The Sma/Mab pathway is one of the TGF-beta related pathways in C. elegans. The loss of function with any known factor (
dbl-1,
daf-4,
sma-6,
sma-2,
sma-3,
sma-4) in this pathway results in small body size and male tail ray fusions. Our model for this pathway is based on biochemical studies in related signal transductions. When the ligand DBL-1 binds on type II receptor DAF-4 and type I receptor SMA-6, the always active DAF-4 phosphorylates SMA-6. SMA-2 and SMA-3 belong to R-smads which are phosphorylated by activated SMA-6 during the signaling. After the activation, they form a heteromeric complex with Co-smad, SMA-4. The complex goes into nucleus and regulates the transcription of target genes. We made two kinds of
sma-3::GFP translational fusion constructs. GFP was inserted at the N-terminus or C-terminus of SMA-3 protein. The N-terminal construct showed the activity of rescuing body length and male tail ray fusions in the mutant
sma-3(
wk30) . By observing the GFP fluorescence distribution, we found that both of the
sma-3::GFP constructs were expressed in pharynx, intestine and hypodermis. They localized in the nucleus. To determine if
sma-3 expression in all of the tissues is critical for body length regulation or not, we did two kinds of analyses. First, with co-injection of
sur-5::GFP and
sma-3 genomic fragment into
sma-3(
wk30) , we got various kinds mosaic worms. We found that with the expression of
sma-3 in hypodermis, the worms have wild type body size. But, without
sma-3 hypodermal expression, all of the worms are small whether
sma-3 is expressed in intestine, pharynx or not. Second, we did the directed expression of
sma-3. In the
sma-3::GFP N-terminal construct, the
sma-3 promoter was removed and tissue specific expression promoters were put in. The promoters were
elt-3,
dpy-7,
vha-7 (hypodermal),
elt-2,
vha-6 (intestinal) and
myo-2 (pharyngeal). We got the co-expression in pharynx and intestine by using
tmy-1 isoform III promoter or the co-injection of
myo-2 and
vha-6 constructs. From the fluorescence images, we found that the constructs were expressed in the regions we expected. The measurement of the worm length was done. The data shows the hypodermal expression of
sma-3 could mostly restore the body length, similar to the
sma-3 promoter. But, the intestinal expression of
sma-3 has no effect. And the
sma-3 expression in pharynx or both in intestine and pharynx can only rescue the body length a little, not significantly. So, the hypodermal expression of
sma-3 is necessary and sufficient to restore the body length of
sma-3 mutant. During the process, we found that the C-terminal
sma-3::GFP, although it has little activity, has much brighter fluorescence by comparing with N-terminal
sma-3::GFP at the same concentration. The co-injection of the two
sma-3::GFP fusion genes gives a result similar to N-terminal construct alone, i.e., it can rescue
sma-3(
wk30) mutant but has weak fluorescence. It indicates that the intensity of GFP fluorescence is negatively regulated by
sma-3 activity. The idea is confirmed by the co-injection of
sma-3 genomic fragment and C-terminal
sma-3::GFP fusion gene. It also gives a weak fluorescence and totally rescues the
sma-3(
wk30) mutant. Currently, we are investigating whether the
sma-3 feed back loop depends on other components in the pathway.