Changes in the ribonucleoprotein constituents of the nucleus during the differentiation of muscle cells in the chick embryo

Biol Cell (1992) 76, 159-165

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© Elsevier, Paris

Original article

Changes in the ribonucleoprotein constituents of the nucleus during the differentiation of muscle cells in the chick embryo Guadalupe Zavala, Xochitl Aguilar, Luis Felipe Jim6nez, Olga M Echeverria, Gerardo V~izquez-Nin * Laboratory of Electron Microscopy, Department of Biology, Faculty of Sciences UNAM, Apartado Postal 70-438, Mexico City, Mexico (Received 16 March 1992; accepted 19 October 1992) Summary - The ribonucleoprotein components of the nucleus of chick embryo muscle cells in different stages of development were

studied by electron microscopic quantitative stereology. The changes of the constituents were related with the appearance of the innervation by means of silver impregnation for light microscope. The numerical density of the perichromatin granules (PCG) is low in mononuclear cells and myotubes. It is noteworthy that the frequency of the PCG does not change during the transition of the cells in mitotic cycle to postmitotic myoblasts and during myofibril differentiation. However, there is an important increment in this parameter when the motor nerve fibers arrive at the muscle and the synaptic contacts are established. This change is correlated with appearance, or at least with a great increase', of the importance of posttranscriptional controls of the expression of some genes. The augmentation in the frequency of PCG is not accompanied by alterations of the abundance of total RNP particles, in close resemblance with the phenomena occurring in neuroblast during the differentiation of synaptic endings. The variations of the nucleolar volume coincide with the changes in rRNA synthesis. interphase / nucleus / cell differentiation I ribonucleoproteins / gene expression

Introduction

Perichromatin granules (PCG) were first described by Watson [37] and unequivocally characterized as a ribonucleoprotein (RNP) component of interphase nuclei of mammalian cells by Monneron and Bernhard [20]. These authors postulated that these granules are composed of pre-messenger or messenger RNA. The study of polytene nuclei of the salivary glands of Diptera demonstrated that the Balbiani ring granules are morphological, and cytochemical similar to the PCG of mammalian cells [30]. Immunocytochemical evidence suggests that PCG [11, 12], as well as Balbiani ring granules are composed of already spliced mRNA [31]. The perichromatin fibrils are RNP structures containing newly synthesized hnRNA (reviewed in [13]; see also [3, 14]). Pulse and chase experiments showed that after completion of transcription these fibrils migrate to interchromatin space [22]. Thus, extranucleolar RNP fibrils contain pre-messenger or messenger RNA. Experiments changing the rate of RNA synthesis by hormone withdrawal and administration caused different alterations in the number of PCG. Cortisol induces a very early increase of transcription accompanied by a small but significant augmentation of the number of PCG in isolated hepatocytes [21]. On the contrary, the administration of estradiol to ovariectomized rats reduces sharply the frequency of PCG in epithelial endometrial cells [32]. The injection of testosterone in castrate rats also induces a rapid decrease of the number of PCG in epithelial prostate cells [10]. In these target cells estradiol and testosterone cause a delayed increase of transcription and a rapid incre* Correspondence and reprints

