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First published online June 27, 2005
doi: 10.1242/10.1242/dev.01906

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Hedgehog signaling controls dorsoventral patterning, blastema cell proliferation and cartilage induction during axolotl tail regeneration

¹ Max Planck Institute for Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307 Dresden, Germany
² Curis Incorporated, 61 Moulton Street, Cambridge, MA 02138, USA

View larger version (42K): [in a new window]   Fig. 1. Shh, Pax6, Pax7 and Msx1 are expressed in the differentiated and regenerating axolotl spinal cord. All panels are cross-sections with the dorsal side up. (A-C) Shh is expressed in the floor plate of the differentiated spinal cord (A) and in the ventralmost ependymal cells 6 dpa (B,C). The overview in C shows that Shh is expressed exclusively in the spinal cord. (D,E) Pax6 is expressed in the lateral cells of the differentiated axolotl spinal cord (D) and in the lateral ependymal cells 8 dpa (E). (F,G) Pax7 is expressed in the dorsolateral cells of the differentiated spinal cord (F) and in the dorsolateral domain of the ependymal tube 6 dpa. In addition, Pax7 is expressed in lateral tail cells (F,G). (H-J) Msx1 is expressed in the roof plate of the differentiated spinal cord (H) and in the dorsalmost ependymal cells 4 and 5 dpa (I,J for overview). Panels A-E,H-J show in situ hybridizations, panels F,G antibody staining. Scale bars: 100 µm in A,B,E-G; 50 µm in C,D,H-J.

View larger version (76K): [in a new window]   Fig. 2. Cyclopamine treatment blocks axolotl tail regeneration and a hedgehog agonist rescues the phenotype. (A-C) Control regenerates 4, 8 and 14 dpa. The blastema cells are surrounding the ependymal tube at 4 dpa (A). Cartilage has differentiated 8 dpa (B) and muscle 14 dpa (C). (D-F) Cyclopamine-treated tail regenerates 4, 8 and 14 dpa. The fin and the ependymal tube have grown, but a blastema is not visible (compare A and D). No cartilage has differentiated 8 dpa (E) and no muscle has formed 14 dpa (F). (G) Quantification of the lengths of control and cyclopamine-treated tail regenerates over time. The length of the regenerating spinal cord is taken as a measurement for overall tail regeneration. Data points represent the average length of three regenerates; error bars are standard deviations. (H-J) Cyclopamine-treated regenerate (H), cyclopamine and agonist-treated regenerate (I), and normal tail regenerate (J) 10 dpa. The tails in I and J are indistinguishable. (K) Quantification of regenerate lengths (as in G) in indicated conditions shows that 40 nmol/l of the agonist are sufficient to rescue the cyclopamine effect, whereas 4 nmol/l are not. The dashed line in A-F and H-J marks the amputation plane. Scale bars: 0.5 mm.

View larger version (124K): [in a new window]   Fig. 3. Dorsal spinal cord progenitor domains are increased in cyclopamine-treated and normal in rescued regenerates. (A-C) Pax7 antibody stainings on cross-sections of cyclopamine-treated regenerate (A), rescued regenerate (B), and agonist-treated regenerate (C). The Pax7 expression domain in the dorsal ependymal tube is expanded ventrally in A, normal in B (compare to Fig. 1G), and absent in C (dashed line in C marks the ependymal tube). The arrows in A,B point to the ventral border of Pax7 expression in the ependymal tube. Note that the Pax7 expression persists in the lateral blastema cells in C. Pax7 staining is in red and nuclear Hoechst staining in blue. (D,E) In situ hybridization of Msx1 on cross-sections of cyclopamine-treated (D) and rescued regenerates (E). Msx1 expression in the ependymal tube of cyclopamine-treated regenerates is strongly expanded laterally (D, compare with Fig. 1I). The rescued tails show normal Msx1 expression (F). Scale bars: 100 µm.

View larger version (58K): [in a new window]   Fig. 4. Ependymal cells and tail blastema cells express the hedgehog receptor Ptc1. (A-F) In situ hybridization of Ptc1 on cross-sections. Sense probe shows no staining of the entire cross-section both in control (A) and agonist-treated (D) regenerates. Ptc1 is expressed in the ventral ependymal cells (dashed line marks the ependymal tube) and in the blastema, but not in the epidermis (B,C). Note that the staining in the blastema is strongest in the cells surrounding the ventral spinal cord. C is taken at twice the magnification of B. A-C is 4 dpa. Ptc1 expression is increased in agonist-treated regenerates (E,F). Now all the ependymal cells express Ptc1 and the vast majority of ventral blastema cells do (compare ventral to dorsal blastema cells). F is taken at twice the magnification of E. D-F is 5 dpa. Scale bars: 100 µm.

View larger version (42K): [in a new window]   Fig. 5. Sox9 and Pax7 expression is reduced in cyclopamine-treated blastemas. (A-C) Sox9 in situ hybridization on cross-sections of control (A), cyclopamine-treated (B) and agonist-treated (C) regenerates 6 dpa. Note the absence of staining in B. The arrows in C point to Sox9-positive dorsal blastema cells. (D) Quantification of Pax7-positive blastema cells in control and cyclopamine-treated regenerates 6 dpa. Columns represent the mean percentage of Pax7-positive blastema cells of three regenerates (total number of counted cells per regenerate is between 704 and 1128). Error bars indicate standard deviations. The t-test value is 0.0007. Scale bar: 100 µm in A-C.

View larger version (13K): [in a new window]   Fig. 6. Hedgehog signaling controls blastema cell proliferation. (A,B) Cumulative BrdU labeling of ependymal cells (A) and ventral blastema cells (B). Each column represents the mean percentage of BrdU-positive cells of 2-4 regenerates at indicated time intervals of BrdU labeling. Between 76 and 255 ependymal and blastema cells were counted in total per regenerate. Error bars are standard deviations. The t-test values are 0.005 at 48 hours and 0.212 at 72 hours in A, and 0.003 at 48 hours and 0.002 at 72 hours in B.

View larger version (48K): [in a new window]   Fig. 7. Cyclopamine treatment of the regenerating axolotl limb does not affect blastema growth but leads to digit loss. (A) The regenerated control limb 15 dpa. (B) The regenerated cyclopamine-treated limb structure 15 dpa. The arrow in B points to the regenerated rod of cartilage. The dashed line in A and B marks the amputation plane. Scale bar: 0.5 mm.

View larger version (50K): [in a new window]   Fig. 8. Hedgehog signaling is not sufficient for tail regeneration in the absence of the spinal cord. (A) Mock operated axolotl tail shows normal 7-day regenerate. (B) Control tail with spinal cord removed does not regenerate. (C) Agonist-treated tail without spinal cord also does not regenerate. Arrows point to the distal tip of the spinal cord in all panels. The dashed line marks the amputation plane. Scale bar: 0.5 mm.