Fate-Restricted Digit Tip Regeneration in Mice

The act of regeneration is a common feat of animal function (Lehoczky 2011) allowing organisms such as fish to regenerate new fins or amphibians regenerating entire limbs. Regeneration in mammals, however, possesses a far less profound role as it is amputation-level-dependent to the digit tip region (Yu et al. 2010), which is no more proximal than the furthest point of the nail bed. Nonetheless, when one compares developmental characteristics of mice and other species such as newts and planaria, one can arrive at hypotheses as to how regeneration is mediated in mice. Beginning from the blastema, which is a post-amputation bud-like structure consisting of cells leading to the musculoskeletal and connective tissues, are its cells actually recruited multiple cell type determinant lineages or is there a pluripotent progenitor pool that may cause transdifferentiation as shown in newt limbs? In addition, are these cells recruited from a preexisting stem cell population or from dedifferentiation of mature tissues?

Researchers at Harvard Medical School and the Pasteur Institute attempted to answer these questions by a series of well-crafted experiments explaining that no transdifferentiation between lineages occurs, and there is regeneration derived specifically from lineage-restricted cells as shown in the following experiments that follow keratinocytes, mesodermally derived tissues, and osteoblasts (required for bone tissue).

In general, digit-tip regenerative studies in mice have the potential to be applied to humans. Applications of regenerative studies could lead to new medical advancements including surgical techniques that enable physicians to induce regeneration given that regenerative ability of the digits is lost in humans with age. This, however, differs with mice, which are able to regenerate their digit tips throughout their lifespan. In addition, a greater understanding of the limitations of digit-tip formation could potentially allow physicians to one day induce regeneration past the current natural limitation of the most proximal point of the nail bed on fingers.

By following tissue-specific and certain lineages throughout development after an amputation has been performed (using inducible Cre alleles to express specific tissues in the neonatal limbs), experiments below were able to make several conclusions.

Skin cells in regenerated digit-tip are derived from pre-existing skin cells

The new skin cells formed on the digit-tip were tracked during the experiment, and were actually found to have their source from skin cells lying more proximal to the amputation site. This additionally provided support for the theory that transdifferentiation between ectodermal and mesodermal lineages during regeneration does not exist. In figure 1, Kertain 14 was used as a marker in order to follow the fate of epidermal tissue. From this experiment, it was determined that most, if not all, of new epithelium originated from pre-existing keratinocytes.  WkPA and dPA define either weeks or days post-amputation as shown in Figure 1.

Fig. 1. Lineage of Krt14-expressing keratinocytes during digit tip regeneration. (A–E) X-gal stained (blue) Krt14-CreESR;R26R-lacZ digit sections from 3 dpa to 1 wkPA. (F and G) TdTomato (red) fluorescence overlaying differential interference contrast brightfield (grayscale) images of Krt14-CreESR1;R26R-CAG-tdTomato digit sections at 2 wkPA and 3 wkPA. Epidermal retraction and proximal attachment to terminal phalanx occurs between 1 and 3 dpa (A and A’, arrows). Dotted line shows approximate plane of amputation. Distally exposed bone becomes integrated into clot (B, B’, C, and C’) as epidermis covers the amputation site under the clot (C–E and C’–E’). Blastema formation occurs after epidermal closure (D and D’). bl, blastema; b, bone; c, clot; ct, connective tissue; e, epidermis. (Scale bars,100 μm.)

Lineage-Restricted Preamputation Osteoblasts are the source of regenerated bone

In addition, the source of the pre-amputation osteoblasts were tested to determine if they result in the regenerative skeletal tissues and whether they were lineage-restricted or dedifferentiated into various tissues in the new digit. Sp7 was used as the marker given that they specifically mark osteoblasts (endochondral and intra-membranous bones).  Post-natal digits show that by 2 wkPa, sp7 expressing osteoblast descendents are found in the blastema, but not the epidermis. By 3 wkPa, the clot was sloughed away with the new digit reappeared, and the preamputation osteoblasts exclusively supporting the regenerated digit as shown in figure 2 B and B’. This indicated that preamputation osteoblasts serve as fate-restricted progenitors, only supporting tissues in which they are typically expressed.

