BMC Dev Biol
July 28, 2008;
Identification of genes associated with regenerative success of Xenopus laevis hindlimbs.
Epimorphic regeneration is the process by which complete regeneration of a complex structure such as a limb
occurs through production of a proliferating blastema
. This type of regeneration is rare among vertebrates but does occur in the African clawed frog Xenopus laevis, traditionally a model organism for the study of early development. Xenopus tadpoles can regenerate their tails, limb
buds and the lens
of the eye
, although the ability of the latter two organs to regenerate diminishes with advancing developmental stage. Using a heat shock inducible transgene that remains silent unless activated, we have established a stable line of transgenic Xenopus (strain N1) in which the BMP inhibitor Noggin
can be over-expressed at any time during development. Activation of this transgene blocks regeneration of the tail
of Xenopus tadpoles. In the current study, we have taken advantage of the N1 transgenic line to directly compare morphology and gene expression in same stage regenerating vs. BMP signalling deficient non-regenerating hindlimb
buds. The wound epithelium
of N1 transgenic hindlimb
buds, which forms over the cut surface of the limb bud
after amputation, does not transition normally into the distal
thickened apical epithelial cap
. Instead, a basement membrane and dermis
form, indicative of mature skin
. Furthermore, the underlying mesenchyme
remains rounded and does not expand to form a cone shaped blastema
, a normal feature of successful regeneration. Using Affymetrix Gene Chip analysis, we have identified genes linked to regenerative success downstream of BMP signalling, including the BMP inhibitor Gremlin
and the stress protein Hsp60
in zebrafish). Gene Ontology analysis showed that genes involved in embryonic development and growth are significantly over-represented in regenerating early hindlimb
buds and that successful regeneration in the Xenopus hindlimb
correlates with the induction of stress response pathways. N1 transgenic hindlimbs, which do not regenerate, do not form an apical epithelial cap
or cone shaped blastema
following amputation. Comparison of gene expression in stage matched N1 vs. wild type hindlimb
buds has revealed several new targets for regeneration research.
BMC Dev Biol
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Figure 1. Histological characterisation of hindlimb bud regeneration in WT and N1 tadpoles Histological characterisation of hindlimb bud regeneration in WT and N1 tadpoles. Representative haematoxylin and eosin stained 5 μm sections showing the phenotypic differences between regenerating WT and non-regenerating transgenic N1 hindlimb buds. Stage 52 limb buds were amputated at knee level and heat shocked as previously. Cartilage condensa- tions are marked with a c. (A-D) WT limb buds, scale bars are 100 μm. Black arrowheads show the approximate plane of amputation. (A'-D') Higher magnification focusing on the distal area, scale bars are 50 μm. (A, A') wound healing has occurred after 24 hours and a wound epithelium (we) covers the cut site. (B, B') 2 days after amputation, blastema (bl) and AEC (aec) are forming. (C, C') 3 days after amputation, a cone-shaped blastema and the AEC are well established. Columnar basal epithelial cells (be) can be seen. Hypertrophic epithelial cells (h) can be seen in the AEC. (D, D') 5 days after amputation, the AEC and blastema are still apparent and outgrowth has begun. Muscle cells (m) can be seen proximally. (E-F) N1 limb buds, scale bars are 100 μm. Connective tissue (ct) is more obvious in these limb buds. E'-F' Higher magnification focusing on the distal area, scale bars are 50 μm. (E, E') wound healing has occurred after 24 hours, and a wound epithelium (we) covers the cut surface. (F, F') No AEC is apparent after 2 days and a rounded pseudoblastema (pb) forms proximal to the wound epithelium. (G, G') 3 days after amputation. (H, H') 5 days after amputation, the pseudoblastema has not expanded and a cell-free area of matrix is visible between the wound epithelium and underlying stump cells. Distal is to the left and posterior uppermost. dr = days of regeneration.
Figure 4. Expression of Gremlin in regenerating WT and N1 limbs and during development Expression of Gremlin in regenerating WT and N1 limbs and during development. Gene expression in regenerating wild type (WT) and N1 limbs and embryo tissue. (A-J) In situ hybridisation showing Gremlin expression in the regeneration bud. (L-O) Unoperated limb buds illustrating Gremlin expression during limb development. (K) In situ hybridisation showing Gremlin expression in a stage 32 embryo, the expression pattern is consistent with previously published Gremlin embryo expression . White arrowheads indicate approximate amputation plane, scale bar in A applies to panels A-J and scale bar in O to pan- els L-O. In limb panels (A-J, L-O) posterior is uppermost, and distal to the left, dr = days of regeneration. In K, anterior is to the left and dorsal uppermost. White asterisks mark areas of Gremlin expression that are developmental and unrelated to regeneration.
Figure 5. Expression of HSP60 in regenerating WT and N1 limbs and during development Expression of HSP60 in regenerating WT and N1 limbs and during development. Gene expression in regenerating WT and N1 limbs and embryo tissue. (A-J) In situ hybridisation showing Hsp60 expression in the regeneration bud. (M-P) Unoperated limb buds illustrating Hsp60 expression during limb development. (K) In situ hybridisation showing Hsp60 expression in stage 57 hindlimb of a WT animal 2 days after amputation. (L) In situ hybridisation showing Hsp60 expression in stage 32 embryo. White arrowheads indicate approximate amputation plane, scale bar in A applies to panels A-J and scale bar in P applies to panels M-P. In limb pictures (A-K, M-P) posterior is uppermost, and distal to the left, dr = days of regeneration. In L, anterior is to the left and dorsal uppermost.
hspd1 (heat shock 60kDa protein 1 (chaperonin)) gene expression in Xenopus laevis embryo, via in situ hybridization, NF stage 32 embryo, lateral view, anterior left, dorsal up.
grem1 (gremlin 1) gene expression in Xenopus laevis embryo, via in situ hybridization, NF stage 32 embryo, lateral view, anterior left, dorsal up.
Figure 2. Design of microarray experiment. A) Timeline showing the treatments used to generate tissue for arrays. hs = heat shock, 30 minutes at 34°C. B) Stage 52 hindlimb buds were bilaterally amputated at the level of the future ankle (dotted line), defined by the anterior indentation, to remove the autopod. Knee level is marked by black arrowhead for orientation. Heat shocks were applied to both WT and N1 tadpoles as depicted in (A). After 3 days the blastemas were removed from the WT limbs and pseudoblastemas from the N1 limbs. BMP signalling is inhibited in the N1 limb buds, due to expression of Noggin from an inducible transgene, preventing successful regeneration. Pools of 20 blastemas or pseudoblastemas were used to extract RNA to generate microarray probes.
Figure 3. Effect of Noggin over-expression on regeneration outcome following amputation at the future knee or ankle level. Histogram of limb regeneration success as defined by the number of toes regenerated by stage 58 following amputation at either the future knee or ankle level of the limb bud at stage 52. WT and N1 animals were both subjected to heat shocks that activate transgene expression of Noggin in N1s. Non heat shocked controls were amputated at knee level. Error bars represent standard error and significant differences between WT and N1 animals are denoted by **).
Ahn, BMPR-IA signaling is required for the formation of the apical ectodermal ridge and dorsal-ventral patterning of the limb. 2001, Pubmed