Figure 1. In situ immunofluorescent detection of
lin-14 nuclear protein in wild-type and heterochronic mutants. Specimens were fixed and incubated with affinity-purified anti-
lin-14 antibodies, followed by a secondary fluorescein isothiocyanate (FITC)-labeled goat anti-rabbit antibody and 4,6-diamido-2-phenylindole (DAPI) stained to visualize all nuclei. Immunofluorescent photographs were taken with FITC (F) filters to show
lin-14 protein staining or DAPI (D) filters to show all nuclei. Representative nuclei of a cell type are identified by arrows; (G) gonad; (H) hypoderm; (I) intestine; (M) body wall muscle; (N) nerve ring; (V) ventral cord neurons. Photomicrographs are generally lateral views, in one of two planes. If a hypodermal cell is pointed out, the plane of focus is lateral and large nuclei are all hypodermal (H) and small nuclei are neuronal (N). If intestinal (I) and ventral cord neurons (V) are labeled, the plane of focus is medial and all large nuclei are intestinal and the line of small nuclei ventrally are the ventral nerve cord and small dorsal nuclei are muscle. The head of the animal is identified in most photomicrographs by the intensely staining nerve ring nuclei (N). (a) (Top) FITC; wild-type early L1 stage (L1), late L1 stage (unlabeled), and L3 stage (L3) animals. Lateral plane showing bright
lin-14 protein staining in most nuclei at early L1, somewhat dimmer staining at late L1, and absence of staining at L3. (Bottom) DAPI; same animals with same plane of focus. We have observed very dim
lin-14 protein staining in some neuronal nuclei of L2 and later animals in some preparations (M. -Finney, unpubl.). (b) (Top left) FITC; lateral view of
lin-14(
n355gf) mutant early L1 stage, showing intense staining of hypodermal, ventral nerve cord, and nerve ring nuclei. (Top right) FITC; ventral view of
lin-14(
n355gf) L2 stage, showing
lin-14 protein in the ventral cord neurons and in muscle nuclei. Both gain-of-function mutants show inappropriate accumulation of
lin-14 protein in L2 and later stages, although the
n536 allele is weaker than the
n355 allele and
lin-14 protein staining is consistently less intense in
lin-14(
n536) mutant animals. (c) (Left) FITC;
lin-4(
e912) L1 stage focused at the midline, showing
lin-14 protein staining in the nuclei of ventral cord neurons, intestine, hypoderm, and tail ganglion neurons. (Right) FITC; lateral view of
lin-4(
e912) L2 stage, showing
lin-14 protein in hypodermal and postdeirid neuronal nuclei. (d) (Top left) FITC;
lin-28(
n719) with three animals, two late stage embryos, and an early L1 stage. All animals show accumulation of
lin-14 protein in nerve ring, ventral cord, hypoderm, intestine, and muscle nuclei, like wild type at these early developmental stages. (Top middle) FITC;
lin-28(
n719) early L1 stage, showing a decrease in
lin-14 protein staining in the hypodermal and intestinal nuclei compared with neuronal and body wall muscle nuclei. (Top right) FITC;
lin-28(
n719) L2 stage, showing no
lin-14 protein staining. (e) (Top left) FITC;
lin-29(
n333) L1 stage that displays
lin-14 protein in the same nuclei as wild type. (Top right) FITC;
lin-29(
n333) L2 stage, showing no
lin-14 staining.