The molecular, genetic and cellular bases for skeletal muscle growth and regeneration have been documented in a number of vertebrate species. Formation of skeletal muscle of fish differs in several aspects when compared with mammals. These include the spatial separation of fast and slow muscle precursor cells during somite formation in embryos. The other unique feature observed in fish muscle is the increase of muscle mass during postembryonic growth by recruitment of new fibers, called hyperplasia. Fish muscle grows by stratified and mosaic hyperplasia at larval and adult stages, respectively. During stratified hyperplasia in larvae, new fibers are formed mainly at the dorsal and ventral extremes of myotome and additionally in a layer between superficial slow and deep fast fibers, whereas mosaic hyperplasia occurs throughout the whole myotome of adult fast muscle. Sarcomeric myosins including skeletal and cardiac ones are composed of two heavy chains (MYHs) and four light chains, whereas fiber characteristics are well correlated with the expression of MYH isoforms. Meanwhile, fish are known to possess highly conserved MYH multigene family, although MYH genes (MYHs) are much more than their higher vertebrate counterparts. However, functional implications for such a high number of MYHs have remained to be elucidated.

The present study was carried out to investigate expression patterns of sarcomeric MYHs in various adult muscles of torafugu Takifugu rubripes, where the total genome database is publicly available. Fiber types were then characterized by histochemical methods in adult skeletal muscles. MYHs were also cloned from embryos and larvae, and analyzed for their expression. Finally, the members of paired box protein (Pax) gene family, Pax3 and Pax7, were characterized for their possible application as a marker for identification of muscle precursor cells.

1. Characterization of fiber types in adult skeletal muscles of torafugu

In the present study, myofibrillar ATPase was demonstrated in adult skeletal muscle of torafugu (body weight 290 g) by selective inhibition or activation of specific fiber groups after preincubation (2 - 3 min) either at acidic or alkaline pH. Fast muscle contained various fibers with different diameters. ATPase of fast fibers with large diameters was inactivated at pH 4.6, whereas that with small diameters was stable to this acidic pH. It was noted that the fibers with small diameters were more stable with those having smaller diameters. Such existence of fibers with small diameters in fast muscle suggests hyperplastic growth in adult fast muscle, since it has been reported that new fibers are formed by hyperplastic process in most adult fish which grow to a large final body size like torafugu. Meanwhile, most fibers in lateralis superficialis (LS) and erector and depressor (ED) slow muscles were resistant to pH 4.6, although some large-sized fibers were found to be slightly acid-labile. In contrast, ATPase of all fast fibers of juvenile torafugu was inactivated at pH 4.6, suggesting that the existence of different fibers in fast muscle is a distinct feature of muscle growth in adult.

NADH-diaphorase staining was performed to identify oxidative fibers in skeletal muscles according to Novikoff et al. (1961). All fibers in LS and ED slow muscles were positive for NADH-diaphorase stain, suggesting that these. muscles have oxidative metabolism. Importantly, fibers in LS slow muscle with large diameters adjacent to fast muscle showed lower NADH-diaphorase reaction compared with those in a superficial region with small diameters, demonstrating that the former fibers have an intermediate oxidative potential. In contrast, none of fibers in fast muscle was stained for NADH-diaphorase.

2. Expression patterns of sarcomeric mvosin heavy chains in adult torafugu muscles

cDNAs encoding sarcomeric MYHs were amplified by RT PCR using MYH-specific degenerate primers. In total, seven sarcomeric MYHs were cloned from adult fast, slow and cardiac muscles of torafugu (body weight 1 kg). The nomenclature of torafugu MYHs found in the present study is described following Ikeda et al. (2007) who found 20 sarcomeric MYHs by in silica approach on the total genome database. Three MYHs, MYHM86.4, MYHM8248 and MYHM880, were cloned exclusively from fast, slow and cardiac muscles, respectively, whereas two MYHs, MYHM2528-1 and MYHMIO34, were cloned from both fast and slow muscles and another two MYHs, MYHM2126-2 and MYHMS, from both slow and cardiac muscles. Evolutionary relationships of torafugu MYHs with those reported from other fish were studied by phylogenetic analysis on the deduced amino acid sequences using the neighbor joining method. MYHM86-1, MYHM2528-1 and MYHM1034 belonged to fast type as they were placed in the same clade representing fast-type MYHs from other fish on the phylogenetic tree. MYHM8248 and MYHM2126-2 belonged to slow and cardiac types, respectively. MYHMS and MYHM880 were found to have appeared in an early evolution of MYHs and thus regarded to belong to ancestral slow/cardiac type.

