We have previously reported that
sma-2,
sma-3, and
sma-4 encode related proteins, the dwarfins, that function in a TGF-b-like pathway with the receptor DAF-4 (WM95 p. 56; Savage et al., PNAS, in press). The phenotypes that place these genes in a common pathway are small body size (Sma) and male tail abnormalities (Mab): crumpled spicules and sensory ray transformations. While
daf-4 mutants have several other defects, most notably including the Daf-c phenotype, these three sma mutants do not seem to share these defects. We wondered whether
sma-2,
sma-3, and
sma-4 have other roles in C. elegans development. To address this question, we are determining the null phenotypes and the expression patterns of these genes. Null alleles of
sma-2 and
sma-3 may be lethal Existing alleles of
sma-2,
sma-3, and
sma-4 have been picked up by either their Sma or their Mab phenotypes, leaving open the question of whether more severe or lethal alleles could be generated. Also, the molecular lesions that we have identified in sequenced alleles are not convincing molecular nulls. To start characterizing the null phenotypes of these genes, we wanted to put existing alleles over a deficiency. After considerable difficulty with nDf17, which deletes all of these genes, we settled on using nDf16, a well-behaved deletion that only removes
sma-3. (Thanks to Theresa Stiernagle for patiently sending numerous deficiency strains!) We crossed
sma-3unc-32/++ males into nDf16/qC1(
dpy-19glp-1) or nDf16/dpy-17unc-32 hermaphrodites. In each case, Sma cross progeny were present but showed reduced viability. For the qC1 strain, we scored 97 wild-type males and only 18 Sma males, instead of the expected 32. For the
dpy-17unc-32 strain, we scored 57 Unc, 92 wt, and 35 Sma males, where Sma and Unc males should have been present in equal numbers. These results suggest that existing
sma-3 alleles may be partial loss-of-function, and that null alleles may be at least partially lethal. The
sma-3unc-32/nDf16 survivors from these crosses appear no worse than
sma-3 homozygotes. Ten Sma males were scored for male tail defects. These look like
sma-3 mutants: all had crumpled spicules, 5/10 sides scored had fusions of rays 4 and 5, 5/10 had fusions of rays 6 and 7, and 3/10 had fusions of rays 8 and 9. Although the
sma-3unc-32/nDf16 hermaphodites appear healthy and no smaller than
sma-3 mutants, they did show reduced fertility as well as higher rates of lethality in the next generation. Of four Sma hermaphrodites picked, one was sterile. The fertile hermaphrodites segregated ~10% Sma (genotype
sma-3unc-32/nDf16) and ~90% SmaUnc (
sma-3unc-32) progeny, where the expected ratios are 2:1 Sma:SmaUnc. This result indicates a maternal effect for
sma-3, since
sma-3/nDf16 progeny of nonSma mothers were ~60% viable while the
sma-3/nDf16 progeny of Sma mothers were only ~5% viable. The deficiency experiments raised the exciting possibility that
sma-2,
sma-3, and
sma-4 may have essential roles in development. Since
sma-3/Df hermaphrodites (from nonSma mothers) are at least reasonably viable and fertile, we decided to isolate null mutations in a non-complementation screen. We are using a
sma-3sma-2 double mutant chromosome to isolate mutations in both genes simultaneously; later we will sort out the complementation groups. We are mutagenizing
unc-32 hermaphrodites with EMS, and mating with
lon-1sma-3sma-2/+++ males. The F1 cross progeny are then screened for Sma animals (sma-?*
unc-32/lon-1sma-3sma-2). From ~2000 genomes screened so far, we have isolated 5 new mutations, for a frequency of ~1/800 per gene. In all cases, no homozygous SmaUnc progeny are being segregated, suggesting a lethal hit on the chromosome. We have looked for dead eggs from these strains, but not found any, so that the mutations may be larval lethal. We are currently doing careful egg lays to determine whether these mutations are in fact larval lethal.