Abstract
BackgroundSalamanders regenerate their tails after amputation anywhere along their length. How the system faithfully reconstitutes the original number of segments and length is not yet known.MethodsTo gain quantitative insight into how the system regenerates the appropriate length, we amputated tails at 4 or 16 myotomes post-cloaca and measured blastema size, cell cycle kinetics via cumulative Bromodeoxyuridine (BrdU) incorporation and the method of Nowakowski, and myotome differentiation rate.ResultsIn early stages until day 15, blastema cells were all proliferative and divided at the same rate at both amputation levels. A larger blastema was formed in 4th versus 16th myotome amputations indicating a larger founding population. Myotome differentiation started at the same timepoint in the 4th and 16 th level blastemas. The rate of myotome formation was more rapid in 4th myotome blastemas so that by day 21 the residual blastema from the two amputation levels achieved equivalent size. At that time point, only a fraction of blastema cells remain in cycle, with the 4th myotome blastema harboring double the number of cycling cells as the 16th myotome blastema allowing it to grow faster and further reconstitute the larger number of missing myotomes.ConclusionsThese data suggest that there are two separable phases of blastema growth. The first is level-independent, with cells displaying unrestrained proliferation. In the second phase, the level-specific growth is revealed, where differing fractions of cells remain in the cell cycle over time.
Highlights
Salamanders regenerate their tails after amputation anywhere along their length
To dynamically monitor the process of tail regeneration including blastema formation and myotome differentiation, we made measurements in cohorts of live, 3 cm long axolotl “white” mutant larvae, expressing the CAGGS:GFP transgene, where the myotome organization is visible under the light microscope (Fig. 1)
The approach to homeostatic rates occurred gradually, with maximum rate from day 15 to approximately day 19
Summary
Salamanders regenerate their tails after amputation anywhere along their length. How animals regulate organ size and proportion is an enduring question in biology. Regeneration of the vertebrate limb or tail, for example, show appropriate level-specific growth control and morphogenesis to replace only the missing structure. Studies in the salamander limb have carefully measured proliferation rates and distributions to investigate. Vincent et al BMC Developmental Biology (2015) 15:48 arm blastemas [2]. How the position of the cut influences growth kinetics on a cellular scale had not been resolved. Analysis of differentiation kinetics in the limb is, complicated since the morphology of the upper arm skeleton and muscle differs from the lower arm and hand, which may lead to intrinsic differences in differentiation kinetic unrelated to a generic proximal/distal position
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