Figure 2. Immunolocalization of CeMyoD in the M lineage. Hermaphrodite larvae were double-stained with antibodies to CeMyoD and to b-galactosidase. b-galactosidase, driven by the
hlh-8 promoter, served as a marker for the M blast cell and its descendants (see Materials and methods). b-galactosidase was visualized using a fluorescein-labeled secondary antibody (green cell), while CeMyoD was visualized using a rhodamine-labeled secondary antibody (red cells). (A) and (C) CeMyoD antibody labeling alone. (B) A photographic double exposure showing CeMyoD and b- galactosidase antibody localization. Filled arrowheads indicate bodywall muscles and open arrowheads, M descendants. (A,B) L1 stage animal: The M blast cell was not immunoreactive with anti-CeMyoD antibody. Of >50 animals examined at this stage (prior to the division of M), none showed CeMyoD immunoreactivity in M. (C) Detectable CeMyoD immunoreactivity in the M lineage first appeared (in a small fraction of animals) after M had divided once. In the animal shown, CeMyoD was detected in both M daughters (M.x, open arrowheads). After the next cell division (four M granddaughters), CeMyoD was detected in 100% of animals (n >100). CeMyoD staining continued at later stages in the M lineage (8-16 M-derived cells); staining in most animals was detected in both muscle and coelomocyte precursors. We also examined the pattern of
hlh-1 promoter activity using an integrated
hlh-1::gfp reporter construct (C. Branda, K. Dej, S. Xu, A. Fire, and M. Stern, unpublished). This reporter construct produced a similar distribution to that observed with the anti-CeMyoD antibody. With reporter fusions and antibody staining we detect de novo postembryonic CeMyoD expression only in the M lineage: no new expression was seen in other (non-myogenic) lineages.