Palmer RE et al. (1994) Worm Breeder's Gazette "Use of an Expression Library to Clone Genes by Complementation"

Palmer RE, Sternberg PW

[Worm Breeder's Gazette, 1994]

Use of an expression library to clone genes by complemcntation. RE. Palmer and P.W. Sternberg, HHMI and Division of Biology, Caltech, Pasadena, CA 91125

Hudson ML et al. (1998) Worm Breeder's Gazette "The Physiological Roles of Soluble Guanylate Cyclase in Caenorhabditis elegans"

O'Shea M, Hudson ML

[Worm Breeder's Gazette, 1998]

Cyclic GMP (cGMP) is an important intracellular signaling molecule in both vertebrates and invertebrates mediating a number of important physiological roles such as cardiovascular tone, neuronal signalling, platelet function and phototransduction (Hobbs, 1997). cGMP is synthesized from GTP by the enzyme guanylate cyclase (GC) which has two main classes; membrane bound or particulate GC (mGC) and the soluble, cytosolic form (sGC). The former generally mediate the action of signaling peptides such as atrial natriuretic peptide whereas sGC is the primary target of nitric oxide (NO), a gaseous intracellular messenger (Palmer et al, 1987). Around thirty GCs have been identified by the C. elegans genomic sequencing program to date (Yu et al,1997), with about five probable sGCs and the remainder being putative mGCs (most closely related to the mammalian natriuretic peptide receptors). We are interested in identifying and characterising the sGCs in C. elegans in terms of their expression, activation and physiological roles. We have already cloned and sequenced a cDNA corresponding to the putative sGC C06B3.8, now identified by Yu et al as gcy-32. By using a combination of RT-PCR and PCR amplification of a cDNA library, the clone was found to use two different polyadenylation signals and also shows a different splicing form to that predicted by the Genefinder software. It is not yet apparent whether this cDNA is trans-spliced. In other systems, sGC has been shown to function as a heterodimeric protein and work is under way to identify which genes are coexpressed in particular cells, using a combination of GFP fusion constructs and immunocytochemistry. A radioimmunoassay has been developed to detect cGMP generation in extracts of nematode tissue. Interestingly, worm cytosolic extract appears to be insensitive to NO stimulation. This is consistent with work carried out in our lab which has failed to demonstrate Ca2+ stimulated turnover of 3H-arginine to 3H-citrulline; a standard assay for NO production by the enzyme nitric oxide synthase (NOS). Also, no genes have yet been identified by the worm sequencing project which show homology to NOS. This has lead us to the intriguing possibility that C. elegans does not possess a classical NO-cGMP signaling pathway. Our ultimate goal is to prove or disprove this theory and to shed light on the physiological and behavioral role of the sGCs that have been found in the worm genome. References. Hobbs, A. J. (1997) Trends Pharmacol. Sci. 18, 484-491 Palmer, R. M. J., Ferridge, A. G. and Moncada, S. (1987) Nature 327, 524-526 Yu, S., Avery, L., Baude, E. and Garbers, D. L. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 3384-3387

Wood WB (1990) Worm Breeder's Gazette "Reversal of L-R Asymmetry by Micromanipulation of 6-Cell Embryos: ABal and ABpl are Equivalent to ABar and ABpr, Respectively"

Wood WB

[Worm Breeder's Gazette, 1990]

