Worm Breeder's Gazette 15(2): 54 (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.

Genetic interaction between dpy-20 and genes affecting development of motor neurons and hypodermal cells in the nematode C.elegans

Y. Shibata, M.A. Shah, M. Umehara, M. Y. Ali, S. S. Siddiqui

Lab. of Molecular Biology, Toyohashi University of Technology, Tempaku-cho,, Toyohashi-441, Japan

In this issue of WBG our colleagues have reported that the dpy-20 gene
expresses in the ventral cord neurons and hypodermal cells during 
larval development (M.Y. Ali,  Z. K. Siddiqui,  D. Janke, D. Baillie,
and S. S. Siddiqui). It has been suggested that the dpy-20 encoded gene
may function as a novel transcription regulator in motor neurons (See
Fukushige and Siddiqui, 1995, and the abstracts by Ali et al., in this
issue of WBG)  

To study the function of dpy-20 gene (Hosono et al.,1982; Clark et
al.,1995), dpy-20::lacZ in different mutants background were constructed
including those mutants that harbor defects in the ventral cord
development. For example, we have constructed dpy-20::lacZ in unc-5(e53)
background. The mutant unc-5(e53) animals  are severe coilers during
larval and adult stages (Brenner, 1973, 1974).  In unc-5 mutants the 
hypodermal cells are defective, and animals lack the dorsal nerve cord,
as seen at the ultrastructural level. The neuronal commissures(axonal
processes) fail to reach target from ventrally located neuronal cell
bodies to their synaptic targets in the dorsal region, such as the
dorsal cord(Siddiqui, 1990; Hedgecock et al., 1990; Leung-Hagesteijn et
al,1992, Hamelin et al.,1993). 

The unc-5::lacZ fusion gene is expressed in the motor neuron cell bodies
in the ventral cord, their axonal processes along the ventral cord,
dorsally directed commissures, and their synaptic targets in the dorsal
cord.  The unc-5::lacZ  fusion gene is also expressed in the lateral
cords, that run from anterior to the posterior along the entire body
length. The fusion gene expression is seen during all larval and adult
stages animals(Hamelin et al.,1993). The expresion of dpy-20::lacZ
reporter gene staining pattern does not change when the dpy-20::lacZ 
is expressed in the unc-5 mutant backgound, but  when the  unc-5::lacZ
reporter gene is expressed in the  dpy-20(e2017) mutant  background;
staining in the motor neurons,lateral cords, dorsal cord and the
dorsally directed commissures is reduced.

In the dpy-20 mutant background most of the animals do not show the
staining in the commissures in the L3,L4 and young adult stages animals.
But in the adult animals reduced staining is seen in the commissures
that run from ventral cord to dorsal cord.  These results suggest that
the dpy-20 gene may regulate the expression of unc-5, whereas the unc-5
gene may not affect the expression of the dpy-20 gene.  To test this, 
we have constructed a double mutant between unc-5(e53) and dpy-20(e2017,
which is severely paralyzed and has very low brood size, suggesting
strong genetic interaction between the dpy-20 and unc-5 genes. Whether
this intercation is at the level of hypodermal cells or motor neurons,
or the germ line is not known.

We have also expressed the dpy-20:: lacZ gene in the background of
unc-25(e156) (Thomas 1990;McIntire et al., 1993; Reiner and Thomas 1995
and unc-30(e191(McIntire et al., 1993; Jin et al 1994)).  Both of these
genes affect locomotion as they are expressed in the motor neurons.
Interestingly, we find that the dpy-20::lacZ reporter gene in the unc-25
and unc-30 mutant background ectopically expresses in a few hypodermal
cells in adult stage animals (in wild type background this expression is
never observed). The pattern of dpy-20 gene expression is unaffected
during larval development in mutant background.  A double mutant between
dpy-20(e2017) and unc-25(e156) is severely paralyzed and has a very low
brood size, producing sick animals of which 95% are sterile. unc-25 gene
expresses in all GABAergic neurons, including the VD 1-6, and DD 1-13,
class of motor neurons. unc-30 gene encodes a 318 aa transcription
factor like protein that contains homeodomain motif and show similarity 
to the unc-4 homeodomain protein(Jin et al.,1994).

Anti beta tubulin antibodies and unc-30 antibodies stain VD and DD
classes of motor neurons(Jin et al., 1994, Siddiqui,1990).  Both the
unc-25(e156) and unc-30(e191) mutants are small in size, shrink,
contract both dorsally and ventrally when touched, move slowly,
normal forward locomotion but have difficulty in backing up. We also
tested the dpy-20::lacZ reporter gene in the unc-4(e120) (White et al
1992; Miller et al 1992), unc-13(e51) (Brenner 1974; Ahmed et al 1992;
Nguyen et al. 1995), unc-86(e1416) (Chalfie et al. 1981; Finney et
al.1988; Baumeister et al. 1996), and unc-104(rh1016*10)(Otsuka et al.,
1991) mutant backgrounds and found no significant effect on the dpy-20
expression pattern. Double mutants dpy-20(e2017);unc-13(e47); 
dpy20(e2017);unc-86(e1416); and dpy20(e2017); unc-104(rh1016*10) are
severely paralyzed and have very low brood size. Most of the animals are
sterile and some animals end up as a bag of worms. Since the dpy-20 gene
also expresses in hypodermal cells, we are examining the dpy-20 gene
expression in various mutants affected in hypodrmal development,
including those encoding molecules that are known to play a role in
signal transduction pathway. 

We thank T. Stiernagle, M. Chalfie, M. Hamelin, J. Culotti, A. Otsuka,
Y.Kohara, for help in this project