ment of the transportation of RNA to the cytoplasm [10, 33]. The differentiation of the matrix cells of the central nervous system successively in neuroblasts and in neurons brings about drastic changes in the RNP components of the nucleus [35]. One of the most interesting modifications is the sharp increase of the density of PCG coinciding with synaptogenesis in the immature motoneurons. This alteration is not accompanied by an important modification of the frequency of extranucleolar RNP fibrils [36]. These changes may involve regulation on the synthesis and transportation of RNA similar to those caused by hormones. It is interesting to know whether these modifications of the RNP particles exist in precise moments of the differentiation of other cell types. The muscle differentiation goes through well-defined stages: mononuclear rounded cells without either striation or myofibrillar proteins; mononuclear cells with myofibrils; elongated multinuclear fibers with longitudinal striation. It was reported that rounded mononuclear cells undergoing mitotic divisions do not synthesize appreciable quantities of muscle specific proteins, and that elongated mononuclear cells and myotubes containing these proteins do not replicate DNA [27]. Electron microscope observations demonstrated that multi-nucleated muscle fibers arise by coalescence of individual cells [26]. The formation of myofibrils was found to begin in myoblasts in very early stages of development [2, 7], well before the axons of the motoneurons become connected to muscle fibers [15]. In fact, a large increase of the transcription of muscle specific mRNA [8, 19, 23, 25] and protein synthesis [8, 25] is reported in postmitotic myoblast and immediately after fusion of in vivo embryonic muscle cell, as well as in cultured mammalian or avian myoblasts.

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The aim o f the present work is to study nuclear R N P constituents during the development o f the chick e m b r y o muscle cells, in order to relate their changes with the large variations in gene expression that occur during cell differentiation and maturation.

Materials and methods Chick embryos (White Leghorn) were used. Their development was estimated according to the classification of Hamburger and Hamilton [16]. The stages were selected taking into account different processes in the differentiation of muscle tissue: 1) Hamburger and Hamilton's stage 11 (about 2 days of incubation), the mesoderm of the neck is organizing in somites; 2) stage 19 (3 to 3.5 days) in the somites of the neck, the myotome is formed and myoblasts are frequently in mitosis; 3) stage 28 (5.5 to 6 days) the fusion of the cell membranes of myoblasts, as well as the synthesis of specific muscle proteins begin; 4) stage 39 (13 days) myotube state of the muscle cells, the synthesis of specific proteins continues; 5) stages 40 and 41 (14 to 15 days of incubation) are intermediate stages with myotubes but without inner'vation; 6) stages 42 and 43 (16 and 17 days of incubation) bundles of myofibrils with their Z lines in register are abundant; 7) new born (22 days) the muscle is completely differentiated and functional. Three specimens of each stage were studied. 2-3-day-old embryos were fixed by dropping 4°70 paraformaldehyde and 2.5°70 glutaraldehyde in 0.2 M phosphate buffer (pH 7.2) on the presumptive neck region. Small pieces of the dorsal portion of the neck of older embryos were fixed by immersion in 2.5°/o glutaraldehyde in same buffer. A few samples were postfixed in osmium tetroxide. Embedding was carried out with an epoxy resin (glycide ether 100, Merck). The sections were stained with uranyl acetate and lead citrate or with Bernhard's EDTA method preferential for RNP [5]. Ten pictures of nuclei myoblasts, myotubes or differentiated muscle cells of each specimen were taken with an EM-10 (Carl Zeiss) electron microscope at fixed magnifications (monoflop on). l-tzm-thick sections stained with toluidine blue were employed for corroborating the stage of development of the cells. A silver impregnation technique [4] was used for the demonstration of synaptic endings.

Fig 1. Embryo of stage 11 (see Materials and methods). The inset shows a light micrograph of a transversal section of a somite. The elongated cells are the myoblasts of the dermatomyotome. 512 x . Electron micrograph of a section stained with EDTA procedure preferential for RNP depicting a nucleus of a myoblast showing a large nucleolus (n) and a cluster of interchromatin granules (i). 20000 x .

Morphometry Nuclear and nucleolar volumes were estimated measuring their larger diameter and their maximal normal diameter in light microscope using these thick sections. The numerical density of PCG and the fraction of the nuclear space occupied by RNP structures were analyzed on electron micrographs of non-tangential sections of nuclei. The latter parameter was estimated by the point fraction method [28]. A transparent grid was superimposed to the pictures of EDTA-stained sections, the number of crosses coinciding with all non-nucleolar dark stained structures was divided by the number of crosses laying inside the nuclear area (nucleolus and compact chromatin areas excluded).

