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First published online May 16, 2007
doi: 10.1242/10.1242/dev.02852

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A clonal analysis of neural progenitors during axolotl spinal cord regeneration reveals evidence for both spatially restricted and multipotent progenitors

¹ Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, D-01307 Dresden, Germany.
² Department of Anatomy, TU Dresden, Fetscherstrasse 74, D-01307 Dresden, Germany.

View larger version (73K): [in this window] [in a new window]   Fig. 1. A 500 µm region of the mature spinal cord provides the progenitor cells for regeneration. Chimeric spinal cords were produced by transplanting a 4 mm-long section of spinal cord from an eGFP-expressing transgenic animal into a normal host. (A) A 3.5 cm axolotl larva containing an implanted eGFP transgenic spinal cord 7 days after implantation. (B) Two days post-amputation of the tail shown in A. The remaining eGFP-positive portion is 600 µm long. (C) The same tail after 16 days of regeneration. The spinal cord in the regenerated tail is wholly derived from eGFP+ cells. The broken line indicates the amputation plane. (D) Another example of an implanted spinal cord 7 days post-transplantation. (E) The tail shown in D two days post-amputation with a 350 µm piece of eGFP+ spinal cord remaining. (F) At 16 days, the distal 70% of regenerated spinal cord is formed from eGFP+ cells. Broken lines, amputation plane. Scale bar: 2 mm.

View larger version (70K): [in this window] [in a new window]   Fig. 2. Rostral-caudal dependence of PAX7 and PAX6 expression in the regenerating spinal cord. Cross-sections of a 7-day regenerating tail in a 2.5 cm-long axolotl larva were immunostained with antibodies against PAX7 and PAX6 proteins. (A,D,G,J) Overlay of PAX6 (red), PAX7 (green) and Hoechst (blue) channels. (B,E,H,K) Overlay of PAX6 (red) and Hoechst (blue) channels. (C,F,I,L) Overlay of PAX7 (green) and Hoechst (blue) channels. (A-C) Cross-section in a distal-most portion of the regenerating blastema close to the terminal vesicle. Note that no cartilage rod is visible ventral to the ependymal tube. PAX6+ signal is not detectable in this portion of ependymal tube (A,B). Very faint PAX7+ signal (arrowhead) is visible in the dorsal-most position of the ependymal tube (A,C). (D-F) Cross-section approximately 75% from the proximal end of the regenerating blastema, in the region where the cartilage rod begins to be visible (cart). Lateral PAX6+ (D,E, red) and dorsal PAX7+ (D,F, green) domains are clearly visible and in distinct expression domains in this portion of ependymal tube. (G-I) Cross-section in a proximal portion of the regeneration blastema close to the amputation plane. Lateral PAX6+ (G,H, red) and dorsal PAX7+ (G,I, green) domains are clearly visible. In this portion of regenerated ependymal tube the cells in a dorso-lateral position co-express PAX6 and PAX7 (G, yellow arrowheads). (J,K,L) Cross-section through the mature part of the same tail cranial to the amputation plane. Notochord (not) rather than cartilage is present ventral to the spinal cord. PAX6 expression is present in both lateral and dorsal domains of the spinal cord (J, red, yellow; K, red), so that the entire PAX7 expression domain is also PAX6+ (J, yellow; L, green). Cart, cartilage; not, notochord. Scale bar: 100 µm.

