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Comparison of the effects of vitamin A on limb development and regeneration in Xenopus laevis tadpoles.
Scadding SR
,
Maden M
.
Abstract
The purpose of these experiments was to compare the effects of vitamin A on developing and regenerating limbs in Xenopus laevis tadpoles. Each tadpole had one hindlimb amputated to induce regeneration while the contralateral developing limb was left intact. Tadpoles at stages 50 through 54 were treated by immersion in retinol palmitate at doses ranging from 0.3 to 75 i.u. ml-1, for periods ranging from 1 to 14 days. Developing limbs usually became hypomorphic as a result of the treatment, with results varying with stage and treatment from slight phalange derangements to total disruption of pattern, or complete inhibition of limb development. Regenerating limbs gave a variety of responses including hypomorphic regeneration, proximodistal or anteroposterior duplication of skeletal elements, or complete suppression of regeneration. The response to retinol palmitate of developing limbs was clearly different from regenerating limbs. Hypotheses which might explain the results were discussed and a hypothesis which proposed a dual action of vitamin A affecting both the cell cycle and the mechanism of pattern regulation was proposed.
Fig. 1. (A) Normal intact lefthindlimb from a stage-59 Xenopus tadpole. The skeleton consists of a femur (Fe), tibia (T), and fibula (F) here fusing into the single tibia-fibula, tibiale (t), fibulare (f), five tarsals (ta) of which three are visible here (the other two lie beneath distal tarsal I and the proximal end of metatarsal I), five metatarsals {mi), and 14 phalanges (ph) arranged on digits I to V (I-V) in the formula 2-2-3-4-3. Magnification x9. (B) Normal intact leftforelimb from a stage-59 Xenopus tadpole. The arm skeleton consists of a humerus (H), radius (R), and ulna (U), here fusing into the single radio-ulna, and a small patella ulnaris (pu) at the elbow. The wrist comprises nine carpals of which seven are clearly visible here. The four larger ones are the ulnare (u), radiale (r), centrale (c), and distal carpal IV (dc). Three smaller distal carpals are also visible. Two other carpals, the radiale externum lying dorsal to the radiale, and the praepollex just medial to distal carpal I are not visible in this photograph (the praepollex is visible in Fig. 2B). The hand consists of four metacarpals (me) and ten phalanges (ph) arranged on digits I through IV (I-IV) in the formula 2-2-3-3. Magnification x24. (C) This righthindlimb was amputated at stage 52 and fixed at stage 65. It was scored as being within the normal range, since it only varies from normal by having a phalange formula of 2-2-3-3-2, i.e. two phalanges are missing, and the lateral metatarsals are slightly bent. Since this specimen wasfixedat a later stage, ossification of the skeleton is advanced, hence there is less cartilage present resulting in fainter Victoria Blue B staining. The claws are visible on digits I, II, and III. Magnification x8. (D) The righthindlimb was amputated at stage 53 and fixed at stage 60. It was slightly hypomorphic having four rather small digits with the phalange formula 0-2-2-3-2. Magnification xll. (E) This righthindlimb was amputated at stage 54. The regenerated foot was extremely hypomorphic (EH-P3) with only two small digits, each with two phalanges, and marked shortening of the tibiale and fibulare. Magnification x9.
Fig. 2. Each photograph shows a dorsal view of both left and right forelimbs of Xenopus tadpole treated with retinol palmitate. (A) Forelimbs treated at stage 53 (0-3i.ii.mn1, 14 days). Both are slightly hypomorphic (SH) showing loss of digit I which is represented only by a vestige of metacarpal I (mcl). Magnification x 14. (B) Forelimbs treated at stage 53 (03i.ii.mn1, 14 days). Both are moderately hypomorphic (MH) with only two digits. Digit IV is missing and is represented by a carpal-like cartilage which is likely to be a vestige of metacarpal IV (mcIV). In both limbs, metacarpals I and II are partially fused and underlie a single set of phalanges. The praepollex (p) is visible in these specimens. Magnification x 17. (C) Forelimbs treated at stage 53 (1-0i.u. ml"1, 7 days). Both are extremely hypomorphic (EH) and hand is reduced to single digit consisting of a single metacarpal. Magnification x 12. (D) Forelimbs treated at stage 51 (0-3 i.u. ml"1, 14 days). Hands are entirely normal, but arms exhibit phocomelia (P2) in that the radius (R) is reduced to a triangular cartilage and the ulna (U) is reduced to a carpal-sized nodule of cartilage. Magnification x 15. (E) Forelimbs treated at stage 51 (3i.u. ml"1, 3 days). Hands are extremely hypomorphic and arm is phocomelic (left - EH-P12; right EH-P1). Note the bend in the humerus in each arm. An increase of this bend, such that the ends of the long bone are brought towards one another, results in a triangular cartilage as seen in radius-ulna of the leftlimb. This pair of limbs is unusual in that left radius-ulna is defective while right radius-ulna is normal. Magnification x 13. (F) Forelimbs treated at stage 51 (0-3i.u. ml"1, 14 days). Both limbs are extremely hypomorphic and phocomelic (EH-P12). Pattern of limbskeleton is quite chaotic. Magnification x 22.
Fig. 4. (A) Intact developing lefthindlimb treated at stage 51 (3i.u. ml"1, 3 days). At this higher dose level, effects of retinol palmitate are more severe. Femur (Fe) and tibia-fibula (TF) are reduced to small triangular cartilages. Fibulare (f) is bent. Metatarsal IV is extremely reduced and lacks phalanges; metatarsal V is thin and bent; metatarsal II and III have fused proximally. Phalanges on digit III have bifurcated. Coded as MH-P123. Magnification x 10. (B) Regenerated righthindlimb treated at stage 51 (0-3i.u. ml"1,3 days). There are thirteen digits arranged as a primary foot and two accessory feet. The digital sequence from dorsal to ventral (right to left in the photograph) is V - I V - I I I - I I - I - I - I I - I I I - I V - V ( v e r y small and not visible in photograph)-IV-IH-II. This is the most extensive AP duplication seen in this investigation. Coded as D7. Magnification x 10. (C) Regenerating righthindlimb contralateral to thai: in A above. It is extremely hypomorphic (EH) with only one digit, but without any duplication of skeletal elements. Magnification x 10. (D) A regenerating righthindlimb treated at stage 53 (0-3 i.u. ml"1,14 days). This limb shows complete duplication of the entire limbdistal to the amputation level (arrowhead). Regenerate included femur (Fe), tibia-fibula (TF), and an extremely hypomorphic foot with only two digits. Coded as D5-EH. Magnification x 10. (E) Regenerated righthindlimb treated at stage 53 (1-0i.u. ml"1, 3 days). Limb exhibited AP dupli- cation. The six digits were arranged in the sequence V-IV-III-III-IV-V indicating a partial mirror-image duplication. Coded D6. Magnification x 6.
