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.

The dpy-20 gene expresses in the hypodermal cells and ventral cord MOTOR NEURONS.

M. Y. Ali1, Z. K. Siddiqui1, Diana Janke2, David Baillie2, Shahid S. Siddiqui1

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.