Worm Breeder's Gazette 14(4): 48 (October 1, 1996)

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.

daf-2 Controls Several Aspects of Dauer Formation Non-Autonomously

Javier Apfeld, and Cynthia Kenyon

Department of Biochemistry, University of California San Francisco

The daf-2 gene acts as a developmental switch. High daf-2 activity leads to normal development, whereas low daf-2 activity leads to the formation of an alternative larval stage, the dauer larva. Multiple signals such as food availability, dauer pheromone concentration, and temperature can induce dauer formation (1). Sensory neurons and a genetic patway have been demonstrated to control dauer formation (2-4). daf-2 functions at a downstream position in this genetic pathway. Dauers have many specialized cells and tissues, including the hypodermis, the pharynx, and the intestine. Is daf-2 function required in each of these tissues to promote their normal development? Or does absence or presence of daf-2 function in some cells control the development of other cells? We are particularly interested in these questions since daf-2 has been implicated in lifespan regulation (5). To answer these questions we are analyzing daf! -2 genetic mosaic worms. We find that the daf-2 state (wild type or mutant) of some cells can control the dauer phenotype of other cells.

Genetic mosaics are isolated from daf-2(e1370) dpy-17(e164) ncl-1(e1865) unc-36(e251)III; sDp3. Animals carrying sDp3 form non-dauers that are non-Dpy, non-Ncl, and non-Unc, while worms lacking sDp3 form dauers that are Dpy, Ncl, and Unc. We find that mosaic worms form both dauers and non-dauers. We distinguish full dauers from non-dauers by the following strict criteria:
* full dauers are thinner than comparable L3 larvae,
* they have specialized cuticular alae which are absent in L3 larvae,
* they have a structurally remodeled pharynx that does not pump, is slimmer and has a more dorsal posterior bulb than that of L3 larvae,
* they have highly refractile intestinal cells instead of the light intestinal cells of L3 larvae,
* they are resistant to 1% SDS.

Some mosaics have a partial dauer phenotype: they express some, but not all, features of a full dauer. The partial dauer mosaics stay in this state only transiently. All partial dauer mosaics are SDS resistant and have continuous dauer alae which sometimes is less refractile than that of a full dauer. Other aspects of the full dauer phenotype are variable or incomplete in the partial dauer mosaics: some partial dauers mosaics have light intestinal coloration, pharyngeal pumping, or an incompletely remodeled dauer pharynx.

We find three classes of mosaics: mosaics that form full dauers, mosaics that form non-dauers, and mosaics that form partial dauers (Table 1). These three classes are described in more detail below:

I. Mosaics that form full dauers: daf-2(+) cells have a dauer phenotype
* The dauer alae is made by a group of hypodermal seam cells called V cells that are present on both sides of the worm. V1, V2, V4 and V6 are ABa-derived, while V3 and V5 are ABp-derived. Some ABa mosaics (worms in which the ABa descendants lost the duplication) form full dauers. In these animals, the V3 and V5 cells form dauer alae even though these cells are daf-2(+). Therefore, daf-2(+) function within V3 and V5 is not sufficient to promote non-dauer development of these cells; instead, the daf-2(-) state of the ABa descendants controls the phenotype of V3 and V5.
* All AB and ABa mosaics that form full dauers have dark refractile intestines even though their intestines are daf-2(+). Therefore, the daf-2(-) state of AB derived cells can control the dauer phenotype of the intestine.

II. Mosaics that form non-dauers: daf-2(-) cells do not have a dauer phenotype
* Full dauers arrest their gonadal development before the ovotestis turns 180 degrees to give rise to the gonad arm bend. The somatic gonad is derived from the MS descendants. MS mosaics form adult worms with a fully developed somatic gonad even though this tissue lacks daf-2 function. Therefore, daf-2 function is not necessary within the somatic gonad for its normal morphogenesis.

III. Mosaics that formed partial dauers
* We find that daf-2 function is necessary within the ABplaa-derived descendants to prevent partial dauer formation.

Our results show that the dauer fate of a number of tissues can be controlled by the state of the daf-2 gene in other tissues. daf-2 function is necessary within the AB-descendants to prevent dauer formation. Because some ABa mosaics do not form dauers, we postulate that daf-2 function within the ABa-descendants may be partially redundant, with a second focus of action within the ABp-descendants. We are continuing the mosaic analysis in order to define more accurately where daf-2 function is required to control dauer formation, and to to investigate where daf-2 function is required to regulate lifespan.

Table 1: Mosaic Analysis of the Constitutive Dauer Formation Phenotype of daf-2
Number of Mosaics Loss Full Dauer Non Dauer Partial Dauer AB 50 0 0 ABa 2 2 0 ABp 0 18 1 MS 0 5 0 ABpl 0 1 0 ABpr 0 1 0 ABplaa 0 0 2 ABar + ABp 0 0 1 ABar + ABpl 0 0 1 ABp + ASJL 0 0 1 ABplaa + MS 0 0 1

We isolated mosaics in multiple ways: we selected for full or partial dauers with 1% SDS, we used the dissecting microscope to identify worms that did not look Dauer, Dpy, Unc or did not look wild type, or we identified ncl-1 mosaic worms directly. We determine the point in the lineage at which the duplication is lost by scoring the Ncl phenotype of diagnostic cells--ncl-1 is a cell-autonomous mutation that causes many cells in the worm to have enlarged nucleoli.

(1) Golden J, and Riddle DL (1984) Dev. Biol. 102:368-378
(2) Bargmann C, and Horvitz HR (1991) Science 251:1243-1246
(3) Gottlieb S, and Ruvkun G (1994) Genetics 137:107-120
(4) Larsen PL, Albert PS, and Riddle DL (1995) Genetics 139:1537-1583
(5) Kenyon C et. al. (1992) Nature 366:461-464