Results

Electron microscopy

nucleolus, dispersed chromatin and a b u n d a n t R N P fibrils and interchromatin granules (fig 2). At this stage the cytoplasm o f myoblasts is mainly occupied by polysomes, few of them b o u n d to the endoplasmic reticulum. There are rod-shaped mitochondria, but no myofibrils could be found.

Stage 28 Myoblasts appear as elongate cells, which are frequently in close contact (fig 3, inset). They have nuclei similar to those o f the preceding stage, with dispersed chromatin, a prominent nucleolus, a b u n d a n t R N P fibrils and interchromatin granules, but few perichromatin granules (fig 3). Polysomes are still the main cytoplasmic c o m p o nent, but in some o f them, myosin and actin filaments are found.

Stage 11 The d e r m a t o m e - m y o t o m e portion o f the somite presents elongated cells (fig 1, inset). The nucleus o f these cells has a very large nucleolus, dispersed chromatin and large groups o f interchromatin granules (fig 1).

Stage 19 Myoblasts are situated in the m y o t o m e (fig 2, inset). They are densely arranged cells with a nucleus with prominent

Stage 39 Myotube stage, fusion is complete, the nuclei occupy a central position, myofibrils are peripheral (fig 4, inset). The nucleus has more compact chromatin than previous stages. Dispersed fibrils and interchromatin granules are the most a b u n d a n t nuclear R N P c o m p o n e n t s (fig 4). Large groups o f interchromatin granules are no longer visible. The nucleoli present frequently large fibrilar centers.

Ribonucleoproteins during muscle differentiation

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Fig 2. Myoblasts of stage 19 in the myotome. Inset l 000 x. Electron micrograph of a section stained with EDTA procedure. The nucleus shows a large nucleolus (n) and clumps of intercbromatin granules (i). In the cytoplasm there are abundant polysomes (p), but not myofibrils. 24000 x .

Fig 3. Stage 28. The inset shows elongated myoblasts in close contact. 384 x. The nucleus presents a nucleolus (n). EDTA staining procedure. 25000 x.

Stages 40 and 41 In these stages the nuclei are in the periphery of the muscle fibers. The myofibrils are abundant and in most of the cells aligned parallel to the.long axis of the fiber (fig 5, inset). Nuclear and cytoplasmic features are intermediary between those of the preceding stage and those of the innervated fibers. Most of the nuclei have a thick layer of dense chromatin, one or two nucleoli smaller than those of the younger embryos, without prominent fibrillar centers, abundant RNP fibrils and interchromatin granules, but few perichromatin granules (fig 5).

nique described by Barroso-Moguel and Costero [4]. In stage 39 the nuclei are central and myofilaments are thin and incipient, and no argyrophil nerve fiber can be seen (fig 8a). Multiple nerve fibers and button shaped nerve endings are impregnated by silver in the stage 43 (fig 8b).

Stages 42 and 43 Bundles of myofibrils with their Z bands in register are densely packed in the cytoplasm (fig 6, inset). The general features of the nucleus are similar to the preceding stage, but perichromatin granules are frequently found (fig 6). New born Morphologically mature muscle fiber. Perichromatin granules are abundant and frequently appear in clusters (fig 7). Silver impregnations In order to determine when differentiating muscle cells are innervated, samples of cervical muscle of embryos of the stages 39 and 43 were silver impregnated with the tech-

Morphometry The numerical density (N//z 2) of perichromatin granules is low in the stages 11, 19, 28 and 39. In fact, there is between 0.22 and 0.41 perichromatin granule per ffm 2. There is a small but significant increase of this parameter between stage 39 and 40. The frequency of perichromatin granules of stage 40 is conserved in stage 41, it increases sharply in stage 42, reaches its peak value in stage 43 and decreases slightly in the new born (fig 9). The nuclear volume does not show large changes during the increase in the numerical density of PCGs (stages 39 to new born), indicating that this variation is an actual augmentation in their quantity per nucleus (fig 10). The nucleolar volume shows a steady decrease between stage 11 and 43, increasing significantly between the stage 43 and the new born (fig 11). Figure 12 shows that during the differentiation of muscle cells there are no important changes in the frequency of RNP structures.

