Lin G , Chen Y , Slack JM .

R01 GM088500 NIGMS NIH HHS , R01GM088500 NIGMS NIH HHS

Xla Wt + Hsa.FGF10 + Hsa.SHH + forelimb amputation (Fig.3.E, F)
Xla Wt + Hsa.FGF10 + Hsa.SHH + TMSB4X + forelimb amputation (Fig.3.M,N,O,P Q)
Xla Wt + Hsa.FGF10 + Hsa.SHH + TMSB4X + forelimb amputation (Fig.3.R)
Xla Wt + Hsa.SHH + forelimb amputation (Fig.3.D)

	Figure 1. Thymectomy Is Necessary for Transplantation Experiments in Xenopus Frogs(A and B) The developing thymus in a control stage 55 tadpole, shown as in whole-mount animal (A) and on cross-section after hematoxylin and eosin staining (B). Black arrows indicate thymus.(C) Limb regeneration after transplantation of a GFP-labeled limb bud to a wild-type frog host. GFP is undetectable in the single spike regenerate, 46 days postamputation (dpa). Inset shows a green limb bud immediately after transplantation. Note the relative size of the donor and host.(D and E) Thymus is not regenerated in thymectomized (T-) host, as shown by the lack of thymus in whole-mount animal (arrows in D) and on cross-section after haematoxylin and eosin staining (E), 36 days after thymectomy.(F) Limb regeneration after transplantation of a GFP limb bud to thymectomized (T-) host. The graft has survived long term and generated a multidigit regenerate. Inset shows GFP fluorescence.White lines in (C) and (F) indicate amputation levels. Scale bars are 100 μm for (A), (B), (D), and (E) and 250 μm for (C) and (F). See also Figures S1 and S2.
	Figure 2. Delivery of Cells to the Limb Stump using a Fibrin Patch(A) A frog limb stump covered with a fibrin patch containing GFP-labeled cells, 1 dpa. The white dotted line indicates the patch. Scale bar, 1 mm.(B) Migration of nGFP (green) cells out of the patch in an early regenerate, 3 dpa. Nuclei stained with propidium iodide (PI) are shown in red. An area far away from the patch (outlined in B) is shown in (B. Scale bar, 100 μm.(C) The patch allows application of growth factor beads to the limb stump. The white dotted line outlines the patch, and the blue arrow indicates Affi-Gel beads. Scale bar, 1 mm.(D and E) Sagittal sections of limb stump with (E) or without (D) fibrin patch. The blue dotted line indicates the boundary of the patch. Scale bar, 500 μm.
	Figure 3. Xenopus Frog Forelimb Regeneration after Cell Transplantation. Left limbs are transplanted, and right limbs are controls with amputation only.(A) Limb regeneration in a frog with a limb bud cell patch, 2 months post-amputation (mpa). Skeletal staining shows that regenerates are still simple spikes (A and C) similar to controls (B), although two cases gave slight extra cartilage (arrows in C).(D) Skeletal preparations of four forelimbs treated with limb bud cell patches and Shh beads, showing slightly disturbed cartilages, 2 mpa.(E) Frogs treated with a βcat∗ limb bud cell patch and Shh + FGF10 beads (BSF), 6 mpa. Multi-digit regenerates are formed (E, F, I, and J) and some proximodistal segmentation of cartilage is evident (blue arrows in G and L). One digit also has a small patch of calcium deposition (red arrow in K). All panels, except (H), are for BSF treatment. (H) shows skeletal preparation of a typical spike as control, 6 mpa.(M) A frog treated with a βcat∗ limb bud cell patch, Shh + FGF10 + thymosin β4 (BSFT), 5 mpa. Multiple digits have regenerated (M), and the X-ray shows the presence of extensive ossification in the regenerate, as confirmed by skeletal staining (O and P). Some proximodistal segmentation is also clear (blue arrows in P).(Q) Regeneration of intermediate skeletal elements in frog shown in (M). White dotted lines indicate individual metacarpal-like structures.(R) Detection of Gdf5 by in situ hybridization in regenerate from frog treated with BSFT. Cross-section of a digit (2 mpa) shows joint-like structure, with Gdf5 expression (between red brackets).(S) A skeletal preparation of a normal forelimb, for comparison.Red dotted lines indicate amputation levels. Scale bars in (A), (E), (I), (J), (M), and (N), 500 μm; scale bars in (B)D), (F)H), (K), (L), and (O)S), 100 μm. See also Figures S3 and S4.
