin purified from 14-day-old chick embryos on the differentiation of the mesenchymal cells dissociated from the limb buds of stage 24 chick embryos, using the micro-mass culture method described previously. When the cells were incubated with the lectin during the initial 12 hr of culture, cell proliferation became slightly activated. The lectin-treated cells formed a greater number of cartilage nodules and incorporated about twice as much as [³⁵S]sulfate per cell than the control cultures. The results of this study show that the chick endogenous lectin promotes cartilage differentiation in vitro and that endogenous lectin may possibly be involved in chondrogenesis in vivo.     

Autoradiographic labeling of spinal cord neurons with high affinity choline uptake in cell culture

Embryonic chick spinal cord neurons grown in dissociated cell culture have a high affinity uptake mechanism for choline. We find that, in addition to acetylcholine synthesis, the accumulated choline is used for the synthesis of metabolites such as lipids that are retained in part by conventional fixation techniques. As a result autoradiographic methods can be used to identify the cells that have the uptake mechanism in spinal cord cultures. About 60% of the neurons are labeled by [³H]choline uptake in cultures prepared with spinal cord cells from 4-day-old embryos, and about 40% are labeled in cultures prepared with cord cells from 7-day-old embryos. Neurons that innervate skeletal myotubes in spinal cord-myotube cultures are consistently labeled by [³H]choline uptake. Neurons unlabeled by the procedure are viable: they exclude the dye trypan blue and accumulate ¹⁴C-amino acids for protein synthesis. Most of the neurons unlabeled by [³H]choline uptake can instead be labeled by uptake of γ-[³H]aminobutyric acid, and vice versa. These results suggest that high affinity choline uptake can be used to label cholinergic neurons in cell culture, and that at least some populations of noncholinergic neurons are not labeled by the procedure. It cannot yet be concluded, however, that all labeled neurons are cholinergic since more labeled neurons are obtained per cord than would be expected from the number of neurons making up identified cholinergic populations in vivo. A three- to fourfold increase in the amount of high affinity choline uptake is observed between Days 3 and 15 in culture for spinal cord cells obtained from 4-day-old embryos. The number of [³H]choline-labeled neurons in such cultures decreases slightly during the same period, suggesting that the increase in uptake reflects neuronal growth or development rather than an increase in population size. Both the magnitude of the uptake and the number of [³H]choline-labeled neurons are the same for spinal cord cells grown with and without skeletal myotubes.     

Chronic stimulation of the spinal cord in developing chick embryo causes the differentiation of multiple clusters of acetylcholine receptor in the posterior latissimus dorsi muscle

Electrodes were implanted around the spinal cord of 7-day-old chick embryos and electric pulses delivered at 0.5-Hz frequency from the 10th to 15th day of incubation. At Day 15, the posterior latissimus dorsi (PLD) muscle, which, in control animals, is focally innervated, was dissected. The number and distribution of AChR clusters revealed by autoradiography after labeling with ¹²⁵I-α-bungarotoxin was quantitatively studied on isolated muscle fiber fragments and on serial sections of the whole muscle. After chronic stimulation, muscle fibers with multiple AChR clusters were observed. The distribution of the clusters appeared less regular than in the anterior latissimus dorsi muscle which, in control embryos, receives a multiple innervation. The total number of AChR clusters per PLD muscle increased about 1.8 times as a consequence of the stimulation without significant change of the total number of muscle fibers.     

