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Figure 1. Transcriptome analysis of three regions of the early Xenopus laevis hindlimb bud proximal to distal axis. (A) Schematic of sample preparation. Stage 51 hindlimb buds were divided into three equal sections along the proximal to distal axis, each part is approximately 200 μM wide. Approximate locations of the apical ectodermal ridge (AER) and zone of polarizing activity (ZPA) are indicated in red and blue, respectively. Location of a potential proximal source of the morphogen retinoic acid is indicated by expression of Aldh1a2 in green. (B) Fate map showing the contribution of developing stage 51 X. laevis hindlimb bud mesenchyme to skeletal elements of the limb, according to Tschumi.8 (C) Venn diagram of all significantly differentially expressed genes using Venny 2.0.86 (D) Dendrogram heatmap showing hierarchical clustering of the differentially expressed genes, selecting for seven clusters. (E) Volcano plot showing the relative distributions of known proximal (red: meis1, meis2) and distal (blue: fgf8, hoxa13, hoxd13) limb genes as well as the genes verified by whole mount in situ hybridization in Figures 2-4). Genes with log2 fold change <3 in either direction are represented by grey dots
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Figure 2. Expression of three cluster 4 proximal genes in X. laevis hind limb buds. (A-I) Whole mount in situ hybridization (purple staining) of stages 50 to 52 hind limb buds as viewed from the dorsal side, posterior uppermost. (A-C) white arrows indicate proximal spot of mesenchymal gata5.L at stage 50 (A), 51 (B), and 52 (C). (D-F) irx5.L in the proximal mesenchyme at stage 50 (D), 51 (E), and 52 (F). Black stellate cells are melanophores in nonalbino tadpoles (D-F), expression in stage 52 is anterior as well as proximal. (G-I) Expression of tnn.L is very strong along the boundary with the body wall, and is epithelial. Stage 50 limb bud dissected to enable visualization of limb bud, as there is strong expression in the ventral fin as well as the limb bud (G), stage 51 (H), and 52 (I). Scale bar in (I) is 250 μM and applies to all panels. (J), Normalized read counts of gata5.L, irx5.L and tnn.L in the three sections of the stage 51 limb bud transcriptome. Red and black dots indicate biological replicate data, where only red can be seen it overlaps completely with black
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Figure 3. Expression of distal epithelial genes in X. laevis hindlimb buds. (A-O) Whole mount in situ hybridization, gene expression is marked by dark purple staining. (A-G) Capn8.3L is expressed in the epithelial cells flanking the AER. (A-E) Dorsal view of limb with distal to the right and posterior uppermost. (A): stage 50, (B): stage 51, (C): stage 52, (D): stage 53, (E): stage 55. (F and G) Views of the distal limb(s) to show the AER (black arrow), (F): stage 53, (G): stage 55. (H-K) Expression of dkk1.S (H): stage 51, dorsal view, (I): distal view, (J): stage 53 dorsal view, (K): distal view. (L-O) expression of fgf8 (L): stage 51 dorsal view, (M) stage 52 dorsal view, (N): stage 53 dorsal view, (O): stage 53 distal view. Black arrows indicate the AER in distal views, and the approximate position of digit IV is indicated by an asterisk from stage 53. Black stellate cells are melanophores in non-albino tadpoles (B, H-M). Scale bar in (O) is 250 μM and applies to all panels. (P) Normalized read counts of capn8.3.L, dkk.S and fgf.S in the three sections of the stage 51 limb bud transcriptome, red and black indicate biological replicate data
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Figure 4. Expression of two distal mesenchymal genes in X. laevis hindlimb buds. (A-L) Whole mount in situ hybridization where expression is indicated by dark purple staining. (A-F) expression of sall1.S. (A-D) Dorsal views of the limb with distal to the right and posterior uppermost. (A): stage 50, (B): stage 51, (C): stage 52, (D): stage 53. (E and F) Views of the distal limb, the ventral midline of the tadpole running left to right in the center of the image. (G-L) Expression of zic5.S indicated by dark purple staining. (G-J) Dorsal views of the limb. (G): stage 51, (H): stage 52, (I): stage 53, (J): stage 54. (K and L) Views of the distal limb, (K): stage 51, (L) stage 54. Asterisk indicates the approximate location of digit IV in stage 53 and above limb buds. Melanophores are present on some limb buds (A-D, F, G, and K) as black stellate cells. Scale bar in (L) is 250 μM and applies to all panels. (M) Normalized read counts of sall1.S and zic5.S in the three sections of the stage 51 limb bud transcriptome, red and black indicate biological replicate data, where only red can be seen it overlaps completely with black
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Figure 5. Relative distribution of read counts for genes in differentially expressed gene ontology gene sets for cell adhesion, Wnt signaling pathway, positive regulation of cell population proliferation, response to fibroblast growth factor, retinoic acid receptor signaling, and retinoid metabolism. Stacked graphs show the percentage of proximal reads is in blue, medial reads in grey, and distal in red. Genes with statistically more reads in distal than proximal are on the left and those more proximal on the right, and are ordered alphabetically within each group.
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Figure 6. Genes previously shown to be upregulated by retinoic acid in axolotl limbs do not show consistent expression distributions in Xenopus limbs. One hundred different genes identified as upregulated by RA in axolotl limb18 were identified one of four categories: unknown (no Xenopus homologue), not expressed (not found in Xenopus stage 51 limb bud), nonsignificant (expression is not significantly different across the three limb bud sections) or differentially expressed. Homologs of the 27 differentially expressed genes in the allotetraploid Xenopus laevis are found in all seven clusters, cluster number is indicated by colored number at the base of each graph, read counts are shown on the y-axis and position on the x-axis. P = proximal, M = medial, D = distal. Replicates are represented by red and black dots and where only one dot can be seen, these overlap completely. Colored boxes are used to group clusters with proximal, distal or medial/terminal expression patterns
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Figure 7. Distal gene expression in stage 51 Xenopus hindlimb bud of genes previously shown to be downregulated by retinoic acid in axolotls. Pie chart: 14 different genes identified as downregulated by RA in axolotls18 were identified as not expressed (not found in Xenopus stage 51 limb bud), nonsignificant (expression is not significantly different across the three limb bud sections) or differentially expressed. Homologs of the 10 differentially expressed genes in the allotetraploid Xenopus laevis are found exclusively in cluster 2. Cluster number is indicated by colored number at the base of each graph, read counts are shown on the y-axis and position on the x-axis. Replicates are represented by red and black dots and where only one dot can be seen, these overlap completely. P = proximal, M = medial, D = distal. Colored box indicates all genes had distal expression patterns
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