Isolation and characterisation of a pax group III gene from the freshwater crayfish cherax destructor: Implications for the evolution of muscle regeneration

Pax genes encode transcription factors that are highly evolutionarily conserved and are vital for animal development. Vertebrate Pax group III genes Pax3 and Pax? are required for proper development of muscular and central nervous systems. In their roles in muscular systems, Pax3 and Pax? specify myoblasts (muscle progenitor cells). Pax3 is predominantly involved in embryonic myogenesis and specifies embryonic myoblasts. In contrast, Pax? specifies adult myoblasts (muscle satellite cells), and has been demonstrated to be vital for adult muscle regeneration. Recent evidence also implicates a role for Pax7 in the conversion of certain stem cells to the myogenic lineage. The Australian freshwater crayfish Cherax destructor (yabby) possesses a phenomenal capability for almost limitless adult muscle regeneration. Interestingly, C. destructor undergoes two distinct types of adult muscle regeneration, and these appear to be driven by two distinct types of muscle progenitor/stem cell. C. destructor has a highly regulated system for normal muscle growth, which is strictly regulated around the periodic shedding of the growth-restricting exoskeleton. The muscle regeneration that occurs in this normal growth is driven predominantly by endogenous muscle satellite cells. C. destructor also possesses the fascinating ability to regenerate entire limbs, including all of the muscle contained within them. This process appears to utilise circulating haematopoietic stem cells as the dominant muscle progenitor cell (myoblast). The role for Pax? in adult muscle regeneration in vertebrates indicates that Pax group III genes may be appropriate candidates to study in muscle regeneration in C. destructor. Pax group III genes have been isolated in fruit fly, grasshopper, mite and jellyfish, and have not been demonstrated to be expressed during embryonic or adult myogenesis in any of these animals. Prior to this research, Pax genes had not been isolated from Crustacea. I have designed nested partially-degenerate primers to complement conserved regions of known arthropod Pax group III gene sequences, and used PCR and RT-PCR of C. destructor genomic DNA and embryonic RNA, respectively. Sequencing of reaction products confirmed the presence of a Pax group III gene in Crustacea. In this research I have isolated a single Pax group III gene from C. destructor which I have designated CdpaxJ/1. Cdpax317 is unequivocally a Pax group III gene, and appears to be the only Pax group III gene present in C. destructor. Cdpax3/7 contains two DNA binding domains characteristic of Pax group III proteins, the paired domain and the homeodomain. Cdpax3/7 also contains an octapeptide motif characteristic of the majority of Pax group III proteins. Interestingly, Cdpax317 is expressed as two alternate transcripts. One alternate transcript lacks a 93 nucleotide section corresponding to approximately one third of the paired domain, indicating that it may have altered DNA binding, and therefore function. RT-PCR expression assays indicate that Cdpax317 is expressed embryonically (in whole C. destructor embryos), in normal non-regenerating adult muscle, in adult muscle undergoing normal regeneration, as well as in limb regeneration (both before and after myogenic differentiation). The gross expression pattern of Cdpax317 in adult muscle regeneration is characteristic of the expression patterns of vertebrate Pax7 in the analogous vertebrate process.