Worm Breeder's Gazette 14(2): 38 (February 1, 1996)

These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.


Cathy Savage, Scott R. Townsend, Huang Wang, Richard W. Padgett

Waksman Institute, Rutgers University, Piscataway, NJ 08855

        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.

sma-2 is expressed widely in larvae and adults

        In conjunction with the genetic and phenotypic analyses
described above, we are also determining the expression patterns of
these TGF-b pathway components. This information should help in the
analysis of the cellular bases of the known and novel phenotypes of
these genes. We have built several reporter constructs for sma-2 and
sma-3 using PCR of genomic DNA to generate the desired promoter
fragments and fusing with lacZ or gfp using Andy Fire¹s vectors. At the
same time, we have tagged the SMA-2 protein with the HA epitope as
another means of verifying the expression pattern of this gene, as well
as to determine the subcellular localization of the protein. We have
begun to transform these constructs into nematodes to assay for sma-2
and sma-3 expression.

        So far, we have preliminary results on the expression pattern of
sma-2. For this experiment, we used a 3kb genomic fragment upstream of
the sma-2 coding region in a transcriptional fusion with cytoplasmic
(not nuclear-localized) lacZ from pPD89.03.  The staining pattern in two
independent (but not integrated) lines shows very high levels of widely
distributed expression, especially in early larval stages. The most
prominent feature at all stages is expression throughout the pharynx:
this expression is the earliest seen, in late embryos, and persists
through adulthood. Since the entire pharynx is stained in these animals,
we hypothesize that pharyngeal muscles may be the source of the
expression. Given the possibility that sma-2 and sma-3 null mutants may
be larval lethal, it is tempting to speculate (but completely
unsubstantiated) that they may have pharyngeal defects. Aside from the
pharynx, it is difficult to identify the other tissues staining in early
larval stages, because the worm appears quite blue throughout. Nuclear
localized staining constructs may help to resolve this issue.

        In adults, the staining pattern is slightly less widespread. As
mentioned above, the pharynx stains quite prominently in adults. We also
see two lateral ribbon-like stripes along the length of the animal that
underlie the cuticular alae, suggesting expression in the seam cells.
This result is very satisfying, as defects in the seam cells could be
responsible for the Sma phenotype. We also see two blobs of staining
near the tail, anterior and posterior of the anus on the ventral side of
the animal. These positions coincide with the locations of the
intestinal and anal depressor muscles. Finally, there is an intriguing
dynamic staining pattern surrounding the vulva in L4 and adult animals.
The staining starts as 4 small spots and 2 larger blobs on the ventral
side of the animal surrounding the developing vulva. The spots later
disappear, and the blobs develop as Y-shaped patches of staining, with
the arms nearly surrounding the adult vulva and the base of each Y
running either anteriorly or posteriorly on the ventral side of the
animal. Our current best guess is that this staining derives from the
ventral hypodermis, although other tissues, such as ventral uterus,
occupy similar positions.

        We are continuing to characterize the sma-2 expression pattern
by integrating the array, and by looking at expression in males and in
mutant backgrounds. As a note of caution, all of the expression results
need to be verified with independent constructs and by comparison with
the sma-3 expression pattern and with the SMA-2-HA tag localization.