Worm Breeder's Gazette 14(2): 36 (February 1, 1996)
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.
Division of Biological Sciences, University of Missouri-Columbia, MO 65211 Mutations in daf-7 result in constitutive formation of dauer larvae. The daf-7 gene encodes a secreted protein that is a novel member of the TGF- beta superfamily (Chang-Su Lim, Ph.D. Thesis, 1994). Laser ablation experiments have determined that amphid sensory neurons ADF, ASG, ASI and ASJ function in the regulation of dauer formation (Bargmann et al., Science 251,1243). Our hypothesis is that daf-7 might be expressed in the amphid neurons to promote non-dauer development in response to food stimuli. Secreted DAF-7 ligand may then bind the DAF-1 and/or DAF-4 receptors which are transmembrane receptor serine/threonine kinases in the TGF-beta receptor family (Georgi et al., Cell 61, 635; Estevez et al.,
Nature 365, 644). Our expression data indicated that daf-1 and daf-4 are expressed in interneurons and in amphid neurons (Georgi and Estevez, unpublished). To study the expression pattern of daf-7, we have generated transgenic worms carrying a gfp reporter gene expressed under the control of the 3 kb daf-7 promoter region from the rescuing genomic DNA fragment (daf- 7p::gfp). In an N2 genetic background, we found that GFP was expressed in one pair of amphid neurons, ASJ. GFP expression can be detected in the four larval stages and adults, but not in eggs or in dauer larvae formed in pheromone or by starvation. During dauer recovery, GFP expression was detected after the dauer larvae were placed in food. Dauer-inducing pheromone suppressed the GFP expression. These data suggested that daf- 7 expression can be regulated by food and pheromone stimuli. The dominant allele of mec-4 , which causes swelling and death of the touch receptor neurons, has been used to define the function of other neurons (Maricq et al., Nature, 378,78). We placed mec-4(d), encoding degenerin, under the control of the daf-7 promoter [daf-7p::mec-4(d)]. This construct, coinjected with rol-6, transformed N2 into a temperature- sensitive(ts) dauer-constitutive (Daf-c) mutant. In synchronous populations, 1%, 5% and 28% of the rollers formed dauers at 15 C, 20 C and 25 C, respectively. However, these dauer larvae recovered spontaneously at 25 C. Since the laser ablation results indicated that ASJ is required for efficient dauer recovery, we again transformed N2 into a ts Daf-c mutant, this time carrying both daf-7p::gfp and daf-7p::mec-4(d). We found GFP expression in dauer larvae, indicating there is sufficient function to allow dauer larvae to recover. The MEC-4 activity may result in sufficient loss of DAF-7 secretion from ASJ to cause entry into the dauer state at 25 C; but in the absence of high pheromone the gfp gene was still expressed. Since in the transgenic animals the expression of mec-4(d) and native daf-7 are driven by daf-7p simultaneously, some animals could have made enough DAF-7 to promote development to the adult before ASJ was sufficiently damaged. This, plus some somatic loss of the transgene, may account for the low percentage (28%) of dauer formation by transgenic animals at 25 C. The daf-7 mutants are all ts, but they form 100% dauers at 25 C. We conclude that ASJ is involved both in preventing entry into the dauer stage at higher growth temperatures and in exiting from the dauer stage in response to food stimuli. Dr. John White helped with identifying neurons, Dr. Monica Driscoll provided mec-4(d) , Dr. Marty Chalfie provided GFP vector and Dr. Pam Hoppe helped with microinjection.