Fig. 5: HepaCAM is essential for early postnatal CST axon growth and expands axon growth cone size.

a Diagram of CM-DiI dye injections at the motor cortex to label layer V pyramidal neurons and descending CST axons; PD pyramidal decussation; green dashed box indicates postnatal CST growth (as shown in c); b Representative (from >10 injected mice) image to show the CM-DiI labeling in the motor cortex 2 days following the injection; Scale bar: 1 mm; Representative images (c) and quantification (d) of CM-DiI-labeled CST axons in the spinal cord of WT (i) and HepaCAM KO (ii) mice. Orange arrows indicate the pyramidal decussation; yellow lines indicate the beginning and ending points for the CST axon length measurement; The image was generated by superimposing images of serial longitudinal sections, which are shown in Supplementary Fig. 5a. Scale bar: 1 mm; n = 8 mice for WT and 9 mice for HepaCAM KO; Representative images (e) and quantification (f) of axon growth cone size of control (i) cortical neurons or cortical neurons treated with WT (ii) or HepaCAM KO (iii) A-Exo. C center ___domain (white circle), P peripheral ___domain (growth cone area outside of the center ___domain); Scale bars: 20 µm. Number of neurons quantified in each group shown in the graph (4–14 neurons/replicate, 3 biological replicates)/group; Representative images (g) and quantification of axon growth cone size (h) of cortical neurons grown on either PDL alone (i) or PDL/HepaCAM-ECD (ii) coating. n = 17 neurons (5–8 neurons/replicate, 3 biological replicates)/group; Scale bar: 20 μm; i Quantification of axon growth rate of cortical neurons treated with HepaCAM ECD and A-Exo. Number of neurons quantified in each group shown in the graph (7–9 neurons/replicate, 3 biological replicates)/group; live-cell imaging was performed at DIV 3 for 2 h; p value in (d, h) determined from two-tailed t-test; p values in (f, i) determined using one-way ANOVA followed by a Tukey post hoc test. Data are presented as mean values ± SEM.