Mouse digit tip regeneration is mediated by fate-restricted progenitor cells

Jessica A. Lehoczkya, Benoît Robertb, and Clifford J. Tabina,1

aDepartment of Genetics, Harvard Medical School, Boston, MA 02115; and bInstitut Pasteur, Génétique Moléculaire de la Morphogenèse, Centre National de la Recherche Scientifique, Unité de Recherche Associée 2578, F-75015 Paris, France

Contributed by Clifford J. Tabin, November 1, 2011 (sent for review September 19, 2011)

Regeneration of appendages is frequent among invertebrates as tation of both the axolotl limb and the zebrafish fin strongly well as some vertebrates. However, in mammals this has been suggest that does not significantly contribute largely relegated to digit tip regeneration, as found in mice and to the regenerates, and that instead the blastemas are made up of humans. The regenerated structures are formed from a mound of lineage-restricted cell populations (9–11). undifferentiated cells called a blastema, found just below the site Digit tip regeneration has been reported in mammals including of amputation. The blastema ultimately gives rise to all of the mice and juvenile humans (12, 13). Amputations of the terminal tissues in the regenerate, excluding the epidermis, and has classi- phalanx through levels associated with the nail organ are capable cally been thought of as a homogenous pool of pluripotent stem of regeneration, whereas more proximal amputations are not. cells derived by dedifferentiation of stump tissue, although this Intriguingly, mesenchymal nail bed cells in neonatal mice and has never been directly tested in the context of mammalian digit tip humans express the Msx1 (14, 15), which is regeneration. Successful digit tip regeneration requires that the a transcription factor expressed in the proliferative, un- level of amputation be within the nail bed and depends on ex- differentiated regions of regenerating newt limbs (16, 17). This pression of Msx1. Because Msx1 is strongly expressed in the expression may reflect a functional importance of Msx1 in blas- nail bed mesenchyme, it has been proposed that the Msx1- tema formation and maintenance. Msx1 induces dedifferentiation expressing cells represent a pluripotent cell population for the and proliferation of cultured myotubes, which are ultimately ca- regenerating digit. In this report, we show that Msx1 is dynamically pable of being redifferentiated down multiple lineages (18). Im- − − expressed during digit tip regeneration, and it does not mark portantly, Msx1 / digits do not regenerate in ex vivo embryo a pluripotent population. Moreover, we show that both culture. Moreover, Msx1 is necessary for Bmp4 activity, which − − the ectoderm and mesoderm contain fate-restricted progenitor could rescue the ability of these Msx1 / digits to regenerate (19). populations that work in concert to regenerate their own lineages Msx1 expression is not observed in the digit tip blastema itself within the digit tip, supporting the hypothesis that the blastema is (20), suggesting that its critical function may be in the nail bed a heterogeneous pool of progenitor cells. mesenchyme. Given the requirement of the nail bed for successful regeneration and the association of Msx1 with undifferentiated, epimorphic regeneration | lineage analysis | transdifferentiation | multipotent cells in both developmental and regenerative set- fibrin clot | mitogenic signal tings, Msx1-expressing nail bed mesenchyme could represent a pluripotent stem cell population necessary for digit tip re- egeneration of appendages occurs widely throughout the generation. Alternately, the Msx1-expressing nail bed mesen- Ranimal kingdom, although it is limited in higher vertebrates. chyme could serve as a signaling center, producing critical factors Although many fish regenerate fins and a number of urodele and required for regeneration. larval anuran amphibians regenerate entire limbs, in mammals To determine whether mouse digit tip regeneration involves such as humans and mice appendage regeneration is limited to the recruitment of multiple cell type determinant lineages or the the distal digits. Regeneration begins with the formation of a formation of a pluripotent progenitor pool, and to answer the specialized regenerative epithelium across the plane of ampu- related question of whether these blastema cells are derived from tation that is required for regeneration to proceed (1, 2). His- a preexisting stem cell population or from dedifferentiation of tological analysis of amphibian limb regenerates suggests that the mature tissues in the digit stump, we undertook a lineage analysis regenerative epithelium forms by the migration of stump epi- using inducible alleles of Cre recombinase expressed in specific dermal cells, although additional contributions from other cell tissues within the neonatal limb. populations have not been ruled out (3). The formation of the regenerative epithelium is followed by an accumulation of mes- Results enchymal cells distal to the stump, which proliferate to form Descriptive Analysis of the Process of Digit Tip Regeneration. To a bud-like structure known as the blastema. These blastema cells develop a context for our lineage studies, we established a time subsequently differentiate into the musculoskeletal and connec- course of the early events of mouse digit tip regeneration. In tive tissues of the regenerated appendage. preliminary experiments, we found that regeneration reliably It is critical to understand the origin of blastema cells and the occurs when the distal-most 400 μm are amputated on postnatal degree to which they represent a multipotent cell population. day 3 (PN3) CD1 neonatal mice (SI Text and Fig. S1). This rep- The blastema could be formed from a preexisting multipotent resents ≈50% of the length of the PN3 distal phalanx and trans- stem cell as found in planaria; however, vertebrate research verses the proximal nail bed, although digit size and regeneration suggests, at least in amphibian limb regeneration, that the ma- jority of blastema cells are from dedifferentiation of the mature tissues of the stump (4, 5). In principle, blastema cells could Author contributions: J.A.L. and C.J.T. designed research; J.A.L. performed research; B.R. remember their origin and only differentiate into the cell types contributed new reagents/analytic tools; J.A.L. and C.J.T. analyzed data; and J.A.L. and C.J.T. from which they arose. Alternately, blastema cells could be wrote the paper. multipotent, capable of transdifferentiation into all cell types of The authors declare no conflict of interest. the regenerate. Indeed, transdifferentiation has been described 1To whom correspondence should be addressed. E-mail: [email protected].