Both the embryo and the embryonic cell lineage are asymmetric from the 6-cell stage onward, particularly with regard to the ABa lineages ( 1). The general bilateral symmetry of the animal is superimposed on the asymmetric lineage by lineally nonhomologous cells adopting analogous fates at equivalent contra lateral positions during embryogenesis (1), but several asymmetries remain throughout larval development, such as the anterior-posterior positions of the left and right coelomocyte pairs (2) and the anterior-r, posterior-l orientation of the primordial and developing gonad (3). These asymmetries normally have the same handedness in all individuals; in N2 populations grown at 16 C, no animals with reversed gonad handedness were found among about 2500 examined. Handed asymmetry first becomes evident in the embryo between the 4- and 6-cell stages, when skewing of the l-r cleavages of ABa and ABp results in positioning of the al and pl daughters anterior to ar and pr, respectively, with al more ventral than ar [see Wood, WBG 11,#3(May) :73, 1990]. Reversal of handedness was accomplished using methods similar to those of Priess and Thomson (4), by rolling a 4-cell embryo to ventral side (EMS) up with a microneedle and then pressing down and back on the left ventral surface of ABa during AB-cell cleavage. When successful, this procedure reverses the normal skewing of both AB spindles and results in ar and pr being anterior to al and pl, respectively, at completion of the cleavage (see diagram). The embryonic development of a reversed embryo was observed and recorded using the '4D Microscope' developed by J. White. (This consists of a Nomarski microscope equipped with a video camera connected to a computer controlled optical disc recorder and focussing drive motor, which automatically records complete sets of serial optical sections at predetermined intervals and allows subsequent playback of consecutive images from any desired optical section to facilitate lineaging.) Cell assignments in the reversed embryo, based on timing of divisions, relative positions, and lineaging to confirm identities of AB descendants, were entirely consistent with a completely enantiomorphic but otherwise normal cleavage pattern. For example, MS descendants were located on the left side of the early embryo and C descendants on the right; MSa and Ca descendants were to the right of MSp and Cp descendants, respectively, and so on. In the L1 that hatched from this embryo, 14 normally asymmetrically placed nuclei scored were all l-r reversed: Z1, Z2, Z3, and Z4 in the gonad primordium, M, the four cc's, Q2, the exc cell, mu sph, hyp11, and PVR. The adults that developed from other reversed embryos showed reversed handedness for the somatic gonad, placement of coelomocytes, and the positions of neuronal cell bodies relative to processes in the ventral nerve cord. The reversed animals were healthy, moved normally, and were normally fertile. These results indicate that ABal is equivalent to ABar and ABpl to ABpr, and, therefore, that the left-right differences in lineage patterns and cell fates exhibited by descendants of ABa and ABp must be dictated by cell interactions, which differ on the two sides as a consequence of the asymmetric positioning of these cells relative to others in the embryo. The respective cell contacts of the ABal-pl and ABar-pr pairs are still equivalent in the 8-cell embryo despite the asymmetry, and earlier laser ablation experiments (1) provide evidence for cell autonomy of many AB-cell fates subsequent to the 51-cell stage. Therefore, the determinative interactions are most likely to occur between the 15-cell and 51-cell stages. [See Figure 1] Thanks to A. Chisolm, J. Rothman, J. Sulston, and J. White for timely coaching and sage advice.

Palmer RE et al. (1994) Worm Breeder's Gazette "Identification of An Egg-Laying Defective Mutant With An Abnormal Contact Between the Anchor Cell and the Vulval Precursor Cells"

Palmer RE, Tietze K, Sternberg PW

[Worm Breeder's Gazette, 1994]

Identification of an egg-laying defective mutant with an abnormal contact between the anchor cell and the vulval precursor cells. R.E. Palmer, K. Tietze, and P.W. Sternberg, HHMI and Division of Biology, Caltech, Pasadena, CA 91125 The development of the hermaphrodite vulva involves many previously characterized cell-cell interactions. One important interaction is between the anchor cell of the somatic gonad and the vulval precursor cells (VPCs). During the late L2 larval stage the anchor cell has been shown to necessary for the induction of the VPCs. Using Nomarski optics we have shown that during the L3 larval stage another cell interaction occurs between the anchor cell and the VPCs. After the second round of cell division of the P6.p VPC (four cell stage) the anchor cell begins to invaginate between the two innermost granddaughters of P6.p (P6.pap and P6.ppa). This process occurs just prior to the invagination of these cells. Throughout the L3 larval stage the anchor cell remains positioned between these cells. We have identified an egg-laying defective mutant, sy275, with an abnormal contact between the anchor cell and P6.pap and P6.ppa. In this mutant the anchor cell fails to invaginate between the P6.pap and P6.ppa and is often observe on top of the two cells. In addition to the anchor cell contact abnormality hermaphrodites have other slight defects. (1) The timing of the last round of cell divisions of the P6.p lineage is delayed. (2) In a small fraction of animals there is a third distal tip cell. As adults, the hermaphrodites are completely egg-laying defective and have a slightly protruding vulva. Using criteria outlined in Trent et al. (Genetics 104; 619 647) sy275 has no hermaphrodite specific neuron defect and behaves as a class A egg-laying defective mutant. sy275 is recessive and both males and hermaphrodites are mating competent. Linkage analyses place sy275 on LGII. Three factor mapping indicates that sy275 is close to unc-52 on the right arm of LGII.

