Figure 7. Anti-CeMyoD Antibody Staining during EmbryogenesisEmbryos were prepared as described in Experimental Procedures and incubated with HM-1 antiserum directed against CeMyoD and a mouse monoclonal antibody (5-6) directed against the body wall muscle-specific myosin heavy chain, MHC A. Antigen recognition was visualized by indirect immunofluorescence using rhodamine-labeled donkey anti-rabbit or fluorescein-labeled goat anti-mouse secondary antibodies for CeMyoD and MHC A, respectively. Embryos are oriented with anterior to the left.(A) An approximately 130 cell embryo with a group of posterior CeMyoD-positive nuclei derived from the C and D cell lineages. (B) An approximately 250 cell embryo with CeMyoD-positive nuclei of the D, C, and MS lineages extending anteriorly along the lateral regions of the embryo. C) A dorsal view of an embryo that has completed cellular proliferation and begun to elongate. CeMyoD-positive nuclei of the dorsal two body wall muscle quadrants are visible; the other two quadrants are in a different focal plane. Each quadrant has two rows of positive nuclei. (D) Fluorescein secondary antibody labeling of the same embryo as in (C) to visualize anti-MHC A antibody staining. MHC A is accumulating in the cells of the body wall muscle quadrants. (E) An early larva that hatched during preparation for antibody staining. Two of the body wall muscle quadrants can be seen; the remaining two quadrants are in a different focal plane. (F) Fluorescein secondary antibody labeling of the same larva as in (E) to visualize anti-MHC A antibody staining. Individual sarcomeres within each body wall muscle cell can be distinguished; the body wall musculature is functional by this stage of development.
Figure 6. CeMyoD Expression in Early Embryos. Embryos were incubated with HM-1 anti-serum directed against CeMyoD and a mouse monoclonal antibody (K-76) directed against germline P granules. Antigen recognition was visualized by indirect immunofluorescence using rhodaminalabeled donkey anti-rabbit or fluorescein-labeled goat anti-mouse secondary antibodies for CeMyoD and P granules, respectively. All embryos are oriented with anterior to the left. (A) An ~80 cell embryo with both D blastomere daughters staining with anti-CeMyoD antibody (arrows). (B) DAPI stain of the embryo in (A). Mitotic figures of dividing MS descendants (arrow) orient blastomeres within the embryo.(C) An embryo at approximately the 180 cell stage stained with the anti-CeMyoD antibody. Solid arrows point to positive nuclei of the four D-derived cells. Open arrows point to a lateral row of three to four nuclei of MS descendants that are beginning to accumulate CeMyoD. A symmetrical row of MS nuclei are present on the other side of the embryo but are in a different focal plane. Unmarked positive nuclei at the posterior of the embryo are C cell descendants.(D) Anti-P granule staining of the blastomeres 22 and 23 (arrows) of the same embryo shown in (C). At this stage of embryogenesis, 22 and 23 are capped by the four D cell descendants and are therefore useful landmarks used to identify other blastomeres.
(G) ufd-2::GFP is expressed in pharyngeal muscles of a L2 larva. (H) L4 larva expressing ufd-2::GFP in the cytoplasm and the nucleus of body wall muscle cells (arrow points to the nucleus of a body wall muscle cell). Scale bars, 10 um.
(G) Images of a transgenic worm that expressed GLR-1::CFP and STG-1::YFP in body wall muscle cells. (H and I) Images of transgenic worms injected with Alexa Fluor 594 conjugated anti-GFP that expressed either GFP::STG-1 (H) or STG-1::GFP (I) in body wall muscle cells.
Figure 4. lmmunofluorescence Localization of Myosin lsoforms in Nematode Body Wall Muscle Cells. (A, B) Polarized light micrographs of body wall muscle cells, Indirect immunofluorescence Image of body wall muscle cells labeled with antibody 26.2 (C, D) and antibody 5-6 (E, F). (A, C, E) are low magnification images and (B, D, F) are high magnification images. Bars are 10 um. The double-headed arrow in (B) depicts the course of a single myosin thick filament in the A-band. Pointers in (8, D, F) denote the center of the A-band.