Can People Regenerate Body Parts? (III of V)

We knew that the epidermis is derived from one of three layers of primitive cells within an early developing embryo, the ectoderm, which is also well known to provide signals that control the outgrowth of limbs from limb buds on the embryo.

Ectoderm cells gather in the bud to form an apical ectodermal ridge (AER), which transiently produces chemical signals that guide the migration and proliferation of the underlying limb bud cells. Although some of the critical signals from the epidermis have not yet been identified, members of the family of fibroblast growth factors (FGFs) are involved. The AER produces a number of FGFs that stimulate the underlying cells of the limb bud to produce other FGFs, fueling a feedback circuit of signaling between the AER and limb bud cells that is essential for the outgrowth of a limb.

A similar feedback circuit spurred by the AEC is thought to function in the same way during limb regeneration, and Hiroyuki Ide of Tohoku University in Japan discovered that the progressive loss of regenerative ability in frog tadpoles is associated with a failure to activate the FGF circuit. By treating older nonregenerating tadpole limbs with FGF10, he was able to jump-start this signaling circuit and stimulate partial regeneration of amputated limbs. The excitement this result inspired was tempered, however, by the fact that the induced regenerates were abnormal, consisting of irregularly placed limb parts, which raises the important issue of how regeneration is controlled so that all the appropriate anatomical structures that are lost when the limb is amputated are accurately replaced. It turns out that the other primary cellular players, the fibroblasts, carry out this function.

Recall from the minimalist accessory-limb experiments that the presence of fibroblasts per se was not sufficient for regeneration because fibroblasts are present at the simple wound site that does not make a new limb. It was the fibroblasts from the opposite side of the limb that proved essential. That discovery illustrates the importance of cellular position in triggering a regeneration response. In an embryo, the sequence of events in limb development always begins with formation of the base of the limb (the shoulder or hip) and is followed by progressive building of more distal structures until the process terminates with the making of fingers or toes.

In salamander regeneration, on the other hand (or foot), the site of amputation can be anywhere along the length of the limb and regardless of where the wound is located, only those parts of the limb that were amputated regrow. This variable response indicates that cells at the amputation wound edge must “know” where they are in relation to the entire limb. Such positional information is what controls the cellular and molecular processes leading to the perfect replacement of the missing limb parts, and it is encoded in the activity of various genes. Examining which genes are at work during these processes helps to reveal the mechanisms controlling this stage of regeneration. Although a large number of genes are involved during embryonic development in educating cells about their position in the limb, the activity of a gene family called Hox is critical.

In most animals, cells in the developing limb bud use the positional code provided by Hox genes to form a limb, but then they “forget” where they came from as they differentiate into more specialized tissues later on. In contrast, fibroblasts in the adult salamander limb maintain a memory of this information system and can reaccess the positional Hox code in the process of limb regeneration. During regeneration the fibroblasts bring this information with them as they migrate across the wound to initiate blastema formation, and once in the blastema, cells are able to “talk” to one another to assess the extent of the injury.

The content of this crosstalk is still largely a mystery, but we do know that one outcome of the conversation is that the regenerating limb first establishes its boundaries, including the outline of the hand or foot, so that cells can use their positional information to fill in the missing parts between the amputation plane and the fingers or toes. Because muscle and bone make up the bulk of a limb, we are also interested in understanding where the raw material for those tissues originates and what mechanisms control their formation. When the regenerative response is initiated, one of the key early events involves a poorly understood process called dedifferentiation.

The term is typically used to describe a cell’s reversion from a mature specialized state to a more primitive, embryonic state, which makes it capable of multiplying and serving as a progenitor of one or more tissue types. In the field of regeneration, the word was first used by early scientists who observed under the microscope that the salamander stump tissues, particularly the muscle, appeared to break down and give rise to proliferative cells that formed the blastema. We now know that those muscle-associated cells are derived from stem cells that normally reside in the muscle tissue and not from dedifferentiation of muscle. Whether or not dedifferentiation is actually happening in the case of every tissue type within a regenerating limb has yet to be proved, although it is clear that a variation of this theme does occur during regeneration. Fibroblasts that enter the blastema and become primitive blastemal cells have the ability to differentiate into skeletal tissues (bone and cartilage) as well as to redifferentiate into the fibroblasts that will form the interstitial meshwork of the new limb, for instance.

Returning to another of the central cellular players in blastema formation, the epidermal cells, we can also pinpoint moments in the regeneration process when it seems these cells are making a transition to a more embryonic state. A number of genes active in the embryonic ectoderm are critical for limb development, including Fgf8 and Wnt7a, but as the ectoderm of the embryo differentiates to form the multilayered epidermis of the adult, these genes are turned off. During regeneration in the adult, the epidermal cells that migrate across the amputation wound and establish a wound epidermis initially begin to display gene activity, such as production of wound-healing keratin proteins, that is not specifically related to regeneration. Later the wound epidermal cells activate Fgf8 and Wnt7a, the two important developmental genes.

For practical purposes, then, the essential definition of dedifferentiation—as it pertains to the epidermis and other cell types—is the specific reactivation of essential developmental genes. Thus, our studies of salamanders are revealing that the regeneration process can be divided into pivotal stages, beginning with the wound-healing response, followed by the formation of a blastema by cells that revert to some degree to an embryonic state, and finally, the initiation of a developmental program to build the new limb. As we move toward the challenge of inducing limb regeneration in humans, we rely on these insights to guide our efforts.

Indeed, the hardest things to discover in science are those that do not already occur, and limb regeneration in humans fits snugly into this category, although that does not mean humans have no natural regenerative capacity.

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