Cumulative indices of DNA synthesizing myocytes in different compartments of the working myocardium and conductive system of the rat’s heart muscle following extensive left ventricle infarction

Ten successive³H-thymidine injections at 12h intervals (which is a little shorter than the adult heart myocyte S phase) were performed for labeling of the majority of cardiac myocytes synthesizing DNA at any moment of such a 5 days experiment. In the hearts of control unoperated rats ten-fold repeated³H-thymidine administration results in labeling of 2–3% myocyte nuclei, in both atria, ca. 1% of the specialized muscle cell nuclei in the atrioventricular conductive system, only occasional muscle cells being labeled in the working ventricular myocardium. When ten successive³H-thymidine injections were made between the 5th and 10th days following extended left ventricle infarction, the percentage of labeled myocytes in left and right atria reaches, respectively, 51.4±4.4% and 34.7±3.6%. In the left ventricle labeled muscle nuclei are accumulated predominantly (9.3±2.1%) within the thin subepicardial layer of the surviving myofibers, while myofibers located in other perinecrotic areas contained only 1.3±0.5% labeled muscle nuclei. The number of these nuclei in the atrioventricular system remains at the level observed in control hearts (up to 2%), approaching closely the zero level in the working myocardium of both the ventricles and interventricular septum, located at the considerable distance from the infarcted region. When similar experiments with ten-fold repeated³H-thymidine injections were performed between 15th and 20th post-infarction days the number of labeled myocyte nuclei was found to be reduced 4–6 times in atria, being changed rather a little in the perinecrotic ventricular myocardium and in the specialized myocardium of the atrioventricular system. Some possible reasons of the observed differences in the proliferative behaviour of cardiac myocytes in terms of their topology and/or specialization are discussed     

Zebrafish Embryonic Slow Muscle Is a Rapid System for Genetic Analysis of Sarcomere Organization by CRISPR/Cas9, but Not NgAgo

Zebrafish embryonic slow muscle cells, with their superficial localization and clear sarcomere organization, provide a useful model system for genetic analysis of muscle cell differentiation and sarcomere assembly. To develop a quick assay for testing CRISPR-mediated gene editing in slow muscles of zebrafish embryos, we targeted a red fluorescence protein (RFP) reporter gene specifically expressed in slow muscles of myomesin-3-RFP (Myom3-RFP) zebrafish embryos. We demonstrated that microinjection of RFP-sgRNA with Cas9 protein or Cas9 mRNA resulted in a mosaic pattern in loss of RFP expression in slow muscle fibers of the injected zebrafish embryos. To uncover gene functions in sarcomere organization, we targeted two endogenous genes, slow myosin heavy chain-1 (smyhc1) and heat shock protein 90 α1 (hsp90α1), which are specifically expressed in zebrafish muscle cells. We demonstrated that injection of Cas9 protein or mRNA with respective sgRNAs targeted to smyhc1 or hsp90a1 resulted in a mosaic pattern of myosin thick filament disruption in slow myofibers of the injected zebrafish embryos. Moreover, Myom3-RFP expression and M-line localization were also abolished in these defective myofibers. Given that zebrafish embryonic slow muscles are a rapid in vivo system for testing genome editing and uncovering gene functions in muscle cell differentiation, we investigated whether microinjection of Natronobacterium gregoryi Argonaute (NgAgo) system could induce genetic mutations and muscle defects in zebrafish embryos. Single-strand guide DNAs targeted to RFP, Smyhc1, or Hsp90α1 were injected with NgAgo mRNA into Myom3-RFP zebrafish embryos. Myom3-RFP expression was analyzed in the injected embryos. The results showed that, in contrast to the CRISPR/Cas9 system, injection of the NgAgo-gDNA system did not affect Myom3-RFP expression and sarcomere organization in myofibers of the injected embryos. Sequence analysis failed to detect genetic mutations at the target genes. Together, our studies demonstrate that zebrafish embryonic slow muscle is a rapid model for testing gene editing technologies in vivo and uncovering gene functions in muscle cell differentiation.     

