Worm Breeder's Gazette 13(1): 48 (October 1, 1993)

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.

The worm without a face: an update on vab-3

Andrew Chisholm, Bob Horvitz

HHMI, Dept. of Biology, MIT, Cambridge, MA 02139

Mutations in vab-3 cause the morphogenesis of the worm's head to be variably disrupted, resulting in the well-known notched head phenotype. vab-3 mutations also cause a number of other intriguing phenotypes(1). The H and G cells in the head execute abnormal postembryonic lineages and in some cases appear to adopt the fates of more posterior homologs(2). The distal tip cells of vab-3 hermaphrodite gonads migrate abnormally, often making supernumerary reflexes. vab-3 males lack spicules as a result of abnormal B cell lineages in the male tail (H. Chamberlin and P. Sternberg, personal communication). Nine viable alleles exist, forming an apparent allelic series from the weakest, c1796 (which does not cause notched heads or the spicule-less phenotype), to the strongest, e1178 .All alleles except e1796 cause embryonic and larval lethality to varying degrees.

Lethal alleles.

We isolated new alleles of vab-3 in a noncomplementation screen. Animals of genotype e1796 / nDf19 are viable and Mig, and have a variable weak Notch phenotype. We therefore screened for mutations that failed to complement e1796 for Mig. From 30,000 mutagenized chromosomes screened, we isolated five such noncomplementing mutations ( n2362 through n2366 ).All five are fully penetrant embryonic to early larval lethals. The phenotype of the arrested embryos is highly variable, ranging from unelongated embryos with incomplete hypodermal closure to blobby or malformed L1 s.Thus vab-3 has essential functions in embryogenesis, as previously suspected. The embryonic lethal alleles may be nulls; however, we cannot exclude the possibility that the true null phenotype is maternal-effect lethality.

Rays in the head: the lin-22 ; vab-3 -3double.

In vab-3 mutants the H cells appear to be partly transformed to behave like posterior hypodermal neighbours. In lin-22 mutants the cells V1 -V4adopt a 'V5-like' fate, producing two rays in males(3). The H cells appear to be slightly abnormal in lin-22 males, in that they often do not make alae, but they do not seem to be completely transformed to the 'V5-like' fate as they do not make rays. Does vab-3 (+)prevent a more complete posterior transformation of the H cells in a lin-22 background? We made the strain lin-22 ( n372 ) him-8 ( e1489 ); vab-3 ( e1796 )and found that such males often make rays in the head. Defining the head as the region anterior to the level of the pharyngeo-intestinal valve, lin-22 him-8 males made no ectopic rays in this region (we scored late L4 or young adult males raised at 20°) whereas lin-22 him-8 ; vab-3 ( e1796 )males made up to 5 ectopic ray papillae in the head (average = 2.6, n = 10). The extent of alae formation was reduced from 10% of wild type in n372 e1489 worms to 1.1% of wild type in n372 e1489 ; e1796 .By position the ectopic rays probably derive from H1 or H2 ,but we need to lineage the males to be sure. We have not checked stronger vab-3 alleles to see if they cause a similar enhancement. We also examined double mutants between the mab-5 gain of function allele e1751 and lin-22 .In e1751 ; n372 e1489 males alae are completely absent, but we did not see ectopic rays in the head, suggesting that vab-3 (+)is still capable of preventing a posterior transformation of the H cells in this background (we are making the triple mutant to test this idea). These results support the notion that vab-3 directs the head-specific fate of the H cells, and in its absence they become partly transformed to more posterior fates.

Cloning vab-3 .

To understand vab-3 further we are cloning it. We took advantage of the proximity of vab-3 to the previously cloned gene mab-18 (4).These two genes had not been separated by recombination in several mapping experiments, placing them within 0.015 map units of one another. In addition, the weak vab-3 allele e1796 causes a Mab-18 phenotype, although stronger vab-3 alleles do not, suggesting that e1796 could be a double mutant or small rearrangement affecting two adjacent genes. We therefore injected vab-3 ( e648 )mutants with cosmids from the mab-18 region and found that C56D7 (which overlaps the mab-18 (+)cosmid) rescued. The male spicule-less phenotype is the easiest to score for rescue and appears to be fully rescued; other phenotypes also appear rescued in the rescuing lines, but it is difficult to be categorical. We have identified a 25 kb subclone of C56D7 that also rescues.

1. Chisholm, WBG 11, 4: p. 83.

2. Hedgecock, TINS 8: 288.

3. Fixsen, Ph.D. thesis, 1985, MIT.

4. Zhang and Emmons, '93 worm meeting abstracts p. 493.