Worm Breeder's Gazette 10(3): 102

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.

Identification of Mutants that Show Ectopic Wheat Germ Agglutinin Binding

Chris Link

I have previously reported that the lectin wheat germ agglutinin and 
the monoclonal antibody Ab117 show specific binding to the vulva of 
adult hermaphrodites and the copulatory bursa of adult males (W.B.G.  
10, #1, p. 117; Link, Ehrenfels, and Wood, Development 103, 485-495).  
Based on the observation that lin-22 males show strong ectopic binding 
of both WGA and Ab117, I devised a non-clonal screen to identify 
mutants that show ectopic WGA binding in hopes of identifying mutants 
with anterior/posterior transformation like lin-22.  As described 
below, I have not yet identified mutants of this class, but have found 
a number of unexpected mutants interesting in their own right.  
Previously I visualized WGA or Ab117 binding to live animals using 
fluorescently-labeled lectin or secondary antibody.  Because this 
method was not feasible for large scale mutant screening, I developed 
a peroxidase-coupled detection system that could be used with live 
animals and easily visualized under the dissecting microscope.  
Briefly, for visualizing WGA binding, animals were sequentially 
incubated in biotin-conjugated WGA and avidin-conjugated horseradish 
peroxidase, then developed using H2O2 and the chromogen 3-amino 9-
ethylcarbazole.  In order to use WGA binding in a non-clonal screen, I 
used semidominant mutations in her-1(n695 or y101) as the genetic 
background.  These mutations variably masculinize XX animals, such 
that a sub-population of the adult animals (5-25%) are sufficiently 
somatically masculinized to show (lin-22 dependent) ectopic staining, 
but still retain a self-fertile hermaphrodite gonad.  In three pilot 
experiments, I have screened approximately 60,000 EMS-mutagenized her-
1(n695) F2 adults and 20,000 EMS-mutagenized wild-type F2 adults for 
ectopic WGA binding, and have identified 15 independent mutations.  
These mutations, which fall into at least four complementation groups, 
appear to be unlike lin-22 in that their ectopic WGA binding is 
observed in both males and hermaphrodites.  I have recently screened 
500,000 EMS-mutagenized her-1(y101) F2 animals and identified an 
additional 50 independent (as yet uncharacterized) mutants.  The most 
interesting of the characterized mutations are the allelic pleiotropic 
mutations ct109 and ct111(V).  ct109 animals bind WGA and Ab117 over 
most of their cuticle surface.  This surface binding is neither stage- 
nor sex-specific, although adult males show somewhat stronger 
posterior WGA binding than adult hermaphrodites.  ct109 animals are 
uncoordinated and have improper body posture reminiscent of unc 
mutants known to have neurological defects.  ct109 males have 
defective bursae, characterized by having crumpled spicules and 
abnormal rays.  These males variably lack diagonal sex muscles.  The 
most surprising ct109 phenotype is its ability to enhance the weak 
semidominant mutation lin-12(n302).     Weak lin-12 dominant mutants 
such as n302 are Vul, while strong lin-12 dominant mutations such as 
n137 are Muv.  lin-12(n302); imals closely 
resemble lin-12(n137); hermaphrodites have at 
least four pseudovulvae and males have ectopic hooks.  All of the 
phenotypes observed for ct109 have also been observed for ct111, 
arguing that these phenotypes are likely to be the result of a single 
mutation.  How can this motley collection of phenotypes be explained? 
A plausible explanation for the ct109 Unc and Mab phenotypes is that 
these animals are defective in cell-cell or cell-matrix interactions.  
Thus, the ct109 Unc phenotype might result from neuronal mis-wiring (
due to improper axon pathfinding) while the Mab phenotype might result 
from defects in migration of the sex muscle precursors and other 
bursal cells.  To speculate wildly, perhaps ct109 defines a gene that 
modifies (glycosylates?) extracellular proteins or protein domains.  
This hypothetical modification would modulate cell-cell interactions 
in a manner akin to the way sialic acid addition modulates N-CAM 
function in vertebrates.  This modification would also modulate the 
function of the lin-12 product (almost certainly a cell surface 
protein) and be involved in cuticle formation.