Muscle tissue regeneration of the lymph hearts in adult Rana temporaria frogs. A study by light and electron microscopy autoradiography methods

The regeneration response of adult frog lymph heart muscle tissue was studied from 2 to 3 weeks after mechanical injury. High resolution autoradiographic studies showed that regenerative necrotic zones have many actively proliferating mononuclear cells deprived of cytoplasmic myofilaments. Some of them have numerous free ribosomes, so they might be identified as myoblasts. On the 13th day after injury newly-formed myotubes with chains of myonuclei and pictures of active sarcomerogenesis were observed. On the other hand, the surviving muscle fibers of the perinecrotic zone were rich in myonuclei at their growing ends. In the vicinity of nuclei, accumulation of a mass of non-differentiated cytoplasm rich in free ribosomes and polysomes, rough endoplasmic reticulum, Golgi apparatus, and centrioles are seen. Tritiated thymidine pulse-labeling showed that only rare myonuclei of the perinecrotic zone muscle fibers were labeled, whereas numerous non-differentiated cells of granulation tissue and myosatellites incorporated thymidine. The number of labeled myonuclei markedly increased 96 hours after 3HTdr administration. These data evidence that the myoblastic mechanism is predominant in the regeneration of adult frog lymph heart muscle tissue. It is necessary to emphasize that during the lymph heart muscle tissue reparative myogenesis some of the perinecrotic myonuclei are able to synthesize DNA and to divide mitotically, which distinguishes this type of muscle from skeletal muscle tissue of vertebrates.     

Growth of limb muscle is dependent on skeletal-derived Indian hedgehog

During embryogenesis, muscle and bone develop in close temporal and spatial proximity. We show that Indian Hedgehog, a bone-derived signaling molecule, participates in growth of skeletal muscle. In Ihh^−/− embryos, skeletal muscle development appears abnormal at embryonic day 14.5 and at later ages through embryonic day 20.5, dramatic losses of hindlimb muscle occur. To further examine the role of Ihh in myogenesis, we manipulated Ihh expression in the developing chick hindlimb. Reduction of Ihh in chicken embryo hindlimbs reduced skeletal muscle mass similar to that seen in Ihh^−/− mouse embryos. The reduction in muscle mass appears to be a direct effect of Ihh since ectopic expression of Ihh by RCAS retroviral infection of chicken embryo hindlimbs restores muscle mass. These effects are independent of bone length, and occur when Shh is not expressed, suggesting Ihh acts directly on fetal myoblasts to regulate secondary myogenesis. Loss of muscle mass in Ihh null mouse embryos is accompanied by a dramatic increase in myoblast apoptosis by a loss of p21 protein. Our data suggest that Ihh promotes fetal myoblast survival during their differentiation into secondary myofibers by maintaining p21 protein levels.     

DNA synthesis, mitosis and fusion of myocardial cells

Myocardial cells obtained from embryonic chick ventricles have been used to investigate (1) whether differentiated cells can undergo DNA synthesis and mitosis and, (2) whether heart cells when grown in culture can fuse with each other and with chick skeletal myoblasts to form heterokaryon myotubes. Electron microscopic observations have shown that myocardial cells of day 3 and day 20 chick embryos did contain myofibrils with defined sarcomeres; these cells have been observed in mitosis. Cells obtained by tryptic digestion of day 12 chick ventricles when grown in culture continued to replicate their DNA as shown by thymidine-³H radioautography with DNase controls and were observed in all stages of mitosis. Electron microscopy showed that myofibrils were present in some of the cultured cells. Bi-, tri- and tetranucleate cells were observed in the cultures. Thymidine-³H radioautography showed that these cells were formed by karyokinesis without cytokinesis and by the fusion of uninucleate cells. Since the heart cells could fuse with each other, we tested the possibility that they could fuse with skeletal myoblasts to form heterokaryon myotubes. This was accomplished by co-culturing thymidine-³H labeled ventricular cells and unlabeled skeletal myoblasts. Radioautography with DNase controls showed that some of the myotubes consisted of unlabeled skeletal muscle nuclei and labeled heart nuclei in varied proportions. The factors initiating the formation of these heterokaryons have not been elucidated.     

