Research paperCharacterization of Pax3 and Pax7 genes and their expression patterns during different development and growth stages of Japanese pufferfish Takifugu rubripes
Introduction
Paired box protein (Paxs) genes play key roles in the formation of tissues and organs during development. This gene family encodes transcription factors that are characterized by the presence of the paired box domain (PD), an octapeptide motif and homeodomain (Chi and Epstein, 2002, Lang et al., 2007). Based on some or all of the above features and sequence homologies between PDs, four subfamilies of paralogous Paxs have been distinguished. These four subfamilies are Pax1/9, Pax2/5/8, Pax3/7 and Pax4/6 in which the last two subfamilies (Pax3/7 and Pax4/6) contain both PD and homeodomain (Gruss and Walther, 1992). Mammals and avians have a single gene each for Pax3 and Pax7, whereas in zebrafish, two distinct genes for Pax3 (Pax3a and Pax3b) and at least four Pax7 variants have been reported, one of which encodes a protein with the sequence close to that of mammalian Pax7 (Seo et al., 1998). Alternatively, spliced variants of Pax3 and Pax7 have been reported in mouse and human, although their exact functions remain poorly understood (Vogan et al., 1996, Ziman et al., 1997, Barber et al., 1999). As in the case of other vertebrates, cDNA clones encoding novel Pax7 splice variants have also been isolated from the brain and skeletal muscle of a tetraploid, Atlantic salmon Salmo salar (Gotensparre et al., 2006).
In vertebrates, the growth and repair of skeletal muscle depends critically upon a pool of tissue specific stem cells, termed satellite cells. These satellite cells are set aside from the proliferative pool of muscle progenitor cells (MPCs) at the end of embryogenesis. In amniote embryos, satellite cells have been shown to arise from a distinct population of dermomyotomal progenitors that express and require the related transcription factors Pax3 and Pax7 for their specification (Kassar-Duchossoy et al., 2005, Relaix et al., 2005). Pax3 is expressed in satellite cells (Conboy and Rando, 2002, Buckingham et al., 2003) and controls precursor cell migration to sites of muscle formation in the limb and body wall (Relaix et al., 2006). During mouse embryonic development, Pax3 is expressed in presomitic mesoderm (PSM) as somites are formed and expression is progressively restricted first to dermomyotome and later to dorsomedial and ventromedial lips. Pax7 has also been identified as a key determinant of satellite cells, as all skeletal muscle groups of Pax7 null mutant mice lack satellite cells and fail to grow or regenerate after birth (Seale et al., 2000). Pax7 expression is restricted to the central region of the dermomyotome (Kassar-Duchossoy et al., 2005, Relaix et al., 2005) and continues to be expressed in fetal and adult satellite cells, although a few Pax3 positive satellite cells also exist in adult skeletal muscles of mouse (Relaix et al., 2006).
As in amniotes, dermomyotome has been described in embryonic development of teleosts and indeed cells previously termed as “external cells” have the morphological and molecular characteristics of dermomyotome (Devoto et al., 2006, Stellabotte and Devoto, 2007). Various studies reported that this epithelium expresses Pax3 and/or Pax7 (Groves et al., 2005, Devoto et al., 2006, Feng et al., 2006, Hammond et al., 2007, Macqueen et al., 2007, Macqueen et al., 2008, Steinbacher et al., 2007), and the external cells in zebrafish give rise to new muscle fibers (Hollway et al., 2007, Stellabotte et al., 2007). The findings of these studies have led to the suggestion that the external cell epithelium is the evolutionary homologue of amniotes dermomyotome generating myogenic cells for myotome growth (Devoto et al., 2006, Stellabotte and Devoto, 2007). These epithelium cells are also labeled with Pax3- and Pax7-specific antibodies in fish such as sturgeon Acipenser ruthenus (Devoto et al., 2006), zebrafish Danio rerio (Maroto et al., 1997, Stellabotte and Devoto, 2007), cichlid Astatotilapia burtoni (Devoto et al., 2006), and common whitefish Coregonus lavaretus (Kacperczyk et al., 2009). In zebrafish embryos, Pax3 and Pax7 genes are initially expressed within the dermomyotome (Devoto et al., 2006, Hollway et al., 2007, Hammond et al., 2007, Minchin and Hughes, 2008), which arises from the anterior portion of the somite and, following an anterior to lateral rotation, comes to cover the lateral surface of the primary myotome (Hollway et al., 2007, Stellabotte et al., 2007). Lineage tracing of individual cells in the Pax7+ ve anterior region of the somite demonstrated that they give rise to muscle progenitors that generate fibers during a secondary period of larval muscle growth (Hollway et al., 2007). In addition, mitotically quiescent Pax7+ ve cells were identified from early larval stages (3 days post fertilization (dpf)), located beneath the basal lamina of individual fibers and accordingly suggested to be satellite cells (Hollway et al., 2007). In a non-model tropical fish Pterophyllum scalare, Pax3 expression was also observed in externally epithelium cells to the myotome and in post-hatching stages, Pax7 expression was observed in mono-nucleated proliferating cells (Kacperczyk et al., 2011). Based on the above information, we can conclude that Pax3 and Pax7 are involved in the specification of MPCs during development of fish.
