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A proliferation gradient between proximal and msxb-expressing distal blastema directs zebrafish fin regeneration

¹ Howard Hughes Medical Institute, Department of Cardiology, Children’s Hospital, Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA
² Department of Human Genetics, University of Utah, Salt Lake City, UT 84112, USA

View larger version (79K): [in a new window]   Fig. 1. Proliferation in non-regenerating caudal fin. Zebrafish (n=5) were incubated in BrdU-containing water for 18 hours and BrdU was detected using immunolabeling (red). Posterior is at left and anterior is at right. (A) Whole-mount caudal fin shows abundant labeling concentrated at the posterior end of the fin. Rectangles indicate the approximate site of longitudinal sections. (B) Longitudinal section from the posterior end of intact caudal fin. Note positive labeling of both epithelial and mesenchymal cells (arrowheads). (C) Longitudinal section through the middle of the caudal fin. Note that both epithelial and mesenchymal cells are labeled (arrowheads). e, epithelium; l, lepidotrichia; m, mesenchyme.

View larger version (117K): [in a new window]   Fig. 2. Fin regeneration blastema consists of two functionally distinct regions, a non-proliferating distal and proliferating proximal blastema. Fish were incubated with BrdU for 6 hours before harvesting their fins. Longitudinal sections were stained with BrdU antibody (brown signal indicated by black arrowheads) and then counterstained with Hematoxylin. (A) 6 hours post-amputation (h.p.a.), a thin layer of wound epidermis covers the stump. The region immediately proximal to the amputation plane shows basal levels of epidermal proliferation; no BrdU-positive cells are detected in the intraray mesenchyme. (B) Early signs of mesenchymal disorganization are seen 12 h.p.a. (bracket). The number of proliferating epithelial cells is at basal levels, but there is no proliferation in the mesenchyme. (C) BrdU-positive mesenchymal cells were first detected 18 h.p.a. (arrowheads). Wound epidermis 24 h.p.a. thickens and the basal epidermal layer is initiated. (D) The number of BrdU-positive mesenchymal cells continues to increase, with some positive cells detected distal to the amputation plane. In addition to fibroblast-like cells, proliferating scleroblasts (D,E and insets) are seen (red arrowheads). (E) 2 days post-amputation, a functional blastema is formed. Note that the number of BrdU-positive cells is high in the proximal blastema, while the distal blastema remains unlabeled. Proliferating scleroblasts are observed immediately distal to bone stumps. (F) 3 days after amputation, newly formed bone is visible distal to old bone stumps. High levels of BrdU incorporation are seen in the proximal blastema, while the distal blastema remains unlabeled. Note that newly formed, BrdU-positive scleroblasts were apparent immediately behind the central zone of proliferation. The amputation plane is indicated by a dashed line. b, blastema; be, basal epidermal layer; e, wound epidermis; dmb, distal-most blastema; l, lepidotrichia; m, intraray mesenchyme; pb, proximal blastema; s, scleroblasts. Original magnifications: (A) 400x, (B) 400x, (C) 250x, (D) 200x, (E) 126x, (F) 126x and (G) 100x.

View larger version (74K): [in a new window]   Fig. 3. G2 shortens dramatically with the shift from blastema formation to regenerative outgrowth. (A-C) Longitudinal sections obtained from fins that were regenerating for (A) 24 hours (n=5), (B) 48 hours (n=3), and (C) 72 hours (n=4). Fins were treated with BrdU for 6 hours, followed by detection of BrdU (red) and anti-phosphohistone (H3P) (green), and counterstaining with DAPI (blue) to reveal nuclei. Double labeling appears yellow (arrowheads). (A) The first mesenchymal mitotic cells are seen 24 hours after amputation. (B) The number of cells undergoing mitosis continues to increase by 48 hours. Note that only a small fraction of H3P-positive cells (arrows) are also BrdU-positive 24 or 48 h.p.a., indicating that the duration of the median blastemal G2 is greater than 6 hours. (C) By 72 hours post-amputation, almost all H3P-positive cells are also BrdU-positive, indicating that the median blastemal G2 is less than 6 hours. (D) The fraction of labeled mitoses is shown for blastema formation (48 hours h.p.a.; 252 H3P-positive cells counted from 3 regenerates, 0.087±0.033) and during regenerative outgrowth (72 h.p.a.; 337 H3P-positive cells counted from 4 regenerates, 0.991±0.005). Note that the fractions are significantly different. Error bars indicate s.e.m. Z value for Mann-Whitney test is 26.759 (Zcritical is 1.645), P<<0.001.

