a before amputation regeneration regenerated limb DERMIS SKELETON MUSCLE SCHWANN CELLS EPIDERMIS DERMIS SKELETON MUSCLE SCHWANN CELLS EPIDERMIS developmental origin: lateral plate mesoderm presomitic mesoderm neural fold lateral ectoderm b amputation wound epidermis formation contribution of dermis-, muscle-, skeletonand Schwann-derived cells to the blastema Supplementary Figure 1. Blastema cells respect their developmental origin. a. Scheme summarizing our results. We found that epidermis-, lateral plate-, presomitic mesoderm- and neural fold- derived lineages (indicated by the respective coloured boxes) remain restricted to their developmental origin during regeneration. b. Scheme illustrating a revised view of blastema formation. Cells coming from different tissues occupy distinct spatial domains and largely remain in distinct lineages during regeneration, except dermis. As the tissue differentiates, dermally-derived cells at the tip of the blastema contribute to the hand cartilage and tendons. www.nature.com/nature 1
ep c n c m ct Supplementary Figure 2. CAGGS-GFP expression is ubiquitous and does not get silenced during regeneration. Cross-section through a regenerated limb of a CAGGS-GFP transgenic animal, 8 weeks after amputation. Apart from blood cells (identified by autoflourescence) most DAPI stained nuclei were GFP positive (see Table S1). c= cartilage, ct= connective tissue, ep= epidermis, m= muscle, n=nerve. Arrowhead points to a nerve. Scale bar: 0.5 mm www.nature.com/nature 2
doi: 10.1038/nature08152 a stage 18 stage 18 2 cm GFP-Epidermis //Fibronectin b c pre-amputation regenerate Supplementary Figure 3. Epidermis only gives rise to epidermis during regeneration. a. Schematic to show the experimental design. b. Cross-section of a mature limb whose epidermis had been labelled as described in a. The basement membrane (visualized by Fibronectin staining, arrow heads) is located between the epidermal and dermal layer of the skin. The image demonstrates that only epidermal cells are GFP+. c. Cross section through the regenerated part of a limb whose epidermal cells had been labelled as described in a. GFP+ cells are only found in the epidermal region (see arrowheads in the inset that point to the Fibronectin-stained basement membrane). 3 limbs from 3 animals. Scale bars in b, c: 50 μm www.nature.com/nature 3
aaa pre-amputation GFP-dermis MHCI b PAX7 c GFP-skeleton MHCI d PAX7 g e GFP-muscle MHCI Supplementary Figure 4. Characterization of fluorescent dermis and skeleton grafts before amputation Cross-sections of mature limbs. Graft type and antibody stainings are indicated. a. Fluorescent cells of a dermal graft do not react with an anti-mhci antibody. b. Fluorescent cells of a dermal graft do not react with an anti-pax7 antibody. c. Fluorescent cells of a cartilage graft do not react with an anti-mhci antibody. d. Fluorescent cells of a cartilage graft do not react with an anti-pax7 antibody. e. Fluorescent presomitic mesoderm grafts gave rise to MHCI+ muscle fibers in 8 cm animals. 5 limbs, 3 animals (a, b); 6 limbs, 4 animals (c, d); 3 limbs, 3 animals (e). Scale bars: 50 µm. www.nature.com/nature 4
PAX7 Supplementary Figure 5. PAX7 expression in a 12-day midbud stage blastema. Longitudinal section of a midbud stage blastema stained for PAX7. Expression domains are highlighted by white rectangles, the amputation plane is depicted by a white line. Note that PAX7 expression is restricted to the periphery. Distal is to the top. Scale bar: 100μm. www.nature.com/nature 5
