FIGURE 5

FIGURE 5

Proliferation of muSCs during regeneration and homeostasis is Notch dependent. Projections of confocal stacks (A–D) of the myotome (A’–D’) and vertical myoseptum (A”–D”) in uninjured (A,C) and injured (B,D) 5 dpf pax7a:eGFP larvae incubated with BrdU for 24 h. Larvae were treated with 1% v/v DMSO (A,B) or 100 μM DAPT (C,D) after injury and detection of BrdU and eGFP was performed by immunolabeling. MuSCs with (red arrowheads) and without BrdU labeling (yellow arrowheads) are recruited to the injury (asterisk) site (B’,D’). The number of cells expressing pax7a:eGFP (E,I), incorporating BrdU (F,J) or both (G,K) were counted in the myotome (E–G) and both vertical myoseptum (I–K) of animals treated with DMSO or DAPT. The proportion of pax7a:eGFP-expressing muSCs incorporating BrdU was calculated for both myotome (H) and vertical myoseptum (L). There were significantly fewer GFP+ muSCs incorporating BrdU after injury in the presence of DAPT compared to DMSO treated control animals (p < 0.05; G). There is no significant change to the number of GFP+ muSCs incorporating BrdU at the vertical myoseptum of injured animals in the presence of DAPT (p > 0.05; K). In contrast, although there are only few cells present, there is a significant increase in the number of muSCs with BrdU labeling at the myoseptum in uninjured animals in the presence of DAPT (p < 0.05; K). Significant differences were tested by 2-way ANOVA (n = 23) with Tukey’s HSD post hoc test or by transforming data by ART and performing 2-way ANOVA followed by a Dunn’s test with Benjamini and Hochberg correction. Error bars display standard deviation, and values above comparison bars indicate significance (p-values). Scale bars: 100 μm (A–D), 50 μm (A’–D’,A’–D”).