Wood WB (1990) Worm Breeder's Gazette "Asymmetry and Antiworms"

Wood WB

[Worm Breeder's Gazette, 1990]

Although the C. elegans larval and adult body plans are generally bilaterally symmetric, they also exhibit several handed asymmetries at gross anatomical as well as cellular levels. Moreover, the embryonic cell lineage and the embryo itself are markedly asymmetric from the 6- cell stage onward. Bilateral symmetry appears to be superimposed on the asymmetric lineage during embryogenesis by lineally nonhomologous cells adopting analogous fates at equivalent positions on the two sides of the animal, as pointed out by Sulston et al. (Dev. Biol. 100:64, 1983). These observations raise questions regarding first, how the polarity (handedness) of the left/right embryonic axis is established, and second, how bilateral symmetry is achieved by contra- laterally analogous programming of nonhomologous cells. Handedness appears to be established during the transverse cleavage of the two AB cells in the 4-cell embryo when their spindles, initially parallel to the left-right axis, become skewed in a clockwise direction as viewed from the dorsal side, so that the resulting daughter cells on the left are anterior to those on the right. The handedness of the gonad in adult hermaphrodites can be scored efficiently under the dissecting microscope after rolling animals through a quarter turn with a pick. Among F3 broods from EMS- mutagenized N2, I have found animals with reversed gonad handedness at an overall frequency of about 0.2%. Some of these are also reversed for other asymmetries such as the placement of the excretory cell nucleus and the handedness of the ventral nerve cord, while others are not. Most showed no increase in frequency of reversed gonads among their progeny. So far 200 F1 clones screened have yielded two strains that produce 10%-15% reversed-gonad animals, but no mutants with higher penetrance effects. I am attempting to test the likelihood, pointed out by zur Strassen on the basis of experiments with Ascaris almost a century ago, that reversed animals can arise from reversed 6- cell embryos via a mirror-image cell lineage.

Wood WB (1976) Worm Breeder's Gazette "dehydration of C. elegans for shipping and storage"

Wood WB

[Worm Breeder's Gazette, 1976]

Several nematode species can survive dehydration in the wild and in the laboratory. A reliable method of dehydrating and resuscitating C. elegans would be useful for shipping worms in letters (see Edgar's note in vol. 1, No. 1 of the Newsletter) and might be an alternative to liquid nitrogen storage of mutant strains. The following technique has been successful in Boulder (a very dry place). A stack of 5-10 circles of Whatman #l filter paper are placed in a plastic petri dish and saturated with Hershey broth obtained from the nearest phage lab. Worms from a starved plate are suspended in Hershey broth and dripped onto each circle to make a sandwich. The plate is closed and placed in 16 C incubator. Complete drying takes about a week. Worms are revived by placing a filter paper from the sandwich on an agar plate with a few drops of E. coli, incubating overnight, and then washing the worms off the paper with buffer. I have obtained 20-40% survival of N2 worms stored in the 16 C incubator for a month using this method. Survival is 10- to 100-fold lower if M9 salts, 80% Ascaris Ringer's, or S medium is substituted for the Hershey broth. Survival of worms dried from broth is decreased by more than 100-fold by overnight storage in a vacuum dessicator over CaSO , suggesting that the Hershey broth may act by preventing complete drying. The survivors are principally dauer larvae and L1's that hatch inside of adult hermaphrodites during the drying process. Both male and hermaphrodite dauer larvae appear to be normally fertile following rehydration and maturation.

Wood WB et al. (1984) Worm Breeder's Gazette "C elegans compensates for differences in X dosage."

Meneely PM, Wood WB

[Worm Breeder's Gazette, 1984]

X-linked hypomorphs provide a genetic assay for X-chromosome expression (Meneely & Wood, Newsletter vol. 8, #1, p.6). To test genetically whether C. elegans compensates for differences in X dosage between males and hermaphrodites, we have used the hypomorphs unc-3(e54) (severely Unc when present over a deficiency in XX animals; moderately Unc as a homozygote) and let-2(mn114) (L1 lethal when present over a deficiency in XX animals; adult sterile as a homozygote) . XO males hemizygous for either of these alleles show the same phenotype as the corresponding homozygous hermaphrodite rather than that of the hemizygous deficiency hermaphrodite, indicating compensation for X dosage, at least for these two genes. To test biochemically for compensation, we compared the activity of the DNase encoded by nuc-1 X (Dew & Sulston, Newsletter, vol. 1, #1 p. 17) in males and hermaphrodites. Enzyme activities were assayed by measuring release of acid-soluble radioactivity during incubation of 100 l reaction mixtures containing a high-speed supernatant fraction of sonicated worms, 100 nmoles of DNA nucleotides in the form of sonicated salmon sperm and [3H]-labelled E. coli DNA, 1mM EDTA, and 40mM sodium phosphate buffer. The assay gave good proportionality with both time and extract concentration up to >40% of radioactivity released. The activity showed a broad pH optimum of 5-6 and was routinely assayed at pH 6.0. Under these conditions E. coli extracts had no detectable DNase activity. Sonicates of nuc-1(e1392) showed <1% the specific activity of N2 sonicates. Sonicates of staged N2 populations showed similar specific activities for L1 - L4 and adult worms, and about 50-fold lower specific activities for early embryos and dauer larvae. Sonicates of young adult males and hermaphrodites, separated (to >95%) from staged populations of him-5(e1490) using Nitex filters, showed the same specific activity +/-5%. Although other interpretations of this result are possible, it is most simply explained by compensation for differences in nuc-1 dosage between males and hermaphrodities.

