Ehrenfels CW et al. (1988) Worm Breeder's Gazette "looking for male-specific proteins."

Link CD, Wood WB, Ehrenfels CW

[Worm Breeder's Gazette, 1988]

We have begun an immunological approach to identifying C. elegans male-specific proteins. We immunized a rabbit with sonicated L4 and adult males, obtaining a high-titer serum that reacted with many antigens common to both males and hermaphrodites as assayed on immunoblots. To increase its male-specificity, we treated this serum with sonicated adult hermaphrodites. When assayed on immunoblots with separated proteins from males and hermaphrodites, the adsorbed serum still showed some reaction with hermaphrodite antigens, but comparisons of male and hermaphrodite lanes nevertheless allowed identification of a male-specific (or highly male-enriched) protein of about 150 kd. We would ultimately like to screen an expression library to identify the gene encoding this 150-kd protein. To enhance the specificity of the adsorbed serum, we have attempted affinity adsorption to and elution from gel blots of male proteins in the 150- kd range. We have had mixed success with this technique and have not yet reproducibly produced an antibody fraction with high specificity for the 150-kd protein.

Link CD et al. (1987) Worm Breeder's Gazette "mutant expression of male copulatory bursa surface markers."

Ehrenfels CW, Link CD, Wood WB

[Worm Breeder's Gazette, 1987]

In a search for molecular markers of male tail development, we have detected two surface markers that are specifically expressed in the copulatory bursa of adult males and the vulva of adult hermaphrodites. One marker is defined by binding of a monoclonal antibody (Ab117) and the other by binding of the lectin wheat germ agglutinin (WGA). Both Ab117 and WGA recognize many proteins on gel blots and bind to multiple tissues in fixed animals; but they show much higher specificity in the staining of live, intact animals. In this case, Ab117 binding is restricted to the bursal cuticle in young adult males and the vulva in adult hermaphrodites; binding to the pharyngeal lining is also observed in all stages of both sexes. WGA staining of live animals is also restricted to the bursa and the vulva; in addition, the phasmid openings are stained in all animals. We have examined the expression of these markers in mutants with specific defects in sex determination, cuticle formation, developmental timing, and bursa formation. We find that: 1) Marker expression is dependent on phenotypic sex: tra-1(e1099) XX pseudomales stain like normal XO males, while her-1(ct22n695) XO hermaphrodites stain like normal XX hermaphrodites. However, the WGA marker can be expressed in her-1(n695) XX intersexual animals with little or no bursal development, so that posterior surface expression of this marker can serve as an indication of subtle masculinization of hermaphrodites. 2) Production of an adult cuticle is neither necessary nor sufficient for marker expression. Adult males and hermaphrodites carrying the retarded heterochronic mutation lin-29(n333) produce larval cuticles but can express both markers, while IA animals carrying the precocious heterochronic mutations lin14(n179) or lin-28( n719) produce adult cuticles but do not express either marker. 3) Some mutants with specific bursal defects show abnormal marker expression: mab-3(e1240) and mab-5(1239) males show reduced or no expression of both markers, while mab-7(e1599) and mab-8(e1250) males show strong bursal over-expression of the WGA (but not the Ab117) marker. Other mutants with specific (mab-1,2,6,9,10) or non-specific ( sma-2, bli-3, vab-8, rsa defects show fairly normal expression. 4) As perhaps our most interesting observation, lin-22 males, which show an anterior to posterior transformation of cell fates in the lateral hypodermis, ectopically over-express the Ab117 marker over much of the cuticle. This ectopic expression represents a novel phenotype for an interesting class of mutations which previously have been difficult to identify. We are using this phenotype as the basis for a mutant screen for more genes involved in establishing or interpreting anterior/posterior positional information.

Link CD (1988) Worm Breeder's Gazette "observations on mab-10."

Link CD

[Worm Breeder's Gazette, 1988]

