Worm Breeder's Gazette 10(2): 39

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.

Pharynx Update: Laser Controls, Serotonin, and Eat Mutants

Leon Avery

I reported previously (WBG 9(2): 57) that coordinated pharyngeal 
pumping continues even after the nuclei of all 20 pharyngeal neurons 
are destroyed in a young larva, although pumping is no longer normally 
regulated.  The obvious interpretation is that the pharyngeal muscles 
are capable of coordinated pumping in the absence of the nervous 
system, and that the function of the nervous system is to regulate 
pumping.  However another possibility was that the neurons continued 
to function to some extent after their nuclei had been destroyed (
Chalfie and Sulston, Dev Biol 82: 358; Avery and Horvitz, Cell 51: 
1071).  I have now done more controls that support the first 
interpretation.  These controls are of two kinds: embryonic kills, and 
electron microscopic verification of the results of larval kills.
Embryonic kills: I killed I4, I6, or MS at late-comma stage (450 min)
, or ancestors of I5, MI, both MCs, or both I2s at late-gastrulation 
stage (200 min).  The results were consistent with larval kill 
experiments.  It is therefore unlikely that any of these 7 classes of 
pharyngeal neurons is individually essential for pumping.
Electron microscopy:  There are two NSM neurons, each of which has a 
subventral process that runs the length of the isthmus.  I killed the 
right-hand NSM neuron in a young larva, leaving the left-hand NSM 
intact.  After letting the worm grow for two days, I gave it to Nichol 
Thomson, who prepared it for electron microscopy and cut a short 
series of sections from the region of the isthmus.  The left-hand (
control) NSM process was present in these sections, but the right-hand 
(killed) NSM process was not.  Thus the process of the killed NSM in 
this worm did not extend the entire length of the isthmus.  If this is 
true for all pharyngeal neurons, the nervous system must not be 
necessary for the coordination of pumping, since the corpus and 
terminal bulb muscles, which are separated by the isthmus, still 
contract in synchrony after the pharyngeal nervous system is killed.
Horvitz et al (Science 216: 1012) discovered that external 
application of the neurotransmitter serotonin stimulates feeding.  I 
have pushed this observation one step further by showing that there is 
a source of serotonin in the worm capable of stimulating pumping, and 
by finding a behavioral phenotype for mutants that lack serotonin.
Imipramine (0.02 mg/ml) stimulates pumping.  Imipramine works by 
preventing serotonin re-uptake, and therefore enhances the action of 
serotonin present in the worm, but should have no effect on worms that 
lack serotonin.  I therefore tested the effects of imipramine on cat-1(
e1111am) X, cat-2(e1112) II, and cat-4(e1141) V mutants (Sulston et al,
J Comp Neurol 163: 215).  cat-1 and cat-4 have reduced serotonin and 
dopamine levels, while cat-2 has no dopamine but approximately normal 
amounts of serotonin (above references and C.  Desai, G.  Garriga, S.  
McIntire, personal communication).  I found that imipramine stimulated 
pumping in wild-type and cat-2 worms, but had little if any effect on 
pumping in cat-1 and cat-4 worms.  Thus imipramine stimulation of 
pumping correlates with serotonin levels, as expected.  There are two 
kinds of serotonergic neurons whose connectivity makes them plausible 
candidates for regulating pumping: RIH (S.  McIntire, personal 
communication) and the NSMs.  It should be possible to find out 
whether these contain the relevant serotonin by testing imipramine 
stimulation in worms whose RIH or NSM neurons have been killed with 
the laser.
unc-31(e928) IV worms are lethargic, egg-laying defective, and 
pumping-constitutive.  Since all these behaviors are regulated by 
serotonin, I hoped that lack of serotonin might produce an obvious 
phenotype in an unc-31 background.  Therefore I constructed an unc-31; 
utant.  Compared to unc-31 the double mutant 
was slightly more active, pumped slightly more irregularly, and was 
severely defective in egg-laying.  Although the activity and pumping 
phenotypes are subtle and difficult to score, the egglaying defect is 
obvious, especially at 15 C, where unc-31 lays eggs almost normally.  
I have mapped this egg-laying defect to between dpy-11 and him-5 on 
chromosome V consistent with the dpy-11  to 
which John Sulston mapped cat-4.  I hope to be able to isolate new 
mutants that lack serotonin by looking for egg-laying-defective 
mutants in an unc-31 background, and then checking whether imipramine 
stimulates pumping.
Although pumping may go on in the absence of the pharyngeal nervous 
system, it becomes slow and irregular.  This slow, irregular pumping 
is especially obvious in an unc-31 background, because it contrasts 
strongly with the rapid, constant pumping of unc-31 worms.  I have 
therefore begun looking for mutations that cause slow, irregular 
pumping in an unc-31 background, in the hope of isolating mutants with 
defective pharyngeal nervous systems.  Although I haven't gotten very 
far yet, I have some preliminary results: 
1.  Such mutants can be isolated, and, more importantly, can be 
manipulated.  The phenotype (which I call Eat) is about as easy to see 
as Egl or Mec, i.e., ES2 on Jonathan Hodgkin's scale.  I have 11 so 
far, all picked up by screening for abnormal pumping in EMS-
mutagenized unc-31(e928).  One (e2335ts) came from a small F1 clonal 
screen, and the remaining 10 (ad418, ad426, ad427, e2337, e2338, e2340 
- e2344) from a screen of 2350 F2s.  All behave as simple Mendelian 
recessives, except for one (ad426) that shows a partial maternal 
rescue effect.
2.  My 11 mutations probably hit at least 10 different genes, so 
there are a lot of genes.
3.  Some eat mutations are pleiotropic.  At least 3/11 are Unc (
e2340-e2342), and 1 is dauer-constitutive (ad418).  e2340 is a new 
allele of unc-26 IV, the Unc phenotype of which is probably due to a 
nervous system defect (Yuan and Horvitz, WBG 9(1): 58).  Therefore I 
am picking up nervous system mutants.
4.  Some eat mutations are not pleiotropic; i.e., they have no 
obvious phenotype other than abnormal pumping.  These mutations 
probably identify new genes.
5.  At least one eat gene, eat(ad418) X, may be redundant with unc-
31.  That is, while unc-31 worms pump rapidly and ad418 worms pump 
normally, unc-31; ad418 worms pump slowly.  The dauer-constitutive 
phenotype of the unc-31; ad418 double mutant is also synthetic: 
neither unc-31 nor ad418 alone causes a dauer-constitutive phenotype.  
I don't know what this redundancy means, but one (testable) guess is 
that the genetic redundancy reflects nervous system redundancy; i.e., 
unc-31 affects some of a redundant set of neurons, and ad418 affects 
the rest, so only in the double mutant is the whole set affected.