	Figure 4. Cell Death and Cell Proliferation Analysis in Cell Transplants(A) Cell death detection by TUNEL in 3 day regenerates after GFP-labeled limb cell patch or bud graft. (A) Cells with GFP label alone. (B) GFP limb cell patch with Shh and FGF10. (C) βcat∗ limb cell patch with Shh and FGF10. (D) βcat∗ limb cell patch with Shh+FGF10+thymosin β4. (E) Whole limb bud transplantation. Sections are through the cell patch or the transplanted bud. GFP shown in green indicates the nGFP or pHsβcat∗GFP donor cells. The TUNEL signal is shown in red, and nuclei are shown in blue with 46-diamidino-2-phenylindole (DAPI) stain. White asterisks (B) indicate positions of Affi-Gel beads. Scale bars, 100 μm.(F) Quantification of cell death in the transplants (G, GFP label only; S, Sonic hedgehog; F, FGF10; B, activated β-catenin; and T, thymosin β4). Error bars are standard deviations; n = 4; single-factor ANOVA shows a significant difference in groups; ∗p < 0.05, BSFT versus BSF; ∗∗p < 0.01, bud versus BSFT, as determined by t test.(G) Cell proliferation determined by EdU incorporation. EdU is shown in red, and nuclei are shown in blue. White asterisks in (H)J) indicate positions of Affi-Gel beads. Scale bars, 100 μm.(L) Quantification of cell proliferation in the transplants; labels as above. Error bars are standard deviations; n = 4; ANOVA and t test analysis show significant differences between groups. ∗∗p < 0.01, as determined by t test.
	Figure 5. Upregulation of Shh, Wnt, and Fgf Genes in Limb Regenerates(A, E, I, M, Q, and U) Whole-mount in situ hybridization shows expression of Shh, Wnt, and Fgf genes in limbs following BSFT treatment, 6 days pa. Scale bars, 500 μm. Numbers indicate frequency of observed expression in limb regenerates.(B, F, J, N, R, and V) Far-red (magenta) image of in situ signals developed with Fast Blue BB, with Y5 filter cube.(C, G, K, O, S, and W) Detection of GFP-positive cells with anti-GFP antibody and AlexaFluor 488 conjugated secondary antibody, with GFP filter cube.(D, H, L, P, T, and X) Merges: the white color indicates gene expression in GFP-positive cells.(A) Shh, (E) Wnt3a, (I) Wnt5a, (M) Fgf8, (Q) Fgf10, and (U) Fgf20. White dotted lines indicate the edges of the regenerates. Black scale bars, 500 μm; white scale bars, 100 μm. See also Figure S5.
	Figure 6. Analysis of Donor Cell Contribution in Limb Regenerates after (pHsβcat∗GFP Donor)/(Wild-Type Host) Transplantations(A) Diagram of cell contribution analysis. Wild-type host frogs were transplanted with a βcat∗ limb bud cell patch with Shh and FGF10 after amputation. Heat shock was given for the first 2 weeks to maintain βcat∗ expression. Frogs were left without heat shock for 4 weeks, and heat shock was given 3 days before sample collection to activate GFP expression (fused to βcat∗) in donor cells. The red line indicates the level of sections shown in (B and C)(B and C) Detection of donor cells (green) in cartilage (B), muscle (red, with 12/101 antibody staining, B and C) and epidermis (C) in a regenerate illustrated as in (A), 45 dpa. m, muscle; c/b, cartilage or bone; and e, epidermis. Muscle tissues are shown in red as revealed by immunostaining with 12/101, a specific muscle marker, and labeled as m in the red channel images; cartilage and epidermis are outlined with white dotted lines in the blue channel images.(D) Quantification of donor and host cell contributions in limb regenerates from (pHsβcat∗GFP donor)/(wild-type host) transplantations. Error bars, standard deviation; n = 9 animals.See also Figures S2 and S3.
