Figure 2. An organized ECM microenvironment rescues fiber resiliency in Dag1-deficient cells.

(A) Anterior left, dorsal top, side-mounted, 3 dpf embryos. Polarized light microscopy shows loss of birefringence in dag1 morphant myotomes (white arrowheads). Birefringence is rescued in NAD+-supplemented dag1 morphants. (B, C, I, J, L) Anterior left, dorsal top, side-mounted, 3 dpf embryos stained with phalloidin (white or green). Fiber detachment is readily observed in dag1 morphants (B, white arrowheads), whereas dag1 morphants supplemented with NAD+ display less fiber detachment (C). (D) Compared to dag1 morphants (gray bars), NAD+ supplementation (blue bar) and vitamin supplementation with Emergen-C (purple bar) significantly reduce fiber detachment; **p<0.01. (E–H) Transmission electron micrographs showing normal BMs (white arrows) and disrupted BMs (red arrows). (I) The dystrophic phenotype of 3 dpf sly/laminin gamma1 mutant zebrafish. White arrowhead points to detached fibers. (J) NAD+ does not rescue the dystrophic phenotype in sly mutants, suggesting that NAD+-mediated amelioration of dystrophy requires laminin. (K) Genetic mosaic cartoon depicting transplantation of fluorescent dextran-labeled dag1 morphant (red) and control (blue) cells into unlabeled, control hosts. Some embryos were stressed (frequently stimulated to swim in a viscous medium), and all hosts were reared to 3 dpf. (L) Transplanted control cells (blue) and dag1 morphant cells (red) remain attached to MTJs, even when hosts are stressed. This suggests that a normal host ECM microenvironment is sufficient for resiliency of dag1 morphant cells and supports that NAD+ functions via augmentation of the ECM microenvironment. (M) The vast majority of Dag1-deficient cells remain attached in unstressed (513/523 Dag1-deficient cells were attached) and stressed hosts (479/491 Dag1-deficient cells were attached), N.S., not significant. Scale bars are 50 micrometers in (B) and (J) and 5 micrometers in (E–H).