The frequencies of cDNA clones encoding above-mentioned MYHs in the cDNA clone libraries and relative mRNA levels determined by Northern blot analysis further revealed their tissue-specific expression in adult skeletal and cardiac muscles. The clones encoding fast-type MYHM86-I were most abundant in the cDNA clone library constructed from fast muscle. Both LS and ED slow muscles contained almost equally the clones of five MYHs including fast-type MYHM2528-1 and MYHM1034, slow-type MYHM8248, cardiac-type MYHM2126-2 and unique, slow/cardiac-type MYHMS. Among three types of MYH clones from cardiac muscle, cardiac-type MYHM2126-2 was most abundant.

In situ hybridization was performed to localize the transcripts of MYHs in skeletal muscles of adult -torafugu (body weight 275 g). The transcripts of fast-type MYHM86-1 were found in all fibers with different diameters in fast muscle. Fast fibers with smaller diameters tended to have transcripts of fast-type MYHM2528-1 more abundantly. Given that such fast fibers with small diameters are generated by hyperplasia, the expression of MYHM2528-1 is thought to be deeply correlated with generation of these fibers and those with the smallest diameter are considered to be most newly formed.

The fibers expressing slow-type MYHM8248 resided a superficial part of LS slow muscle with small diameters. Fibers expressing cardiac-type MYHM2126-2 also occupied a superficial layer in LS slow muscle with small diameters. Interestingly, fast-type MYHM2528-1 was expressed in fibers of LS and ED slow muscles with large diameters which showed an intermediate oxidative potential as described above, implying their possible involvement in muscle generation by hyperplasia.

The expression levels of fast-type MYHs were also investigated in both wild and farm-cultured torafugu individuals (body weight 0.8 - 1 kg). Among three fast-type MYHs, the relative mRNA levels of MYHM2528_1 were significantly higher in wild than farm-cultured fish.

3. Expression patterns of myosin heavy chain genes in torafugu at embryonic and larval stages

Six sarcomeric MYHs were cloned from embryos and larvae of torafugu laboratory-reared at 18°C by using the same method as described for adult torafugu. These included four fast-type MYHs, MYHM743, MYHM86-2, MYHM2528-1 and MYHM1034, cardiac-type MYHM2126-1 and slow/cardiac-type MYHMS. MYHM743 and MYHA186-2 have been reported by Ikeda et al. (2007) using gene specific primers. Among all fast-type MYHs, the cDNA clone encoding MYHM743 was most abundant in all clone libraries from embryos and larvae, followed by those encoding MYHM86-2 in embryos [5 and 7 days post fertilization (dpf)] and MYHM2S28-1 in larvae (10 and 16 dpf). The cDNA clone encoding MYHi111f34 was marginally observed in clone libraries from larvae. While cDNA clones of slow/cardiac-type MYHmS were identified in all clone libraries from embryos and larvae, their abundance in larvae was found to be much lesser than in embryos.

RT-PCR using highly specific primers based on the 3' untranslated region nucleotide sequences of MYHs showed that the transcripts of MYHM743 appeared in embryos at 3 dpf, whereas those of MYHM86.2 in embryos at 4 dpf. These two MYHs continued to be expressed during embryonic and larval development, suggesting their involvement in muscle development. The transcripts of fast-type MYHM2528-1 appeared in embryos at 7 dpf and continued to be expressed at successive embryonic and larval stages, as well as in adult fast and slow skeletal muscles. The transcripts of slow/cardiac-type MYHM5 1S continued to be expressed from embryos at 3 dpf to larvae and as well in adult slow and cardiac muscles. Such expression patterns of MYHSI2528-1 and MYHMS in adult muscles were consistent with those described in the previous section

Whole mount in situ hybridization for embryos at 4 dpf with probes specific to fast-type MYHA186-2 and slow/cardiac-type MYHMS revealed that the former transcripts were localized in the whole embryonic myotome, whereas the latter transcripts were restricted to the superficial slow muscle as well as to the horizontal myoseptum. The transcripts of cardiac-type MYHAl2126-1 were localized adjacently to the notochord of embryos at 3 dpf.