Palmer RE et al. (1995) Worm Breeder's Gazette "sy275 Males Have a Defect in the Migration and Attachment of the Linker Cell."

Palmer RE, Sternberg PW

[Worm Breeder's Gazette, 1995]

sy275 males have a defect in the migration and attachment of the linker cell. R.E. Palmer and P.W. Sternberg, HHMI and Division of Biology, Caltech, Pasadena, CA 91125 We have previously described the isolation of an egg-laying defective mutant with an abnormal contact between the anchor cell and the vulval precursor cells (WBG 13:3). L3 hermaphrodites homozygous for the sy275 mutation have an abnormal contact between the anchor cell and P6.pap and P6.ppa, i.e., the anchor cell fails to invaginate between the P6.pap and P6.ppa and is often observe on top of the two cells. In mid-L4 hermaphrodites the connection between the uterus and the vulva appears obstructed suggesting that the connection between these two structures is disrupted in this mutant. Hermaphrodites with sy275 in trans to the translocation mnT11 (used as a deficiency, see Herman et. al. Genetics 1982) are egg-laying defective and their brood size is decreased by ~10 fold. In addition, approximately 5percent of the animals are sterile. Other evidence suggesting that sy275 is not a complete loss-of-function allele is that 5percent of sy275 hermaphrodites grown at 25 C are sterile (n=275). We are presently, conducting a non-complementation screen to identify additional alleles. We had previously reported that sy275 males are mating competent, however, quantitative mating experiments indicate that only 30percent of the males tested were able to sire cross progeny (n=50). Examination of sy275 males with Nomarski optics suggests that there may be two linker cell specific defects. In about 30percent of the adult males examined the linker cell had not migrated more than 1/2 way down the ventral side of the animal. Interestingly, the intestines of these worms were completely distended and full of bacteria suggesting that the animals were unable to defecate. In approximately another 30percent of the adult males examined ectopic sperm were present in the body cavity. Ectopic sperm were only observed in mated males but not in virgin adult males. Anatomical observation suggests that in about 30percent of late L4 or young adult males the linker cell, while it has migrated into the tail, has not attached to the proctodeum properly. Thus, sy275 disrupts two analogous cell attachments - the anchor cell to vulva and linker cell to proctodeum.

Prasad SS et al. (1989) Worm Breeder's Gazette "mapping of the dpy-14 gene using cosmid rescue."

Rose AM, Prasad SS

[Worm Breeder's Gazette, 1989]

