Fig. 5: Axin2^TdTom cell identity is maintained through autocrine Wnt signaling.

A Cartoon showing the experimental set-up used for the analysis with TAM treatment at 3 months and scRNA-seq of total Achilles tendon cells in homeostasis at 4 months. Single cell RNA-seq dot plot of canonical Wnt pathway components expressed in the different cell clusters during homeostasis; Scx, Tnmd, and TdTom expression is shown. B–E UMAP feature plots of Axin2, TdTom, Scx, and Wnt9A show that these genes are expressed in the MBT cluster during homeostasis (MBT, MTJ, and Ent clusters are outlined in red). F, H smFISH images of one cell containing Axin2 (green), Wnt9a (white), and PolyA (red) puncta in 4-month-old Achilles tendons from wild type (WT) (F) and Axin2:Cre^ERT2; Porcn^fl/fl mutants (H; note scale bars are 2 μm). G Quantification of Axin2, Wnt9a, and PolyA RNA puncta per cell in Achilles tendons shows expression of Axin2 and Wnt9A in WT and a decrease in RNA puncta in Axin2:Cre^ERT2; Porcn^fl/fl and in Scx:Cre^ERT2; Porcn^fl/fl mutants at 4 months of age (n > 8 Achilles tendon per group with n > 20 tendon cells analyzed per tendon; groups consisted of 4-month-old WT, and Axin2^TdTom; Porcn^fl/fl, Scx^TdTom; Porcn^fl/fl with TAM given at 3 months; multiple T-test with Holm–Sidak correction was used for statistical analysis; n.s. not significant; ****p < 0.0001). I Representative cell co-expressing AXIN2 (green) and WNT9A (white) in human semitendinosus (hamstring) tendon (I). PolyA (red) was used as an internal expression control (F–I).