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Progress of Cell Proliferation in Striated Muscle Tissues during Development of the Mouse Tongue
J. Nagata and
A. Yamane*
Department of Pharmacology, Tsurumi University School of Dental Medicine, 2-1-3 Tsurumi, Tsurumi-ku, Yokohama 230-8501, Japan;
Correspondence: * corresponding author, yamane-a{at}tsurumi-u.ac.jp
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ABSTRACT
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The developmental stages of and places for the proliferation of tongue muscle cells have not yet been determined. To determine the stages of and places for proliferation between embryonic day (E) 9 and birth, we analyzed the expression of cyclin D1 mRNA and the immunolocalization for proliferating cell nuclear antigen (PCNA). The ratio of PCNA-positive nuclei to total nuclei (PCNA-labeling index) was obtained in the anterior, middle, and posterior regions. Cyclin D1 mRNA was highly expressed between E11 and E13, but decreased thereafter until birth. The distribution of PCNA-positive cell nuclei was consistent with that of myogenic cells in the occipital somites at E9. The PCNA-labeling index was highest at E11, then decreased until birth without a significant difference among the 3 regions. These findings suggest that some tongue muscle progenitor cells begin proliferation in the occipital somites at E9, and that the proliferation in the whole tongue region occurred most actively between E11 and E13, then decreased until birth without regional differences.
Key Words: proliferation • differentiation • mouse • tongue • PCNA
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INTRODUCTION
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The development of skeletal muscle consists of 5 phases (Buckingham et al., 2003): phase 1 (commitment)—muscle progenitor cells are committed to becoming muscle cells in somites; phase 2 (migration)—the muscle progenitor cells migrate to presumptive places where muscles are formed; phase 3 (proliferation)—the muscle progenitor cells proliferate, increase in number, and become myoblasts; phase 4 (differentiation)—the myoblasts fuse to become multinucleated myotubes; and phase 5 (maturation)—the multinucleated myotubes mature to myofibers, such as fast- or slow-twitch myofibers.
In the development of striated muscle in the mouse tongue, all 5 phases, except for proliferation, have been studied extensively. Tongue muscle progenitor cells undergo commitment in the occipital somites (somites 2 ~ 5) between embryonic days (E) 8 and E11 (Mayo et al., 1992). They migrate to the first branchial arch between E9 and E11 (Mayo et al., 1992), and hepatocyte growth factor (HGF) plays an important role in the migration (Amano et al., 2002). Tongue myoblasts fuse and become myotubes between E13 and E15 (Yamane et al., 2000). Insulin-like growth factor-I and transforming growth factor- promote the differentiation of tongue myoblasts, whereas HGF inhibits it (Yamane et al., 2003). The myotubes mature to fast-twitch myofibers between E15 and birth (Yamane et al., 2000).
Little is known about cell proliferation in striated muscle tissues in the tongue, except for the stimulation of proliferation by HGF (Amano et al., 2002). It would be useful if we had better understanding of the progression profile of cell proliferation in the developing muscle tissues of the mouse tongue. To identify the stages and places in which the proliferation of tongue muscle cells begins and occurs most actively during the development of tongue muscles, we measured the mRNA expression of cyclin D1, a marker for cell proliferation, and determined the distribution of the proliferating cells in the tongue muscle tissues by immunostaining for proliferating cell nuclear antigen (PCNA) in the somites 2 ~ 5 at E9 and the tongue at E11 ~ birth. We chose these developmental stages to observe the complete development of tongue muscle.
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MATERIALS & METHODS
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Tissues
Pregnant ICR mice were purchased from Nippon Clea (Tokyo, Japan). Tongues for PCR analysis were removed from E11, E13, E15, E17, and newborn mice, immediately frozen by liquid nitrogen, and then stored at -80°C until use. For PCR analysis, 5 samples were collected at each developmental stage. Four tongues at E11 and 2 at E13 were collected because of small tongue size and treated as 1 sample. One tongue at E15 - birth was treated as 1 sample. Tongues at E9 - birth were immediately fixed in Bouin’s solution for immunohistochemistry. Our experimental protocols concerning animal handling were approved by the Institutional Animal Care Committee of Tsurumi University School of Dental Medicine.
RNA Extraction, Reverse Transcription, and Competitive Polymerase Chain-reaction (competitive PCR) Amplification
RNA extraction, treatment with deoxyribonuclease I, and competitive RT-PCR amplification were performed as previously described (Yamane et al., 2000). The specificity of the primer pair for cyclin D1 was previously determined by Qian et al.(1998). The amplification products were separated by electrophoresis on an agarose gel containing ethidium bromide. The logarithmic values of the fluorescent intensity ratios of the cyclin D1 bands (lower bands in Fig. 1A ) to their respective competitor bands (upper bands in Fig. 1A) were plotted against the logarithmic values of cyclin D1 cDNA concentrations (a standard curve, Fig. 1B). We used the logarithmic value of the fluorescent intensity ratio of cyclin D1 to competitor bands in each sample to calculate the quantity of endogenous cyclin D1 mRNA, based on the line formula derived from the standard curve (Fig. 1B). The quantity of cyclin D1 mRNA in each tongue sample was normalized by the quantity of glyceraldehyde-phosphate dehydrogenase (GAPDH) mRNA (Yamane et al., 2000). The resulting ratio value was expressed as a percent value relative to the mean value at E11.
