Worm Breeder's Gazette 13(5): 35 (February 1, 1995)

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.

Site of action of genes that affect pharyngeal muscle excitability: The LEFH

Leon Avery (leon@eatworms.swmed.edu)

Biochemistry Department, University of Texas Southwestern Medical Center,
5323 Harry Hines Blvd, Dallas, TX 75235-9038.

Several genes and drugs affect pharyngeal muscle excitation.  The
acetylcholine agonist arecoline and loss-of-function mutations in eat-6
(the Na,K-ATPase alpha subunit; WBG 13(2): 52) depolarize the muscles.  An
eat-11 mutation causes hypersensitivity to eat-6(lf) and arecoline. 
egl-30 mutations suppress the eat-11 hypersensitivity (WBG 12(5): 64). 
These genes could act directly in pharyngeal muscle, or they might act in
the nervous system.  To distinguish these possibilties, I designed the
following experiment: kill the pharyngeal nervous system (PhNS) in mutant
and wild-type worms, then compare them.  If the phenotypes are still
different, the gene has an effect outside the PhNS.  This experiment,
which eventually resulted in the senseless slaughter of thousands of
pharyngeal neurons, became known as the Laser Experiment From Hell (LEFH).

In practice all pharyngeal neurons except the essential neuron M4 were
killed.  As an objective measure of feeding, I determined whether each
worm became a fertile adult, and if so the number of days from hatching to
the production of the first progeny.  By this test eat-6 (compare rows 2-4
to 1), eat-11 (row 9 vs 8), and egl-30 (row 10 vs 9) all act outside the

row     Relevant        [arecoline]     number  number  days to          
        genotype                        animals fertile fertility 
1       +                   0 mM          13      13      4.3 
2       eat-6(ad601)        0 mM          11       5      8.4* 
3       eat-6(ad792)        0 mM           2       1     16*
4       eat-6(ad467)        0 mM          13       4      7.3* 
5       snt-1(md290)        0 mM           6       0      *
6       snt-1(ad596)        0 mM          14      10     10.5* 
7       eat-11(ad541)       0 mM          11      11      5.4 
8       +                   5 mM          12      10      6.4 
9       eat-11(ad541)       5 mM          10       0      *
10      egl-30(ad803)       5 mM          14      14      5.1^           

*Significantly different from wild-type at the same [arecoline], P < 1%.
^Significantly different from eat-11, P < 1%.

The surprise was snt-1.  snt-1 encodes the synaptic vesicle protein
synaptotagmin, and within the pharynx antibodies were not reported to
recognize anything other than neurons (Nonet et al, Cell 73: 1291).  I
expected that PhNS- snt-1 worms would be similar to PhNS- wild-type. In
fact, they pumped little or not at all, and their growth was drastically
slowed (rows 5 and 6 vs 1; md290 is a null, and ad596 a hypomorph).  This
result suggested that snt-1 has a site of action outside the PhNS capable
of affecting pharyngeal muscle function.  The snt-1 expression pattern,
however, suggested that snt-1 does not act in pharyngeal muscle.  Guessing
that the depression of pumping in PhNS- snt-1 worms resulted from an
inhibitory effect of cells outside the pharynx, I dissected pharynxes out
of PhNS- snt-1(ad596) worms. In fact, the average firing rate increased
from 1.1/min to 9.7/min, consistent with an extrapharyngeal inhibition.

This experiment showed that the logic of the LEFH was flawed. Although it
can exclude pharyngeal neurons as the sole cells affected by a gene, the
LEFH cannot show that pharyngeal muscle is the site of action, since
something outside the pharynx can also drastically affect pharyngeal
muscle function.  To get around this problem I killed the PhNS in mutant
and wild-type, then dissected the pharynx out of the PhNS- worms that
survived to adulthood.  This experiment has been done only on wild-type,
snt-1(ad596), and eat-6(ad467).  (And if it is ever done again, it will
not be me doing it.)  The results are consistent with a nervous system
site of action for snt-1, and support a muscle site of action for eat-6. 
After dissection 4 PhNS-eat-6 pharynxes hypercontracted, the
characteristic eat-6 phenotype. 7 PhNS- snt-1 and 5 PhNS- wild-type didn't
hypercontract, and weren't strikingly different from each other.

There are two potential problems in interpreting the eat-6 results. First,
since M4 was spared in the LEFH, it might in principle be the sole site of
eat-6 action.  However, I showed previously that eat-6 mutation suppresses
the lethality of M4- worms (WBG 13(2): 53), implying a site of action
outside M4.  Second, it could be that eat-6 acts only in the PhNS, but
that the mutant nervous system changes muscle physiology in a way that
can't be reversed after removal of the PhNS.  This is unlikely, because
Wayne Davis has shown that the effects of a cold-sensitive eat-6 mutation
are rapidly reversible.

In conclusion, the LEFH strongly supports but doesn't prove the following
two points: (1) A cell or cells outside the pharynx can profoundly inhibit
pharyngeal muscle excitation.  (2) eat-6 acts directly in pharyngeal