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.