Circulatory system in chicken skeletal muscle in the second half of embryogenesis: Shape,blood flow,and vascular reactivity

A restructuring of the capillary bed—from the embryonic structure with a three-dimensional network of wide and long protocapillaries to the mature structure with high density of thin and short capillaries along the fibers—has been demonstrated in the chick skeletal muscle on embryonic days 10–19 by morphometric analysis. In this case, the specific blood flow and capillary luminal area per cm³ of the muscle remained unaltered, while the blood volume in it significantly dropped. The response of muscle circulation to nitroprusside (increase) and noradrenaline (decrease) appeared in 19-day-old embryos, but this response could develop only under conditions of initially low or high blood flow, respectively. We propose that the arterial trunk lumen area to the total capillary lumen area remains constant as the intraorganic circulation is formed, which provides for the required linear blood velocity in capillaries.     

Scanning electron microscopy of nerve-muscle contacts in embryonic cell culture.

Scanning electron microscopy (SEM) of cell cultures of dissociated nerve and muscle from chick embryos has shown that developing muscle fibers can be contacted at many sites by one or more than one neuron, and that a single nerve can send branches to several myofibers. At these contact regions of nerve with muscle, the neurons send out terminal or lateral sprouts with fine tips which initially lack terminal swellings, but later acquire small “bouton”-like structures in contact with the sarcolemma, which resemble embryonic synapses. At these points, the sarcolemma does not appear to differ in ultrastructure from other surface regions of the myofiber. Transmission electron microscopy (TEM) has revealed the presence of both electron lucent and dense-cored vesicles at some nerve terminals. However, fluorescence histochemistry (Falck-Hillarp technique) failed to detect the presence of catecholamines in these cultures. The SEM pictures at substantially higher resolutions than the light microscope, and the enhanced three dimensional perspective of this technique, provide additional information about the developmental morphology of the nerve-muscle cell culture system. The results are correlated with previous findings by light microscopy, TEM and electrophysiology, and discussed in relationship to proposed innervation processes of skeletal muscle fibers in vivo.     

Controlled differentiation of myoblast cells into fast and slow muscle fibers

Skeletal muscles are classified into fast and slow muscles, which are characterized by the expression of fast-type myosin heavy chains (fMyHCs) or slow-type myosin heavy chains (sMyHCs), respectively. However, the mechanism of subtype determination during muscle fiber regeneration is unclear. We have analyzed whether the type of muscle is determined in the myoblast cells or is controlled by the environment in which the muscle fibers are formed from myoblast cells. When myoblast cells from 7-day-old chick embryo were cultured and formed into muscle fibers, more than half of the fibers produced only fMyHCs, and the remaining fibers produced both fMyHCs and sMyHCs. However, when myoblast cells were cultured in medium supplemented with a small amount of slow muscle extract, the expression of sMyHCs in muscle fibers increased, whereas the expression of fMyHCs increased in the group supplemented with fast muscle extract compared with the control group. The same results were obtained when cloned mouse myoblast cells (C₂C₁₂ cells) were cultured and formed into muscle fibers. The data presented here thus show that the subtype differentiation of muscle fiber is controlled by the environment in which the muscle fiber forms. This work was funded by the Sasakawa Scientific Research Grant of the Japan Science Society.     

An analysis of dorsal root ganglia differentiation using three tissue culture systems

Summary The histogenesis of the dorsal root ganglia of chick embryos (ages 3 to 9 days) was followed in three different tissue culture systems. Organotypic explants included dorsal root ganglia connected to the lumbosacral segment of the spinal cord or isolated explants of the contralateral ganglia. Additionally, dissociated monolayer cultures of ganglia tissue were established. The gradual differentiation of progenitor neuroblasts into distinct populations of large ventrolateral and small dorsomedial neurons was observed in vivo and in vitro. Neurites developed after 3 days in the presence or absence of nerve growth factor in the medium. In contrast, autoradiographic analysis indicates that [³H]thymidine incorporation in neuronal cultures differed significantly from intact embryos. In vivo, the number of neuronal progenitor cells labeled with [³H]thymidine decreased in older embryos; in vitro, uptake of [³H]thymidine label was not observed in ganglionic progenitor cells regardless of the age of the donor embryo or the type of culture system. Lack of proliferation in ganglionic progenitor cells was not due to degeneration because vital staining and uptake of [³H]deoxyglucose indicated that neurons were metabolically active. Furthermore, the block in mitotic activity in vitro was limited to presumptive ganglionic neuronal cells. In the ependyma of the spinal cord segment connected to the dorsal root ganglia, neuronal progenitor cells were heavily labeled as were non-neuronal cells within both spinal cord and ganglia. Our results suggest that in vitro conditions can promote the differentiation of sensory neurons from early embryos (E3.5–4.5) without proliferation of progenitor cells.     