View larger version (59K): [in a new window]   Fig. 4. Rapid G2 during regenerative outgrowth. (A-C) Representative sections of fins treated with BrdU for (A) 40 minutes, (B) 70 minutes, and (C) 140 minutes and stained for BrdU (red) and H3P (green). Double labeling appears yellow (arrowheads). Note the absence of double-labeled cells after 40 minutes, presence of some double-labeled cells after 70 minutes, and labeling of all H3P-positive cells with BrdU after 140 minutes. (D) Plot of the fraction of labeled mitoses over time. Each time point represents measurements ±s.d. from 40 sections obtained from three or four regenerates. Scale bars: 100 µm.

View larger version (61K): [in a new window]   Fig. 5. Steep proliferation gradient is formed between distal-most and proximal blastema at the onset of regenerative outgrowth. (A-C) Longitudinal sections obtained from fins regenerating for (A) 36 hours, (B) 48 hours, and (C, E, F) 72 hours. BrdU incubations were for 6 hours (A-C), 140 minutes (E) and 40 minutes (F). BrdU labeling is red, DAPI is blue. A 20 µm scale bar is aligned with the most distal blastemal cells. (A) Distal portion of 36-hour regenerate showing proliferation in distal blastemal cells (n=4, 39 sections examined). (B) Distal-most blastema is established at the end of blastema formation and the beginning of regenerative outgrowth, 48 h.p.a. (n=3, 36 sections examined). Note the absence of BrdU labeling in the distal blastema. (C) During regenerative outgrowth, 72 h.p.a., the distal-most blastema is limited to 20 µm (n=4, 34 sections examined). Note the absence of proliferation within the distal 10 µm of DMB, and limited proliferation within the next 10 µm of DMB. (D) Differences (±s.e.m.) in the fraction of BrdU-positive cells of between 0-10 µm (left, blue bars) and 10-20 µm (red, right bar) indicate that DMB is established at the beginning of blastema formation and maintained throughout regenerative outgrowth. Z and P values for Mann-Whitney test are shown above each time point. (E,F) Representative fields of short BrdU treatments, (E) 140 minutes (n=4, 35 sections examined) and (F) 40 minutes (n=4, 36 sections examined) are shown. Note with progressively shorter BrdU treatment fewer distal blastema cells are labeled. (G) Fraction of BrdU-positive blastemal cells in 72-hour regenerates during 40 minutes, 140 minutes, and 6-hour treatments are plotted as a function of length from the distal tip of the blastema. Note the decrease in the zone of limited proliferation (double headed arrows of the corresponding color) with increased length of BrdU treatment. (H) Number of H3P-positive cells shown as a function of length from the distal tip of the blastema. Note that the trend closely follows that observed during BrdU incorporation experiments. Stars indicate the most distal scleroblasts. b, blastema; be, basal epidermal layer; dmb, distal-most blastema; m, intraray mesenchyme; pz, blastema proliferation zone; s, scleroblasts.

