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Dev Biol
2010 Feb 15;3382:148-57. doi: 10.1016/j.ydbio.2009.11.026.
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Analysis of hoxa11 and hoxa13 expression during patternless limb regeneration in Xenopus.
Ohgo S
,
Itoh A
,
Suzuki M
,
Satoh A
,
Yokoyama H
,
Tamura K
.
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During limb regeneration, anuran tadpoles and urodele amphibians generate pattern-organizing, multipotent, mesenchymal blastema cells, which give rise to a replica of the lost limb including patterning in three dimensions. To facilitate the regeneration of nonregenerative limbs in other vertebrates, it is important to elucidate the molecular differences between blastema cells that can regenerate the pattern of limbs and those that cannot. In Xenopus froglet (soon after metamorphosis), an amputated limb generates blastema cells that do not produce proper patterning, resulting in a patternless regenerate, a spike, regardless of the amputation level. We found that re-expression of hoxa11 and hoxa13 in the froglet blastema is initiated although the subsequent proximal-distal patterning, including separation of the hoxa11 and hoxa13 expression domains, is disrupted. We also observed an absence of EphA4 gene expression in the froglet blastema and a failure of position-dependent cell sorting, which correlated with the altered hoxa11 and hoxa13 expression. Quantitative analysis of hoxa11 and hoxa13 expression revealed that hoxa13 transcript levels were reduced in the froglet blastema compared with the tadpoleblastema. Moreover, the expression of sox9, an important regulator of chondrogenic differentiation, was detected earlier in patternless blastemas than in tadpole blastemas. These results suggest that appropriate spatial, temporal, and quantitative gene expression is necessary for pattern regeneration by blastema cells.
Fig. 1. hoxa11 and hoxa13 expressions in tadpolelimb buds. (A) hoxa11 expression in a stage 50 limb bud. hoxa11 expression was observed broadly in the distal half of limb bud mesenchyme. (B) hoxa13 expression in a stage 50 limb bud. Its expression was observed in the distal region. (C) hoxa11 expression in a stage 52 limb bud. hoxa11 was expressed in the presumptive zeugopod region. (D) hoxa13 expression in a stage 52 limb bud. hoxa13 was expressed in the presumptive autopod region. Distal is to the top in all figures. Scale bar = 400 μm.
Fig. 2. hoxa11 and hoxa13 expressions in tadpole blastemas. Blastemas were derived from amputations at the ankle level (A, B, E, F, I and J) or at the knee level (C, D, G, H, K and L). (A, C) hoxa11 expression in 3 days post amputation (dpa) blastema. (B, D) hoxa13 expression in 3 dpa blastema. (E, G) hoxa11 expression in 5 dpa blastema. (F, H) hoxa13 expression in 5 dpa blastema. (I, K) hoxa11 expression in 7 dpa blastema. (J, L) hoxa13 expression in 7 dpa blastema. Note that expression patterns of hoxa11 and hoxa13 during tadpolelimb regeneration were similar to those of a developing limb bud (Fig. 1). Lines indicate the estimated amputation planes. Distal is to the top in all figures. Scale bar = 400 μm.
Fig. 3. hoxa11 and hoxa13 expressions in froglet blastemas. Blastemas were derived from amputation at the wrist level (A, B, E and F) or at the elbow level (C, D, G and H). (A, C) hoxa11 expression in 7 days post amputation (dpa) blastema. (B, D) hoxa13 expression in 7 dpa blastema. (E, G) hoxa11 expression in 14 dpa blastema. (F, H) hoxa13 expression in 14 dpa blastema. Note that hoxa11 and hoxa13 were reexpressed in the froglet blastema, but expression domains were never separated. Lines indicate the estimated amputation planes. Distal is to the top in all figures. Scale bar = 400 μm.
Fig. 4. Expression of cartilage maker gene, sox9, in tadpole and froglet blastemas. Limbs were amputated at the ankle level of tadpolehindlimb bud (A–C) or at the wrist level of froglet forelimb (D, E). (A) Expression in tadpole 3 days post amputation (dpa) blastema. Expression was observed in the proximal to amputation plane but not in the blastema. (B) Expression in tadpole 5 dpa blastema. Expression was observed in the proximal to amputation plane but not in the blastema. (C) Expression in tadpole 7 dpa blastema. Expression was observed distal and proximal to the amputation plane. (D) Expression in froglet 7 dpa blastema. Expression was observed in the blastema. (E) Expression in froglet 14 dpa blastema. Expression was observed in a broad region of the blastema. Lines indicate the estimated amputation planes. Distal is to the top in all figures. Scale bar = 400 μm.
Fig. 6. Expression of EphA4 in limb bud and tadpole and froglet blastemas. Blastemas were derived from the ankle level of tadpolehindlimb bud (B, C) or at the wrist level of froglet forelimb (D, E). (A) Expression in stage 53 limb bud. (B) Expression in tadpole 3 dpa blastema. (C) Expression in tadpole 5 dpa blastema. (D) Expression in froglet 7 dpa blastema. (E) Expression in froglet 14 dpa blastema. Note that expression is not observed in the froglet blastema. Lines indicate the estimated amputation planes. Distal is to the top in all figures. Scale bar = 400 μm.