The region of chromosome I, defined by the breakpoints of hDp19 (K. McKim, personal communication) and sDp2 (Rose et al, 1984), contains the dpy-14 gene, other visibles and at least nineteen essential genes. Two other duplication breakpoints, hDp58 and hDp62 (K. McKim, personal communication), are located in this region and are being used to position the genes. The dpy-14 region is located within a large contig on linkage group I (A. Coulson and J. Sulston, personal communication). Within a portion of this region ten coding regions have been identified in 170 kb. of DNA flanking the polymorphisms sP1 and hP9 (Starr et al., 1989) . The N2/B0 polymorphic fragment sP1, which is included in the cosmid T21G5 has been positioned very close to dpy-14 by Starr et al.(1989). However, the dpy-14 gene had not been recombinationally separated from sP1. We have used the cosmids T13C1, T14D10 and T21G5 mixed together to rescue the Dpy-14 phenotype. Rescue was achieved by microinjecting the cosmid DNAs into the mature oocytes of 'wild type' nematodes with the genotype dpy-5 y-5 p2. One Dpy-5 animal indicative of dpy-14 rescue was identified in the F1 progeny. In the F2 both Dpy-5 and Dpy-5 were scored which indicated the cosmid rescue to be extrachromosomal (hEx1). A similar attempt with the individual cosmid T13C1, which contains a coding region homologous to col-4 (T. Starr, personal communication), did not rescue the Dpy-14 phenotype. When the cosmid T14D10 was used extrachromosomal rescue of the Dpy-14 phenotype was obtained (hEx2). Since this cosmid does not have any homology to the collagen genes, dpy-14 gene is not likely to be a collagen. The observation that the dpy-14 gene lies in the cosmid T14D10 has positioned the sP1 polymorphic site to the right of dpy-14. Four of the identified ten coding regions lie in the cosmid T14D10, one of which encodes the snRNA U1-1 (T. Blumenthal, personal communication; T. Starr, personal communication). An attempt to rescue the Dpy-14 phenotype with another coding region was negative. Therefore, we believe that one of two coding regions in T14D10 is the dpy-14 gene. Attempts are underway to determine which of these coding regions contains the dpy- 14 gene. This work was supported by the grant from Medical Research Council of Canada to AR.

Pilgrim DB (1988) Worm Breeder's Gazette "zinc finger proteins in worms ?"

Pilgrim DB

[Worm Breeder's Gazette, 1988]

In an attempt to identify genes involved in sex determination which may be evolutionarily conserved, I have tried looking for a worm homologue to mammalian Testis Determining Factor (TDF). TDF is thought to be the 'master' sex-determining locus in mammals. The putative human gene has been cloned (Page et al., Cell 51:1091 [1987]), and the predicted protein sequence revealed the presence of repeated Cys/His 'zinc finger' domains, indicating a DNA binding function. There are homologues found on the sex chromosomes of most mammals tested. (The human X and Y homologues are apparently now referred to as zfx and zfy, respectively.) A full length TDF cDNA, isolated from a human testis library, was kindly provided by Mark Palmer (ICRF, London) . This was used at low stringency to probe a Southern Blot of N2 genomic DNA. 6-7 bands were seen, depending upon the restriction enzyme. The same probe was used to isolate 9 clones from a genomic lambda library, kindly provided by Alan Coulson. Four of the clones were chosen for further study. Two overlap, the other two appear to be discrete. The clones were fingerprinted by Alan Coulson, and compared with the physical map. The two overlapping inserts (CB#DP004, 005) lie on an unmarked contig which has since been localized by Rita Fishpool to LG V, using in situ hybridization. The third clone (DP002) lies on the rsn-8/myo-2/myb-1/col-9 contig on the right half of X ( although none of these genes has been genetically mapped). The last clone (DP003) has a small insert, and didn't match anything in the data base. The cross-hybridizing regions of each clone are being subcloned for further analysis. So far I have begun characterization of the DP004/005 clone. This insert accounts for three of the bands seen on the southern blot. A fragment of 3.5 kbp, which cross- hybridizes with the human cDNA, was subcloned, and used to probe a Northern Blot. A band of 3.9 kb was detected in pA+ RNA prepared from him-8 worms in L3, L4 and a mixed stage prep, but no band was detected in L2 pA+ or in total RNA. I have not yet looked to see if the message is sex-specific. When this subclone is used as a probe against the human cDNA, it cross reacts most strongly to the portion of the cDNA coding for 6 of the 'zinc-finger' repeats, as well as some nonfinger-coding sequence. I have begun to sequence this region of the DP004/005 clone to see if it also codes for a zinc-finger protein, and if so, if it has a role in sex in worms.

Nelson GA et al. (1985) Worm Breeder's Gazette "effects of heavy ion irradiation on C elegans."

