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Figure 1. Raldh2 shows dynamic expression in the mesenchyme during limb development. Roman numerals I–V indicate forming digits of the autopod, and lowercase “h†following st. indicates hindlimb whereas “f†indicates a forelimb. Limbs are proximal to the left and posterior uppermost and viewed from the dorsal side, except in B where both hindlimbs are viewed from the ventral side of the tadpole. A, B: In very early hindlimb buds, expression is proximal (black arrow). C: By st. 51, expression is limited to proximal-most cells (black arrow) with lower levels of expression distally (white asterisk). D: Additional expression domains appear in the anterior at st. 52, one either side of the hindlimb (white arrows) and one posteriorly (yellow arrow). Distal expression is resolving into interdigital staining (black arrowheads). E: Interdigital expression is in four domains at st. 53 (black arrowheads). A small area begins to express Raldh2 in the anterior zeugopod region (yellow arrow). F: In st.54 hindlimbs, expression is interdigital (black arrowheads), and is still seen in proximal (black arrow), proximal/anterior (white arrow), and anterior and posterior zeugopod patches (yellow arrows). G: As the hindlimb digits form, Raldh2 is limited to cells surrounding cartilage and proximal expression disappears. H, I: In early stage forelimbs, expression is also only proximal (black arrow). J: Forelimb expression at st. 53. Proximal (black arrow), proximal/anterior (white arrow), and interdigital domains (arrowhead). K: As the forelimb digits form, Raldh2 is limited to cells surrounding cartilage and proximal expression disappears.
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Figure 2. Raldh3 is expressed during mid to late limb development. Hindlimbs are proximal to the left and posterior uppermost and viewed from the dorsal side, except in E, which is viewed from the ventral side. A: Expression is first seen at st. 52 in a faint patch of cells on the anterior, marked by a yellow arrow. B: Two spots of expression appear in the autopod at st. 53 (black arrows, also shown in C and D). C: Viewed from the ventral side of the tadpole, expression of Raldh3 can be seen in two triangular patches on the ventral side of the limb at st. 53, marked by white arrowheads. D,E: Expression in muscle blocks is shown in the dorsal view of the st. 54 limb (D) and ventral view of st. 55 limb (E), marked by white asterisks.
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Figure 3. Cyp26a is expressed interdigitally during mid to late limb development. Dark blue stain indicates expression, roman numerals indicate digit identity. A–D:Cyp26a expression in hindlimbs at stages 51 to 55. A:Cyp26a is not expressed at st. 51. B: At st. 53, a diffuse staining can be seen in the distal 2/3 of the limb, with the proximal limit marked by white arrows. C: At st. 54 Cyp26a is expressed between digits IV and III and III and II, marked by black arrowheads. D:Cyp26a is expressed between all five digits at st. 55 (black arrowheads), in two stripes at the base of each forming digit (yellow arrows), and in two stripes in the anterior limb (white arrows).
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Figure 4. Cyp26b expression is distal in early limbs and restricted to the forming autopod skeleton and claws later. Cyp26b expression in hindlimbs (h) and forelimbs (f). Limbs are oriented with proximal to the left and posterior uppermost viewed from the dorsal side except L, which is a ventral view. A:Cyp26b is not expressed in early st. 51 hindlimbs. B, H: Strong expression (white asterisk) in the distal half of mesenchyme is seen by late st.51/early st. 52 hindlimbs (B) and st. 52 forelimbs (H) with the exception of the distal-most cells. From st. 52, the extent of expression in the most distal mesenchyme cells underlying the AER is marked by black arrows (C, H). Note that this never reaches as far as digit V. A more proximal domain of expression appears at stage 52 in hindlimbs (yellow asterisk in C). D: Yellow arrows mark expression in metatarsals in st. 53 hindlimbs. I: St. 53 forelimbs show no expression of Cyp26b in the autopod. E: Expression in hindlimb metatarsals of digits IV and II and phalanges of digits V and IV at st. 54 (yellow arrows). J: Forelimbs at st. 54 show no expression of Cyp26b in the autopod. F:Cyp26b is expressed in the most distal mesenchyme of toes I, II, and III (black arrowheads) but is absent from toes IV and V and from forelimb digits (K). G: At stage 56, Cyp26b is expressed in forming claws of toes I–III (arrowheads) and in distal most phalanges of digits I–IV (yellow arrows). L: Ventral view of st. 56 forelimb reveals expression in all four digits.
