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.
Laser controls 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. Serotonin 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. Eat mutants 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.