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Homeobox genes are important in the regulation of outgrowth and pattern formation during limb development. It is likely that homeobox genes play an equally important role during limb regeneration. We have isolated and identified 17 different homeobox-containing genes expressed by cells of regenerating axolotl limbs. Of these, nearly half of the clones represent genes belonging to the HoxA complex, which are thought to be involved in pattern formation along the proximal-distal limb axis. In this paper we report on the expression patterns of two 5' members of this complex, HoxA13 and HoxA9. These genes are expressed in cells of developing limb buds and regenerating blastemas. The pattern of expression in developing axolotl limb buds is comparable to that in mouse and chick limb buds; the expression domain of HoxA13 is more distally restricted than that of HoxA9. As in developing mouse and chick limbs, HoxA13 likely functions in the specification of distal limb structures, and HoxA9 in the specification of more proximal structures. In contrast, during regeneration, HoxA13 and HoxA9 do not follow the rule of spatial colinearity observed in developing limbs. Instead, both genes are initially expressed in the same population of stump cells, giving them a distal Hox code regardless of the level of amputation. In addition, both are reexpressed within 24 hours after amputation, suggesting that reexpression may be synchronous rather than temporally colinear. Treatment with retinoic acid alters this Hox code to that of a more proximal region by the rapid and differential downregulation of HoxA13, at the same time that expression of HoxA9 is unaffected. HoxA reexpression occurs prior to blastema formation, 24-48 hours after amputation, and is an early molecular marker for dedifferentiation.
Fig. 3. Expression of HoxA13 and HoxA9 in developing axolotl forelimbs as visualized by whole-mount in situ hybridization. The yellow coloration of the limbs is due to the Bouin postfixative, which we do not completely remove so as to provide enhanced contrast with the blue reaction product. Anterior is to the right in all limb buds. (A-F) Hox A9 expression; (G-L) HoxA13 expression; (A,G) stage 36; (B,H) stage 37; (C,I) stage 38; (D,J) stage 39; (E,K) stage 40; (F, L) stage 41/42 (stages after Harrison, 1969). Large dots indicate the base of the bud; small dots in A and H outline the edge of the bud where it overlaps the body. Scale bar, 100 μm.
Fig. 4. Expression of HoxA13 and HoxA9 during early stages of axolotl limb regeneration as visualized by whole-mount in situ hybridization. All samples are forelimbs amputated at either the level of the humerus (B,D,G and I) or the level of the carpals (A,C,E,F,H and J). The limbs illustrated were collected 1 day (1d), 2 days (2d) or 4 days (4d) after amputation. Limbs are viewed from the dorsal side and are matched left limbs (HoxA9, anterior to the right) and right limbs (HoxA13, anterior to the left) from the same animal. Arrows in E and J indicate regions of stump tissues not expressing HoxA9 or HoxA13. Scale bar, 200 μm.
Fig. 5. Expression of HoxA13 and HoxA9 during blastema stages of axolotl limb regeneration as visualized by whole-mount in situ hybridization. All samples are forelimbs amputated at either the level of the humerus (C,E,H and J) or the level of the radius/ulna (A,B,D,F,G and I). The amputation plane is indicated by hash marks. All limbs are viewed from the dorsal side and are either left limbs with anterior to the right (HoxA9) or right limbs with anterior to the left (HoxA13). The limbs illustrated represent progressively later stages of regeneration; early bud (EB), medium bud (MB), late bud (LB) and palette (PAL). Scale bar, 200 μm.
Fig. 6. Effects of retinoid treatment on the expression of HoxA13 and HoxA9 as visualized by whole-mount in situ hybridization. (A) HoxA13 expression in a medium bud blastema on an animal treated with retinol palmitate for 1 day. (B) HoxA13 expression in a medium bud blastema on an animal treated with retinol palmitate for 3 days. (C) A medium bud blastema that was hybridized with the sense probe from the 5′ region of HoxA13. (D) HoxA13 expression in a medium bud blastema into which a retinoic acid-containing bead was implanted 23 hours earlier. (E) HoxA9 expression in a medium bud blastema into which a retinoic acid-containing bead was implanted 23 hours earlier. Scale bar, 200 μm