Wood WB et al. (1994) Worm Breeder's Gazette "Low Temperature Affects Embryonic Handedness Reversal."

Bergmann DC, Florance A, Wood WB

[Worm Breeder's Gazette, 1994]

LOW TEMPERATURE AFFECTS EMBRYONIC HANDEDNESS REVERSAL Bill Wood, Arny Florance and Dominique Bergmann Department of MCD Biology, University of Colorado, Boulder C. elegans embryos become left-right asymmetric at the 6-cell stage witlf a handedness which is essentially invariant among N2 animals reared at 20, and which persists into adulthood, determining the asymmetric placement of the gonad, intestine, the coelomocytes, and several other cells. Reversal of embryonic handedness at the 6-cell stage by micromanipulation results in development of healthy adults with all left-right asymmetries reversed. l Spontaneous reversal occurs at a frequency of a few percent among animals developing embryos treated with chitinase at the 2-cell stage to remove the egg shell.2 We have now found that when N2 are reared at 10, the frequency of animals with reversed gonad handedness as adults increases to above 0.5%. Those examined by Nomarski microscopy exhibited handedness reversal of the ventral nerve cord (cell bodies on the left) and reversed placement of the coelomocytes as well as the intestine and gonad, suggesting that these animals developed from reversed embryos. To determine the cold-sensitive period for handedness reversal, young gravid adults reared at 20 were placed at 10 for 2 hours, then transferred to new 20 plates and subsequently transferred to fresh 20 plates at 2-hr intervals. Eggs laid during the cold pulse (interval l; exposed to 10 after fertilization), the first 2 hr after return to 20 (interval 2; exposed to 10 either just before or just after fertilization), and each of the four subsequent 2-hr intervals at 20 (3-6, exposed to 10 before fertilization) were allowed to hatch and develop at 20 into adults, which were then scored for gonad handedness. Among animals developing from embryos exposed to 10 after fertilization (interval 1), no reversals were' observed (N=1,956), whereas among animals developing from oocytes and sperm exposed to 10 before fertilization (intervals 3-6), the frequency of reversal was 0.21% + 0.09% (N=9,223). Surprisingly, although left-right asymmetry first appears at the 6-cell stage, and the embryo's left and right are highly unlikely to be specified until the 4-cell stage, I the cold-sensitive period for reversal is before fertilization. This result suggests that the effect is on the gametes. We can imagine two possible explanations: 1) Production of some eggshell precursor component, synthesized during oogenesis, could be cold sensitive, such that embryos with "soft" eggshells are produced at 10, leading to a low frequency of reversal as seen in chitinased embryos.2 2) Some handed structure in a gamete, such as the sperm centriole for example, may normally dictate the skewing of the ABa and ABp spindles that leads to handed asymmetry in the embryo;l when this structure is exposed to 10 its subsequent ability to dictate handedness could be impaired, leading to a low frequency of reversal. We are currently carrying out experiments with chilled males to determine whether egg or sperm is the cold-sensitive gamete, as well as examining effects of other treatments on handedness reversal. Finally, we are continuing small scale screens for maternal-effect mutations that result in a high frequency of embryonic reversal. None have been found, but so far we have screened only about 1000 EMS-mutagenized genomes. I Wood, W. B. (1991) Nature 349: 536-538. 2 Wood, W. B. and Kershaw, D. (1991) In Biological Asymmetry and Handedness, CIBA Foundation Symposium 162: 143-164.

Wood WB (1976) Worm Breeder's Gazette "maternal effects among ts zygote-defective (zyg) mutants."

