Worm Breeder's Gazette 14(1): 46 (October 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.
|1||Department of Biochemistry, UT-Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75235-9038|
|2||HHMI, Department of Biology, MIT, 77 Massachusetts Ave., Cambridge, MA 02139|
Loss-of-function mutations in eat-4 (aka. not-1) lead to abnormal behaviors like defective feeding (Eat; Avery, Genetics 133: 897), insensitivity to light touch on the nose (Not), abnormal foraging (Fab) (Kaplan, et al., WBG 12.3: 105, J Kaplan, pers. comm.), defective chemotaxis (Che; C Bargmann, pers. comm.), and defective thermotaxis (Ttx; Mori, et al., WBG 12.5:73). At least two of these phenotypes (Eat and Not) may be attributed to loss of glutamatergic neurotransmission. Therefore, it was proposed that eat-4 may be necessary for all glutamatergic neurotransmission (Avery, et al., WBG 13.4:72). eat-4 has been cloned. It shares 46% amino acid sequence identity with a neural-specific Na-Pi cotransporter found in rat brain (Lee and Avery, WBG 13.5:36). To learn whether eat-4 is expressed in the neurons that it affects and if there are other cells where it might have a function, we have started characterizing eat-4::GFP and eat-4::lacZ translational fusion expression. eat-4::lacZ and eat-4::GFP are expressed in more than 20 extrapharyngeal neurons. In addition, we could detect lacZ expression in the pharyngeal M3 and NSM neurons. (We were unable to see GFP expression in the pharynx, probably because the expression level is too low.) The expression in M3s is consistent with our hypothesis that eat-4 is necessary for M3 synaptic transmission. NSM, however, is known to be serotonergic. We don't yet know the significance of NSM expression. The extrapharyngeal eat-4::GFP expressing neurons include the mechanosensory touch neurons ALM, PLM, and AVM. The fact that eat-4 is expressed in the mechanosensory touch neurons suggests that eat-4 might be required for touch cell function. However, eat-4 animals are sensitive to gentle touch to the body, indicating that the touch cell circuits in eat-4 animals are able to mediate the touch response. In adults the anterior touch response is mediated by two neural pathways that function redundantly (Chalfie et al., J. Neurosci 5: 956). One pathway depends upon the gap junctions between ALM and AVD. The second pathway depends upon the outputs of AVM which makes chemical synapses to AVB, BDU, PVC and gap junctions to AVD (White et al., Philos. Trans. R. Soc. Lond. B314:1). Chalfie et al. reported that adults lacking either the AVD or the AVM neurons remained touch sensitive but that animals lacking both classes of neurons were touch insensitive. This result suggests that either the gap junction pathway from ALM to AVD or the chemical pathway from AVM can mediate anterior touch sensitivity. This feature of the circuitry allowed us to test the model that eat-4 was defective in chemical transmission from AVM. We created animals in which anterior touch senstivity was mediated only by the AVM-dependent pathway by killing AVD with a laser microbeam. To confirm that we were successfully killing the appropriate neurons, we first performed an experiment similar to that of Chalfie et al., killing AVD, AVM or AVD and AVM in L2 animals and measuring the touch response 2 days later by scoring the animals' response to anterior touch. As shown below, we found that ablation of AVD or AVM caused a decrease in the percentage of trials in which the animal backed in response to touch. However, animals lacking both AVM and AVD were more defective than animals lacking only one class of neuron. genotype/cells killed # animals #trials % of touches resulting in backing (1) N2/none 23 157 96 (2) N2/AVD 10 66 62 (3) N2/AVM 8 55 76 (4) N2/AVD+AVM 7 47 23 We used the same approach to test the function of AVM in eat-4 animals. As shown below, mock ablated eat-4 animals were slightly less sensitive to touch than N2, and eat-4 animals lacking AVD responded very poorly. genotype/cells killed # animals #trials % of touches resulting in backing (5) eat-4(n2474)/none 8 42 86 (6) eat-4(ky5)/none 13 100 86 (7) eat-4(n2474)/AVD 8 42 19 (8) eat-4(ky5)/AVD 11 84 24 These results can be summarized as follows: intact eat-4 mutants have anterior touch sensitivity equivalent to AVM- wild type (compare 5&6 to 3), and AVD- eat-4 have anterior touch sensitivity equivalent to AVM-AVD- (compare 7&8 to 4) wild type. That is, an eat-4 mutation has the same effect as eliminating the chemical synapse-dependent touch response pathway, consistent with the hypothesis that eat-4 is necessary for chemical transmission from AVM. We further tested the effect of an avr-15 mutation on the AVM pathway since avr-15, like eat-4, is required for normal M3 function (Avery, et al., WBG 13.4:72). As shown below, we found that avr-15(ad1051) shows AVM defects similar to those seen in eat-4, but slightly weaker. genotype/cells killed # animals #trials % of touches resulting in backing (9) avr-15(ad1051)/none 12 89 98 (10) avr-15(ad1051)/AVD 12 87 31 Although eat-4 appears to express in both ALM and AVM, the laser ablation experiments argue that eat-4 specifically affects the chemical transmission. Since avr-15 has been implicated in the glutamatergic transmission in the pharynx and since avr-15 is required for AVM synaptic transmission, it seems likely that AVM is also glutamatergic. Taken together, our results suggest that eat-4 may indeed be necessary and possibly specific for glutamatergic neuron function. We thank Marty Chalfie and Andy Fire for providing GFP and lacZ vectors.