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Figure 1. Electrophoretic gel pattern (A) of cyclin D1 cDNA standard and its competitor after competitive PCR, and the standard curve (B). Chronological changes in the expression level of cyclin D1 mRNA in mouse tongues at embryonic days (E) 11, 13, 15, 17, and birth as analyzed by competitive RT-PCR (C). Each point with its vertical bar represents the mean ± 1 SD of 5 samples. The vertical axis is expressed as a percentage of the mean value at E11.
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Immunohistochemistry
Tongues from 3 embryos were used at each developmental stage. We used a cryostat to prepare sagittal sections (at a 10-µm thickness) of whole embryos at E9 and whole tongues at E11 ~ birth. To detect the proliferating tongue cells and to identify myogenic cells, differentiating myoblasts, myotubes, and myofibers, we performed immunolocalizations for PCNA, desmin, a marker for myogenic cells, and fast myosin heavy-chain (fMHC), a marker for muscle differentiation, using antibodies purchased from Progen Biotechnik GmbH (Heidelberg, Germany), Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA), and Sigma-Aldrich Inc. (St. Louis, MO, USA), respectively.
The numbers of PCNA-positive and -negative cell nuclei were counted in a rectangular area of 92,400 µm2 in the whole portion of the tongue at E11 (Fig. 2E) and in rectangular areas of 154,000 µm2 in the anterior, middle, and posterior regions of tongues at E13, E15, E17, and birth (Fig. 2F). We calculated the ratios of PCNA-positive cell nuclei to total cell nuclei (PCNA-labeling index) and averaged them to obtain a mean value for 3 tongues in each region at each developmental stage. For control staining, the primary antibody was replaced with PBS or normal mouse or rabbit IgG (Fig. 2J).
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Figure 2. Immunostaining images for PCNA (A-C) and desmin (D) in the somites 2, 3 (A), 4, 5 (B), and 3 (C,D) of the E9 mouse embryo. The diagrammatic representations show sagittal sections of tongues at E11 (E) and E13 ~ birth (F), viewed from the buccal side. The dotted rectangles indicate the regions in which proliferating cell nuclear antigen (PCNA)-labeling indices were obtained. Immunostaining images for PCNA (G,H,K-N) and fast myosin heavy-chain (I,O-R) in the whole portion of the E11 tongue (G,I), and in the anterior (K,L,O,P), middle (M,Q), and posterior (N,R) regions of E13 tongues. A control staining image of a whole portion of an E11 tongue treated with normal mouse IgG instead of the antibodies (J). H, L, and P are higher-magnification images of the regions indicated by the rectangles in G, K, and O, respectively. White lines in L contour the areas which lack the PCNA-positive cell nuclei.
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Statistical Analysis
For multiple comparisons, we used Tukey-Kramer’s method to compare the mean values between 2 groups.
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RESULTS
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The electrophoretic gel pattern of cyclin D1 and its competitor (after competitive PCR) and the standard curve obtained for the cyclin D1 mRNA are shown (Figs. 1A , 1B, respectively). The formula for the standard curve was y = 0.575x - 1.413, and its correlation coefficient was 0.986 (p < 0.01). Cyclin D1 mRNA was expressed at a high level between E11 and E13 (Fig. 1C), and decreased thereafter by 43% (p < 0.01) until birth. However, more than 50% of the expression levels at E13 were maintained, even at birth.
At E9, most of the cell nuclei in the somites 2 ~ 5 were immunostained brown with an antibody for PCNA (Figs. 2A, 2B). Only a few cells were immunostained for desmin (Fig. 2D), and their distributions appear to be consistent with the distributions of PCNA-positive cell nuclei (Fig. 2C). At E11, most of the cell nuclei were immunostained for PCNA, and the distribution of the PCNA-positive cell nuclei appeared to be uniform in the whole portion of tongue (Figs. 2G, 2H). Immunostaining for fMHC was not detected in the whole portion of the E11 tongue (Fig. 2I). At E13, a great many PCNA-positive cell nuclei were observed in the whole portion of the tongue (Figs. 2K–2N). Areas that lacked the PCNA-positive cell nuclei emerged in the anterior region (Figs. 2K, 2L) (the typical examples of such areas were contoured by white lines in Fig. 2L), and these areas appeared to be consistent with the areas where fMHC was expressed (Figs. 2O, 2P). Such areas were not clearly evident in the middle and posterior regions (Figs. 2M, 2N, 2Q, 2R).
