En1 and Lmx1b do not recapitulate embryonic dorsal-ventral limb patterning functions during mouse digit tip regeneration

Abstract

The mouse digit tip regenerates following amputation. How the regenerate is patterned is unknown, but a long-standing hypothesis proposes developmental patterning mechanisms are re-used during regeneration. The digit tip bone exhibits dorsal-ventral (DV) polarity, so we focus on En1 and Lmx1b, two factors necessary for DV patterning during limb development. We investigate whether they are re-expressed during regeneration in a developmental-like pattern and whether they direct DV morphology of the regenerate. We find that both En1 and Lmx1b are expressed in the regenerating digit tip epithelium and mesenchyme, respectively, but without DV polarity. Conditional genetics and quantitative analysis of digit tip bone morphology determine that genetic deletion of En1 or Lmx1b in adult digit tip regeneration modestly reduces bone regeneration but does not affect DV patterning. Collectively, our data suggest that, while En1 and Lmx1b are re-expressed during mouse digit tip regeneration, they do not define the DV axis during regeneration.

Keywords: CP: Developmental biology; En1; Lmx1b; digit tip regeneration; dorsoventral patterning; limb development.

Figure 1.. Expression of En1 and Lmx1b during digit tip development

(A) Schematics of mouse digit tip cross-sections at E16.5 (embryonic; top), PN4/5 (neonatal; middle), and 8 weeks old (adult; bottom), including En1 expression in green and Lmx1b expression in magenta. Lettered boxes correlate with position of in situ images on right. (B–J) HCR RNA-FISH for En1 (green puncta) and Lmx1b (magenta puncta) in embryonic (B–D), neonatal (E–G), and adult (H–J) digit tip sections. DAPI is shown in grey. Arrowheads show examples of positive puncta; neg denotes no detected expression. Scale bars, 50 μm. Asterisks (*) denote blood vessel and red blood cell autofluorescence. Dashed lines show epithelial border. Epi, epithelium; mes, mesenchyme. Representative images from at least two biological replicates are shown. See also Figure S1.

Figure 2.. Expression of En1 and Lmx1b during digit tip regeneration

(A and B) Gene expression of En1 (A) and Lmx1b (B) in regenerating and unamputated digit tips shown by violin plot. Black dots represent individual cells. (C) Schematic of mouse digit tip regeneration. Digits are amputated at the plane of the dashed line (top), resulting in blastema formation (middle) and regeneration of distal digit tip structures (bottom). (D–I) HCR RNA-FISH for En1 (green puncta) (D–F) and Lmx1b (magenta puncta) (G–I) in 11 dpa regenerating digit tips. Schematics of mouse digit tip cross-sections at 11 dpa to the left include En1 expression in green and blastema-specific Lmx1b expression in magenta. Lettered boxes correlate with position of in situ images on right. DAPI is shown in grey. Scale bars, 50 μm. Dashed lines show epithelial border. Epi, epithelium; mes, mesenchyme. Representative images from at least two biological replicates are shown. See also Figures S2 and S3.

Figure 3.. Developmental loss of En1 or Lmx1b perturbs digit tip regeneration

(A–C) Ventral view (A) of control En1-wt/wt;K14-cre digits and lateral view of alizarin red-stained unamputated (B) and 3 wpa (C) En1-wt/wt;K14-cre digit bones. (D–F) Ventral view (D) of En1-fl/fl;K14-cre digits and lateral view of alizarin red-stained unamputated (E) and 3 wpa (F) En1-fl/fl;K14-cre digit bones. (G–I) Dorsal view (G) of control Lmx1b-fl/wt;Prx1-cre digits and lateral view of alizarin red-stained unamputated (H) and 3 wpa (I) Lmx1b-fl/wt;Prx1-cre digit bones. (J–L) Dorsal view (J) of Lmx1b-fl/fl;Prx1-cre digits and lateral view of alizarin red-stained unamputated (K) and 3 wpa (L) Lmx1b-fl/fl;Prx1-cre digit tip bones. Dashed lines denote amputation plane. Scale bars, 200 μm. (M and N) Boxplot of digit tip bone percent regeneration length in En1-wt/wt;K14-cre or En1-fl/fl;K14-cre 3 wpa digits (M) or Lmx1b-fl/wt;Prx1-cre or Lmx1b-fl/fl;Prx1-cre 3 wpa digits (N). n = individual digits.

Figure 4.. Conditional knockout of En1 and Lmx1b has a small effect on bone regeneration

(A) Schematic of the En1 and Lmx1b conditional knockout experiment. (B–I) Representative 3D models of uCT scanned digits. (B and C) En1-fl/fl control UA (B) and 4 wpa (C) digits. (D and E) En1-fl/fl;K14-creERT2 + tamoxifen UA (D) and 4 wpa (E) digits. (F and G) Lmx1b-fl/fl control UA (F) and 4 wpa (G) digits. (H and I) Lmx1b-fl/fl;Msx1-creERT2 + tamoxifen UA (H) and 4 wpa (I) digits. (J–M) Boxplots showing quantification of digit length and volume for En1 control and +tamoxifen digits (J and K) and Lmx1b control and +tamoxifen digits (L and M). Number of individual digit tip bones in each group is noted in methods and Figure S5. *p < 0.05, **p < 0.01, ***p < 0.001. See also Figure S4.

Figure 5.. Loss of En1 during development, but not regeneration, increases circularity of the digit tip bone

(A) Example width and height measurements for slices shown in blue. (B) Example 3D rendering of a neonatal En1-fl/fl;K14-cre digit. Numbered red lines indicate approximate position of correspondingly numbered slices through the digit bone. (C) Boxplot of aspect ratio measurement for slices at 25%, 35%, 45%, 55%, 65%, and 75% of the length of the digit in unamputated (top) and 3 wpa (bottom) digits. n = individual digits. (D) Example 3D rendering of an adult En1-fl/fl digit. Numbered red lines indicate approximate position of correspondingly numbered slices through the digit bone. (E) Boxplot of aspect ratio measure for slices at 25%, 35%, 45%, 55%, 65%, and 75% of the length of the digit starting from the distal most part of the ventral hole in unamputated (top) and 4 wpa (bottom) digits. UA En1-fl/fl n = 48, UA En1-fl/fl;K14-creERT2 n = 24, UAEn1-fl/fl;K14-creERT2+tam n = 28. 4 wpa En1-fl/fl n = 48, 4 wpa En1-fl/fl;K14-creERT2 n = 22, 4 wpa En1-fl/fl;K14-creERT2+tam n = 27. n = individual digits. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 6.. Conditional knockout of En1 or Lmx1b does not affect overall morphology of regenerated digit tip bones

(A) Schematic of data and analysis in (B–E). An unamputated digit is shown with dorsal area in white and ventral area in blue (top left), with a slice at the approximate position of the black line (top right). A ratio is computed by taking the ventral area divided by the dorsal area at many slices along the length of the digit. The plotted ratios (black points) are within the area shown by the inset, with the fitted model shown as gray lines. s1, first segment; cp, change point; s2, second segment. (B and D) Boxplot showing mean change point predictions for each digit in the En1 (B) or Lmx1b (D) cohort. (C and E) Boxplot showing mean slope predictions for the second segment for each digit in the En1 (C) or Lmx1b (E) cohort. Number of individual digit tip bones in each group is noted in STAR Methods and Figure S5. **p < 0.01.