4. Characterization of paired box protein genes as mvogenic precursor cell markers in torafugu

Paired box protein (Pax) genes play pivotal roles in the formation of. tissues and organs during development. This gene family encodes transcription factors characterized by the presence of paired box domain (PD), octapeptide motif and homeodomain. It has been reported that Pax3 and Pax7 regulate survival, proliferation and migration of myogenic precursor cells. In this context, Pax3 and Pax7 were cloned from torafugu embryos and adult fast skeletal muscle by using degenerate primers based on highly conserved amino acids in PD and homeodomain. Subsequent in silico analysis with the Fugu genome database (ver. 4.0) yielded two distinct genes each for Pax3 (Pax3a and Pax3b) and Pax7 (Pax7a and Pax7b). The 75th amino acid, glutamine (G1u75), from the N-terminus was replaced by proline in PD of Pax3b. Mammalian Pax3 and Pax7 both have alternatively spliced isoforms, differing in the presence or absence of G1u75 (Q+/Q-) in PD which affects the DNA-binding specificity (Vogan et al., 1996). One single cDNA clone encoding Pax3a had deletion of G1u75 in PD, suggesting the presence of alternatively spliced variants (Q+/Q-) for torafugu Pax3a. This was further supported by identification of two adjacent alternative 3' splice acceptor sites for torafugu which produce Pax3a Q+ (aagCAGGGA) and Q-(aagcagGGA) variants. Interestingly, torafugu Pax7b, but not Pax7a, had an insert encoding five amino acid residues (GEASS) in a C-terminal region of PD in two out of three cDNA clones. Genomic analysis showed two alternate splice donor cites at exon 4 of Pax7b which is responsible for forming two alternately spliced variants.

RT-PCR revealed that the transcripts of Pax3a, Pax3b, Pax7a and Pax7b were found to appear in embryos at 3 dpf and later developmental stages, suggesting their key roles during development. Interestingly, the transcripts of Pax7b were observed in adult skeletal muscles. Thus, Pax3 and Pax7 can be used to monitor muscle precursor cells.

Conclusion

Expression patterns of seven sarcomeric MYHs were determined in adult muscles of torafugu. While three MYHs were specifically expressed in either fast, slow or cardiac muscle of adult torafugu, four showed a mixed expression pattern, suggesting the functional significance of each MYH. Furthermore, six MYHs were cloned from embryos and larvae and found to be expressed sequentially during development. Fiber-type diversity was also demonstrated by in situ hybridization for MYH transcripts. This is an important step ahead in understanding fiber type diversity and associated muscle growth by hyperplasia specifically observed in larval and adult fish having an intermediate body size. Our study also greatly helps to understand functional significance of higher number of MYHs in fish.

魚類における筋形成は、速筋と遅筋の分離した構造や成体での筋線維数の増大など、哺乳類と比較して多くの違いがある。骨格筋や心筋の主要構成成分である横紋筋型ミオシンは2本の重鎖(MYH)と4本の軽鎖からなるが、筋線維の性質はMYH遺伝子(MYH)の発現により規定される。他の脊椎動物同様、魚類のMYHもマルチジーンファミリーを形成しているが、その構成数は高等脊椎動物よりはるかに多く、筋形成の過程でそれらがどのように発現し、筋肉の機能とどのように関連するか、その詳細は明らかでない。本研究は、トラフグTakifugu rubriipesを対象に、成体および発生過程胚および仔魚の筋肉におけるMYHの発現パターンを明らかにするとともに、筋芽細胞の維持や増殖に関わるPaxファミリーの構造と発現様式を解析した。

まず、酸性(pH4.6)処理に対する筋原線維ArPase活性の感受性の違いから、速筋には様々な直径の筋線維が含まれており、太い線維は酸性処理でArPase活性が不活化し、細い線維は安定であることが示された。一方、体の表層と背鰭下の遅筋の筋線維は同処理でも安定であった。また、仔魚では、速筋の筋線維は全て酸性処理で不活化された。さらにNADH-diaphorase染色では、背鰭下遅筋の筋線維は全て染色され、これらは酸化的代謝を行うと考えられた。一方、表層遅筋では、内側の比較的太い筋線維は、より表層にある細い線維に比して染色の程度が弱く、これらは中間的な酸化的代謝能を持つことが示唆された。速筋では全ての筋線維が染色されなかった。

