Worm Breeder's Gazette 12(2): 89 (January 1, 1992)

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.

Reach out and touch someone

Judith Austin, Cynthia Kenyon

Figure 1

UCSF, San Francisco, CA 94143

We are interested in understanding the cell interactions required for formation of the postdeirid. Previous work (Sulston and White, Dev. Biol. 56:110; Fixsen, Ph.D Thesis; Waring, Wrischnik and Kenyon, in preparation) has shown that formation of the postdeirid during L2 by descendents of V5 requires the presence of neighboring V cells. If V6 is ablated or if V2 , V3 and V4 are ablated, V5 gives rise to an extra seam cell instead of the postdeirid neuroblast. These results suggest that V5 (or its descendents) requires contact with cells both to the anterior and posterior in order to form a postdeirid. Who takes part in this interaction, V5 , V5 .por the postdeirid neuroblast, V5 .pa?Is the requirement for cell interaction continuous or is there a critical period during which the decision to make a postdeirid can be influenced? To address these questions we have begun to look at the connections between the cells of the lateral epidermis and how these connections are influenced by cell ablation.

We began by ablating V6 and V6 .pat different points during L1 . V6 normally divides between 4 and 5 hours after hatching at 20°C. When V6 was ablated at 1, 3, or 4 hours after hatching, no postdeirid was made (0/15 animals). Likewise, when V6 .pwas ablated at 6 or 7 hours after hatching, no postdeirid was made (0/15 animals). In contrast, when V6 .pwas ablated at 8 hours after hatching a postdeirid was made in a majority of the animals (8/11 made a postdeirid; 1/11 made a hybrid postdeirid). Similar results were seen after the triple ablation of V2 .p, V3 .pand V4 .p.

What is happening to V5 .pbetween 7 and 8 hours after hatching? We used the antibody MH27 (kindly supplied by Bob Barstead) to visualize the V cell boundaries. At hatching, the undivided V cells form a continuous connected line of cells. After the V cells divide, the Vn.a cells fuse with hyp7 ,which leaves the Vn.p cells unconnected to each other. Subsequently, the Vn.p cells extend processes both anteriorly and posteriorly along the lateral midline and reconnect with their lateral neighbors by approximately 8 hours after hatching. A comparison of the MH27 labeling patterns with the ablation data for the same points in development suggests the hypothesis that the reconnection of V5 .pto its neighbors (normally V4 .pand V6 .p)is crucial for the decision to make a postdeirid. After these cell contacts are formed, further cell interaction is not required and the ablation of V6 .phas no effect If V5 .pcannot contact its neighbors in a timely fashion (due to the removal of V cells by ablation), no postdeirid is formed.

To test this hypothesis, it will be crucial to determine whether V5 .pcan reconnect to cells other than V4 .pand V6 .pafter these cells are ablated. We have recently come up with a simple procedure that makes the outlines of the lateral hypodermal cells visible and should allow us to answer this question. Animals are incubated in a solution of 0.25% SDS at room temperature for 3 minutes (the timing is critical; the trick is to catch them just as they stop moving), placed on an agar pad and viewed by Nomarski (sometimes the cell separation will not develop until the worms have been on the slide for a while). When this technique works, the V cells can be clearly distinguished from the surrounding hypodermal cells and have a slightly puffed up appearance. The pattern observed at different times during L1 using this technique is similar to that seen with MH27 .

Our hypothesis predicts that after ablation of V6 , V5 .pwill either not connect to the next posterior cell, T.ap, or connect to it later than it would normally connect to V6 .p.When animals in which V6 had been ablated were treated with SDS ~8 hours after hatching, we saw that V5 .pwas connected to V4 .pbut not to T.ap (4/4 animals). Similarly, when animals m which V2 , V3 and V4 were ablated were treated with SDS ~9 hours after hatching, we saw that V5 .pwas connected to V6 .pbut not to V1 .p(5/5 animals).

Together these results support the hypothesis that the parent of the postdeirid neuroblast, V5 .p,must touch its neighbors by a critical time in order to generate a postdeirid rather than an extra seam cell. Following ablation of V6 or V2 -V4, V5 .pdoes not make this contact at the normal time and therefore does not generate a postdeirid. We will be interested to see when V5 .pconnects to V3 .pafter ablation of V4 .When V4 alone is ablated, V5 almost always makes a postdeirid (80-90% of the time). We would predict therefore, that after ablation of V4 we would see a connection between V5 .pand V3 .p,close to the time that V5 .pwould normally connect to V4 .p.

Diagram of V cell behavior during L1 (MH27 staining pattern) time after hatching (20°C)

[See Figure 1]

Figure 1