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Fig 4. Stage 39. The light micrograph shows several myotubes with central nuclei and longitudinal striation in the cytoplasm. l 000 x. The electron micrograph depicts a nucleolus (n), abundant interchromatin granules (i), and masses of compact chromatin (c) bleached by the EDTA staining procedure. In the cytoplasm there are bundles of myofibrils (m). 24000 x.

Fig 5. Stage 40. The inset is a low power electron micrograph of an uranyl acetate-lead citrate stained section, showing the cytoplasm of two developing muscle fibers filled with parallel bundles of myofibrils. The nucleus is located in the periphery of one fiber. 6 300 x. The larger picture shows a section stained with EDTA method preferential for RNP. The compact chromatin (c) is clear gray. RNP are darkly stained, i, interchromatin granules. Arrows, perichromatin granules. 25 000 x.

Discussion

The synaptic influences on the development of the postsynaptic cell are well known [6]. The present results suggest that the variation in the number of PCG is due to synaptogenesis. Similar changes in the frequency of PCG have been seen in motoneurons during the development of the synaptic endings [35]. These modifications involving preand post-synaptic cells are probably due to an alteration of the rate of transcription to transportation to the cytoplasm of m R N A similar to that caused in the target cells by experimental alterations of hormonal concentrations [10, 32, 33]. Transport of m R N A from the nucleus to cytoplasm was considered to be one of the possible events controlling the availability of messenger for translation in eukaryotes [24]. The lack of significant changes in the total non-nucleolar RNP components and in the nuclear volume during the increase of P C G demonstrates a close resemblance of the phenomena occurring in muscle cells and motoneurons during synaptogenesis [35, 36]. The experiments changing the relation transcription/transportation by deprivation and administration of estradiol or testosterone, also bring about changes of the frequency of PCG without large alterations in the abundance of other RNP constituents of the nuclei of target tissues [10, 33]. All these data suggest that perichromatin granules represent a component

The results demonstrate a large increase in the frequency of perichromatin granules concomitant with the innervation of the muscle and the development of the effector synapsis, the motor end plate. At these stages the development of myotubes into myofibers is very advanced or complete and the periodic structures of the myofibrils are clearly visible. Posttranscriptional regulation of muscle-specific genes has been reported to occur by different mechanisms (see [29]). Medford et al [19] suggested the existence of a control affecting the stability of newly transcribed m R N A . Translational control RNAs have been isolated in chick embryo maturating muscle [17, 18]. They have been found in rat embryonic muscle at advanced stages of maturation, a translation inhibition caused by a poly A - RNA [29]. However, several reports describe coinciding emergence of muscle-specific protein m R N A being mainly transcriptionally controlled [l, 8, 23, 25]. However, a posttranscriptional modulation was also found [1]. It is of interest to note that the transition of mitotic dividing myoblasts to postmitotic cells in the process of fusion into myotubes, which takes place between stages 19 and 28, does not bring about any change in the abundance of PCG.

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of nuclear R N P submitted to a strong transportation control, while other nuclear RNP structures as peri- and interchromatin fibrils are not so sensible to variations of the transportation. This interpretation is in accordance with the finding that in the developing muscle there are two simultaneous types of gene expression controls; 'housekeeping' proteins are subject to transcriptional regulation, and muscle-specific ones are transcriptionally and posttranscriptionally controlled [9]. The changes in nucleolar volume are similar to those found during neuroblast development [36], that is, large nucleoli at the stages in which cells are in mitotic cycle, a decrease during morphological and functional differentiation and a final increase coincident with functional maturation. These findings are in agreement with the changes of the rate of synthesis of r R N A in differentiating rat striated muscle cell line L6 [38]. The transcription of r R N A is large in proliferating tnyoblasts and decreases following myotube formation [38]. The ensemble of these results supports the well known direct relation between the nucleolar volume changes and the rate of transcription of r R N A [34].