	Figure 7. Analysis of Cell Contribution in Limb Regenerates after (pHsβcat∗GFP Donor)/(pCMVnGFP Host) Transplantations(A) Diagram of cell contribution analysis. pCMVnGFP host frogs were used, and GFP expression in pHsβcat∗GFP donor cells was turned off by not giving heat-shock treatments. Thus, GFP+ cells shown in (B)D) are from the host. The red line indicates the level of sections shown in (B)D).(B) Detection of host cells (green) in cartilage (B), muscle (C), and epidermis (D). Cartilage, muscle, and epidermis are shown as in Figure 6. Scale bars, 100 μm.(E) Quantification of donor and host cell contributions in limb regenerates from (pHsβcat∗GFP donor cells)/(pCMVnGFP host) transplantations. Error bars, standard deviation; n = 9 animals.See also Figures S2 and S3.
	Figure S1. Comparison of graft survival and limb regeneration in normal and thymectomized hosts. (related to Fig.1). When a pCMVnGFP limb bud is grafted to a wild type host, expression of the GFP is detectable 6 days post amputation (A) but disappears soon after, so that at 20 dpa, it is no longer visible (B). In thymectomized (T-) hosts, GFP expression persists long term (E, F). Histology shows spaces in the tissue and lymphocyte accumulation in the limb bud grafted into a wild type host (C) but a normal mesenchymal appearance of the limb bud grafted into a thymectomized host (G). Two examples of limb regenerates with skeletal preparation are shown in (D) and (H). Arrow heads indicate amputation levels. Scale bars for (A, B, D, E, F, H) 500 um; Scale bars in (C, G) 100 um.
	Figure S2. Contribution of donor and host cells in regenerates formed after limb bud grafting (related to Fig. 1) Muscle tissues are shown in red as revealed by immunostaining with 12/101, a specific muscle marker, and labeled as m in the red channels; Cartilage (C/B) is outlined with white dotted circles in the blue channels. Epidermis (e) is outlined with white dotted lines in the blue channels. Scale bars 100um. (A-C) Detection of donor cells by GFP antibody staining (green) in cartilage, muscle (with 12-101 antibody, red), and epidermis, in regenerate from (pCMVnGFP donor) / (wild type host) transplantations. (B, C) are higher power images for outlined areas in (A). White arrows in (B) indicate examples of GFP-positive cells in the cartilage. (D) Quantification of donor and host cell contributions in (pCMVnGFP donor) / (wild type host) transplantations. Error bars are standard derivations (n=9 animals). (E-G) Detection of host cells by GFP antibody staining in regenerate from (wild type bud donor) / (pCMVnGFP host) transplantations in regenerating cartilage/bone (E), muscle (F) and epidermis (G). White arrows in (E) indicate examples of GFP-positive cells. (H) Quantification of donor and host cell contributions in (wild type donor) / (pCMVnGFP host) transplantations. Error bars are standard derivations (n=9 animals).
	Figure S3. Characterization of pCMVnGFP and pHsBeta-cat*GFP transgenics (related to Fig. 3, 6, 7). (A-F) GFP expression in pCMVnGFP transgenic frog limb. Expression of GFP is detected with anti-GFP and Alexa Fluor 488 conjugated secondary antibody shown in green in muscle (Mus, A), cartilage or bone (Cart/bone, D). Nuclear staining with propidium Iodide is shown in red (B, E). (C, F) merged images. White arrows indicate cells that are negative or very low in GFP expression. Scale bars 100 um. (G-J) Heat shock induced expression of Beta-cat*. Detection of activated beta-catenin (shown in green) by anti-active--catenin antibody (alpha-ABC) in limb cells isolated from pHscat*GFP limb buds, with (I) or without (G) heat shock treatment. Nuclei are shown in red (H, J). Nuclear localization of beta-catenin is apparent after the heat shock induction (J). Scale bars 50 um. (K-P) GFP expression in pHsBeta-cat*GFP tadpole limb bud. Red arrow shows an example of a limb bud cell with very low GFP expression. (N-P) are higher power images for outlined area in (M). Scale bars, 25 um. (Q-V) Illustration of methods for cell counting. (Q, R, S) Contribution of GFP-positive donor cells to regenerating cartilage in one of the sections obtained from an experiment involving transplantation of beta-Cat*GFP donor cells into a wild type host, 13 out of 25 cartilage cells are GFP-positive. The GFP-positive cells are circled in the green channel, and the boundary of the cartilage tissue is shown with the purple dotted line. Scale bar: 10 um. (T, U, V) Contribution of GFP-positive host cells to regenerating muscle tissue. GFP-positive muscle cells are circled in the green channel. Muscle fibers are indicated with 12/101 antibody staining. Of 75 muscle fibers (red channel), 29 are GFP-positive. Scale bar 10 um.
