Retinoic acid breakdown is required for proximodistal positional identity during amphibian limb regeneration

Abstract

Regenerating limbs retain their proximodistal (PD) positional identity following amputation. This positional identity is genetically encoded by PD patterning genes that instruct blastema cells to regenerate the appropriate PD limb segment. Retinoic acid (RA) is known to specify proximal limb identity, but how RA signaling levels are established in the blastema is unknown. Here, we show that RA breakdown via CYP26B1 is essential for determining RA signaling levels within blastemas. CYP26B1 inhibition molecularly reprograms distal blastemas into a more proximal identity, phenocopying the effects of administering excess RA. We identify Shox as an RA-responsive gene that is differentially expressed between proximally and distally amputated limbs. Ablation of Shox results in shortened limbs with proximal skeletal elements that fail to initiate endochondral ossification. These results suggest that PD positional identity is determined by RA degradation and RA-responsive genes that regulate PD skeletal element formation during limb regeneration.

Keywords: Limb regeneration; positional identity; proximodistal axis; retinoic acid.

Figure 1:. PD patterning genes are dynamically expressed during limb regeneration

(A) Schematic of PD amputation plane qRT-PCR experiment. (B-C) qRT-PCR of Meis1 (B) and Meis2 (C) at different PD amputation locations (n = 3-6, 4-5 blastemas per sample, 3.5 cm (HT) animals aged 2.5 months, 10 DPA). Each gene was normalized to Ef1a and analyzed with a one-way ANOVA using a Tukey-Kramer multiple comparison test. R² and p values from linear regression analysis are shown. * = p < 0.05. (D) HCR-FISH for Meis1 and Meis2 in PBs and DBs at 10 and 14 DPA. Dashed lines indicate amputation plane. Scale bars = 200 μm or 20 μm (inset). (E) HCR-FISH dot quantification for mesenchymal Meis1 and Meis2 in PBs and DBs at 10 and 14 DPA (n = 3-6, 3.5 cm (HT) animals aged 2.5 months). Expression is the square root of RS-FISH dots within ROIs. Groups were analyzed using a clustered Wilcoxon rank sum test according to the Datta-Satten method. * = p < 0.05. (F) PD intensity plots for mesenchymal Meis1 and Meis2 in PBs and DBs at 10 and 14 DPA (n = 3-6, 3.5 cm (HT) animals aged 2.5 months). Lines represent average signal intensity (expression) along a normalized PD axis across each sample. (G-I) qRT-PCR of Hoxa9 (G), Hoxa11 (H), and Hoxa13 (I) at different PD amputation locations (n = 3-6, 4-5 blastemas per sample, 3.5 cm (HT) animals aged 2.5 months, 10 DPA). Analyses as in Fig. 1B-C. ** = p < 0.01, *** = p < 0.001. (J) HCR-FISH for Hoxa9, Hoxa11, and Hoxa13 in PBs and DBs at 10 and 14 DPA. Dashed lines indicate amputation plane. Scale bars = 200 μm or 20 μm (inset). (K) HCR-FISH dot quantification for mesenchymal Hoxa9, Hoxa11, and Hoxa13 in PBs and DBs at 10 and 14 DPA (n = 3-6, 3.5 cm (HT) animals aged 2.5 months). Axes and analyses as in Fig. 1E. * = p < 0.05. (L) PD intensity plots for mesenchymal Hoxa9, Hoxa11, and Hoxa13 in PBs and DBs at 10 and 14 DPA (n = 3-6, 3.5 cm (HT) animals aged 2.5 months). Axes and analyses as in Fig. 1F.

Figure 2:. Cyp26b1 is differentially expressed in PBs and DBs and correlates with Meis1, Hoxa11, and Hoxa13 expression

(A-B) qRT-PCR of Cyp26a1 (A) and Cyp26b1 (B) at different PD amputation locations (n = 3-6, 4-5 blastemas per sample, 3.5 cm (HT) animals aged 2.5 months, 10 DPA). Analyses as in Fig. 1B-C. * = p < 0.05, *** = p < 0.001. (C) HCR-FISH for Cyp26a1 and Cyp26b1 in PBs and DBs at 10 and 14 DPA. Dashed lines indicate amputation plane. Scale bars = 200 μm or 20 μm (inset). (D) HCR-FISH dot quantification for mesenchymal Cyp26a1 and Cyp26b1 in PBs and DBs at 10 and 14 DPA (n = 3-6, 3.5 cm (HT) animals aged 2.5 months). Axes and analyses as in Fig. 1E. * = p < 0.05. (E) PD intensity plots for mesenchymal Cyp26b1, Meis1, Hoxa11, and Hoxa13 in PBs and DBs at 10 and 14 DPA. Axes and analyses as in Fig. 1F.

Figure 3:. CYP26 inhibition phenocopies exogenous RA during limb regeneration

(A) Timeline of TAL experiments and tissue collection timepoints. (B) Brightfield images of regenerates and skeletal structures of PBs and DBs treated with DMSO or 0.1, 1, or 5 μM TAL. Dashed lines indicate amputation plane. Scale bar = 2 mm. (C) 10 DPA DBs from RA reporter animals treated with DMSO or 1 μM TAL (n = 8, 3 cm (HT) animals aged 2 months). Dashed lines indicate amputation plane. Scale bar = 500 μm. (D) qRT-PCR of Gfp in tissue from RA reporter animals. (n = 4, 4 blastemas per sample, 3.5 cm (HT) animals aged 2.5 months, blastemas collected at 10 DPA). Analyses as in Fig. 1B-C. *** = p < 0.001. (E) HCR-FISH for Cyp26a1 and Cyp26b1 in DBs administered 1 μM TAL at 14 DPA (n = 3-6, 3.5 cm (HT) animals aged 2.5 months). Dashed line indicates amputation plane. AF = autofluorescence. Scale bar = 200 μm or 20 μm (inset). (F) HCR-FISH dot quantification for mesenchymal and epithelial Cyp26a1 and Cyp26b1 in DBs treated with DMSO or 1 μM TAL at 14 DPA (n = 3-6, 3.5 cm (HT) animals aged 2.5 months). Axes and analyses as in Fig. 1E. * = p < 0.05. (G) 14 DPA DBs from Hoxa13:mCHERRY reporter animals treated with DMSO or 1 μM TAL (n = 8, 7.5 cm (HT) animals aged 6 months). Dashed lines indicate amputation plane. Scale bar = 500 μm.

