Hunter CP et al. (1991) International C. elegans Meeting "NON-AUTONOMY OF HER-1 FUNCTION IMPLICATES CELL INTERACTIONS IN C. ELEGANS SEX DETERMINATION."

Hunter CP, Wood WB

[International C. elegans Meeting, 1991]

Activity of the her-l gene is required for normal male development in C. elegans. Loss-of-function her-l mutations cause feminization of XO animals (normally male) to produce fertile hermaphrodites, while gain-of-function her-l mutations cause masculinization of XX animals ( normally hermaphrodite) to produce pseudo-males. Genetic analysis indicated that her-l functions early in the hierarchy of regulatory interactions among the major sex determination genes (1). We have analyzed her-l genetic mosaics to determine which cells must express her-l for wild-type male development. If her-l functions cell- autonomously, then in XO animals her-l (+) cells will follow male fates, and her-1(-) cells will follow hermaphrodite fates. If her-l or a her-l regulated activity functions non-autonomously, then the her-l genotype of a cell may not determine its sexual phenotype. The phenotypes of XO her-l genetic mosaics are variable, but clearly show that both her-1(-) and her-l (+) cells can follow either hermaphrodite or male fates. We conclude that her-l is neither necessary nor sufficient to cell-autonomously determine male sexual phenotypes and, therefore, that her-l or a her-l regulated activity must be able to function non-autonomously. The observed patterns of non-autonomous effects suggest that many or all cells express and respond to her-l activity. These findings implicate cell interactions in C. elegans sex determination. Earlier genetic analysis indicated that her-l might directly control tra-2 activity (1). Molecular characterization of the gene products of her-l and tra-2 (see abstracts by M. Perry et al. and P. Kuwabara et al) is consistent with a model in which a secreted ligand encoded by her-l interacts with a cell-surface receptor encoded by tra-2. We will discuss the possible role of such an interaction in coordinating the sex determination decision.

Birsoy, Bilge et al. (2009) International Worm Meeting "Right Way to Have Sex."

Bloss, Tim, Birsoy, Bilge, Rothman, Joel H., Downes, Joanna C., Choi, Shin Sik

[International Worm Meeting, 2009]

While the mechanism that breaks left-right (L-R) anatomical asymmetry (handedness) has been described in C. elegans, we have found that at least one additional mechanism must exist to establish other handedness cues that generates L-R differences in the deployment of alternative cell death pathways (see abstract from our lab by Choi et al.). To assess whether such a system might affect a behavior in the worm, we investigated whether male mating behavior exhibits L-R handedness. Upon recognizing a hermaphrodite, males initiate backward locomotion and continuously scan along the hermaphrodite''s body. When their tails reach either the head or tail of the hermaphrodite, males turn to maintain contact and then continue backward locomotion on the opposite side of the hermaphrodite. We found that this turning behavior shows a distinct right-handed bias: when individual males were allowed to mate with lin-2(e1309) vulvaless hermaphrodites, >70% of the turns when made over the hermaphrodites'' body (away from the agar surface) and >55% of turns when made under the hermaphrodites'' body (toward the agar surface) are right-handed. To determine whether this bias in turning direction correlated with L-R asymmetry in the male mating structure, which results from stochastic EGL-1-dependent loss of sensory rays (Choi et al. abstract), we examined the turning bias in egl-1(n1084n3082) mutants. We were surprised to find that, although wild-type males lack rays more frequently on the right, the right-hand turning bias is actually increased in the egl-1 mutant, in which no rays are lost and therefore rays are always symmetrically arranged. Thus, the intrinsic turning bias is even more apparent in males with symmetric mating structures. To assess whether this L-R behavioral bias is dependent on anatomical handedness, we analyzed gpa-16(it143) mutants in which the L-R asymmetry of the internal organs is reversed as a result of the reversal in the early embryonic symmetry break. Males with reversed anatomical asymmetry showed a virtually identical right hand bias in turning, demonstrating that a handedness-determining system that is independent of the previously described L-R symmetry breaking event must control this behavior. These findings raise the possibility that, analogous to brain laterality in humans, functionally L-R asymmetry in the C. elegans motor nervous system may be independent of anatomical handedness.