	Figure S4. Joint marker expression and nerve innervation in limb regenerate (related to Fig.3). (A-C) Gdf5 is expressed in digit regenerates from Beta-cat* cell patch and Shh, Fgf10, thymosin Beta-4 treatment (BSFT, B), but not expressed in the control cartilaginous spike (A). The white rectangle in (B) indicates Gdf5 expression in a digit regenerate that is similar to the joint of a developing tadpole digit (C). Scale bars 500 um. (D-K) Nerve regeneration in limb regenerates and spikes. Immunofluorescence staining with anti--tubulin III antibody (green) indicates abundant innervation in limb regenerate from Beta-cat* cell patch and Shh, Fgf10, thymosin Beta-4 treatment (D, BSFT). White arrows in (D) indicate nerve bundles. (E) Shows a high magnification of nerve bundles (n) that are Beta-tubulin III-positive and a glandular structure (g) that is Beta-tubulin III-negative. (F, G) Histology of a section from the same regenerate in (D) shows the distinct morphology of a nerve bundle (n) and a glandular structure (g). Scale bars in (D-G), 50 um. (H, I) Transmission electron microscopy (TEM) images of axons in limb regenerate from BSFT treatment showing multiple small (2-5 um) myelinated axons at 15K magnification (H) and a single axon (a) with numerous closely packed myelin lamellae (m) at high magnification (30K, I) (G) Immunofluorescence detection of Beta-tubulin III in a control spike, 2 mpa. The spike is innervated but there is a lack of larger nerve bundles. (H) High magnification (30K) TEM image of a single axon in spike.
	Figure S5. Gene expression analysis in limb regenerates (related to Fig.5). (A-F) Detection of Shh, Fgf8, Fgf10, Fgf20, Wnt3a, Wnt5a in frog limb regenerates after limb bud transplantation and amputation, 6 dpa. Black arrows indicate the implanted limb bud. All genes are expressed in the regenerating limb bud. Scale bar 500 m. (G-L) Detection of Shh, Fgf8, Fgf10, Fgf20, Wnt3a, Wnt5a in frog limb stumps after beads (containing Shh and FGF10) are implanted, 6 dpa. Short blue arrows indicate some of the implanted beads. Only Fgf8 and Fgf10 are weakly expressed in the limb stump (red*). Scale bar 500 um. (M) RT-PCR analysis in control limb regenerates (C. 6dpa), limb regenerates with GFP labeled limb bud cell transplants (G. 6dpa), B-cat* limb bud cells with Shh, FGF10 beads (BSF. 6dpa), and 2 month old controls (Spike) shows that Shh, FGF and Wnt pathways are active in BSF limb regenerate. T, non-reverse transcriptase control.(Xenbase Curators Note: typo: 'Mpk3' [sic]= mkp3/dusp6) (N-U) In situ detection of hoxa13 and hand2 in frog limb regenerate. hoxa13 is expressed in limb regenerate after Beta-cat* cell patch together with Shh, FGF10, thymosin Beta-4 (BSFT), in whole mount specimens 6 dpa (N) and 20 dpa (O). Sagittal section shows expression of hoxa13 in the limb regenerate (P) and limb stump with beads (Shh, Fgf10) implantation only (Q). Hand2 is expressed in limb regenerate after BSFT treatment, 6 dpa (R), 20 dpa (S). Sagittal section shows expression of hand2 in the limb regenerate (T) but not in limb stump with bead (Shh, Fgf10) implantation only (U). Scale bars in (N, O, R, S) 500 um; Scale bars in (P, Q, T, U) 100 um.
	Figure S6. Failure of limb regeneration in frog transplanted with cells from pHsBeta-cat*GFP tadpole tail regeneration bud (related to Table 1). (A, B) The majority of the regenerates show no difference from controls (B). (C-F) Two cases showed slight extra cartilage formation (insets in C, E). (D, F) are spikes regenerated from the control side. dpa, day post amputation. Scale bars 250 um.

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