Acknowledgments This work was partially supported by grants PADEP FC-9119 and FC-9131. The authors would like to thank M and C Alejandro Martinez Mena for light microscope photography.

References 1 Affara N, Robert B, Jacquet M, Buckingham M, Gros F (1980) Changes in gene expression during myogenic differentiation. I. Regulation of messenger RNA sequences expressed during myotube formation. J Mol Biol 140, 441-458 2 Allen E, Pepe F (1965) Ultrastructure of developing muscle cells in the chick embryo. A m J A n a t 116, 115-148 3 Bachellerie JP, Puvion E, Zalta JP (1975) Uitrastructural organization and biochemical characterization of chromatinRNA-protein complexes isolated from mammalian cell nuclei. Fur J Biochem 58, 327-337 4 Barroso-Moguel R, Costero I (1962) Argentaffin cells of the carotid body tumor. A m J Pathol 41, 389-403 5 Bernhard W (1969) A new staining method for electron microscopical cytology. J UItrastruct Res 27, 250-265

Ribonucleoproteins during muscle differentiation 6 Black I, Geen S (1973) Trans-synaptic regulation of adrenergic neuron development: inhibition by ganglionic blockade. Brain Res 63,281-302 7 Dessouky D, Hibbs R (1965) An electron microscope study of the development of the somatic muscle of the chick embryo. A m J Anat 116, 523-566 8 Devlin R, Emerson C (1979) Coordinate accumulation of contractile protein mRNA during myoblast differentiation. Dev Biol 69, 202-216 9 Endo T, Nadal-Ginard B (1987) Three types of musclespecific gene expression in fusion-blocked rat skeletal muscle cells: translational control in EGTA treated cells. Cell 49, 515-526 l0 Echeverria O, Pag~in-Santini R, V~izquez-Nin GH (1991) Effects of testosterone on ribonucleoprotein components of the nucleus of prostate epithelial cells. Biol Cell 72, 223-229 I l Fakan S, Leser G, Martin TE (1984) UItrastructural distribution of nuclear ribonucleoproteins as visualized by immunocytochemistry on thin sections. J Cell Bio198,358-363 12 Fakan S, Leser G, Martin TE (1986) Immunoelectron microscope visualization of nuclear ribonucleoprotein antigens within spread transcription complexes. J Cell Biol 103, 1153-1157 13 Fakan S, Puvion E (1980) The ultrastructural visualization of nucleolar and extranucleolar RNA synthesis and distribution, lnt Rev Cytol 65, 255-299 14 Fakan S, Puvion E, Spohr G (1976) Localization and characterization of newly synthesized nuclear RNA in isolated rat hepatocytes. Exp Cell Res 99, 155-164 15 Hamburger V (1975) Cell death in the development of the lateral motor column of the chick embryo. J Comp Neurol 160, 533-546 16 Hamburger V, Hamilton AL (1951) A series of normal stages in the development of the chick embryo. J Morpho188, 49-92 17 Heywood S (1986) tcRNA as a naturally occurring antisense RNA in eukaryotes. Nucleic Acids Res 14, 6771-6772 18 Heywood S, Kennedy D, Bester A (1974) Separation of specific factors involved in the translation of myosin and myoglobin messenger RNAs and the isolation of a new RNA involved in translation. Proc Natl Acad Sci USA 71, 2428- 2431 19 Medford R, Nguyen H, Nadal-Ginard B (1983) Transcriptional and cell cycle-mediate regulation of myosin heavy chain gene expression during muscle cell differentiation. J Biol Chem 258, 11063-11073 20 Monneron A, Bernhard W (1969) Fine structural, organization of the interphase nucleus of mammalian cells. J Ultrastruct Res 27, 266-288 21 Moyne G, Nash RE, Puvion E (1977) Perichromatin granules and other nuclear components in isolated hepatocytes treated with cortisol and cycloheximide. Biol Cell 30, 5-16 22 Puvion E, Moyne G (1978) Intranuclear migration of newly synthesized extranucleolar ribonucleoproteins. A high resolution quantitative autoradiographical and cytochemical study. Exp Cell Res 115, 79-88