in both axolotl tail regeneration (6) and amphibian limb re- edu. BIOLOGY

generation experiments in which certain tissues were removed or This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. DEVELOPMENTAL irradiated (7, 8). However, recent lineage analyses after ampu- 1073/pnas.1118017108/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1118017108 PNAS | December 20, 2011 | vol. 108 | no. 51 | 20609–20614 Downloaded by guest on September 29, 2021 can vary with strain (Fig. S2). CD1 mothers were used in all sub- steps of epidermal closure are the same from digit to digit, sequent experiments, except where specifically noted. whereas the timing is variable (21). It is not unusual to observe 1- Over the first few days after amputation of the distal 400 μm to 2-d variability in each regenerative stage among littermates or of a PN3 digit, the epidermis closes across the wound. As has between different genetic backgrounds or drug treatment groups. previously been noted for adult mouse digit tip regeneration, the Fig. 1 shows an idealized course of events. Immediately after amputation a superficial blood clot forms at the amputation site. One to three days after (post) amputation (dpa), the existing epidermis retracts and attaches to the terminal phalanx at a level proximal to the amputation plane (Fig. 1 A and A’). The epi- dermal retraction leaves the distal tip of the stump bone pro- truding into the clot (Fig. 1 A and A’), and this bone sub- sequently fragments distally. Between 3 dpa and 7 dpa the clot is maintained and integrates the residual bone fragment while the epidermis closes over the amputation, yet underneath the clot (Fig. 1 B–E and B’–E’). The regenerative blastema forms under the new epithelium by 6 dpa (Fig. 1 D and D’). By 2 weeks after amputation (wkPA) (Fig. 1 F and F’) the clot begins to slough off, and the majority of the bone regeneration is complete, and by 3 wkPA regeneration is complete (Fig. 1 G and G’). This course of regenerative events corroborates the postamputation events of adult digit tip regeneration (21), underscoring the similarity in these two processes.

There Is No Transdifferentiation Between Ectodermal and Mesodermal Lineages During Digit Tip Regeneration. Transdifferentiation be- tween ectoderm and mesoderm has been described during am- phibian tail regeneration (6). To determine whether epidermally derived cells contribute to the regenerating digit tip blastema, we used the Krt14-CreESR1 tamoxifen-inducible Cre allele (22) and either the R26R-lacZ Cre reporter allele (23) or the R26R- CAG-tdTomato Cre reporter allele (24) to fate-map the stump keratinocytes. Keratin 14 is a well-established marker expressed in mitotically active keratinocytes in the basal epidermis. Ta- moxifen was administered to PN0 pups. PN3 400 μm digit tip amputations were carried out, and litters were analyzed throughout regeneration to follow the fate of the keratinocyte descendants (Table S1).