Fig. 2. Lineage of Sp7-expressing osteoblasts during digit tip regeneration. X-gal stained Sp7-tTA-tetO-EGFP::Cre;R26R-lacZ digit sections. (A and A’) Images at 2 wkPA show descendants in clot (arrow), bone, and blastema, but not the epidermis. (B and B’) Images at 3 wkPA show descendants in bone and connective tissue but not epidermis. Staining resembles normal 3 wk nonregenerative digit osteoblast contribution (C and C’). Insets in A’–C’ show 40× magnification. bl, blastema; b, bone; ct, connective tissue; e, epidermis. (Scale bars, 100 μm.)


Expression pattern of Msx1 shows further evidence that there is no trans-differentiation between the mesodermal and ectodermal germ layers

Given that the marker, Msx1, has been shown to induce dedifferentiation and proliferation of cultured myotubes, which are then able to redifferentiate into multiple lineages (Odelberg et a. 2000), the specific expression of Msx1 was also studied. The work essentially displays that although Msx1 contributes broadly to the blastema and their own lineages in the regenerate digit, these efforts are not in isolation, and are accompanied by additional progenitor populations that participate as well. In figure 4 B-B’, the clot due to the amputation is almost sloughed away, but Msx1 descendents are found within the bone and dermis, but not the epidermis. At 3 wkPa Msx1 descendants heterogeneously populated bone and dermis of the digit, which shows no abnormal expression of Msx1 (figure 3F). Once again, Msx1 descendents were not found in the epidermis, which indicates that there is no transdifferentiation between ectodermal and mesodermal germ layers. Additionally, this shows that the cell types expressing Msx1 before amputation matched only to the cell types of their derivatives.

Fig. 3. Expression of Msx1 during digit tip regeneration. Msx1-nlacZ x-gal stained digit sections during regeneration. PN3 (A) and 3 wk (B) unamputated digits show expression in dorsal and ventral dermis and throughout bone and sweat glands. (C and C’) Expression at 3 dpa is in normal stump domains and in the clot. (D and D’) Expression at 5 dpa is in the clot and a few bone cells where epidermis is closing under the clot (arrow). (E and E’) Expression at 1 wkPA remains in the clot but is absent from the blastema. (F and F’) Expression at 3 wkPA returns to a nonregenerative expression pattern and resembles B. Epidermis is outlined in higher-magnification pictures. bl, blastema; b, bone; c, clot; e, epidermis; np, nail plate; sg, sweat gland. (Scale bars, 200 μm.)

Fig. 4. Lineage of Msx1-expressing cells during digit tip regeneration. TdTomato fluorescence from Msx1-CreERT2;R26R-CAG-tdTomato digit tips. (A–A’’’) Descendants at 1 wkPA are in blastema (A’’) and clot (A’’’) but not epidermis (A’–A’’’). (B–B’’) Descendants at 2 wkPA are found in bone and dermis but not epidermis. (C–C’’) Descendants at 3 wkPA have reestablished normal Msx1 expression pattern of the distal tip. (A–C) TdTomato expression with no overlay. (A’–A’’’, B’, B’’, C’, and C’’) Differential interference contrast brightfield images (grayscale) with tdTomato (red) fluorescent overlay. bl, blastema; b, bone; cl, clot; d, dermis; e, epidermis; np, nail plate. (Scale bar, 100 μm.)

Conclusion

Though the practice of standardizing results via the use of post-natal mice with weeks-post-amputation for a scale seems to be the most practical method, this experiment presents a weakness by disregarding the effects of digit tip regeneration in adult mice when attempting to relate scientific evidence to humans, an intention expressed by the authors. Indeed, mice are unlike humans in the sense that humans lose regenerative ability with age (Lehoczky 2011), but as to whether any changes occur to the mechanism of regeneration during the adult lifespan should be studied given that this could also provide comparative insight as to the reason for the loss of regenerative ability in humans, and provide further applications of regenerative studies.

SOURCE:

Lehoczky, JA, B Robert, and CJ Tabin. “Mouse Digit Tip Regeneration is Mediated by Fate-restricted Progenitor Cells.” Proceedings of the National Academy of Sciences of the United States of America, 108.51 (2011): 20609-20614.

Odelberg SJ, Kollhoff A, Keating MT (2000) Dedifferentiation of mammalian myotubes induced by msx1. Cell 103:1099-1109

Yu, L, M Han, M Yan, EC Lee, J Lee, and K Muneoka. “BMP Signaling Induces Digit Regeneration in Neonatal Mice.” Development (Cambridge, England), 137.4 (2010): 551-559.

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