a DAPI GFP PAX7 Merge b average intensity (A.U) 2,000 1,800 1,600 1,400 1,200 1,000 800 600 400 200 0 Dermis (CarAct) Satellite cells (CarAct) Satellite cells (CAGGS) Dermis (CAGGS) Muscle fibers (CAGGS) c average intensity (A.U) 2,000 1,800 1,600 1,400 1,200 1,000 800 600 400 200 0 Dermis GFP+ satellite cells GFP+ muscle fibers d DAPI GFP Autofluorescent erythrocytes Supplementary Figure 6. Fluorescent muscle grafts harbor GFP+ satellite cells a. Representative cross-section of a mature limb prior to amputation immunostained with an anti-pax7 antibody (3 limbs, 3 animals). PAX7+, GFP+ cells (white arrow head) and PAX7+, GFP- cells (red arrow head) were present. b. Comparison of the average GFP fluorescence in PAX7+ satellite cells in transgenic donor animals (CAGGS) to satellite cell and dermis cell fluorescence in a different transgenic line (as a negative control) where only the muscle fibres express GFP (CarAct). Error bars show standard deviation. n = for CAGGs::GFP, dermis 6; muscle fibers 6; satellite cells 4; for CarAct::GFP, dermis 5; muscle fibers 5; satellite cells 8. c. Quantification of average GFP-fluorescence signal found in PAX7+ satellite cells in the presomitic mesoderm (muscle) grafts compared to negative dermis cells. Error bars show standard deviation. n= dermis 11; muscle fibers 15; satellite cells 30. Similar results were obtained from another replicate. d. Some GFP+ cells outside muscle in limbs prior to and after regeneration exhibit blood vessel morphology and often surround autofluorescent erythrocytes. Scale bar: 50 μm. 6
a b HoxA13 MEIS Supplementary Figure 7. MEIS and HoxA13 expression in the blastema a. Longitudinal section of a 12- day blastema on which immunostaining using an anti-meis antibody had been performed. Note accumulation of nuclear MEIS in the cells in the proximal part of the blastema while cells in the distal domain of the blastema do not exhibit MEIS signal. b. Longitudinal section of a 12- day blastema on which in situ hybridization using a HoxA13 antisense probe was performed. Note that the signal concentrates in the distal domain. a,b. Distal is to the top. Scale bars: 100 µm. www.nature.com/nature 7
a skeleton-derived (distal region) MEIS GFP Merge b skeleton- derived (proximal region) MEIS GFP Merge c Schwann cell-derived (proximal region) MEIS GFP Merge d 300 250 200 150 GFP+, Meis- GFP+, Meis+ 100 50 0 skeleton Schwann cells dermis muscle Supplementary Figure 8. MEIS is expressed in skeleton-, but not in Schwann- derived blastema cells. a-c. MEIS immunofluorescence on 12-day upper arm blastema sections harbouring indicated GFP+ grafts. Scale bar: 50µm. d. Different fractions of blastema cells from different sources express MEIS. Number of cells/blastemas/animals examined- skeleton: 203/6/6, Schwann: 281/7/7, dermis: 234/10/7, muscle: 187/2/2. www.nature.com/nature 8
a - reverse transcription cdna amplification dissociation FACSorting of single GFP+ cells - detection of genes using gene-specific primers b total number of sorted, blastema cells 450 400 350 300 250 200 150 100 50 0 skeleton Schwann cells dermis muscle Rp4+, HoxA13- Rp4+, HoxA13+ Supplementary Figure 9. Single Cell PCR analysis of HoxA13 expression in blastema cells derived from GFP+ grafts a. 12-day blastemas harbouring fluorescent cells were dissociated and single GFP+ cells were FACSed into 96-well plates. After extraction, reverse transcription and cdna amplification, the generated cdnas were analysed by QPCR using gene specific primers. Only cells that displayed a low Ct value for ribosomal protein4 (Rp4) were considered for further analysis. b. Histogram showing the expression of the distal marker HoxA13 in GFP+ skeleton-, Schwann-, dermis-, and muscle-derived blastema cells. Number of cells/blastemas/ animals/ experiments analysed- skeleton: 152/8/8/4, Schwann cells: 402/6/6/6, dermis: 230/12/12/6, muscle: 184/6/6/3. www.nature.com/nature 9