Fig. 1. Lineage of Krt14-expressing keratinocytes during digit tip re- generation. (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- Fig. 2. Lineage of Sp7-expressing osteoblasts during digit tip regeneration. CreESR1;R26R-CAG-tdTomato digit sections at 2 wkPA and 3 wkPA. Epider- X-gal stained Sp7-tTA-tetO-EGFP::Cre;R26R-lacZ digit sections. (A and A’) mal retraction and proximal attachment to terminal phalanx occurs between Images at 2 wkPA show descendants in clot (arrow), bone, and blastema, but 1 and 3 dpa (A and A’, arrows). Dotted line shows approximate plane of not the epidermis. (B and B’) Images at 3 wkPA show descendants in bone amputation. Distally exposed bone becomes integrated into clot (B, B’, C, and connective tissue but not epidermis. Staining resembles normal 3 wk and C’) as epidermis covers the amputation site under the clot (C–E and C’– nonregenerative digit osteoblast contribution (C and C’). Insets in A’–C’ show E’). Blastema formation occurs after epidermal closure (D and D’). bl, blas- 40× magnification. bl, blastema; b, bone; ct, connective tissue; e, epidermis. tema; b, bone; c, clot; ct, connective tissue; e, epidermis. (Scale bars,100 μm.) (Scale bars, 100 μm.)

20610 | www.pnas.org/cgi/doi/10.1073/pnas.1118017108 Lehoczky et al. Downloaded by guest on September 29, 2021 contribution from a distinct cell pool. The epidermis continues to consist of marked cells (tdTomato) at 2 wkPA and 3 wkPA (Fig. 1 F, F’, G, and G’), indicating that the marked keratinocytes that form the initial regenerative epithelium continue to maintain the epidermis as the digit tip regenerates. Hence, epidermal stem cells as well as mature keratinocytes of the regenerate derive from the ectodermal tissue overlying the stump. Most importantly, our analysis of postamputation digits up to 3 wkPA does not show significant keratinocyte descendants in any tissue type other than the epidermis. Krt14-expressing keratinocyte descendants are not found in the clot, bone, or connective tissue, demonstrating that no transdifferentiation occurs from the ectodermal germ layer during digit tip regeneration. Conversely, we never see contribution to the regenerative epidermis from the various mesodermally derived cell populations analyzed below.