Ellis RE et al. (1991) International C. elegans Meeting "PROGRESS IN CLONING fog- 1"

Kimble JE, Ellis RE

[International C. elegans Meeting, 1991]

The fog-l gene plays an important role in the decision of germ cells to differentiate as either spermatocytes or oocytes (Doniach 1986, Barton and Kimble 1990). In fog-l mutants, all germ cells develop into oocytes. This is true for both males and hermaphrodites. However, these fog-l mutations do not appear to affect other aspects of sexual development. Furthermore, the fog-l mutant phenotype is not suppressed by mutations in any of the other genes known to control sex- determination. Thus fog-l might function to initiate spermatogenesis in response to other genes that regulate general sexdetermination. There are 42 fog-l mutations. These mutations arise at a frequency typical for knockout mutations in other C. elegans genes, and four of these fog-l alleles were isolated from a screen capable of recovering deletions. Thus it is likely that these mutations cause a loss of fog- l function. In order to study the mechanisms by which fog-l functions, and to determine how fog-l expression is regulated by the genes that control sex-determination, we are now cloning this gene. The fog-l gene is located on the left arm of LGI, between sup-ll and unc-ll, a region spanned by a large contig of about one megabase in size. By mapping DNA polymorphisms with respect to fog-l, we have narrowed down the region in which fog-l must be located, and are now injecting YAC DNA that covers this region in an attempt to rescue fog-l mutants.

Lackner MR et al. (1995) International C. elegans Meeting "The role of mpk-l in vulval development"

Kim SK, Lackner MR

[International C. elegans Meeting, 1995]

The mpk-l gene encodes a MAP kinase protein that is approximately 75% identical to vertebrate MAP kinases. Previously, we have shown that mpk-l plays an important role in ras-mediated induction of vulval cell fates, as reduction-offunction mutations in mpk-l completely suppress excess vulval differentiation caused by a gain-of-function mutation in let-60 ras. We have now characterized a strong reduction-offunction allele of mpk-l that results in temperature-sensitive vulvaless phenotype (84% Vul at 25 degrees Celsius) and a completely penetrant sterile phenotype at all temperatures. Epistasis analyses suggest that mpk-l acts downstream of lin-15 and let-60 ras, but upstream of the putative transcription factors lin-l and lin-31. To determine whether mpk-l is absolutely essential for vulval induction, or whether some induction may occur in the absence of mpk-l activity, we are in the process of isolating a null allele of mpk-l. We used mutant activated forms of mpk-l and Drosophila MEK to address the question of whether activation of these two kinases is sufficient to promote vulval fates in the absence of upstream signaling. Coexpression of both activated mpk-l and activated D-MEK causes a strong Muv phenotype (~90% Muv), whereas coexpression of wild-type mpk-l and wild-type D-MEK results in a weak Muv phenotype (~10% Muv). The strong Muv phenotype is unaffected by ablation of the anchor cell or a reduction-offunction mutation in lin-45 raf, consistent with activation of mpk-l and MEK being at least partially independent of upstream signaling. Our future plans are to identify targets of mpk-l by isolating suppressors of this strong Muv phenotype.

Mango SE et al. (1991) International C. elegans Meeting "A NOVEL glp-1 ALLELE MIMICS THE EFFECT OF lin-12(d) ON VULVAL DEVELOPMENT."

Kimble JE, Maine EM, Mango SE

[International C. elegans Meeting, 1991]

The lin-12 and glp-l genes encode homologous transmembrane proteins ( l) that may act as receptors for cell interactions during development ( 2,3). The glp-l gene is required zygotically for germ line proliferation and maternally for embryogenesis (2, 4); lin-12, in contrast, is involved in various postembryonic cell interactions in the soma, including those between vulval precursor cells (VPCs) that specify vulval fates (5). The novel allele glp-l (q35) has a semidominant Multivulva phenotype (Muv) in addition to the typical recessive Glp phenotypes. We find that the Muv phenotype of glp-l (q35) is similar to that of lin-12(d) in three ways. First, VPCs that would normally fuse with the hypodermal syncytium (3 fate) follow a 2 -1ike vulval lineage instead. Second, the Muv phenotype is not dependent on the anchor cell. Third, the Muv phenotype results from increased or novel, rather than decreased, glp-l activity. Because glp-l (q35) remains Muv in animals lacking lin-12, this effect does not depend on lin-12 activity. We conclude that the glp-l (q35) product is functionally very similar to that of lin-12. The glp-l (q35) gene bears a nonsense mutation predicted to truncate the glp-l protein 122 amino acids from its carboxy terminus. The region which should be missing from the glp-l (q35) protein includes a PEST sequence thought to destabilize proteins. A simple explanation of these results is that the carboxy terminus bears a negative regulatory domain that normally keeps glp-l activity off in the VPCs. In glp-l ( q35) animals, inappropriate glp-l activity may drive these cells to proliferate, and thereby channel them toward a 2 -like fate.