At E15, a great many PCNA-positive cell nuclei were distributed in the whole portion of the tongue without a marked regional difference (Figs. 3A–3D). At E17, the density of both the PCNA-positive and -negative cell nuclei (Figs. 3E–3H) appeared to be lower than that of nuclei at the earlier stages. The distribution pattern of PCNA-positive cell nuclei in the anterior region appeared to be different from that of those in the middle and posterior regions, due to wider spaces among the myofibers immunostained for fMHC in the anterior (Fig. 3I) region relative to those in the middle (Fig. 3J) and posterior (Figs. 3K, 3L) regions.
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Figure 3. Immunostaining images for PCNA (A-H) and fMHC (I-L) in the anterior (A,E,I), middle (B,F,J), and posterior (C,D,G,H,K,L) regions of E15 (A-D) and E17 (E-L) tongues. Chronological changes in the PCNA-labeling index in mouse tongues between E11 and birth (M). D, H, and L are higher-magnification images of the regions indicated by the rectangles in C, G, and K, respectively.
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The PCNA-labeling index was 81.2 ± 0.5% in the whole portions of E11 tongues, but gradually and significantly decreased by more than 20% (p < 0.01 ~ 0.001) in the 3 regions until birth (Fig. 3M). However, the labeling indices in the 3 regions exceeded 50%, even at birth. No statistically significant difference in the PCNA-labeling index was found among the 3 regions throughout the developmental stages studied, indicating that the termination of cell proliferation progresses almost simultaneously throughout the tongue muscle.
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DISCUSSION
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In the present study, the consistency of immunolocalization for desmin-positive cells and PCNA-positive cell nuclei in the occipital somites at E9 (Figs. 2C, 2D) suggests that some tongue muscle precursor cells begin proliferation in the occipital somites at E9. The analyses of cyclin D1 mRNA expression (Fig. 1C) and the PCNA-labeling index (Fig. 3M) indicated that cell proliferation in the muscle tissues of the mouse tongue occurs most actively between E11 and E13. In addition, the expression of fMHC (Figs. 2O–2R) verified our previous result, that tongue myoblasts begin to differentiate and become myotubes at around E13 (Yamane et al., 2000). Proliferation and differentiation are regarded as discrete processes, in which the myogenic cells stop dividing, become myoblasts, then enter into the differentiation process. Thus, it seems reasonable that proliferation occurs most actively before the onset of myoblast differentiation.
In the present study, the PCNA-labeling index (Fig. 3M) and distribution of PCNA-positive cell nuclei (Figs. 2G, 2H, 2K–2N, 3A–3H) indicated that cell proliferation progresses without a marked regional difference in the whole portion of tongue muscles, except that the distribution pattern of proliferating cells in the anterior regions differs slightly from that of those in the middle and posterior regions at E13, E17, and birth. Previous studies have reported several differences in the developmental origin and innervation between the anterior and middle regions (anterior two-thirds) and the posterior region (posterior one-third) of the mouse tongue (Kaufman, 1992). In addition, since tongue myogenic cells are reported to be derived from the somites 2 ~ 5 (Noden, 1983), the distance of migration from the somites seems different among the tongue regions. The progress of cell proliferation in the muscle tissues of the mouse tongue may be independent of developmental origins, innervation, and the distance of migration.
At the newborn stage, the PCNA-labeling indices in the 3 regions exceeded 50% (Fig. 3M), suggesting that more than half of the cells in tongue muscle tissues maintain a proliferative activity. During post-natal development, the mouse tongue more than doubles in weight (Yamane et al., unpublished data). The tongue myogenic cells with a proliferative activity at the newborn stage seem to contribute to the post-natal growth of the mouse tongue by increasing the number and/or size of myofibers by several means: increasing numbers by proliferation, fusion, and becoming myofibers by themselves; and/or increasing size by proliferation and fusion to pre-existing myofibers that are formed during embryonic development (Donoghue and Sanes, 1994).
Previous studies have identified the most active stages of the commitment, migration, differentiation, and maturation phases of the development of striated muscle in the tongue (Fig. 4). Further, since the present study found that some tongue muscle precursor cells begin proliferation at E9, and that proliferation occurs most vigorously between E11 and E13, all 5 phases of skeletal muscle development have now been identified (Fig. 4). These findings will allow us to better focus on the most suitable and key developmental stage for studies of the development of striated muscles in the tongue.
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ACKNOWLEDGMENTS
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We thank Professor M. Chiba, Tsurumi University School of Dental Medicine, for his support and encouragement throughout the present study. The present study was supported by grants-in-aid for funding scientific research (Nos. 13671955 and 16591871 to A.Y.), Bio-ventures and High-Technology Research Center from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, and by the Science Research Promotion Fund from the Promotion and Mutual Aid Corporation for Private Schools of Japan.
Received for publication November 11, 2003.
Revision received September 3, 2004.
Accepted for publication September 7, 2004.
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Journal of Dental Research, Vol. 83, No. 12,
926-929 (2004)
DOI: 10.1177/154405910408301207
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ABSTRACT
INTRODUCTION