View larger version (63K): [in this window] [in a new window]   Fig. 3. The adult axolotl spinal cord contains PAX7+ and PAX6+ cells in distinct domains from ßIII-tubulin+ and NeuN+ cells. Cross-sections through a 30 cm-long adult axolotl tail and body spinal cord were immunostained with antibodies against PAX6, PAX7, ßIII-tubulin and NeuN. (A) PAX7/PAX6 double immunostaining of gray matter in the uninjured body spinal cord, overlaid with Hoechst staining of nuclei (blue). PAX7+ cells (green) are found in a dorsal sub-ependymal zone (arrowheads). PAX6+ cells (red) are located in a lateral ependymal and sub-ependymal layer. A few PAX6+ cells are also found in the neuronal zone (arrows). (B) PAX7/ßIII-tubulin double immunostaining in an adult, uninjured body spinal cord section. ßIII-tubulin staining highlights the neuronal layer of the gray matter. PAX7+ cells are distributed in the sub-neuronal layer and appear not to express ßIII-tubulin. (C) NeuN staining in an adult, uninjured body spinal cord section. Similar to ßIII-tubulin staining, NeuN+ cells (green) are found outside the PAX7+ sub-ependymal zone. (D) PAX7/PAX6 double immunostaining in adult, uninjured tail spinal cord. PAX7+ cells (green) are found in dorsal ependymal and sub-ependymal zones (arrowheads). PAX6+ cells are mostly located in a lateral ependymal layer, with a few cells in the neuronal layer (arrows). (E) PAX7/ßIII-tubulin double immunostaining in an adult, uninjured tail spinal cord transverse section. Anti-ßIII-tubulin stains the neuronal layer and axonal tracts in the tail spinal cord. PAX7+ cells are localized dorsally in the ependymal tube as a sub-neuronal layer that does not express ßIII-tubulin. (F) NeuN in an adult, uninjured tail spinal cord section. NeuN+ cells (green) are found outside the sub-ependymal zone. The PAX6 and PAX7 expression patterns do not overlap in the adult, uninjured body or tail spinal cord. Scale bar: 100 µm.

View larger version (46K): [in this window] [in a new window]   Fig. 4. Lineage tracing of a single ventral spinal cord cell during tail regeneration. A single spinal cord cell was electroporated with a nuclear-eGFP expression plasmid directly after tail amputation. (A) On the third day after tail amputation and electroporation, a single eGFP+ cell (arrowhead) was visible in the ventral spinal cord. The fluorescence image is overlaid with a DIC image of the tail tissue. (B) Day 5. The cell has divided into two cells. (C) Day 7. One daughter cell moves from a ventro-lateral to a more lateral position. (D) Day 9. Four cells are visible, two of them in a lateral position. (E) Day 15. Eight cells spread along the ventro-lateral region of the spinal cord. (F) Day 23. The clone consists of at least 12 cells spanning ventral, lateral and dorso-lateral domains of the regenerating spinal cord. Broken lines denote walls of the spinal cord; cart, cartilage; d, dorsal; not, notochord; v, ventral. Arrow denotes amputation plane. Inset: 40x fluorescence image of the eGFP+ cell. Scale bars: 50 µm.

View larger version (82K): [in this window] [in a new window]   Fig. 5. The ventrally derived clone generated PAX6- and PAX6+ daughter cells. The tail shown in Fig. 4 was fixed at day 23 and sectioned transversally. Sections containing nuclear GFP+ cells (A,D) were double immunostained for PAX6 (B,E; rhodamine fluorescence) and PAX7 (C,F; Cy5 fluorescence, shown in red, arrowheads). (A,B) The ventrally located nuclear-eGFP-expressing cell is PAX6- (arrows). (D,E) The GFP+ cells in the lateral domain are PAX6+ (arrows with asterisk). Arrows, eGFP+ and PAX6- cells; arrows with asterisk, eGFP+ and PAX6+ cells; arrowheads, PAX7-expressing cells.

View larger version (55K): [in this window] [in a new window]   Fig. 6. Lineage tracing of a dorsal spinal cord cell. (A) Three days after tail amputation and electroporation a single dorsal cell expresses nuclear-eGFP (white arrow). The fluorescence image is overlaid with a DIC image of the tail tissue. Broken lines denote walls of the spinal cord; cart, cartilage; d, dorsal; not, notochord; v, ventral. Arrow denotes amputation plane. Inset: 40x fluorescence image of the eGFP+ cell. (B) Day 8. The cell has divided into two dorsal cells. (C) Day 10. The two cells have separated from each other. (D) Day 14. One daughter cell (white arrow with asterisk) moves to the lateral domain of the spinal cord, whereas the other cell remains dorsal. Scale bars: 50 µm.

