Worm Breeder's Gazette 15(2): 51 (February 1, 1998)
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||Lab. of Molecular Biology, Toyohashi University of Technology, Tempaku-cho, Toyohashi 441, Japan.|
|2||Institute of Molecular Biology & Biochemistry, Department of Biological Sciences, Simon Fraser University, Burnaby,British, Columbia, Canada V5A 1S6|
Previously the dpy-20 gene has been reported to encode a non collagen novel protein(Clark et al, 1995). We have suggested that the dpy-20 may encode a novel transcription factor that may regulate transcription of a variety of genes in a set of ventral cord motor neurons (See the accompanying abstract by M. Y. Ali, O. S. Siddiqui and S. S. Siddiqui in this issue of WBG. To study the temporal and spatial expression of the dpy-20 gene, a dpy-20::lacZ fusion gene was constructed. A 2351 bp XbaI-BcnI dpy-20 gene fragment was inserted into a lacZ expression vector pPD95.57(A. Fire, 1995)which includes nuclear localization signal. The inserted fragment which contains 995 bp promoter and 1356 bp coding region, was obtained from plasmid pMN86, encoding dpy-20 gene. Four independent germlines were obtained by microinjection and all of them showed identical pattern of lacZ staining. We have found that the dpy-20 fusion gene is expressed in the ventral cord and hypodermal cells. The expression is restricted to the late L1, L2, L3 and L4 developmental stages animals. There is no lacZ staining found in the embryos and adult animals. This result is consistent with the temperature shift experiments on the dpy-20(e1282)ts allele and Northern blot analysis results (Clark et al., 1995). Expression in the ventral cord neurons is quite remarkable as it shows that the dpy-20 gene is expressed both in the hypodermal cells (as expected since the mutants are defective in hypodermis) and also in motor neurons (that was unexpected as the dpy-20 mutants are only partially defective in locomotion, and not immediately apparent as uncoordinated). We tested the locomotion of dpy-20 alleles and found that they are defective in locomotion which is consistent with our lacZ staining result. We find that there are only 5-7 ventral cord neurons that are staining in the L1 and L2 stage animals whereas 11-13 neurons are staining in L3 and L4 animals. We have identified that these neurons could belong to several subsets, perhaps at least two different classes of motor neurons. Tentatively these neurons are members of the VA, VB, VD and DD classes. We have also found that the neurons which are staining in the L1 stages, do not stain in the L3 and L4 stages. In the L3, L4 stages, some new neurons are stained. There is also an increase in the hypodrmal cell staining during larval development. We have studied expression of the reporter gene dpy-20::lacZ in different mutant background. e.g. in the dpy-20(cn142) background, and found that there is no change in the staining which suggests that in this allele dpy-20 gene transcription is normal, and the mutant may be defective in translation or some other process. We have also tested the expression of alpha-3 tubulin gene(T. Fukushige, Z. K. Siddiqui, C. Gogonea, J. Culotti, S. S. Siddiqui and M. Hamelin, Unpublished), in the dpy-20(e2017) mutant background and found that the staining in ventral cord neurons is reduced significantly. Staining in the touch neurons and in embryos remains unaltered which suggests that the dpy-20 controls the expression of alpha-3 tubulin gene primarily in the motor neurons. It will be worthwhile to examine the expression of dpy-20 in live animals to see the changing pattern of motor neuron staining during larval development. Therefore we are in the processes of constructing dpy-20::GFP to identify the cells, their developement and dpy-20::lacZ staining in different mutants background. We thank T. Fukushige, R.Hosono, K. Harada, Y. Kohara, J. Miwa, K. Nishiwaki, A. Fire, A.Coulson, and T. Stiernagle for their valuable help and cooperation in this project.