The recessive mutation mab-10(e1248) II was identified by J. Hodgkin in his screen for male-infertile mutants (Genetics 103:43-64, 1983). mab-10(e1248) males have rather subtle bursal defects, are completely infertile, and have been reported to occasionally have supernumerary molts. I have extended these observations and find that mab-10(e1248) causes a highly penetrant, male-specific retarded heterochronic defect in addition to affecting bursal development. Age-synchronized 248); 90) males and hermaphrodites were followed from the L4/adult molt at 20 C. Male animals were found to synchronously begin a supernumerary molt 18-20 hrs after L4 lethargis. Hermaphrodites of the same age (n=32) were carefully observed under DIC optics and showed no signs of a supernumerary molt. The male-specific supernumerary molt phenotype was found to be highly penetrant (>95%) at 16 C, 20 C, and 25 C. The male supernumerary molt appears to result from the production of a second adult cuticle. Young (presupernumerary molt) mab-10(e1248) adult males appear to have normal 'first' adult cuticles, as judged by the presence of adult lateral alae and wild-type staining with WGA and Ab117. The supernumerary cuticles also have adult lateral alae. To my knowledge, none of the known retarded heterochronic mutations (e.g., in lin-29, s pattern of adult supernumerary molts. The mab-10(e1248) supernumerary molt is not affected by passage through the dauer larvae stage. I have mapped mab-10(e1248) (scoring the supernumerary molt phenotype) to the right of the gene cluster on linkage group II, between unc-4 and rol-1 and near lin-29. (Male infertility has co- segregated with the supernumerary molt phenotype in these mapping experiments.) Interestingly, this places mab-10 in a region of the chromosome where the nearby known morphological mutations (in the genes lin-29, ifically affect the adult cuticle (see figure). mab-10(e1248) is complemented by lin-29 and the deficiencies mnDf87 and mnDf77, but not by the deficiencies mnDf89 or mnDf83. I have carefully observed the phenotype of mnDf89/mab-10(e1248) heterozygous males and hermaphrodites, and find the same pattern of male specific supernumerary molts as seen in mab- 10(e1248) homozygotes. This argues against the possibility that the male specificity of mab-10(e1248) results because this is a partial loss-of-function mutation and that a null mutation would affect both sexes. However, mnDf89/mab-10(e1248) males have significantly more abnormal bursae than mab-10(e1248) homozygotes, suggesting that mab-10( e1248) is not a null mutation. (mnDf89/+ males are wild-type.) Because the bursal defects (and male infertility) of mab-10(e1248) are observed before the supernumerary molt, the relationship between these phenotypes is unclear. It is also not obvious why a heterochronic defect would appear to be so sex-specific. In this regard, it is interesting to note that neither retarded nor precocious heterochronic mutations affect the timing of expression of male- specific bursal surface markers (Link, Ehrenfels, and Wood, Development 103:485-495, 1988). Perhaps some portion of male or bursal development involves an independent temporal program. [See Figure 1]

Link CD (1988) Worm Breeder's Gazette "identification of mutants that show ectopic wheat germ agglutinin binding."

Link CD

[Worm Breeder's Gazette, 1988]

I have previously reported that the lectin wheat germ agglutinin and the monoclonal antibody Ab117 show specific binding to the vulva of adult hermaphrodites and the copulatory bursa of adult males (W.B.G. 10, #1, p. 117; Link, Ehrenfels, and Wood, Development 103, 485-495). Based on the observation that lin-22 males show strong ectopic binding of both WGA and Ab117, I devised a non-clonal screen to identify mutants that show ectopic WGA binding in hopes of identifying mutants with anterior/posterior transformation like lin-22. As described below, I have not yet identified mutants of this class, but have found a number of unexpected mutants interesting in their own right. Previously I visualized WGA or Ab117 binding to live animals using fluorescently-labeled lectin or secondary antibody. Because this method was not feasible for large scale mutant screening, I developed a peroxidase-coupled detection system that could be used with live animals and easily visualized under the dissecting microscope. Briefly, for visualizing WGA binding, animals were sequentially incubated in biotin-conjugated WGA and avidin-conjugated horseradish peroxidase, then developed using H2O2 and the chromogen 3-amino 9- ethylcarbazole. In order to use WGA binding in a non-clonal screen, I used semidominant mutations in her-1(n695 or y101) as the genetic background. These mutations variably masculinize XX animals, such that a sub-population of the adult animals (5-25%) are sufficiently somatically masculinized to show (lin-22 dependent) ectopic staining, but still retain a self-fertile hermaphrodite gonad. In three pilot experiments, I have screened approximately 60,000 EMS-mutagenized her- 1(n695) F2 adults and 20,000 EMS-mutagenized wild-type F2 adults for ectopic WGA binding, and have identified 15 independent mutations. These mutations, which fall into at least four complementation groups, appear to be unlike lin-22 in that their ectopic WGA binding is observed in both males and hermaphrodites. I have recently screened 500,000 EMS-mutagenized her-1(y101) F2 animals and identified an additional 50 independent (as yet uncharacterized) mutants. The most interesting of the characterized mutations are the allelic pleiotropic mutations ct109 and ct111(V). ct109 animals bind WGA and Ab117 over most of their cuticle surface. This surface binding is neither stage- nor sex-specific, although adult males show somewhat stronger posterior WGA binding than adult hermaphrodites. ct109 animals are uncoordinated and have improper body posture reminiscent of unc mutants known to have neurological defects. ct109 males have defective bursae, characterized by having crumpled spicules and abnormal rays. These males variably lack diagonal sex muscles. The most surprising ct109 phenotype is its ability to enhance the weak semidominant mutation lin-12(n302). Weak lin-12 dominant mutants such as n302 are Vul, while strong lin-12 dominant mutations such as n137 are Muv. lin-12(n302); imals closely resemble lin-12(n137); hermaphrodites have at least four pseudovulvae and males have ectopic hooks. All of the phenotypes observed for ct109 have also been observed for ct111, arguing that these phenotypes are likely to be the result of a single mutation. How can this motley collection of phenotypes be explained? A plausible explanation for the ct109 Unc and Mab phenotypes is that these animals are defective in cell-cell or cell-matrix interactions. Thus, the ct109 Unc phenotype might result from neuronal mis-wiring ( due to improper axon pathfinding) while the Mab phenotype might result from defects in migration of the sex muscle precursors and other bursal cells. To speculate wildly, perhaps ct109 defines a gene that modifies (glycosylates?) extracellular proteins or protein domains. This hypothetical modification would modulate cell-cell interactions in a manner akin to the way sialic acid addition modulates N-CAM function in vertebrates. This modification would also modulate the function of the lin-12 product (almost certainly a cell surface protein) and be involved in cuticle formation.