Worm Breeder's Gazette 12(3): 103 (June 15, 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.

pha-4 :A New Gene Required for Pharyngeal Development

Susan E. Mango, Eric J. Lambie, Judith Kimble

Laboratory of Molecular Biology, 1525 Linden Dr., Univ. of Wisconsin, Madison, WI, 53706

We have isolated four alleles of a zygote lethal that we call pha-4 .Animals homozygous for pha-4 die either during the first larval stage (L1 ) or during embryogenesis. By Nomarski DIC microscopy, pha-4 L1 shave two striking defects: there is no visible pharynx (Pha-) and the intestine is not connected to the rectum. Other tissues appear normal. Homozygous pha-4 embryos arrest at the beginning of morphogenesis, and are also Pha-. We have mapped pha-4 to the right end of V, next to rol-9 . pha-4 complements three genes in this region ( rol-9 , sel-6 and zen-3 )suggesting that it is a novel gene.

Three criteria suggest that the pharyngeal and elongation defects are due to a reduction in gene function. The mutant alleles are obtainable at loss-of-function frequency (3 x10 -4with 50mM -MS). pha-4 ( q400 )homozygotes look like pha-4 ( q400 )/ ozDf2 hemizygotes with respect to the ,percentage of animals that hatch and their terminal L1 phenotype. Moreover, arrested ozDf2 embryos do not elongate and are Pha-.

Using immunocytochemistry, we have examined the Pha- larval phenotype in greater detail. The wild-type C.elegans pharynx is made up of five cell types (muscles, neurons, glands, epithelial cells and structural cells called marginal cells) that arise from ABa and EMS (1, 2). Strong loss-of-function alleles of pha-4 appear to be missing at least three pharyngeal cell types that derive from both of these progenitors: muscles (e.g. 9.2.1 (3)), glands (e.9.1CB4 (4)) and, probably, marginal cells (MH4 (5)). The pharyngeal-intestinal valve, which is both lineally and physically close to the pharynx, is also missing (e.g. MH27 (6)). Moreover, unlike many other lethal mutations, an antibody that recognizes an epitope found in early pharyngeal muscle precursors (3NB12 (7)), does not stain any pharyngeal cells in pha-4 homozygotes. Although we do not yet know what happens to the pharyngeal cells, antibody stains and Nomarski microscopy suggest that they are unlikely to follow a hypodermal fate (as occurs in skn-1 embryos (8)).

Antibody stains for several non-pharyngeal cell types appear normal, indicating that these cells are neither missing, nor duplicated. For example, we have identified the IL2 interneurons, the amphid neurons, the excretory cell, pore and duct, hypodermal cells, the intestinal and anal depressor muscles. pha-4 L1 s move well, and appear to have the full complement of muscles, suggesting that the muscle cells that derive from EMS are present. Thus, the pha-4 pharyngeal defect appears to be specific for an organ (the pharynx), rather than for a lineage (for example, EMS) or for the anterior.

We would like to thank Andrew Chisolm, who very kindly sent us a pha-4 allele he had isolated ( n2498 ).Many thanks also to everyone who sent us antibodies and strains.

Literature Cited:

Albertson and Thomson, Phil. Trans. R. Soc. Lon. B. 275:299-324 (1976)

Sulston et a/., Developmental Biology 100:64-119 (1983)

Miller et al/., Cell 34:477-490 (1983)

Okamoto and Thomson, J. Neurosci. 5:643-653 (1985)

Francis and Waterston, J. Cell Biol. 114:465-479 (1991)

Francis and Waterston, unpublished; Priess and Hirsh, Dev. Biol. 117:156-173 (1986)

Priess and Thomson, Cell 48:241-250 (1987)

Bowerman et al, Cell 68:1-20 (1992)