Regenerated Bone Derives from Lineage-Restricted Preamputation Osteoblasts. The distal 400 μm of the PN3 mouse digit consists primarily of bony and connective tissues, with no muscle or tendon present at this level. Moreover, there is no cartilage: the terminal phalanx forms during limb development by two discrete processes, endochondral ossification via chondrocytes limited to the growth plate of its proximal end, and direct ossification via osteoblasts at its distal tip (25). Although muscle, tendons, and cartilage are all absent from the amputation plane, it was nonetheless possible that these proximal tissues could contribute pools of cells that would migrate into the blastema after amputation injury. To test this, we addressed the possibility that chrondrocytes contribute to the regenerated bone with the Col2a1-CreERT allele (26) with the R26R-lacZ Cre reporter line and found that Col2a1- expressing chondrocytes do not contribute to any tissues of the regenerated digit (SI Text and Fig. S3). We turned our attention to the descendants of the pre- amputation osteoblasts, to verify that they give rise to the regen- erating skeletal tissues and to test whether they remain lineage- restricted or dedifferentiate to give rise to multiple tissues in the regenerated digit. To address the contribution of preamputation osteoblasts to the regenerate, we used the Sp7-tTA-tetO-EGFP:: Cre allele (27) with the R26R-lacZ Cre reporter line. Sp7 is known to specifically mark osteoblasts in both endochondral and intra- membranous bones. In this cross, Cre is expressed in osteoblasts throughout development until doxycycline is administered on PN0 Fig. 3. Expression of Msx1 during digit tip regeneration. Msx1-nlacZ x-gal and throughout the rest of the experiment to repress Cre ex- stained digit sections during regeneration. PN3 (A) and 3 wk (B) unampu- pression. PN3 digits were amputated and regenerates were ana- tated digits show expression in dorsal and ventral dermis and throughout lyzed 2 wkPA and 3 wkPA. (Mice developed and regenerated bone and sweat glands. (C and C’) Expression at 3 dpa is in normal stump significantly slower than animals from the other crosses in this domains and in the clot. (D and D’) Expression at 5 dpa is in the clot and ’ report. In this set of experiments, all regenerative events a few bone cells where epidermis is closing under the clot (arrow). (E and E ) are slightly delayed.) By 2 wkPA, Sp7-expressing osteoblast 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 pat- descendants are found in the blastema, the newly forming bone, fi and the clot at the distal tip of the regenerate, but not the epi- tern and resembles B. Epidermis is outlined in higher-magni cation pictures. A A’ bl, blastema; b, bone; c, clot; e, epidermis; np, nail plate; sg, sweat gland. dermis (Fig. 2 and ). The large concentration of osteoblast (Scale bars, 200 μm.) descendants in the clot likely reflects the observed trapping of a fragment of the distal bone into the clot as the epidermis closes underneath it (Fig. 1). By 3 wkPA the clot has sloughed off, and Because the Krt14-dependent Cre genetically causes re- the digit is largely regenerated, with preamputation osteoblasts combination and hence activation of the reporters exclusively contributing exclusively to the regenerated bone and the adjacent in the PN0 epidermal cells and is not active during regeneration, periosteum (Fig. 2 B and B’). In unamputated contralateral digits, x-gal staining allows for lineage analysis of stump keratinocytes Sp7-expressing osteoblasts are also found to contribute to these C C’ throughout regeneration. At 3 dpa, marked cells are seen two tissues by 3 wk (Fig. 2 and ), thus indicating that pre- throughout the epidermis surrounding the lateral stump and amputation osteoblasts serve as fate-restricted progenitors, only extending to the retracted edge of the epidermis, where it reat- repopulating tissues in which they were normally expressed. Im- portantly, no x-gal stained cells were found in the epidermis, in- taches to the bone, just proximal to the amputation plane (Fig. 1 A–C dicating that there is no transdifferentiation of mesodermally ). By 6 to 7 dpa, stump-derived keratinocytes are clearly derived osteoblasts into the ectodermal lineage. incorporated into the newly formed epidermis (Fig. 1 D, E, D’, E’ fi and ). From these experiments we nd that most, if not all, of Msx1 Is Expressed More Broadly than Previously Appreciated. The the regenerated epithelium is descended from preamputation necessity of Msx1 for successful digit tip regeneration (19) led us

keratinocytes. As seen at 3 dpa, there are a few unlabeled cells in to evaluate its expression pattern throughout the process, to BIOLOGY fl

the regenerative epidermis, likely re ecting incomplete pene- ensure that we have a complete understanding of the tissues DEVELOPMENTAL trance of the Cre activity, although we cannot rule out a minor involved. Using the Msx1-nlacZ knockin line (28), we analyzed

Lehoczky et al. PNAS | December 20, 2011 | vol. 108 | no. 51 | 20611 Downloaded by guest on September 29, 2021 Fig. 4. Lineage of Msx1-expressing cells during digit tip regeneration. TdTomato fluorescence from Msx1-CreERT2;R26R- CAG-tdTomato digit tips. (A–A’’’) Descend- ants 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’’) Dif- ferential 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.)

