Analysis of Gene Expressions during Xenopus Forelimb Regeneration

Xenopus laevis can regenerate an amputated limb completely at early limb bud stages, but the metamorphosed froglet gradually loses this capacity and can regenerate only a spike-like structure. We show that the spike formation in a Xenopus froglet is nerve dependent as is limb regeneration in urodeles, since denervation concomitant with amputation is sufficient to inhibit the initiation of blastema formation and fgf8 expression in the epidermis. Furthermore, in order to determine the cause of the reduction in regenerative capacity, we examined the expression patterns of several key genes for limb patterning during the spike-like structure formation, and we compared them with those in developing and regenerating limb buds that produce a complete limb structure. We cloned Xenopus HoxA13, a marker of the prospective autopodium region, and the expression pattern suggested that the spike-like structure in froglets is accompanied by elongation and patterning along the proximodistal (PD) axis. On the other hand, shh expression was not detected in the froglet blastema, which expresses fgf8 and msx1. Thus, although the wound epidermis probably induces outgrowth of the froglet blastema, the polarizing activity that organizes the anteroposterior (AP) axis formation is likely to be absent there. Our results demonstrate that the lost region in froglet limbs is regenerated along the PD axis and that the failure of organization of the AP pattern gives rise to a spike-like incomplete structure in the froglet, suggesting a relationship between regenerative capacity and AP patterning. These findings lead us to conclude that the spike formation in postometamorphic Xenopus limbs is epimorphic regeneration.     

Dual-regulated myoD- and msx1-based interventions in C2C12-derived cells enable precise myogenic/osteogenic/adipogenic lineage control

BACKGROUND: Advanced gene therapy, tissue engineering and biopharmaceutical manufacturing require sophisticated and well-balanced multiregulated multigene interventions to reprogram desired mammalian cell phenotypes. METHODS: We have combined the streptogramin (PIP)- and tetracycline (TET)-responsive gene regulation systems for independent expression control of the differentiation determinants myoD and msx1 in C2C12-derived cells. RESULTS: Different dual-regulated expression scenarios which induce either both, only one or none of the lineage control genes triggered differential differentiation and precise control of myogenic, osteogenic or adipogenic cell phenotypes. CONCLUSIONS: Our findings substantiate the use of multiregulated multigene interventions in reprogramming cellular differentiation pathways in a desired manner.