Figure 4:. CYP26 inhibition reprograms DBs into a more PB-like identity

(A) PCA of bulk transcriptomes from DBs treated with DMSO, 0.1, or 1 μM TAL and PBs treated with DMSO. (B) Heatmap of the top 371 (padj < 0.01, FC = 1.5) genes expressed in each sample type. Cluster numbers are next to the dendrogram. (C) Bar graphs of significantly upregulated and downregulated genes (padj < 0.1) within each comparison. (D) Venn diagram of overlapping DEGs (padj < 0.1) from DMSO treated DBs vs DMSO treated PBs and DMSO treated DBs vs 1 μM TAL treated DBs. Full gene lists are in Table S6. (E) Selected shared DEGs from (D). (F) HCR-FISH for Meis1 and Meis2 in DBs administered 1 μM TAL at 14 DPA. Dashed line indicates amputation plane. Scale bars = 200 μm or 20 μm (inset). (F) HCR-FISH dot quantification for mesenchymal Meis1 and Meis2 in DBs treated with DMSO or 1 μM TAL at 14 DPA (n = 3-6, 3.5 cm (HT) animals aged 2.5 months). Axes and analyses as in Fig. 1E. * = p < 0.05.

Figure 5:. Shox and Shox2 mark proximal and posterior positional identity

(A) Whole mount HCR-FISH for Shox, Shox2, and Hoxa13 in stage 44-47 developing limb buds. Scale bars = 100 μm. (B-C) qRT-PCR of Shox and Shox2 in DMSO or 1 μM TAL treated DBs (n = 4, 4 blastemas per sample, 3.5 cm (HT) animals aged 2.5 months, 10 DPA). Each gene was normalized to Ef1a and the groups were analyzed using a two-tailed t-test. * = p < 0.05. (D-E) qRT-PCR of Shox (D) and Shox2 (E) at different PD amputation locations (n = 3-6, 4-5 blastemas per sample, 3.5 cm (HT) animals aged 2.5 months, 10 DPA). Analyses as in Fig. 1B-C. * = p < 0.05, *** = p < 0.001. (F) HCR-FISH for Shox and Shox2 in PBs and DBs at 10 and 14 DPA. Dashed lines indicate amputation plane. Scale bars = 200 μm or 20 μm (inset). (G) HCR-FISH dot quantification for mesenchymal Shox and Shox2 in PBs and DBs at 10 and 14 DPA (n = 3-6, 3.5 cm (HT) animals aged 2.5 months). Axes and analyses as in Fig. 1E. * = p < 0.05. (H) PD intensity plots for mesenchymal Shox, Shox2, Meis1, and Hoxa13 in PBs and DBs at 10 and 14 DPA. Axes and analyses as in Fig. 1F. (I) HCR-FISH for Shox, Shox2, and Hoxa13 in PBs at 14 DPA. Dashed line indicates amputation plane. Scale bar = 200 μm or 20 μm (inset). (J) UMAP of Shox⁺ and Hoxa13⁺ cells in DBs from reanalyzed scRNA-seq dataset (Li et al., 2021).

Figure 6:. Shox crispants show defects in endochondral ossification of proximal limb skeletal elements

(A) Schematic of the Shox genomic landscape. Introns reduced 50X for visibility. Scale bar = 100 bp. (B) Brightfield images of control and Shox crispant limbs (3.5 cm (HT) animals aged 2.5 months). Scale bar = 1 mm. (C) Skeletal element quantification in control and Shox crispant limbs (n = 8 per group, 7.5 cm (HT) animals aged 6 months). n.s. = no statistical difference, * = p < 0.05. (D) Alcian blue and alizarin red stain of adult control and Shox crispant limbs (12 cm (HT) animals aged 10 months). (E) H&E&A of whole stylopods, proximal epiphyses, and digits from controls and Shox crispants (8 cm (HT) animals aged 7 months). RZ = resting zone, PZ = proliferative zone. Stylopod scale bar = 1 mm, Digit scale bar = 0.5 mm. (F) Whole mount HCR-FISH for Shox and Sox9 in a stage 46 developing limb. Scale bar = 100 μm.

Figure 7:. Shox is dispensable for limb regeneration but required for PD patterning

(A) Regeneration time course of PBs and DBS in Shox crispants. Scale bar = 1 mm. (B) HCR-FISH for Shox and Sox9 in PBs at 21 DPA. Dashed line indicates amputation plane. Scale bars = 200 μm or 20 μm (inset). (C) UMAP of Shox⁺ and Sox9⁺ cells in DBs from reanalyzed scRNA-seq dataset (Li et al., 2021). (D) HCR-FISH for Meis1 and Hoxa13 in Shox crispant PBs and DBs at 10 DPA. Dashed lines indicate amputation plane. Scale bars = 200 μm or 20 μm (inset). (E) Brightfield images of regenerates and skeletal structures of control or Shox crispant limbs treated with 1 μm TAL. Scale bar = 2 mm. (F) Model for PD patterning during limb regeneration.