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Figure 5. Expression of Crabp1 and Crabp2 in limb development. In situ hybridisation of Crabp1 and 2 in hindlimbs (h) and forelimbs (f); dark blue stain indicates expression. A:Crabp1 is expressed weakly in the skeleton of digits I to III, as seen in the ventral view of a hindlimb at st. 56. B–J: Crabp2 expression in developing limbs. B: At st. 50, Crabp2 is expressed in the distal half of the limb bud (white asterisk) except the most distal mesenchyme cells and the epithelium. C: At st. 51, expression is limited to the central third of the mesenchyme (white asterisk). D: By st. 52, Crabp2 is expressed in patches of cells in the proximal half of the limb (black arrow), with expression also in the forming knee region (yellow arrow). E, F: Expression at the level of the knee (yellow arrow) and autopod mesenchyme (black arrowhead), in dorsal (E) and ventro-anterior (F) views of the st. 53 hindlimb. G, H: At st. 54 and 55, interdigital expression of Crabp2 (black arrowheads) is strong and there is also expression at the knee level in the anterior (yellow arrow). I, J: Dissected forelimbs showing no clear expression of Crabp2 at st. 53 and interdigital expression beginning at st. 54.
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Figure 6. Expression of RA-modifying genes during hindlimb regeneration. Dark blue stain indicates expression, white arrowheads mark the level of amputation; tissue to the right of this is regrowth. A–C:Cyp26b is expressed strongly in mesenchymal blastema cells, but is absent from the AEC. D–F:Crabp2 is also strongly upregulated in the blastema although it appears absent from the distal tip (white arrow in E). D′–F′: Expression of Crabp2 in non-regenerating, transgenic N1 tadpole hindlimbs to show residual expression. G–I:Raldh2 is not expressed in regenerating tissue but note that it is expressed in the anterior stump tissue (marked with a white asterisk) providing a potential source of RA to the blastema. Limbs are viewed from the dorsal side and oriented with proximal to the left and posterior uppermost, the tadpole's head is to the left. Scale bar in I = 0.5 mm (applies to all panels).
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Supporting Information Figure 1. Whole mount in situ hybridisation showing expression of Crabp2 and Raldh2 in st. 56 hindlimbs and equivalent 3-day regenerates amputated at knee level at st. 56. Arrows indicate Crabp2 and Raldh2 expression in the anterior knee (A, C). Crabp2 (B) but not Raldh2 (D) shows expression in the pseudoblastema, which will not regenerate. White arrowheads mark the plane of amputation. Scale bars = 1 mm. Limbs are oriented with proximal to the left and posterior uppermost, and viewed from the dorsal side.
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Supporting Information Figure 2. Whole mount in situ hybridisation showing expression of Crabp2 and Raldh2 in 3-day tail regenerates for wildtype st. 50 (WT) transgenic st. 50 (N1) and refractory st. 47 (st 47) tadpoles. The animals were subjected to a heat shock at 34 for 30 min and the posterior 50% of the tail was removed 3 hr later. Daily heat shocks were used to maintain transgene expression. Black arrowheads indicate Crabp2 and Raldh2 expression in the regenerating tail blastema (A, D). There is no regeneration and no expression of either gene in N1 tail stumps (B, E) or in refractory st. tails (C, F). White arrowheads, plane of amputation; yellow arrow in A, a pigment cell (melanophore). Scale bars = 1 mm. Tadpoles are oriented with anterior to the left and dorsal uppermost.
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Supporting Information Figure 3. Wholemount in situ hybridisation for Crabp2 and Raldh2 expression in wounded tadpole tails. Neither gene is expressed as a result of wounding. The yellow arrow indicates a melanophore and white arrows indicate the position of the wound site. Scale bars = 1 mm. Tadpoles are oriented with anterior to the left and dorsal uppermost.
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crabp2 (cellular retinoic acid binding protein 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 54, hind limb only, lateral view, dorsal view, anterior up.
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crabp1 (cellular retinoic acid binding protein 1) gene expression in Xenopus laevis embryo via in situ hybridization, NF stage 56, hind limb only, lateral view, dorsal view, anterior up.
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aldh1a2 (aldehyde dehydrogenase 1 family, member A2) gene expression in Xenopus laevis embryo via in situ hybridization, NF stage 54, hind limb only, lateral view, dorsal view, anterior up.
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