Fig. 2: Genetic ablation of TTLL1 delays motor axon remodeling.

a–k Quantitative immunostainings for microtubule markers and neurofilament heavy polypeptide on triangularis sterni muscles at postnatal day (P) 8–12 from TTLL1^mnKO vs. TTLL1^mnWT littermate controls. Confocal stacks of terminal axons leading to NMJs depicting immunostainings against (a) tubulin beta-3 (Tubb3, green), (c) alpha-tubulin (Tuba, cyan), (e) neurofilament (NF, white), (g) polyglutamate chains (PolyE, magenta), (i) monoglutamate on beta-tubulin (βmonoE, pink), and (k) acetylated tubulin (Ac-Tub, red). b–l Corresponding quantifications of immunostaining intensities in TTLL1^mnKO terminal motor axons (right; relative to TTLL1^mnWT, left, which is set to 1). b Tubb3, normalized to NF (WT n = 70 axons, 3 mice; KO n = 72 axons, 3 mice, p-value = 7,04949E-13). d Tuba normalized to NF (WT n = 46 axons, 3 mice; KO n = 50 axons, 4 mice, p-value = 0.0036). f NF (WT n = 108 axons, 7 mice; KO n = 100 axons, 6 mice). h polyE normalized to Tubb3 (WT n = 70 axons, 3 mice; KO n = 72 axons, 3 mice, p-value = 1,23212E-24). j βmonoE, normalized to Tubb3 (WT n = 58 axons, 3 mice; KO n = 54 axons, 3 mice). l ac-tub, normalized to Tubb3 NF (WT n = 68 axons, 4 mice; KO n = 177 axons, 8 mice, p-value = 0.00546). m Confocal stacks showing NMJs of P12 TTLL1^mnKO and TTLL1^mnWT littermates crossbred to Thy1-YFP or stained for Tubb3 (green; BTX, blue), depict singly (gray arrowheads) and polyinnervated synapses (light red arrowheads). n Percentage of polyinnervated NMJs in TTLL1^mnWT vs. TTLL1^mnKO littermates at postnatal day (P) 6-7, 8-9, 10-11, 12-13. (P6-7 WT n = 5, KO n = 3, p-value = 0.0357; P8-9 WT n = 5, KO n = 3, p-value = 0.0357; P10-11 WT n = 5, KO n = 5 mice, p-value = 0.0159, WT n = 4, KO n = 7, p-value = 0.0242; ≥ 197 NMJs per animal). Graphs: 25− 75% quantiles shown as a box with top and bottom black lines, mean as middle black line, and SEM as white lines (left); data representing single axons as dots (middle); half violin (right) in (b, d, f, h, j, l) or mean + SEM, with data representing single animals as dots in (n). A two-sided Mann-Whitney test determined significance: *, P < 0.05; **, P < 0.01; ****, P < 0.0001. Scale bar in (a) 5 µm also applies to (c, e, g, i, k), in (m) 10 µm. Source data are provided as a Source Data file.