Biochemical characterization of [3H]norepinephrine uptake in dissociated brain cell cultures from chick embryos

The uptake of [³H]norepinephrine ([³H]NE) was studied in dissociated brain cell cultures prepared from 8-day-old chick embryos using the whole brain (minus optic lobes). Uptake of [³H]NE, 5×10^–9 M, 10 min incubation, in freshly dissociated noncultured embryonic chick brain cells, was detected in 6-day-old embryos; it was temperature and drug (cocaine, metanephrine) sensitive and increased with brain development. In cultured cells, which were assayed at various days in culture, the increase in [³H]NE accumulation per culture was less than that seen in freshly dissociated noncultured embryonic cells. When [³H]NE uptake was expressed per mg protein, a decrease with days in culture was observed, reflecting perhaps a dilution of growth or proliferation of cells not accumulating NE. Metanephrine, 5×10^–6 M, an inhibitor of extraneuronal uptake, inhibited [³H]NE in 5-day-old cultures whereas desmethylimipramine, an inhibitor of neuronal uptake, inhibited [³H]NE uptake in 15- and 20-day-old cultures. Cocaine, another neuronal inhibitor, inhibited [³H]NE at 10 and 15 days only. We interpret these findings to suggest that during early growth in culture most neuroblasts accumulate NE nonspecifically and, as neuronal maturation proceeds, NE accumulation becomes specific.     

Structural and functional properties of ryanodine receptor type 3 in zebrafish tail muscle

The ryanodine receptor (RyR)1 isoform of the sarcoplasmic reticulum (SR) Ca²⁺ release channel is an essential component of all skeletal muscle fibers. RyR1s are detectable as “junctional feet” (JF) in the gap between the SR and the plasmalemma or T-tubules, and they are required for excitation–contraction (EC) coupling and differentiation. A second isoform, RyR3, does not sustain EC coupling and differentiation in the absence of RyR1 and is expressed at highly variable levels. Anatomically, RyR3 expression correlates with the presence of parajunctional feet (PJF), which are located on the sides of the SR junctional cisternae in an arrangement found only in fibers expressing RyR3. In frog muscle fibers, the presence of RyR3 and PJF correlates with the occurrence of Ca²⁺ sparks, which are elementary SR Ca²⁺ release events of the EC coupling machinery. Here, we explored the structural and functional roles of RyR3 by injecting zebrafish (Danio rerio) one-cell stage embryos with a morpholino designed to specifically silence RyR3 expression. In zebrafish larvae at 72 h postfertilization, fast-twitch fibers from wild-type (WT) tail muscles had abundant PJF. Silencing resulted in a drop of the PJF/JF ratio, from 0.79 in WT fibers to 0.03 in the morphants. The frequency with which Ca²⁺ sparks were detected dropped correspondingly, from 0.083 to 0.001 sarcomere⁻¹ s⁻¹. The few Ca²⁺ sparks detected in morphant fibers were smaller in amplitude, duration, and spatial extent compared with those in WT fibers. Despite the almost complete disappearance of PJF and Ca²⁺ sparks in morphant fibers, these fibers looked structurally normal and the swimming behavior of the larvae was not affected. This paper provides important evidence that RyR3 is the main constituent of the PJF and is the main contributor to the SR Ca²⁺ flux underlying Ca²⁺ sparks detected in fully differentiated frog and fish fibers.