Nelson GA, Hammen RF

[Worm Breeder's Gazette, 1985]

We have been investigating the effects of heavy ion irradiation on C. elegans using the Lawrence Berkeley Laboratory's Bevalac accelerator as a source of ions of various charge, energy and fluence. To date, ions of C, Ne, Ar, Fe and La have been used to irradiate N2 and JP10 ( eT1 balanced over dpy-18 and unc-46, of Rosenbluth, et. al., Genetics 109: 493) in order to characterize: 1) lethal mutation rates and structures vs. fluence and LET (linear energy transfer), and 2) fertility and gonad development vs. fluence, LET and target size (cell number in gonad primordia). The goals are to understand how heavy ion mechanisms of mutagenesis and cell killing are related to their LET, charge, velocity and tract structure and to compare these mechanisms to those for gamma & X-rays, which deposit energy in a more uniform fashion. For example, with respect to forward lethal mutation rate, 495 MeV/amu Fe ions at a fluence of 3x10+E7/cm^2 (three to four hits per gonad nucleus) are roughly equivalent to 2x10+E10 Co-60 gamma photons (2400 hits per gonad nucleus or 1000 rads). A brief summary of our findings to date is: (1) Mutation rate in the eT1 balanced region of LG III and LG V (strain JP10) is strongly dependent on LET below 100 KeV/ m and at three hits per cell reaches 8% of F1's at LET = 1100 KeV/ m. (2) At LET = 198 KeV/ m the lethal mutation rate in JP10 in the dose range 32 to 1600 'rad equivalents' ( Note that rads is a misleading unit for heavy ion radiation as it takes no account of track structure) is linear and equal to approximately 0.6% of F1's per 'rad'. (3) A large fraction of ion- induced lethals have altered segregation patterns. (4) Fertility in N2 irradiated as larvae is strongly dependent on LET and gonad size. The LET effect is maximum at about 100 KeV/ m and gonads with approximately 30-50 cells are most sensitive. Larvae with gonads of greater than 100 cells produce many males indicating survival of cells with sublethal damage to LG X. HELP! We are trying to find ways of immobilizing worms for extended periods of time (e.g., one week) in order to correlate particle tracks with worms and worm parts. The best we can do is to keep dauers in 3% agarose at 11 C, but we want to hold worms at 20-22 C. Suggestions would be most welcome-call GN @ (818) 354-4401.

Vandana Rajakumar et al. (2002) Worm Breeder's Gazette "Suppressor Screen of egl-38 egg-laying defect to study the vulva to uterus signalling pathway"

Helen M Chamberlin, Vandana Rajakumar

[Worm Breeder's Gazette, 2002]

Communication between tissues in an organ system during their development is vital to ensure that they function as a unit. The egg-laying system in Caenorhabditis elegans offers a good model system to study this paradigm. Here the reciprocal interactions from the AC to the vulva and from the vulva to the uterus coordinate development between these tissues. The AC initially induces 3 of 6 multipotential Vulval Precursor Cells (VPCs) to form the vulval cells by the LIN-3-LET-23 mediated signalling pathway. Then the 1 deg vulval cells reciprocally signal to the uterine cells to induce uv 1 cell formation via LIN-3 again. Some genes important for the AC to vulva signal also mediate the vulva to uterus signal, whereas others function in only one or the other developmental pathway. For example, only the vulval transcription of lin-3 requires EGL-38, a PAX transcription factors while the earlier AC expression is EGL-38- independent (1). Although the AC to vulva signal pathway has been studied in detail, the vulva to uterus pathway is less well characterized. To identify additional genes in the reciprocal pathway from the vulva to the uterus, we have devised a mutagenesis screen for temperature sensitive supressors using egl-38(n578 ) animals. These animals are egg-laying defective (2,3). We reasoned that mutations in genes that act as negative regulators of the reciprocal pathway may supress the egg-laying defect. Identification and characterization of these suppressor genes would enable us to better understand at a molecular level, the signalling pathway occuring between tissues during their development to form an organ system. We have carried out two genetic screens using EMS as a mutagen wherein the mutagenized animals are subjected to different temperatures during their development. To identify mutations in genes that may have essential additional functions one screen incorporates a temperature shift during the egg-laying system development: 1.Screen --1: Mutagenized worms allowed to grow at 15 deg C throughout their development 18,783 gametes were screened and eight candidate supressors were recovered. Two supress at all temperatures, five are cold sensitive and one supresses at 20 deg C. 2.Screen -- 2: Mutagenized worms allowed to grow at 15 deg C at all stages but exposed t 25 deg C during vulval development.12,375 gametes were screened, wherein five candidate supressors were isolated.Two have been verified to be strong supressors at 15 deg C while two more are weaker. One candidate seems to require exposure to both 15 deg C and 25 deg C during development to exhibit supressor phenotype.Results from screening another 12,408 gametes are still awaited. Chang, C., Newman, A.P and Sternberg, P.W. (1999) Current Biology 9: 237-246 Trent.C., Tsung ,N and Horvitz, H.R (1983) Genetics 104 : 619-647 (3) Chamberlin, H.M., Palmer, R.E., Newman, A.P., et al (1997) Development 124: 3919-3928