続いて、成体トラフグ筋肉におけるMYHsの発現パターンを解析した。ランダムクローニングの結果、速筋、遅筋および心筋から7種類のMYHが得られ、うちMYHM86-1、MYHM8248およびMYHM880はそれぞれ速筋、遅筋および心筋特異的に、.MYHM2528-1、MYHM1034、MYHM2126-2およびMYHM5は複数の筋肉で検出された。分子系統解析の結果、MYHM86-1、MYHM2528-1およびMYHM1034は速筋型、、MYHM8248は遅筋型、MYHM2126-2は心筋型、MYHM5とMYHHM880は遅筋/心筋型にそれぞれ分類された。クローンの出現頻度分析とノーザンブロット解析の結果、速筋ではMYHM86-1が、心筋ではMYHM2126-2が最も優先的であり、遅筋ではMYHM2528-1、MYHM1034、MYHM8248、MYHM2126-2およびMYHM5がそれぞれほぼ等量ずつ発現していた。In situ hybridizationの結果、MYHM86-1の転写産物は、速筋の全ての筋線維で検出された。また、速筋中、細い筋線維ほどMYHM2528-1を多く発現する傾向がみられた。細い筋線維は成体で新しく形成されたものと考えられ、MYHM2528-1の発現と成体でのhyperplasiaとの関連が示唆された。MYHM8248と,MYHM2126-2を発現する筋線維は遅筋に分布した。興味深いことに、遅筋の中で比較的太く、中間的な酸化的代謝能を示す筋線維は速筋型のMYHM2528-1を発現していた。

また、トラフグ胚および仔魚についても、同様にMYHsの発現パターンを明らかにした。トラフグ胚および仔魚からは6種の横紋筋型.MYHがクローニングされ、ここには速筋型MYHM743、MYHM86-2、MYHM2528-1およびMYHM1034、心筋型MYHM2126-1、遅筋/心筋型MYHM5が含まれた。クローンの出現頻度は、胚から仔魚期まで通じてMYHM734が最も多く、受精後5日目と7日目の胚ではMYHM86-2が続いて、受精後10日目と16日目の仔魚ではMYHM2528-1が続いて多かった。MYHM1034は仔魚でのみ僅かに検出され、MYHM5は胚から仔魚期まで検出された。RT-PCRで各MYHの発現時期を調べたところ、MYHM743は受精後3日目、MYHM86-2は受精後4日目の胚で発現が始まり、その後仔魚期まで発現し、成体では検出されなかった。これらMYHは筋発生過程での働きが示唆される。一方、MYHM5は受精後3日目、MYHM2528-1は受精後7日目の孵化期の胚から発現が始まり、その後継続的に発現し、成体でも発現が認められた。これらは筋成長過程での働きが示唆される。In situ hybridizationの結果、受精後4日の胚では、MYHM86-2は筋節全体で、MYHM5は表層遅筋と水平筋隔で特異的に発現することが明らかになった。

Paxファミリーは様々な組織の発生や器官形成に関わるが、このうちPax3とPax7は筋芽細胞の増殖や維持等に関与する。そこで、トラフグについてもPaxの構造と発現様式を検討した。In silico解析で、トラフグゲノムにはそれぞれ2種類のパラログPax3aとPax3bおよびPax7aとPax7bが含まれることが明らかになった。哺乳類のPax3とPax7にはペアードドメイン(PD)の一次構造が異なるスプライシングバリアントがあり、その結果、DNA結合の特異性に違いが生じる。cDNAクローニングとゲノム解析の結果、トラフグPax3bとPax7bについても同様に、選択的スプライシングでPDの構造の異なるバリアントが形成されていた。RT-PCRの結果、4種のPax転写産物は受精後3日目以降仔魚期まで継続的に検出され、それらが筋発生過程で働くことが示された。さらにPax7bは成体でも発現し、同遺伝子が成体での筋形成に関与していることが示唆された。

以上、本研究は、トラフグ成体、胚および仔魚の筋肉の構造と機能的特徴を明らかにするとともに、それら筋肉で発現するMYHを同定し、その発現パターンの詳細を明らかにした。各MYHは筋肉の部位によって、あるいは発生過程において特徴的な発現様式を示し、それぞれに機能的分担があり、筋肉の性質や成長と密接に関わることが考えられた。本研究の成果は、魚類の筋肉の特徴的な構造と成長様式を理解するうえで、きわめて重要な基礎的知見であり、学術上、応用上貢献するところが少なくない。よって審査委員一同は本論文が博士(農学)の学位論文として価値あるものと認めた。