The ontogeny of development and growth in fish, however, is much different from mammals. In mammals, hyperplasia ceases near the time of birth and further muscle growth results mainly from hypertrophy (Rowe and Goldspink, 1969). In contrast to mammals, the growth of trunk skeletal muscle in adult fish results from both increase in hypertrophy and hyperplasia. The majority of fish species pass through two stages of muscle hyperplasia during development and growth: stratified and mosaic hyperplasia. In stratified hyperplasia, a distinct region of hyperplastic nascent fibers develops in the epaxial myotome (Johnston and Mclay, 1997). Mosaic hyperplasia occurs later in life and involves the recruitment of nascent myofibers throughout the myotome. The distinction among embryonic, stratified and mosaic hyperplasia in fish suggest distinct mechanisms underlying muscle development and involvement of MPCs. Therefore, the identification of fish MPCs is a key factor in developmental biology in determining early events in myogenesis and fiber generation in adult muscle.
Japanese pufferfish, commonly known as torafugu, Takifugu rubripes has the smallest genome size among the vertebrates, which is only around 400 Mb (about an eighth the size of the human genome) and publicly available after the human genome. Torafugu was suggested as appealing “model” vertebrates for genomic analysis in part because, although compact, their genomes have essentially the same genes and regulatory sequences as other vertebrates. Thus less effort is needed to obtain a comparable amount of information. Although Pax3 and Pax7 play an important role in regulating survival, proliferation and migration of MPCs in mammals and zebrafish, information regarding them in torafugu, has remained unknown. Therefore, as an initial step in the present study, partial cDNAs encoding Pax3 and Pax7 genes were cloned from embryos and fast skeletal muscle of torafugu and characterized, whereas their expression patterns were investigated by reverse transcription (RT)-PCR during different stages of development.
Section snippets
Fish
Artificially fertilized eggs of torafugu were collected from Oshima Fisheries Hatchery, Nagasaki, Japan, and brought to The University of Tokyo, where they were reared in a 500 L tank in seawater at 18 °C. About 100 individuals each from embryos at 1 to 7 dpf and an equal number of larvae at 8, 10 and 16 dpf were collected, snap-frozen in liquid nitrogen and kept at − 80 °C for cDNA cloning and RT-PCR. In an adult torafugu (275 g body weight), fast and slow muscle in the trunk were identified and
Characterization of Pax3 and Pax7 genes
RT-PCR with the degenerate primers based on conserved amino acids at PD and homeodomain resulted in the amplification of DNA fragments with about 600 bp from torafugu embryos at 3 dpf and adult fast skeletal muscle (data not shown). It is noted that cloning of Pax3 (Pax3a and Pax3b) and Pax7 (Pax7a and Pax7b) genes was successful from torafugu embryos at 3 dpf but cloning of only Pax7a was successful from adult fast muscle samples. Blast search by using the partial cDNA sequences of Pax3 and Pax7
Discussion
In the present study, cDNAs encoding Pax3 and Pax7 genes were cloned from embryos and adult fast skeletal muscles of torafugu. Two types of cDNA clones each encoding Pax3 and Pax7 genes showed a high identity in their deduced amino acid sequences (Table 2, Table 3) and subsequent in silico analysis on the fugu genome database revealed two separate genes each for Pax3 (Pax3a and Pax3b) and Pax7 (Pax7a and Pax7b) genes (Fig. 1).
Deletion in glutamine residue at the 75 amino acid from the
Conclusion
In this study, we characterized Pax3 and Pax7 from torafugu and investigated their expression patterns during different developmental stages by RT-PCR. There are two distinct genes each for Pax3 (Pax3a and Pax3b) and Pax7 (Pax7a and Pax7b) genes in torafugu and they have high syntenic relationship with other teleosts and mammals. Pax3 might be the most important during embryonic (primary) stages of myogenesis and Pax7 might be important for secondary growth by hyperplasia that leads to
Acknowledgments
This study was partly supported by a Grant-in-Aid for Scientific Research (S) from the Japan Society for the Promotion of Science (JSPS) to SW (No. 19108003). The authors also thank Mr. Koda, Oshima Fisheries Hatchery, Nagasaki, Japan, for supplying the torafugu embryos.
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These authors contributed to this work equally.