Abstract
The regenerating region of an amputated salamander limb, known as the blastema, has the amazing capacity to replace exactly the missing structures. By grafting cells from different stages and regions of blastemas induced to form on donor animals expressing Green Fluorescent Protein (GFP), to non-GFP host animals, we have determined that the cells from early stage blastemas, as well as cells at the tip of late stage blastemas are developmentally labile such that their positional identity is reprogrammed by interactions with more proximal cells with stable positional information. In contrast, cells from the adjacent, more proximal stump tissues as well as the basal region of late bud blastemas are positionally stable, and thus form ectopic limb structures when grafted. Finally, we have found that a nerve is required to maintain the blastema cells in a positionally labile state, thus indicating a role for reprogramming cues in the blastema microenvironment.
Highlights
For over a century, regeneration biologists have puzzled over the ability of the regenerating region of an amputated salamander limb, known as the blastema, to replace exactly the lost distal structures
Grafted early bud blastema cells survive but do not form supernumerary limb structures To test whether the cells of the early blastema have stable positional information or if they are positionally labile, we grafted proximal Early bud (EB) blastemas to distal amputation stumps to observe whether regenerates with duplicated proximal-distal (P-D) structures formed (Figure 2A-C, Figure S1A)
The presence of stump tissues in the EB blastema grafts presumably provided P-D information corresponding to the level of the donor tissues from which the EB blastema cells formed more distal pattern [1,2,37] resulting in a limb with a duplicated P-D pattern
Summary
Regeneration biologists have puzzled over the ability of the regenerating region of an amputated salamander limb, known as the blastema, to replace exactly the lost distal structures. When cells that are normally non-adjacent (i.e. come from different positions and have different positional information) are grafted next to each other, their subsequent interactions lead to proliferation and the formation of new pattern that normally lies between those cells. This process of pattern formation is referred to as “intercalation” [1,2,6,7], and leads to reestablishment of the proximal-distal (P-D) limb axis during regeneration [8], and appears to generate the P-D limb axis during limb development in the mouse embryo [9]. Intercalation results in formation of the normal pattern, but in others can lead to formation of supernumerary limb structures
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