The
aph-1 gene was first identified in the Priess lab, based on its
glp-1-like maternal-effect embryonic lethal phenotype. Embryos that are deficient in either
glp-1 (Notch receptor), Presenilins (
sel-12 and
hop-1),
aph-1, or
aph-2 cannot carry out Notch signaling in the early stages of blastomere fate determination. Both genetic and biochemical data now point to a model in which all of these membrane-associated proteins are involved in activation of the Notch receptor in response to ligand. This activation involves a presenilin-mediated intramembranous cleavage of the Notch receptor at the cell surface. Curiously, removal of
aph-1 or
aph-2 activity does not cause obvious defects in all Notch signaling events. This may be due in part to differential requirements for the activity of these proteins in different developmental contexts. We are investigating the requirement for APH-1 protein in the post-embryonic Notch signaling events that mediate vulval development. We have found that
aph-1(
or28) animals, which show a fully penetrant egg laying defect, correctly specify a single anchor cell (AC), but are defective in the specification of the uterine pie cells. This was demonstrated by observing expression of the pie-cell specific
cog-2::gfp reporter, and by observing the differentiation of the pie-cell derived uterine-seam cell (utse), which normally forms a thin laminar process between the uterine and vulval lumens and ultimately fuses with the AC. In
aph-1(
or28) animals, we observe a thick tissue in place of the thin utse process, and concomitant persistence of an unfused AC. This defect is identical to that described previously for mutations in
sel-12 (Cinar et al., 2001) or
aph-2 (Levitan et al., 2001), which also do not interfere with the AC-VU cell interaction. The fully penetrant and specific vulval phenotype of
aph-1(
or28) provides an ideal situation in which to test the cell autonomy of APH-1. To this end we have generated transgenic lines that express
aph-1 under the control of either the
cdh-3 promoter [expresses in the inducing cell (AC), but not in the responding cells (pie cells)] or the
cog-2 promoter (expresses in the responding cells). We are currently assaying for rescue of the
aph-1(
or28) Egl phenotype by either of these constructs. We are also setting up the same assay for
aph-2 to test cell autonomy of
aph-2 in the context of the AC-pie cell interaction. Under these same circumstances,
sel-12 expression under the
cog-2 promoter was shown to be sufficient to rescue the
sel-12 defect in pie cell specification (Cinar et al., 2001). We will also present our progress in using
aph-1::gfp transgenes in an attempt to detect
aph-1 expression in the vulva.