Figure 1.
Description of early blastema and apical late blastema tissue grafts.
Images on the left are of an early bud (EB) blastema (A) and a late bud (LB) blastema (B). Cartoons to the right highlight landmarks in the EB blastema and LB blastema that were used to determine where the graft (donor) tissue was taken from. (A) Distal view of an EB blastema, where carbon (dark spots) was used to mark anterior stump and anterior blastema. The intact basement membrane made the stump appear lighter in color than the blastema tissue, which was covered with a wound epithelium and did not have a basement membrane. Additionally, the blood vessels in the stump were highly branched (honeycomb), while the blastema tissue had fewer visible blood vessels that aligned along the proximal/distal axis. The thick blue dotted line indicates the boundary between the stump tissue and the early blastema tissue. The thin blue dotted line marks the proximal boundary of the graft. (B) Anterior view of a late blastema with carbon marks identifying the anterior stump, and anterior apical tissues. The basal region of the LB blastema typically had blood vessels that were aligned along the P/D axis, while the apical region had very few visible blood vessels. The apical graft tissue (thin blue dotted line) was taken above these blood vessels to decrease the chances of including basal tissue.
Figure 2.
Grafted early bud blastema cells survived but did not form supernumerary limb structures
(A-C) Proximal EB blastemas grafted to the stump of a limb amputated at a distal level. Graft cells contributed to connective tissue (A) Live images of proximal EB blastema with stump grafted to the stump of a limb amputated at a distal level, 1 and 5 weeks post surgery, and whole mount skeletal preparations from 7 weeks post surgery. Red arrows indicate the distal amputation plane on the host limb. Green arrows point to extra elbow joint and radius/ulna. Duplicated proximal structures were observed in 5/6 grafted limbs. (B-C) Proximal EB blastemas without stump grafted to the stump of a limb amputated at a distal level. None of the grafted blastemas (6/6) formed duplicated proximal-distal limb patterns. (B) Live images at 1 and 5 weeks post surgery, and tissue section 7 weeks post surgery where the regenerated skeletal tissues are outlined in white. Nuclei are stained with DAPI (blue), and grafted cells are GFP positive (green). Red line demarks the host amputation plane (higher magnification in (C)). The grafted cells contributed to a variety of different tissues including connective tissue (arrow 1), muscle (arrow 2), and cartilage (arrow 3). (D-I) Distal EB blastemas grafted to the stump of a limb amputated at a proximal level. (D) Live images of distal EB blastema with 1 mm of stump grafted to the stump of a limb amputated at a proximal level. The grafted cells remain viable and contributed to regenerated tissues as evident in sections of regenerated limbs 7 weeks post surgery in all grafts with (5/5) or without (E) (8/8) stump tissue included. Grafted cells contributed to nerves (arrow 1), bone (arrow 2, magnified in F), fibroblast-like cells in the dermis (G) connective tissue surrounding cartilage and cartilage (H), and muscle (I).
Figure 3.
Forelimb (FL) EB blastema cells were reprogrammed and expressed hind limb markers when grafted to the stump of an amputated hind limb (HL).
(A) Illustration describing how GFP+ (graft) cells were isolated from GFP- (host) cells in a mosaic blastema created by grafting a GFP+ blastema to a GFP- host. (B) Validation by q-rtPCR of Tbx4 and Tbx5 as markers for HL and FL respectively. Histogram is fold change of expression in FL blastemas relative to HL blastemas. Error bars are SE (N = 3 technical replicates). (C) Representative histogram of relative Tbx4 expression in GFP+ FL grafts to FL or HL (FL+, HL+, respectively) and GFP- FL or HL host tissue (FL-, HL-). Error bars are SEM (N = 3 technical replicates). P-values were determined by T-test with 2 tails and unequal variance (N = 3 biological replicates for which cells were pooled from 8 blastemas for each sample). (D) RT-PCR (35 cycles) for Tbx4, Tbx5, and GAPDH performed on graft and host cells. The sequence of the high molecular weight Tbx5 band can be found on GenBank (Accession # KC920480). Illustrations describing the surgical manipulations performed in this study are in Figure S1C,D.
Figure 4.
Apical and basal regions of LB blastema grafts had different levels of positional stability/lability.
(A) The basal region from a proximal LB blastema grafted to the stump of a limb amputated at a distal level (imaged 1 and 7 weeks post-grafting) gave rise to a limb with a duplicated proximal-distal limb pattern as evident in whole mount skeletal preparations. Red arrows indicate the distal amputation plane on the host limb. Green arrows point to extra elbow joint and radius/ulna. Duplicated proximal structures were observed in 5/6 grafted limbs. (B) In contrast, when the apical region from a proximal LB blastema was grafted as in (A), almost no proximal pattern duplications were observed (N=5/6). (C) Longitudinal section of a regenerated limb derived from apical LB or basal-LB (D) blastema grafts from a distal amputation (green) to a proximal host site. GFP+ graft cells live and differentiate into a variety of tissues in the regenerate (6/6 apical grafts, 5/5 basal grafts). (E-J) Grafted cells from the apical and basal region of a LB blastema contributed to a variety of tissues including (E) blood vessels, (F) cartilage, (G) connective tissue surrounding skeletal elements, (H) nerve-associated cells (red stain is for acetylated β-tubulin in neural axons), (I) muscle, and (J) cells in the dermis.
Figure 5.
Innervation was required to maintain positional lability of EB blastema cells and apical-LB blastema cells.
(A) An ectopic limb was formed when posterior skin was grafted to a nerve-deviated wound on the anterior side of the arm (7/9) [11]. Consistent with our previous results, 7 of 8 grafts of basal LB blastemas formed well-patterned distal limb structures (F); whereas, 25 of 30 grafts of either EB blastemas (B) and 14 of 17 apical LB blastemas (D) did not form ectopic cartilage structures. In contrast, 9 of 14 grafts of EB blastemas from denervated donor limbs (C) and 6 of 9 apical LB blastema grafts from denervated donor limbs (E) formed ectopic skeletal elements. Experimental limbs were imaged 1 week and 3 weeks post-grafting, and samples were stained for the skeletal patterns in whole-mount preparations (right) 9 weeks post-surgery. Red arrows in (C) and (E) indicate the distal tip of the ectopic structures. Illustrations describing the surgical manipulations performed in this study are in Figure S1E. A more detailed quantification of the regenerated phenotypes in the present study is presented in Table S1.
Figure 6.
Lability of positional information was nerve dependent in the limb blastema.
(A) (top panel) The “Distal-First” hypothesis is based on the idea that the early blastema is composed entirely of cells with the distal-most (“D”) identity. The intermediate (corresponding to the pattern between the distal tip and the proximal level of amputation) positional information (“I”) is intercalated as these distal cells interact with the proximal information (“P”) in the stump. (A) (bottom panel) The distal positional information in the early blastema and the apical-tip of the late blastema is labile (“L”). The proximal and intermediate information in the stump and basal region of the late blastema, respectively, is stabile (“S”). (B) Removal of signals from the nerve by denervation results in the loss of lability and premature stabilization of the distal-most positional information before the intermediate identities have been intercalated.