% of GFP+ cartilage cells found in the regenerated hand 100 90 80 70 60 50 40 30 20 10 p=0.000174 ïïï ïï ïïïïï ï ïïï ï ï ï ï ïï ïï ïï ï ïïï ï ï cartilage graft dermis graft 0 Tissue source 1of the GFP graft 2 Supplementary Figure 10. Dermal cells converting to skeletal fates preferentially contribute to the hand Histogram quantifying the fractional contribution of fluorescent skeleton- derived versus dermis- derived cells to the hand skeleton. Six fluorescent grafts of upper arm dermis or skeleton, respectively, were transplanted to the upper arm of normal hosts. Amputation was performed through the transplants. The fully regenerated limbs were sectioned, and fluorescent progeny in the skeletal elements were quantified. The graph shows the percentages of fluorescent cells found in the regenerated hand over the total number of fluorescent cells in lower arm and hand of the regenerate (n>1000 in each regenerate). Error bars indicate standard deviations. The p-value was determined using the Student s t-test (Welch; unpaired). www.nature.com/nature 10
Supplementary Table 1. CAGGS-GFP transgene expression is not silenced after regeneration a. CAGGS-GFP transgenic animals express the transgene ubiquitously in the limb muscle, nerves and skeleton (4 limbs, 4 animals) Sample % GFP+ satellite cells (PAX7+) % GFP+ cells within the nerve % GFP+ muscle cells (MHCI+) % GFP+ cells in the skeleton 1 98.4% (n=62) 100% (n=91) 100% (n=456) 100% (n=137) 2 100% (n=80) 100% (n=93) 100% (n=95) 100% (n=93) 3 97.1% (n=102) 100% (n=74) 100% (n=112) 100% (n=150) 4 100% (n=54) 100% (n=73) 100% (n=326) 100% (n=150) b. Regenerated limb cells maintain CAGGS-GFP expression (4 limbs, 4 animals) Sample % GFP + satellite cells (PAX7 + ) % GFP + cells within the nerve % GFP + muscle cells (MHCI + ) % GFP + cells in the skeleton 1 100% (n=100) 100% (n=72) 100% (n=219) 100% (n=290) 2 98.7% (n=80) 100% (n=57) 100% (n=230) 98.9% (n=88) 3 98.5% (n=198) 100% (n=157) 99.8% (n=618) 100% (n=282) 4 100% (n=96) 100% (n=210) 100% (n=1265) 100% (n=110) GFP-expressing cells from different tissues were counted in mature, previously not amputated limbs of the 10-12 cm long CAGGS-GFP transgenic animals (a) and in the 8 weeks old limb regenerates (b). Left and right arms of the same animal were used as experimental and control samples, respectively. There is no indication of silencing of the transgene during regeneration. n= number of cells counted. www.nature.com/nature 11
Supplementary Table 2. Tracking of fluorescent dermal cells during regeneration a. Skin-derived cells do not express PAX7 in the blastema (5 blastemas from 3 animals) Sample GFP +, PAX7 - GFP -, PAX7 + GFP +, PAX7 + 1 121 83 0 2 800 15 0 3 16 30 0 4 79 250 0 5 115 118 0 b. Skin-derived cells do not express MHCI or PAX7 in the regenerated limb but contribute to skeletal cartilage (6 limbs from 5 animals) Sample GFP +, GFP -, GFP +, MHCI +, PAX7 - PAX7 + PAX7 + GFP + Contribution to skeleton? 1 185 110 0 0 Yes 2 450 45 0 0 Yes 3 290 90 0 0 Yes 4 730 120 0 0 Yes 5 320 47 0 0 Yes 6 209 55 0 0 Yes www.nature.com/nature 12
