Worm Breeder's Gazette 10(3): 147

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.

Development of the Egg-Laying Circuit

C. Li and Marty Chalfie

The VC motoneurons branch from the ventral nerve cord to innervate 
the vulval muscles with a complex tuft of processes or arborization.  
We have been examining the assembly of this egg-laying circuit.  We 
have previously reported that the formation of this arborization (as 
assayed immunocytochemically with an anti-FMRFamide antibody) is not 
dependent on neuron-target muscle or neuron-neuron (HSN) interactions, 
but is dependent on the presence of vulval cells.  Vulval cells, 
irrespective of their position or their lineage, are capable of 
inducing VC branching into the vulval region.  Only vulval cells 
derived from a primary lineage, however, are necessary to induce the 
branching and form the arborization.  The presence of vulval cells 
alone, however, is not sufficient for the formation of the 
We have found that the somatic gonad also plays a role in the 
formation of the arborization.  Because the anchor cell normally 
induces the vulva (1), gonadal ablations were performed in lin-15(n309)
Multivulva mutants in which vulval lineages can be expressed 
independent of the gonad (2).  Ablation of Z1 and Z4, the somatic 
gonad precursors, in lin-15 results in animals in which VC cell 
branches are present at the vulva and in pseudo vulvae, but the 
arborization is absent (n=12).  By contrast, ablation of Z2 and Z3, 
the germline precursors, has no effect on the arborization (n=4).  
Induction of VC branching, therefore, can occur independently of the 
gonad, but the presence of the gonad is required for the formation of 
the arborization.
We have begun to examine the question of how the vulval muscles are 
organized into their final positions around the vulva.  Previous work 
suggested that the final position of the sex myoblasts, the vulval 
muscle precursors, is regulated by the somatic gonad (3, P.  Sternberg,
pers.  comm.).  We examined whether the vulval muscles were under the 
same instructive cues as the VC neurons.  Vulval muscles were 
visualized immunocytochemically with an antimyosin antibody (kind gift 
from D.  Miller), and were scored for their position around the vulva 
and/or between VC4 and VC5 and for their orientation.
Our results suggest that the position of the sex myoblasts largely 
determines the position of the vulval muscles.  The final position of 
the muscles, however, is dependent on gonadal and vulval cues.  When 
the somatic gonad is ablated in wild-type animals, the sex myoblasts 
migrate anteriorly to a variable extent (3) but usually to between P6.
pp (about VC4) and P7.pp (about VC5) (M.  Stern and H.R.  Horvitz, 
pers.  comm.).  In similarly ablated animals (n=9), the muscles are 
observed scattered throughout the length of the animal, as far 
anteriorly as VC2 and as far posteriorly as halfway between VC6 and 
the anus.  In one animal muscles were detected dorsally.  These 
muscles do not appear to be body wall muscle and look like vulval 
muscles, and will be referred to as vulval-like muscles.  In Vulvaless 
mutants, the sex myoblasts migrate to their normal position (P.  
Sternberg, pers.  comm).  In these animals vulval-like muscles develop 
in the correct position between VC4 and VC5, but are randomly oriented.
The muscles, therefore, can appear in their correct position with 
cues from the gonad and without input from the vulval cells.   Whether 
vulval muscle orientation is lost due to the loss of the vulval 
attachment point or whether vulval cues are also important for 
orientation can not be distinguished from these experiments.
The vulval cells, however, can also play a role in the positioning 
of the vulval muscles.  Ablation of the somatic gonad precursors in 
lin-15(n309) Multivulva animals results in a protruding vulva.  In 
such ablated animals (n=14) vulval-like muscles in their correct 
orientation are detected at the protruding vulva and at 2-3/4 pseudo 
vulvae.  No vulval-like muscles are detected except at the pseudo 
vulvae.  The vulval-like muscles are derived from the M lineage.  
Ablation of Z1, Z4, and M in lin-15 animals (n=2) results in the 
absence of any vulval or vulval-like muscles.  lin-15 animals in which 
no ablations have been performed or in which the germline precursors 
were ablated (n=3) have correctly positioned vulval muscles and no 
ectopic vulval-like muscles.  These results suggest that in the 
absence of gonadal cues, the vulval cells are capable of directing the 
position and orientation of the vulval muscles.
Nematode muscle differs from muscle in other animals since it sends 
muscle arms to nerve cells rather than nerve processes seeking the 
muscle.  The mispositioning of the muscles in these experiments could 
be caused by muscles projecting to ectopic VC branches in the pseudo 
vulvae.  We think this possibility unlikely, however, because vulval-
like muscles can be present at pseudo vulvae in the absence of any 
ectopic VC branches, and, conversely, ectopic VC branches are seen 
projecting into pseudo vulvae with no vulval-like muscles.  These 
results are consistent with the hypothesis that the VC cells do not 
provide cues to the vulval muscles.
We propose that the components of this neural circuit, the VC cells 
and the vulval muscles, are organized by the somatic gonad and the 
vulva.  Preliminary experiments suggest that the HSNs may be under 
similar control.