Schedl TB et al. (1991) International C. elegans Meeting "GLD-1, A GENE NECESSARY FOR MULTIPLE STEPS IN GERMLINE DEVELOPMENT"

Kimble JE, Barton MK, Maine EM, Kahn S, Schedl TB, Francis GR, Jones AR

[International C. elegans Meeting, 1991]

Three crucial aspects of germline development are the switch from proliferation to meiosis, sex determination, and gametogenesis. An important question is how these processes are controlled and coordinated. Mutations in previously identified genes effect only one of these processes. We describe a novel gene gld-l (defective in germline development, formerly called fog-3), in which mutations effect each of these processes in the hermaphrodite germline. Twenty- five mutations were isolated and demonstrated to be gld-l alleles by complementation tests, mapping, and intragenic reversion. 8 mutants are defective in the switch from proliferation to meiosis: homozygotes have a tumorous germline phenotype (Tum), with major proliferative regions proximally and diqtally, and no gametogenesis. gld-l(Tum) mutations are probably null as they behave like a deficiency in complementation tests. 11 other mutants are defective only in germline sex determination: homozygotes for 10 alleles display masculinization of the germline (Mog), while one allele displays feminization of the germline (Fog). The semi-dominant Mog mutations were shown to be gain- of-function alleleg of gld-l by intragenic reversion to gld-l recessive phenotypes at high frequency (1/2300 haploid genomes). 6 mutants are defective in gametogenesis: homozygotes for two alleles produce small oocytes that can not be fertilized, while 4 other alleles display germline nuclei arrested in meiotic prophase with no obvious signs of gametogenesis. Somatic phenotypes have not been observed. The role of gld-l in germline proliferation has been analyzed by: l) laser ablation of somatic gonad cells or their ancestors which promote germline proliferation [distal tip cell (DTC) or anchor cell (AC)], and 2) interactions in double mutants with glp-l (which causes premature entry into meiosis). Ablations demonstrate that gld-l proximal proliferation is independent of the AC or DTCs, but distal proliferation remains dependent on a DTC. The gld-l(Tum); glp-l(null) double mutant undergoes two additional rounds of proliferation followed by entry into meiosis and differentiation of sperm. As gld-l(Tum) can increase germline proliferation in the absence of the glp-l product, gld-l is likely to act downstream or on a pathway parallel to glp-l. However, full expression of the gld-l tumorous phenotype is dependent on some glp-l activity.

Crittenden SL et al. (1991) International C. elegans Meeting "LOCALIZATION OF THE glp-1 PROTEIN IN THE GERMLINE"

Kimble JE, Crittenden SL

[International C. elegans Meeting, 1991]

The glp-l protein mediates several cell interactions during C. elegans development. In particular, glp-l activity is required zygotically for maintenance of germline mitosis by the somatic distal tip cell and maternally for embryonic induction of the anterior pharynx (1,2). Mosaic analysis (l) and sequence data (3) together suggest that the glp-l protein may be a transmembrane protein that acts as part of the receiving mechanism in regulatory cell interactions. As a first step in characterizing the glp-l protein, we have raised rat polyclonal antibodies to glp-l fusion proteins. Following affinity purification, we have used these antibodies to visualize the glp-l protein in dissected gonads. Specific staining appears to outline the membrane around each germ 'cell', giving the gonad a honeycomb like appearance. There is extremely strong staining at the distal end of the gonad, in the region of mitotic germline nuclei. The staining is much weaker proximally and undetectable in maturing oocytes. This pattern of expression supports the idea that the glp-l protein is required in the membrane of mitotic germ cells to receive and transduce a signal from the distal tip cell. In addition, it suggests that maternal glp-l RNA, not protein, is packaged into oocytes. We are currently optimizing procedures to examine glp-l protein localization in embryos and in larval and adult somatic tissues.