c. Dermis- derived cells do not express MHCI, PAX7 or MBP in the regenerated limb but contribute to skeletal cartilage (3 limbs from 3 animals). Sample GFP +, GFP +, GFP +, MHCI +, PAX7 - PAX7 + MBP + GFP + Contribution to skeleton? 1 528 0 0 0 Yes 2 273 0 0 0 Yes 3 568 0 0 0 Yes a. Limb blastemas harbouring fluorescent skin-derived cells were sectioned longitudinally 12 days post amputation. 16 µm sections were stained with an anti-pax7 antibody. Note that we did not find GFP, PAX7 double positive cells. b. Labelling of dermis was achieved by transplanting fluorescent full-thickness skin from GFP-transgenic adult animals (8 cm snout-tail) to limbs of normal hosts. Fully regenerated limbs harbouring fluorescent dermis-derived cells were sectioned longitudinally 30 days post amputation. 16 µm sections were stained with antibodies directed against PAX7 or MHCI. Contribution of fluorescent cells to skeletal elements was tested by morphological observation. Note that we did not find GFP, PAX7 or GFP, MHCI double positive cells, however, in each case we found fluorescent cells being present in the skeleton. c. Labelling of dermis was achieved by transplanting lateral plate mesoderm from GFPtransgenic embryos to normal host embryos. Operated embryos were allowed to grow to a size of 8 cm. They were then used as donors to transplant full-thickness skin harbouring GFP + dermal cells to normal hosts. Fully regenerated limbs harbouring fluorescent dermis- derived cells were sectioned longitudinally 30 days post amputation. 16µm sections were stained with antibodies directed against PAX7, MHCI or MBP. Contribution of fluorescent cells to skeletal elements was tested by morphological observation. Note that we did not find GFP,PAX7; GFP, MHCI or GFP, MBP double positive cells, however, in each case we found fluorescent cells being present in the cartilage. To determine total numbers of values in a- c, every section containing GFP + cells was included into our analysis. www.nature.com/nature 13
Supplementary Table 3. Tracking of cartilage- derived cells a. Cartilage- derived cells do not express PAX7 in the blastema (2 blastemas from 2 animals). Sample GFP +, PAX7 - GFP -, PAX7 + GFP +, PAX7 + 1 84 130 0 2 40 64 0 b. Skeleton- derived cells mainly give rise to skeleton (3 limbs from 3 animals). Sample Cells in Cells outside PAX7 +, GFP + GFP +, MHCI + skeletal skeletal structures structures (likely tendon and dermis) 1 253 10 0 0 2 757 20 0 0 3 3338 327 0 0 a. Limb blastemas harbouring fluorescent cartilage-derived cells were sectioned longitudinally 12 days post amputation. 16µm sections were stained with an antibody against PAX7. b. Fully regenerated limbs harbouring fluorescent cartilage-derived cells were sectioned longitudinally 30 days post amputation. 16µm sections were stained with antibodies directed against PAX7 and MHCI. Contribution of fluorescent cells to skeletal elements was tested by morphological observation. To analyze all GFP + cells of the regenerate, every section containing fluorescent cells was counted. Note that we did not find GFP, MHCI or GFP, PAX7 double positive cells. www.nature.com/nature 14
Supplementary Table 4. Tracking of muscle- derived cells a. Fluorescent muscle grafts harbour GFP + satellite cells (3 limbs from 3 animals) Sample % GFP + satellite cells at amputation plane before regeneration 1 50% (n=38) 2 70% (n=43) 3 37.5% (n=56) b. Muscle derived cells express PAX7 in the blastema (2 blastemas from 2 animals) Sample GFP +, PAX7 - GFP -, PAX7 + GFP +, PAX7 + 1 53 297 135 2 13 20 493 c. Muscle- derived cells contribute to muscle, but not to cartilage or epidermis of the regenerated limb (6 limbs from 6 animals) Sample No. of nuclei GFP + cells in GFP + cells in GFP + cells in GFP + fibres epidermis/ total no. cartilage/ total no. outside muscle of epidermal cells of cartilage cells (blood vessels) 1 3172 0/26094 0/13838 224 2 3733 0/31554 0/14664 925 3 4012 0/52617 0/27378 1465 4 5034 0/49476 0/22843 1498 5 6616 0/52894 0/17849 848 6 5717 0/40887 0/18897 1522 www.nature.com/nature 15
