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Recent studies in Xenopus laevis have begun to compare gene expression during regeneration with that of the original development of specific structures (e.g., the hindlimb and lens), while other studies have sought differences in gene expression between regeneration-competent and regeneration-incompetent stages. To determine whether there are any similarities between the regeneration of different structures, we have used a differential screen to seek shared early gene expression between hindlimb regeneration and cornea-lens transdifferentiation in the Xenopus tadpole. We have isolated 13 clones representing genes whose expression is up-regulated within the first few days of both regenerating processes and which are not demonstrably up-regulated in the context of basic wound healing. Furthermore, all of these genes also show prominent late embryonic expression. The expression patterns and putative identities of all 13 genes are presented, and a model is considered that allows us to characterize and profile important changes in gene expression, which might be shared among various regenerating and developmental systems.
Figure 1. Spatiotemporal expression of clones W001 through W007 during selected posthatching stages. Embryos and early larvae at the stages indicated in the top row were probed with ldquo antisense rdquo riboprobes against the clones indicated in the leftmost column. All specimens are shown with the anterior end to the left and the dorsal surface toward the top of the figure. Where indicated, white arrowheads indicate the position of the presumptive lens ectoderm or lens and black arrowheads indicate the position of the otic vesicle. The inset in O (W005) is a twofold enlargement of the boxed area in another specimen that underwent longer color development after in situ hybridization to reveal mRNA localization in discrete groups of migrating somite-derived cells (open arrowheads) described by Martin and Harland (), which presumably represent hypaxial myoblasts. Scale bar = 1 mm in U (applies to A-U), 0.5 mm in O inset.
Figure 2. Spatiotemporal expression of clones W008 through W013 during selected posthatching stages. Embryos and early larvae at the stages indicated in the top row were probed with ldquo antisense rdquo riboprobes against the clones indicated in the leftmost column. All specimens are shown with the anterior end to the left and the dorsal surface toward the top of the figure. Where indicated, white arrowheads indicate the position of the presumptive lens ectoderm or lens and black arrowheads indicate the position of the otic vesicle. Open arrowheads indicate the position of the pronephros in F, I, and L. Prominent staining of the cloacal region by W009 is evident adjacent to asterisks in D-F. Scale bar = 1 mm in R (applies to A-R).
Figure 3. Expression during hindlimb regeneration. Each clone's expression during hindlimb regeneration was examined using in situ hybridization 24, 48, and 72 hr after amputation through the left presumptive ankle joint at stages 51-53. The right limb in each case was also examined for expression in the unoperated, developing hindlimb. The paired limbs are shown with the distal (or amputated) end toward the top of the figure. Each sample may be staged by the morphology of the control limb on the right; differences in regeneration blastema expression were not observed among the three stages chosen for amputation. Clones used as riboprobe are shown as follows: A, W001; B, W002; C, W003; D, W004; E, W005; F, W006; G, W007; H, W008; I, W009; J, W010; K, W011; L, W012; M, W013. Each panel displays a representative limb pair from either a 24 hr or 48 hr regenerate ( ldquo early rdquo time point) and a limb pair from a 72 hr regenerate ( ldquo later rdquo time point). In some, but not all, cases background staining was observed at the base of a limb, corresponding to the site where the limbs were detached from the flank tissue after fixation (e.g., B, left limb pair); this staining was also observed in ldquo sense rdquo probe controls (not shown) and is an artifact relating to tissue preparation. N: Illustrations of limb morphology used to stage each set of limbs are shown; the dashed lines indicate amputation sites at each stage (illustrations modeled after Nieuwkoop and Faber, ). The stage at which each sample was amputated may be ascertained by comparison with these staging diagrams (recovery times were not sufficient for specimens to advance in developmental stage). Scale bar = 1 mm in N (applies to A-N).
Figure 4. Expression of selected clones during cornea-lens transdifferentiation. mRNA expression was examined by means of in situ hybridization 24, 48, 72, and 96 hr after lentectomy in tadpoles at stages 47-51. Representative examples and regeneration times are shown as follows: A, W001 at 48 hr; B, W002 at 24 hr; C, W003 at 24 hr; D, W004 at 24 hr; E, W007 at 24 hr; F, W011 at 96 hr. White dotted lines illustrate the approximate boundary of staining in the cornea epithelium. Open arrowheads in B and F indicate prominent staining of lateral line organs by probes W002 and W011, respectively. G: The contralateral, unoperated lens from F (probed with W011). H: An eye 96 hr after lentectomy probed with a ldquo sense rdquo control riboprobe for W011. All specimens were examined during developmental stages 47-51 (Nieuwkoop and Faber, ); eye morphology does not demonstrably differ during these stages. Scale bar = 300 mu m in H (applies to A-H).