View larger version (32K): [in this window] [in a new window]   Fig. 7. The dorsal clone generated one PAX7+ and one PAX6+ cell. The tail shown in Fig. 6 was fixed on day 16, sectioned and double immunostained for PAX6 and PAX7. (A) Nuclear-eGFP+ cells overlaid with Hoechst showing the two cells. (B) PAX6 staining (rhodamine) overlaid with eGFP and Hoechst. The lateral eGFP+ cell (arrow with asterisk) is PAX6+. (C) PAX7 staining (Cy5, shown in red). The lateral eGFP+ cell is PAX7- and the dorsal eGFP+ cell is PAX7+. Arrow with asterisk, lateral cell from Fig. 6; arrow, dorsal cell from Fig. 6. Scale bar: 25 µm.

View larger version (17K): [in this window] [in a new window]   Fig. 8. Schematic of ventral and dorsal neural plate grafts between an eGFP transgenic donor and normal host axolotl embryo. Small pieces of eGFP+ tissue were removed from prospective posterior ventral or dorsal neural tube regions of germline eGFP transgenic embryos at stage 15-16 (A) and orthotopically grafted into white hosts (B and C, respectively). After growth to a 2 cm-long larva, eGFP+ cells are restricted to the ventral spinal cord domain in ventral graft embryos (D) or to the dorsal spinal cord domain in roof plate grafts (E). (A) eGFP transgenic axolotl donor embryo (d/d alleles). (B) Host embryo harboring a ventral eGFP transgenic graft (green). (C) Host embryo with a dorsal eGFP transgenic graft (green). (D) Larval tail with ventrally restricted eGFP+ cells. (E) Larval tail with dorsally restricted eGFP+ cells.

View larger version (44K): [in this window] [in a new window]   Fig. 9. Growth and distribution of dorsal domain cells during spinal cord regeneration. EGFP+ prospective dorsal spinal cord tissue was transplanted at stage 15-16 as described in Fig. 8 and the embryo grown to the 2 cm-long larval stage. The tail was amputated in a region where clear dorsal labeling was visible. Panels are fluorescence and DIC images overlaid. (A) Day 3 after tail amputation. eGFP+ dorsal and lateral cells are located behind the amputation plane. Arrows mark the borders of this cell group. (B) Day 9. The eGFP+ cell group proliferated and distributed along the rostral-caudal axis. Cells remained in dorsal and lateral positions. (C) Day 20. The large expansion of the cell group was restricted to the dorsal and dorso-lateral side of the spinal cord. Broken lines, dorsal (d) and ventral (v) walls of the spinal cord; arrows, start and end points of eGFP+ cell group; arrowheads, terminal vesicle; cart, cartilage; not, notochord. Scale bar: 100 µm.

View larger version (71K): [in this window] [in a new window]   Fig. 10. Division and distribution of ventral domain cells during spinal cord regeneration. EGFP+ prospective ventral spinal cord tissue was transplanted at stage 15-16 as described in Fig. 8 and the embryo grown to the 2 cm-long larval stage. The tail was amputated in a region where clear ventral labeling was visible. Images are fluorescence and DIC images overlaid. (A) Day 1 after tail amputation. The tail spinal cord contains a group of eGFP+ ventral and ventro-lateral cells (green). (B) Day 9. EGFP+ cells proliferated and expanded restricted to ventral and ventro-lateral domains. (C) Day 21. EGFP+ cells remain in ventral and ventro-lateral positions in the proximal portion of regenerated spinal cord. By contrast, in the region close to the terminal vesicle, cells also distribute to the dorsal side (arrowheads). (D) Cells leaving the spinal cord from the terminal vesicle (arrowheads). (E) Black and white image of the terminal vesicle containing dorsally located eGFP+ cells (arrowheads, cells in dorsal domain). Broken lines delineate dorsal (d) and ventral (v) walls of the spinal cord; arrows, start and end points of eGFP+ cell group extension; cart, cartilage; not, notochord. Scale bars: 100 µm.