a. 5 sections (16 µm) next to the amputation plane were taken from the amputated limbs in samples 1-3 to assess GFP, PAX7 colocalization. Note that we detected GFP +,PAX7 + cells in all three samples. n= number of cells counted. b. Limb blastemas harbouring fluorescent muscle-derived cells were sectioned longitudinally 12 days post amputation. 16 µm sections were stained with an antibody directed against PAX7. For each sample, at least 3 representative sections were analyzed. Note that we found GFP, PAX7 double positive cells among the fluorescent progeny. c. Fully regenerated limbs harbouring fluorescent muscle-derived cells were sectioned longitudinally 30 days post amputation. 16 µm sections were stained with antibodies against PAX7 and MHCI. Contribution of fluorescent cells to epidermis and skeletal elements was determined by morphological observation. Here, every limb section was included in our analysis. Note that we did not detect GFP + cells in cartilage or epidermis. www.nature.com/nature 16
Supplementary Table 5. Tracking of Schwann cells a. GFP-labelled Schwann cells are closely associated with MBP and/or BIII-tubulin after regeneration (4 limbs, 3 animals) Sample GFP +, cells next to MBP or BIII-tubulin GFP + cells not next to MBP or BIII-tubulin 1 464 6 2 1310 13 3 992 0 4 1842 0 b. GFP-labelled Schwann cells remain lineage restricted when implanted into irradiated limbs. Sample GFP + cells GFP + cells outside GFP + Implant type / recipient next to MBP cartilage and not cells in the treatment or BIII- next to MBP or BIII- cartilage tubulin tubulin 1 259 2 0 Schwann cell-gfp / P 2 322 2 0 Schwann cell-gfp / P 3 223 2 0 Schwann cell-gfp / P 4 226 5 0 Schwann cell-gfp / X 5 632 1 0 Schwann cell-gfp / X 6 1505 14 0 Schwann cell-gfp / X 7 665 5 0 Schwann cell-gfp / X 8 470 1657 2467 All-GFP / X 9 165 90 43 All-GFP / X www.nature.com/nature 17
a. Fate of Schwann cell derived GFP+ progenitors after regeneration was determined by staining sections of the regenerates with the antibodies against Myelin Basic Protein (MBP) and BIII-tubulin. The GFP+ cells next to BIII-tubulin or colocalizing with MBP were counted as being Schwann cells. 19 cells that scored as GFP +,MBP - /BIII-tubulin - were neither cartilage nor muscle cells and appeared to be immature glia with weak immunostaining. b. Regeneration of the X-rayed limbs was rescued by implanting non-irradiated GFPlabelled nerve. P=protected from X ray, X= X-rayed limbs. Samples 1-7: only Schwann cells in the implanted nerve were GFP+; samples 8,9: all cells in the implanted nerve were GFP + (All-GFP). The presence of GFP + cartilage in these samples but not in the Schwann cell-gfp samples shows that the cartilage derives from the GFP + nerve sheath but must come from a non-schwann cell source. Animal numbers used: Samples 1-3, N=3; 4-7, N=4; 8-9, N=2. N= number of animals www.nature.com/nature 18
Supplementary Table 6. Proximo-distal location of fluorescent cartilage grafts from different limb levels Graft type No. of grafts Location of fluorescent cells Lower arm only Lower arm + hand Hand only Upper arm skeleton 14 6 8 0 Finger skeleton 20 0 1 19 Upper arm Schwann Cells 15 0 15 0 Hand Schwann Cells 15 0 15 0 Fluorescent tissues were grafted to the upper arm level of sibling host animals and allowed to heal. Amputation was performed through the transplants. The final location of fluorescent progeny in hand and lower arm of the fully regenerated limbs was determined. www.nature.com/nature 19