preamputation PN3 digit tips and found the nail bed mesen- however, represent a cell population that does not actively express chymal expression to be the largest and most homogenous do- Msx1 (Fig. 3E), indicating a change in and per- main of expression (Fig. 3A). Msx1 was also expressed in the haps cell type as the cells enter and proliferate in the blastema. By lateral dermis (still contiguous with the nail) as well as, to a lesser 2wkPA(Fig.4B–B’’) the clot is mostly lost, and Msx1 descend- degree, the ventral dermis (not associated with the nail) (Fig. ants are found within the bone and dermis, but not the epidermis. 3A), domains that were not previously reported. Additionally, At 3 wkPA (Fig. 4 C–C’’) Msx1 descendants have heterogeneously many lacZ-positive cells are found within the proximal articular populated the bone and dermis of the regenerate, resembling cartilage, the growth plate, tendon attachment points, and the the normal expression pattern of Msx1 at this stage (Fig. 3F). periosteum (Fig. 3A), all of which have been previously noted for Importantly, Msx1 descendants were not found within the epi- long bones (29). Msx1 is also expressed within the sweat glands dermis, once again indicating that there is no transdifferentiation of the ventral fat pad, blood vessels, and hair follicles, but these between the mesodermal and ectodermal germ layers. Because tissues are not directly analyzed in this study. Msx1 is expressed in a diverse set of cell types before amputation, During regeneration there are dynamic changes to the ex- we cannot conclude that Msx1-expressing cells do not trans- pression of Msx1 at the amputation plane. By 3 dpa the expression differentiate into another Msx1-expressing cell type. However, it is of Msx1 in the stump seems unchanged, but a new expression clear that the set of cell types expressing Msx1 before amputation domain forms in the clot surrounding the piece of trapped bone, is congruent to the cell types to which their derivatives contribute. in which there are also sparse Msx1-expressing cells (Fig. 3 C and Because Msx1 is expressed more broadly than just the nail bed C’). At 5 dpa Msx1 expression is still maintained in the clot, and mesenchyme, and because no alternative unambiguous markers additional expression can now be found in the bone, perhaps exist for this tissue, we cannot determine the fate of these specific osteoclasts (29), specifically where the epidermis is closing under cells in the regenerate. Thus, it remains unclear whether the nail the clot (Fig. 3 D and D’). By 7 dpa the epidermis has closed, and bed mesenchymal cells give rise exclusively to the regenerated nail the blastema has formed. Msx1 expression remains in the clot and bed mesenchyme and whether they represent a true cell type or is absent from the blastema (Fig. 3 E and E’). By 2 wkPA the clot simply a dense accumulation of dermal cells. has sloughed off, and by 3 wkPA (Fig. 3 F and F’) regeneration is complete and the expression of Msx1 once again resembles that of Regeneration Is Impaired by Removal of Fibrous Clot. In our lineage an unamputated control digit (Fig. 3B). analyses we found that Sp7 and Msx1 descendants populate the clot after amputation (Fig. 2A and 4A). To address whether the Cells Expressing Msx1 in the Preamputation Digit Contribute Broadly fibrin clot at the distal tip of the regenerating digit serves a re- to the Regenerate. To explore the contribution of Msx1-expressing generative function in addition to classical , we cells to the regenerate, we used the Msx1-CreERT2 tamoxifen- removed the clot at time points correlating with the closure of inducible Cre allele (30) with the R26R-CAG-tdTomato Cre re- the regenerative epithelium. For technical reasons the clot could porter line* (24). Intraperitoneal tamoxifen was given to PN0 not be removed cleanly before 6 dpa. Regeneration, as assayed pups, followed by digit tip amputation on PN3. By 1 wkPA (Fig. 4 by bone outgrowth, is compromised compared with control A–A’’’), descendants of Msx1-expressing cells are found in the clot regenerates, when the clot was removed at 6 dpa or 7 dpa (P < − (Fig. 4 A’ and A’’’) as well as the blastema (Fig. 4A’’), but not the 2.205e 14 and P < 0.0001, respectively), but not 8 dpa (P < 0.416) epidermis. The descendants found in the clot are consistent with (Fig. 5). Moreover, removal of the clot at 6 dpa often produces the Msx1 clot expression domain (Fig. 3 C–E) and may be the small regenerates that near the size of a nonregenerated digit same population of cells. The descendants found in the blastema, (Fig. 5 B and G, red line), consistent with the clot serving an essential regenerative function. Although the results of these manipulations were statistically significant, there was consider- *The R26R-lacZ reporter allele used in some of the previous experiments generally dis- able variability in the outcome (Fig. S5). The newly formed plays robust expression when activated via Cre-mediated recombination and was used epidermis that lies just under the clot structure is fragile and interchangeably with the R26R-CAG-tdTomato reporter. However, in some tissues re- easily punctured or removed. Mechanical removal of the clot, porter expression is much weaker and hence the use of the R26R-CAG-tdTomato allele fi was favored, as was the case here (Fig. S4). Although less dramatic, the staining from the although possible, is dif cult and can result in fresh bleeding. R26R-lacZ reporter allele is consistent with the R26R-CAG-tdTomato results These instances were documented, and the data were left in the described below. analysis. Thus, the data, as presented, may underemphasize the