Goshima Y et al. (2000) Japanese Worm Meeting "Effects of L-3, 4-dihydroxyphenylalanine (L-DOPA) on Caenorhabditis elegans behavior, and isolation and characterization of L-DOPA response abnormal mutants"

Goshima Y, Sasaki Y, Ogura K-i, Tsukamoto S, Katsura I, Ishihara T

[Japanese Worm Meeting, 2000]

We previously proposed that L-3,4-dihydroxyphenylalanine (L-DOPA) is a neurotransmitter and/or a neuromodulator in the mammalian central nervous system (CNS). Receptor or transporter molecules of L-DOPA, however, have not been determined. In an attempt to understand the molecular events involved in the L-DOPA response, we investigated the effect of L-DOPA on C. elegans behavior, and further have isolated nine L-DOPA response abnormal (dpa) mutants of C. elegans. The C. elegans Bristol strain N2 on agar plate spread with E. coli strain OP50 with L-DOPA added (5 - 20 mM) exhibited characteristic behaviors such as back and forward or twist and turn movements. Some aspects of these behaviors appeared to resemble osmotic avoidance response. Cultures on NGM plates of EMS (ethyl methanesulfonate)-mutagenized worms containing many F1 gravid adults (about 2000) were transferred to new plates. F2 worms were put on NGM plate containing L-DOPA 20 mM, which had been seeded with E. coli OP50. Nine mutant worms (dpa), which did not exhibit L-DOPA response, were obtained. They exhibited a moderately diminished chemotaxis response to Na+ and/or Cl-, and some of them exhibited a diminished response to an odorant 2-butanone. We are now characterizing dpa mutants in comparison to several chemotaxis and thermotaxis mutants.

Spence AM (1991) International C. elegans Meeting "Expression of fem-1."

Spence AM

[International C. elegans Meeting, 1991]

Loss-of-function mutations in thefem-l gene transforrn animals of both chromosomal sexes into fertile females, indicating thatfem-l function is essential for male development in both soma and gerrnline( l) . The DNA sequence of the gene predicts that its product is an intracellular protein containing six tandemly arranged 33 arnino acid cdclO/SWI6 motifs near its N-terminus(2) . Northern analysis shows that the 2.4 kb fem-l mRNA is expressed throughout hermaphrodite development at approximately constant levels. Althoughfem-l acts in hermaphrodites only to influence the sexual fate of the germline, its mRNA is present in mutant hermaphrodites lacking a germline [glp-l( e2144) or SS104(bn2)] suggesting that in contrast to fem-3(3), the gene is expressed in herrnaphrodite somatic tissues. Reduced levels offem-l transcripts in animals lacking germ cells suggest that the gerrn line is however a major site offem-l expression. In adult males, a sub-population of transcripts slightly larger than lhose seen in hermaphrodites is evident. Whether the change in size is due to regulation of polyadenylation as has recently been observed infem-3(gf) mutants(4) is under investigation.

Jongeward GD et al. (1991) International C. elegans Meeting "THE GENETICS OF sli-l, A CANDIDATE NEGATIVE REGULATOR OF VULVAL INDUCTION."

Lee JH, Sternberg PW, Jongeward GD

[International C. elegans Meeting, 1991]

We have isolated more than 40 alleles of sli-1 (suppressor of lineage defect) as silent suppressors of the Vulvaless (Vul) phenotype of weak alleles of let-23. sli-l mutations suppress the Vul, Male Abnormal and Lethal phenotypes of non-null alleles of let-23, but do not suppress the lethality of a null allele of let-23. Since these mutations are silent and do not suppress nulls, we believe that sli-l is a negative regulator of let-23, rather than a target of let-23 activity. Certain sli-l and let-23 alleles show allele-specific interactions, suggesting that these two gene products directly interact. Iet-23; sli-l double mutants are generally Hyperinduced (or Hin; more than three of the vulval precursor cells respond to the inductive signal in a gonad-dependent manner). Hin is believed to be a reduction-of-function phenotype of some of the Vul genes. sli-l suppresses both hypomorphic and null alleles of lin-2 and lin-7 to Hin. Since the sli-l allele used suppresses a strong hypomorphic let-23 allele to wild-type, sli-l is not simply increasing the dosage of let- 23 or another component of the vulval induction pathway. sli-l suppresses weak alleles of lin-3 but does not cause these animals to become Hin. sli-l does not suppress let-60 or lin-46, two genes acting after let-23 in the vulval induction pathway. Therefore sli-l acts near let-23 in the pathway. sli-l and unc-101, another suppressor of the vulval defect of weak alleles of let-23, show a synthetic vulval phenotype, such that unc-101; sli-l double mutants can display as much as 130% induction, where 100% is wild-type induction. We believe that sli-l is a partially redundant negative regulator of let-23 or some other early component of the vulval induction pathway.