Extended Data Fig. 1
a, Cells expressing >1 UMI of Acta2 from (Fig. 1a). b, Cartoon summary (right) from cell atlas (Fig. 1a) and immunofluorescence (Fig. 1b); midsubstance (maroon), sheath (gray), and key (left). c, Fluorescence images of Tppp3CG/+;R26RtdT tendon; upper, no TMX control with anti-GFP antibody (Ab.); middle, +TMX and Ab.; lower, +TMX and no Ab. control–eGFP expressed by the Tppp3CG driver is only detectable with Ab. staining; 3. Tppp3CG driver labels 38.3±2.6 (Mean±SEM)% of sheath cells. d, Tppp3CG driver labeling efficiency is 79.5±3.4 (Mean±SEM)%; n=3 animals. e, Fluorescent images of Tppp3CG/+;R26RtdT tendons: digit flexor (leftmost), tail (left), Achilles (right) or Patellar (rightmost); TMX pulsed at embryonic day (E)15.5 and chased to E17.5; Col I, collagen-I antibody stained; dashed line, midsubstance-sheath boundary; 3 animals/tendon. f, Fluorescent images of Tppp3CG/+;R26RtdT Patellar tendon; TMX pulsed at postnatal day (P)5 and chased to P8; dashed line, midsubstance-sheath boundary; 3 animals. g, Sheath cell fractions (key; right) over time; n=3 animals/time point; all ns. h, Midsubstance cell fractions (key same as g) over time; n=3 animals/time point; all ns. i, Midsubstance cell fractions (key; right) over time; n=3 animals/time point; all non-significant by Chi-square test. j, Wholemount multiphoton images of uninjured and biopsy punched (immediately after (T0)) Patellar tendon; 3 animals; collagen fibers visualized by second harmonic generation (SHG). k, Regenerated tendon immuno-stained for Tenascin-C (TNC); asterisks, proliferated Tppp3-lineage cells in TNC matrix of midsubstance; ^, self-renewed Tppp3-lineage in sheath with lower TNC signal; 3 animals. (l,m) Zoomed in FMOD images from Fig. 1g; 3 independent repeats; asterisks, proliferated Tppp3-lineage in midsubstance (l); ^, self-renewed Tppp3-lineage in sheath (m). Scale bar = 30 (c), 15 (flexor) 50 (tail, Achilles, Patellar) (e), 40 (f), 200 (j), 50 (k), 10 (l,m) μm. Error bars = SEM (d,g,h,i). Two-tailed Student’s t-test (g,h).