20612 | www.pnas.org/cgi/doi/10.1073/pnas.1118017108 Lehoczky et al. Downloaded by guest on September 29, 2021 genitors derived from the preamputation tissue. First we estab- lished that there is no transdifferentiation between tissue layers during regeneration. Following Krt14-marked keratinocytes during regeneration, we were able to show that although the regenerative epithelium is derived from preexisting stump ker- atinocytes, these cells do not contribute to any other lineage in the regenerate and remain fated to the epidermis. In comple- mentary experiments, we show that mesodermally derived tis- sues, marked by Sp7 and Msx1, contribute only to mesoderm lineages, never ectoderm. Moreover, Sp7-expressing osteoblasts from the limb only contribute to skeletal tissues (bone and periosteum) in the regenerate, indicating that at least these blastema cells are lineage restricted. Altogether, the lineage data we present are consistent with and support reports of fate-re- stricted progenitor cells contributing to the regenerates of zebrafish fin, Xenopus tail, and axolotl limb (9–11, 31); and the emerging model that transdifferentiation between germ layers or tissue types does not occur during appendage regeneration. Additionally, these results indicate that digit tip regeneration may be more analogous to other examples of appendage re- generation than previously believed. During the preparation of this manuscript, Rinkevich et al. (32) published a report focusing on cell lineage restriction during adult mouse digit tip regeneration, using a similar approach to that taken here. Their conclusion, that lineage-restricted progenitor cells repopulate the digit tip without transdifferentiation, cor- roborates the findings in our report. As in our study, Rinkevich et al. demonstrated that the ectoderm of the regenerate is derived from preamputation progenitor cells and that these cells do not contribute to any other lineage or germ layer. Moreover, they showed that no mesodermally derived tissue transdifferentiates into an ectodermlal fate and all cells remain faithful to their original lineage.

Insight into Human Adult Digit Tip Regeneration. Appendage re- generation occurs in teleost fish and urodele amphibians throughout life, yet only larval anurans are capable of limb re- generation. Similarly, mice can regenerate their digit tips throughout life, whereas humans lose the ability with age. Hy- potheses on the lack of human adult digit tip regeneration include the following: preferential inflammation response over regener- ative response in adults, -mediated in- hibition of hyperproliferation in postneonatal tissues, age-dependent progenitor cell depletion, and neonatal and adult digit tip regeneration occurring by two discrete pro- cesses (of which humans have lost the latter). Although the ma- jority of these hypotheses are beyond the scope of this work, our data combined with other mouse digit tip reports, can provide insight into whether the neonate and adult processes, as exem- plified in the mouse, are the same. It is becoming increasingly clear that neonatal and adult mouse Fig. 5. Clot removal impairs bone regeneration. (A) Brightfield image of fibrin clot at 6 dpa. Inset: PN3 amputated digit tip, with dotted line depicting digit tip regeneration are much more similar than previously ap- – preciated. Fernando et al. (21) showed that during regeneration amputated tissue. (B F) Alizerin red/alcian blue stained digit tip skeletons at fi 3 wkPA representing average extents of regeneration for the group. (B) in adult mice the terminal phalanx regenerates via direct ossi - Nonregenerate digit after nail removal. (C) Control digit after no clot re- cation, consistent with the study by Han et al. (20), in which the moval. Digits after clot removal at 8 dpa (D), 7 dpa (E), and 6 dpa (F). (G) same was found during neonatal regeneration. Our report builds Boxplot analysis of clot removal experiment shows significant decrease in upon these commonalities and reveals that the regenerative epi- regeneration when the clot was removed at 6 dpa or 7 dpa, as measured by thelium of the neonatal postamputation digit tip forms at a vari- 2D area in pixels. Open circles represent 1.5 interquartile range outliers. able rate and includes a fragmentation of the terminal bone at a Average nonregenerate digit tip area is denoted with a red line. more proximal level, highly similar to what is described for the adult. Moreover, our data provide evidence against a single plu- ripotent blastema cell type and support a mechanism of multiple impact of clot removal because puncturing the skin may lead to fate-restricted progenitor populations participating in neonatal new clot formation, possibly rescuing regeneration. digit tip regeneration, which is consistent with what Rinkevich et al. (32) found during adult mouse digit tip regeneration. Col- Discussion lectively, the data support a single process used both during

Fate-Restricted Progenitor Model of Mouse Digit Tip Regeneration. neonatal and adult mouse digit tip regeneration, and the question BIOLOGY Using tissue-specific, inducible Cre alleles, we were able to show remains what the fundamental difference is in adult human digit DEVELOPMENTAL that the neonatal digit tip regenerates via fate-restricted pro- tips that prevents successful regeneration.