Palmer RE et al. (1995) Worm Breeder's Gazette "Search For Exploding Vulvae."

Palmer RE, Sternberg PW

[Worm Breeder's Gazette, 1995]

Search for exploding vulvae. R.E. Palmer and P.W. Sternberg. HHMI and the Division of Biology, Caltech. Pasadena CA 91125. The hermaphrodite vulva is formed from three of six posterior ectodermal cells (Pn.p cells). The three Pn.p cells (P5.p, P6.p, and P7.p) have two distinct fates. One difference between the two fates is the attachment of the cells to the ventral hypodermis. After three rounds of division, all the progeny of P6.p are not attached to the ventral hypodermis while half the progeny of P5.p and P7.p remain attached to the ventral hypodermis. Certain mutations and mutant combinations result in worms in which all three of Pn.p cells adopt the fate of P6.p, i.e. their progeny are not attached to the ventral hypodermis. In many instances, these mutants are lethal because when the vulval cells are not attached to the ventral hypodermis the worms explode at the vulva during the L4-adult molt. We sought to identify additional genes that when mutated would result in this phenotype. We performed an F1 clonal screen looking for dead worms in which the worms had exploded at the vulva during the L4 - adult molt. We screened 12,000 haploid chromosome sets and identified three mutants that exhibited an exploding vulval phenotype. All three mutants were recessive and incompletely penetrant for the vulval defect with between 40-75% of the homozygotes exploding. Two of the three mutants were allelic to the lin-1 mutant and the third mutation, sy329, defined a new gene. For both lin-1 alleles approximately 5percent lethality was observed, however, the penetrance of the exploding vulval phenotype varied between the alleles (70percent of sy321 homozygotes exploded and 25percent had a multi-vulval phenotype while in sy289 45percent of the homozygotes exploded and the other 50percent had a multi-vulval phenotype). Linkage analysis placed the sy329 mutation on LGI. Deficiency and three-factor mapping placed the sy329 between unc-13 and lin-10. sy329 in trans to the deficiencies nDf24 or nDf25 was indistinguishable from sy329 homozygotes. Approximately 40percent of sy329 hermaphrodites had exploding vulvae. An additional 15percentof the worms had protruding or multi-vulvae. Vulval lineage analysis of sy329 revealed that 40percent of the hermaphrodites had extra non-adherent vulval tissue (P6.p-like). Often additional Pn.P cells also formed vulval tissue. These vulval defects were suppressed by mutations in the well characterized signaling pathway required for vulval formation, i.e. lin-3, lin-7, and lin-45. sy329 hermaphrodites and males are sterile and exhibit several somatic gonad defects. There is no germ cell differentiation in sy329 hermaphrodites and males. Microscopic examination of sy329 hermaphrodites with Nomarski optics revealed that the germ cell nuclei, and presumably the cells, would fuse and form extremely large cells. DAPI staining of sy329 gonads showed that these large germ cells contained a massive excess of chromosomal DNA. In sy329 hermaphrodites, the distal tip cells (DTC) and the anchor cell were noticeably enlarged. The enlarged DTC phenotype was highly penetrant whereas the enlarged AC phenotype was less penetrant. In addition, it was not uncommon to observe 2 DTC per gonad arm and in some animals 2 AC were also observed. Since the AC is required for the formation of the vulva we postulate that the enlarged AC may be responsible for the extra vulval induction observed in sy329.