165

23 Saidapet C, Munro H, Valgeirsd6ttir K, Sarkar S (1982) Quantitation of muscle-specific mRNA by using cDNA probes during chicken embryonic muscle development in ovo. Proc Natl Acad Sci USA 79, 3087-3091 24 Schwartz H, Darnell J (1976) The association of protein with the polyadenylic acid of HeLa cell messenger RNA: evidence for a 'transport' role of a 75 000 molecular weight polypeptide. J Mol Biol 104, 833-851 25 Shani M, Zevin-Sonkin D, Saxel O, Carmon Y, Katcoff D, Nudel U, Yaffe D (1981) The correlation between the synthesis of skeletal muscle actin, myosin heavy chain, and myosin light chain and the accumulation of corresponding mRNA sequences during myogenesis. Dev Bio186, 483-492 26 Shimida Y (1971) Electron microscope observations on the fusion of chick myoblasts in vitro. J Cell Bio148, 128-142 27 Stockdale F, Holtzer H (1961) DNA synthesis and myogenesis. Exp Cell Res 24, 508-520 28 Underwood E (1970) Quantitative stereology, AddisonWesley, London, 23-30 29 Vanderburg C, Nathanson M (1988) Posttranscriptional control of embryonic rat skeletal muscle protein synthesis Control at the level of translation by endogenous RNA. J Cell Biol 107, 1085-1098 30 V~izquez-Nin GH, Bernhard W (1971) Comparative ultrastructural study of perichromatin- and Balbiani ring granules. J Ultrastruct Res 36, 842-860 31 V~zquez-Nin GH, Echeverria OM, Fakan S, Leser G, Martin TE (1990) Immunoelectron microscope localization of snRNPs in the polytene nucleus of salivary glands of Chironomus thummi. Chromosoma 99, 44-51 32 V~izquez-Nin GH, Echeverria OM, Molina E, Fragoso J (1978) Effects of ovariectomy and estradiol injection on nuclear structures of endometrial epithelial cells. Acta Anat 102, 308-318 33 V~izquez-Nin GH, Echeverria OM, Pedron J (1979) Effects of estradiol on ribonucleoprotein constituents of the nucleus of cultured endometrial epithelial cells. Biol Cell 35, 221-228 34 V~izquez-Nin GH, Echeverria OM, Zavala G, Jim6nezGarcia LF, Gonz~ilez MA, Parra R (1986) Relations between nucleolar morphometric parameters and pre-rRNA synthesis in animal and plant cells. Acta Anat 126, 141-146 35 V~izquez-Nin GH, Ortega-Rangel JA, Echeverria OM (1980) Nuclear aspects of neuroblast differentiation in the chick embryo. Biol Cell 39, 143-146 36 V~zquez-Nin GH, Ortega-Rangel JA, Echeverria OM, Parra MR, Jim6nez-Garcia LF (1983) Changes in the nuclear ribonucleoprotein constituents and chromatin disposition during neuronal differentiation and maturation. Biol Cell 48, 17-24 37 Watson ML (1962) Observations on a granule associated with chromatin in the nuclei of cells of rat and mouse. J Cell Biol 13, 162-167 38 Zahradka P, Larson DE, Sells LB (1991) Regulation of ribosome biogenesis in differentiated myotubes. Mol Cell Biochem 104, 189-194