Lehoczky et al. PNAS | December 20, 2011 | vol. 108 | no. 51 | 20613 Downloaded by guest on September 29, 2021 Why Is Msx1 Necessary for Digit Tip Regeneration? The requirement formation is typically associated with a wound healing response, it of Msx1, coincident with its expression in the nail bed, led to the will be interesting to further explore the specific function(s) the hypothesis that the Msx1-expressing nail bed mesenchymal cells clot serves during digit tip regeneration. could serve as a stem cell population for the regenerate. Our data show that although Msx1 cells do broadly contribute to the Materials and Methods blastema and their own lineages in the regenerate, they do not Full methods are provided in SI Materials and Methods. act in isolation, and multiple other progenitor populations ac- tively participate in the process. This suggests that the necessity Mouse Strains and Alleles. Msx1-nlacZ mice were described previously (28). The of Msx1 during regeneration may be due to the signaling prop- Msx1-CreERT2 allele was generated by introducing the CreERT2 coding se- erties of the cells rather than the strict necessity of the de- quence (35) (a kind gift of Pierre Chambon, Institut de Génétique et de Biol- scendant population, although a combination of the two ogie Moléculaire et Cellulaire, Illkirch, France) at the initiator ATG site of Msx1 remains possible. by homologous recombination (30). Additional inducible Cre mouse strains Msx1 has been shown to be an important mediator of Bone Sp7-tTA-tetO-EGFP::Cre (27), Krt14-CreESR1 (22), and Col2a1-CreERT (26) were Morphogenetic Protein (BMP) signaling during digit tip re- obtained from Jackson Laboratories (strains 006361, 005107, and 006774). Cre generation (19), and it follows that Msx1-expressing cells may be reporter alleles R26R-CAG-tdTomato (24) and R26R-lacZ (23) were also necessary to mediate BMP activity after amputation. In this re- obtained from Jackson Laboratories (strains 007905 and 003474). All crosses port we show that Msx1 is expressed in the distal clot. The clot were performed with compound heterozygous males carrying both the in- has not received much attention in regard to digit tip regeneration ducible Cre driver and Cre reporter alleles and a CD1 wild-type female with the because it is a structure that forms routinely upon injury to the exception of the Sp7-tTA-tetO-EGFP::Cre allele, which was crossed directly to mammalian skin, but by lineage analyses we show that Msx1 and the R26R-lacZ reporter. Sp7 descendants reside within the clot, suggesting a functional role during regeneration, possibly beyond typical wound healing. Digit Manipulations. All mouse breeding and manipulations were done in By removing the clot, we provide preliminary data in support of accordance with the Harvard Medical School Institutional Animal Care and this hypothesis. In urodele amphibians, successful limb re- Use Committee. Mouse digit tip amputations were done on PN3 as previously generation depends on mitogenic signals provided by the nerve. reported (20). A dissection microscope (Leica MZ6) fitted with an eyepiece In contrast, mouse digit tip regeneration proceeds even in the reticule was used for digit amputations. Pups were cryoanesthetized before absence of innervation (33), suggesting that if these are analogous 400 μm of hindlimb digits 2, 3, and 4 were amputated using microdissection processes, the necessary signals are coming from another source. spring scissors (FST 15003-08). In all animals, right hindlimb digits were left Agrawal et al. (34) provide support for this hypothesis by showing unamputated as controls. One dose of 0.05 mg/kg s.c. buprenorphine was that at nonregenerative levels in digits of adult mice, application given as postsurgical analgesia. of ECM degradation products to the wound, as an artificial source ACKNOWLEDGMENTS. We thank Andrew McMahon and Susan Dymecki for of signals, led to the accumulation of stem-like cells at the plane helpful conversations regarding expression from the R26R-lacZ allele. This of amputation. Collectively there is clearly a delicate balance research was funded by National Institutes of Health Grants F32 AR56149 (to between wound healing and regeneration, and although clot J.A.L.) and R01 ND045499 (to C.J.T.).

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