Figure 3. (a) Expression of
nos-3 mRNA. Polyadenylated C. elegans mRNA was separated through a 1% agarose glyoxal gel, transferred to a filter and probed with a
nos-3 cDNA. Lane 1, wild-type adults, which possess approximately 2,000 germ cells; lane 2,
glp-1(
q224) mutant adults, which possess no more than eight germ cells; lane 3, mixture of developmental stages (from first larval stage through adults with embryos); lanes 4-9, embryos (E), the four larval stages (L1-L4) and adults (A), respectively. In lane 2, note that very little
nos-3 mRNA is detectable; the small amount remaining may represent somatic expression. To verify equal loading of lanes, the blot was probed for expression of elongation factor-1 alpha (
eft-3). (b-g) NOS-3 protein expression in the gonads of L3 hermaphrodites. (b-d) Wild type and (e-g)
nos-3(delta) L3 larvae stained with (b,e) anti-NOS-3 and (c,f) anti-PGL-1 antibodies; (d,g) are the merged images of (b,c) and (e,f), respectively. At this stage, the germ line is proliferating; the
nos-3 mutant is slightly older than the wild type and thus has a slightly larger germ line. (b) NOS-3 protein was uniformly distributed throughout the cytoplasm in the anterior and posterior germ-line tubes of wild-type animals (arrowheads), and there was faint staining in the somatic gonadal tissue (arrow). (e) The protein was absent in
nos-3 mutants even though germ-line cells were present, as evidenced by (f) staining with anti-PGL-1 antibody, which is specific for P granules in the germ line. (h,i) NOS-3 protein expression in the L4 hermaphrodite germ line. The germ-line tube of a wild-type late L4 hermaphrodite was extruded for staining. At this stage, spermatogenesis has begun. (h) NOS-3 protein was found in the germ-line cytoplasm and was most prominent in germ cells in mitosis and meiotic pachytene. (i) Mitotic nuclei were stained with an antibody against a phosphorylated form of histone H3 (anti-phospho-H3 antibody, green), and spermatogenic cells with an anti-sperm antibody called SP56 (blue). Sperm with highly condensed nuclei also stain with anti-phospho-histone antibody. (j-l) NOS-3 protein expression in the early embryo. A wild-type four-cell embryo is shown stained with antibodies against (j) NOS-3 and (k) PGL-1; (l) is the merged image with overlapping signals in yellow. (j) Each of the four blastomeres is indicated. NOS-3 was localized to the cytoplasm of P blastomeres, which generate the germ line. (k) PGL-1 is localized to P granules in the early embryo.