Body length in C. elegans is regulated by a conserved signalling pathway activated by a protein in the TGF-beta superfamily, DBL-1. Loss-of-function mutations in genes encoding components of the pathway cause a marked decrease in body size, the Sma phenotype. Conversely, constitutive activation of the pathway results in a dramatic increase in body length. The molecular mechanisms by which the TGF-beta pathway affects body length are not yet known however. One of us (A.L.) has previously reported that mutations in
sma-2 and
daf-4 reduce the extent of endoreduplication of hypodermal nuclei. We found that hypodermal ploidy was also reduced in
dbl-1(0) mutant hermaphrodites. Ploidy was not increased, however, in worms that overexpressed
dbl-1 indicating that while it is a regulator of body length,
dbl-1 is permissive but not instructive for hypodermal ploidy. A mutation in
lon-3 ,
e2175 , gives rise to a Lon phenotype that is strikingly similar to that caused by overexpression of
dbl-1 . In a screen of 50,000 haploid genomes for new Lon mutants, we isolated three new
lon-3 alleles,
sp5 ,
sp6 and
sp23 . The strongest alleles cause worms to be more than 20% longer than wild type. All alleles are recessive to wild type and behave genetically as though they reduce or eliminate gene activity. We cloned
lon-3 by transformation rescue and showed that it is predicted to encode a collagen with a structure typical of that of cuticle collagens. Consistent with the genetic analysis, characterization of the sequence changes associated with
lon-3 mutant alleles suggests that loss-of-function phenotype is Lon.
sp6 and
sp23 are associated with sequence changes that introduce stop codons very early in the open reading frame. Analogous mutations in other collagen genes, such as
sqt-1 and
rol-6 , behave as null alleles. Interestingly we found that while the loss-of-function phenotype is Lon, higher than wild-type levels of
lon-3 cause a Dpy phenotype. Thus
lon-3 can function as a regulator of body length. The hypodermal ploidy in both
lon-3(0) mutants and worms overexpressing
lon-3 was wild type indicating that
lon-3 , like
dbl-1 , can regulate length independently of hypodermal endoreduplication. Defects in
lon-3 expression have no effects on dorso-ventral patterning of the male tail or on dauer development, processes that are controlled by the TGF-beta ligands DBL-1 and DAF-7 respectively. In addition, a
lon-3-lacZ fusion gene is expressed in many hypodermal cells; cells that are known to secrete the cuticle. Thus it seems likely that
lon-3 regulates worm length by altering the shape or elasticity of the cuticle rather than by altering the activity of genes in the TGF-beta pathway. Previously it has been shown that mutations in
sqt-1 , which also encodes a cuticle collagen, can also affect body length. However
sqt-1 does not seem to be a prime regulator of body length since mutations in
sqt-1 giving rise to longer or shorter worms appear to be neomorphic, and the
sqt-1 null phenotype is wild type. LON-3 may nevertheless function together with SQT-1 to regulate body length: the
lon-3 overexpression phenotype is completely suppressed by
sqt-1(0) , and
sqt-1(0) strongly suppresses the Lon phenotype of
lon-3(0) . Since
sqt-1 null mutations do not suppress the Dpy phenotype caused by mutations in any of the known collagen genes we have tested, overexpression of
lon-3 cannot cause a Dpy phenotype simply by interfering indiscriminately with the activity of the proteins encoded by these genes. The relationship between
lon-3 and the TGF-beta pathway regulating body length is presently unclear. The expression of a
lon-3-lacZ reporter is not affected by mutations in genes in the TGF-beta pathway. Experiments with a LON-3 antiserum have revealed that LON-3 protein levels are altered in worms lacking
dbl-1 or that overexpress the protein. However, LON-3 protein levels do not change in a simple reciprocal fashion in response to changes in
dbl-1 activity. Thus
lon-3 could either function as one (of two or more) targets downstream of the TGF-beta pathway to regulate body length or in a parallel pathway. To learn more about how body length and body size are regulated in C. elegans we are presently characterizing four new Lon genes that we identified in our screen.
lon-4 maps between
egl-29 and
lin-29 on chromosome II, whereas
lon-5 ,
lon-6 and
lon-7 all map to the X chromosome.