lin-4 is a heterochronic gene that controls the timing of a number of developmental events in C. elegans. The
lin-4 allele (
e912) causes a retarded phenotype that is virtually identical to that caused by
lin-14(sd) mutations. Genetic epistasis experiments have suggested that
lin-4 acts upstream of
lin-14 in the heterochronic regulatory pathway. Gary Ruvkun's laboratory has shown that the temporal gradient of
lin-14 protein, high to low from early to later in development, is disrupted in
lin-4(
e912) animals. In
lin-4(
e912) animals, the level of
lin-14 protein remains high throughout development causing the reiteration of early cell fates. These data suggest that
lin-4 is, at least in part, responsible for generating the temporal gradient of
lin-14 activity. In order to further understand the role of
lin-4 in the temporal control of development we have begun to isolate new alleles of this gene. The original
lin-4(
e912) allele was generated by [32P] decay over 10 years ago. Because no new alleles of
lin-4 have been identified in numerous general screens for egl or vul animals (where they would have been expected to turn up) we wanted to establish the null phenotype of
lin-4 prior to screening for point mutations in this gene. Unfortunately, no deficiencies existed that covered the
lin-4 region of chromosome II. Therefore our first non-complementation screen was aimed at isolating a deficiency of the
lin-4 locus. Wild type N2 males were mutagenized with gamma rays, mated by
lin-4(
e912)
dpy-10/mnc1; 79ts) hermaphrodites and the F1 hermaphrodite progeny were screened at 20 C for non-dpy
lin-4 like animals. Out of 7,000 F1 hermaphrodites screened, one
lin-4 deletion mutant (maDf4) was identified. maDf4 is homozygous inviable and fails to complement
lin-4,
bli-2, analysis using a dissecting microscope, the phenotype of
lin-4(
e912) Df4 animals is indistinguishable from that of
lin-4(
e912)/lin-4
(e912) animals (with the exception of an enhanced sterility of
lin-4(
e912) Df4 animals). These data suggest that the original
lin-4(
e912) allele is not a gain-of-function allele and indicates that
lin-4(
e912)/null is fertile and viable. Since lin- 4
(e912)/maDf4 is not significantly different from
lin-4(
e912)/lin-4(
e912),
lin-4(
e912) is probably a null allele. However, maDf4 does not delete
spe-3, a gene that maps only 0.1 mu to the right of
lin-4 and thus we are not yet certain that maDf4 completely deletes
lin-4. Now that we have a
lin-4 clone it will be possible to test this directly ( see Lee, Feinbaum and Ambros, this WBG). Encouraged that we knew the expected phenotype of
lin-4(
e912)/lin-4 null we have initiated a new series of non-complementation screens to identify EMS induced
lin-4 alleles. A non-complementation screen identical to the one outlined above, except that the males were treated with EMS instead of gamma rays, was undertaken. Out of 8,000 F1 hermaphrodites screened we have identified 1 new
lin-4 allele (
ma161).
ma161 looks phenotypically similar to
e912 but Southern blot analysis with our
lin-4 clone has confirmed that it is in fact a new allele of
lin-4. We intend to continue isolating new
lin-4 alleles utilizing this non-complementation strategy.