View larger version (46K): [in a new window]   Fig. 6. Early proliferating mesenchymal cells contribute to the blastema. Fin sections from BrdU (red) pulse-chase experiments are shown. Fish were pulsed with BrdU for 2 hours at (A) 16 h.p.a., (B) 36 h.p.a. and (C) 70 h.p.a. and chased until harvesting at 72 hours. Fluorescent sections were counterstained with DAPI (blue) to reveal nuclei. (A) When a 2-hour BrdU pulse was given during early blastema formation (16 h.p.a.), staining is most apparent in the DMB and apical epidermis (arrows), including the basal epidermal layer. Note that BrdU label is diluted, indicating that blastemal cells divided before segregating into the DMB. BrdU label is even more diluted in the proximal parts of the regenerate (small dots, indicated by arrowheads) during outward growth of the regenerate. (B) When fish were pulsed at the end of blastema formation (36 h.p.a.) BrdU label in the DMB is mostly undiluted, while it is diluted in the proximal blastema. (C) By contrast, a late pulse of BrdU did not result in staining in the DMB (72 h.p.a.), indicating the absence of DNA synthesis during outgrowth. (D) A bar graph of the fraction of BrdU-positive cells in the DMB (0-20 µm) for the various 2-hour pulse times, 12 h.p.a., 16 h.p.a., 28 h.p.a., and 36 h.p.a.. Note that the fraction of BrdU-positive DMB cells is greatest when a pulse occurs late in blastema formation (36-38 h.p.a.), indicating that blastemal cells segregate into DMB late in blastema formation. For each case, the number of cells in the DMB from pulse-chase experiments was compared to 70-72 hour BrdU-treated fins using a Mann-Whitney test. P and Z values are: P<0.05, Z=1.649; P<<0.001, Z=7.558; P<<0.001, Z=9.518; P<<0.001, Z=11.249 for pulses administered at 12, 16, 28, and 36 hours respectively (Zcritical=1.645). Error bars indicate s.e.m. Scale bars: 50 µm.

View larger version (89K): [in a new window]   Fig. 7. Fin blastema is not formed from slow cycling stem cells. Representative fins from long-term pulse-chase experiments are shown. All the fins had continuous BrdU treatment for 4 days, followed by chases of various times. BrdU is indicated by red and H3P by green. (A) Intact caudal fin after a 4-week chase showing a high number of labeled transit-amplifying (TA) cells, mostly in the epidermis. In whole-mount (B) and longitudinal sections (C) of intact caudal fins after an 8-week chase, most of the BrdU label is lost from TA cells and concentrated in basal epidermal cells (arrowheads in C). (D) Longitudinal section through regenerating fin 2 days post-amputation. Note that BrdU-positive cells in the regenerate are rare and strongly labeled (arrows), indicating that these cells had not likely undergone division. H3P-positive cells are not BrdU-positive, suggesting that proliferating blastemal cells in the regenerate are not derived from slow-cycling cells. Original magnification (in A and B): 50x.

View larger version (133K): [in a new window]   Fig. 8. Msxb expression is restricted to non-proliferating DMB. Representative sections from 30- and 72-hour fin regenerates (n=4-6) were processed for detection of msxb (A-H) and shh (I-L) mRNA (red) and PCNA protein (green). Fluorescent sections are counterstained with DAPI to reveal nuclei (blue). (A-D) Note that during blastema formation, a large number of distal blastemal proliferating cells are positive for msxb, while during regenerative outgrowth (E-H), msxb expression is restricted to a small number of non-proliferating DMB cells. Although, msxb and PCNA domains partially overlap, there is no PCNA staining in the DMB. (I-L) The level of shh staining correlates with the end of the blastema proliferation zone and the onset of differentiation (appearance of new scleroblasts, indicated by stars).

View larger version (48K): [in a new window]   Fig. 9. A cellular model of zebrafish fin regeneration. During stage 1 (0-12 hours; wound healing), the wound epidermis is formed by migrating epithelial cells. In stage 2 (12-48 hours; blastema formation), the basal epidermis is formed, mesenchymal tissue proximal to the amputation plane begins to disorganize and intraray mesenchymal cells proliferate and move upward. A subset of early mesenchymal proliferating cells (red) expresses msxb (blue nuclei). A number of msxb-positive proliferating cells continue to increase throughout blastema formation. Just before the onset of regenerative outgrowth, blastemal cells segregate into msxb-positive, non-proliferating DMB and msxb-negative proliferating PZ, with a gradient of proliferation between the two domains. During stage 3 (48 hours 1 week; regenerative outgrowth), the gradient of msxb expression and proliferation is maintained, controlling the direction of outgrowth. Cells in the PZ proliferate vigorously and move in the proximal direction to differentiate. A zone of negative proliferation in the DMB maintains the directionality of the outgrowth by inhibiting proliferation.