Wood WB

[Worm Breeder's Gazette, 1976]

We have studied maternal effects in 23 zyg ts mutants to estimate the times of expression of genes whose products are required in embryogenesis. We have used the following three tests, called arbitrarily A, B, and C. A test: Heterozygous (m/+) L4's are shifted to 25 C and allowed to self-fertilize. If 100% of their eggs yield larvae (25% of which express the mutant phenotype as adults), then the mutant is scored as maternal (M). If 25% of the F1 eggs fail to hatch, then the mutant is scored as non-maternal (N). An M result indicates that expression of the + allele in the parent allows m/m zygotes to hatch and grow to adulthood. A result of N indicates the opposite: that the + allele must be expressed in the zygote for hatching to occur. Out of 23 zyg mutants tested, 3 were scored N and 20 were scored M in the A test. Therefore, for most of the genes defined by these mutants, expression in the parent is sufficient for zygote survival, even if the gene is not expressed in the zygote. B test: Homozygous (m/m) hermaphrodites reared at 25 C are mated with N2 (+/+) males. If eggs fail to hatch at 25 C, but mated hermaphrodites shifted to 16 C produce cross progeny to give proof of mating, then the mutant is scored M. If cross progeny appear in the 25 C mating, then the mutant is scored N. An M result indicates that expression of the + allele in the zygote is not sufficient to allow m/+ progeny of an m/m hermaphrodite to survive. Conversely an N result indicates either that zygotic expression of the + allele is sufficient for survival, or that a sperm function or factor needed for early embryogenesis can be supplied paternally (see C test below). Out of the 23 zyg mutants tested, 11 were scored M and 12 were scored N. The combined results of A and B tests and their simplest interpretation are as follows. Ten mutants are M,M; the genes defined by these mutants must be expressed in the hermaphrodite parent for the zygote to survive. Ten mutants are M,N; these genes can be expressed either in the parent or in the zygote. Two mutants are N,N; these genes must be expressed in the zygote. One mutant is N,M; this gene must be expressed both in the maternal parent and in the zygote. C test: Homozygous (m/m) hermaphrodites reared at 25 C are mated with heterozygous (m/+) males. If rescue by a +/+ male in the B test depends on the + allele, then only half the cross progeny zygotes of a C test mating (m/+ male x m/m hermaphrodite) should survive. However, if rescue depends on a function or cytoplasmic component from the male sperm, then all the cross progeny zygotes in a C test should survive. Of the 10 M,N mutants, 6 have been C tested; one exhibited paternal rescue independent of the + allele. The A and B tests also were carried out on 16 mutants that arrest before the L3 molt (acc mutants). In the A test on 2 of these mutants, all m/m progeny of m/+ parents grew to adulthood at 25 C. Therefore, parental contributions are sufficient to overcome a progeny mutational block as late as the L2 stage. All 16 acc mutants scored N in the B test.

Wood WB et al. (1985) Worm Breeder's Gazette "suppression of dpy-22 male inviability by X duplications."

Wood WB, Meneely PM

[Worm Breeder's Gazette, 1985]

XO males carrying the dpy-22(e652) X mutation generally do not survive to adulthood; those that do are small and sickly. We have postulated that the low viability is due to apparent under-expression of the X chromosome caused by this mutation (Meneely and Wood, 1985 C. elegans Meeting Abstracts, p. 90; Meneely et al., in preparation). Presence of X duplications generally appears to increase overall X expression (Meneely and Wood, 1983, Genetics 106:29; Meneely, this issue). To determine how these two effects interact, we have begun testing the viability of dpy-22 XO males that carry X duplications derived from either the maternal or the paternal parent. In N2(male) x dpy-22(hermaphrodite) crosses, the progeny male ratio (PMR: identifiable Dpy progeny males to total outcross progeny) ranges from about 10% to 30%. For the initial duplication experiments we used mnDp25 (X;I), a duplication of about 25% of the X including the unc-3 locus but not the dpy-22 locus. The cross mnDp25/+;unc-3/0(male) x dpy-22 rodite) gave PMR of 16% +- 5% (39/244 in two experiments), compared to 19% +- 6% (31/165) in the control cross N2(male) x dpy-22 maphrodite). Therefore, introduction of this duplication paternally has no significant effect. To test the effect of introducing it maternally, we crossed N2(male) x mnDp25;unc-20 unc-3(hermaphrodite) (rather than the simpler mnDp25;dpy-22 maphrodite), which turned out to have unexpectedly low viability). We obtained a PMR of 46% +- 5% (158/341), compared to 5% +- 2% (21/389) in the control cross N2( male) x unc-20 unc-3(hermaphrodite). Similar results have been obtained in preliminary experiments with mnDp8 (-15% of X) and mnDp9 (-25% of X), neither of which include the dpy-22 locus. Further experiments with these and additional duplications are in progress. So far it appears that X duplications not including the dpy- 22 locus can suppress the inviability of dpy-22 XO males if supplied from the maternal parent, but not if supplied from the paternal parent, consistent with a maternal effect of X duplications on early X- chromosome expression in the embryo.

Wood WB (1982) Worm Breeder's Gazette "two maternal-effect embryonic lethals that are suppressed by sup-7(st5) X."

Wood WB

[Worm Breeder's Gazette, 1982]

Five absolute embryonic lethal mutant alleles showing a strict maternal effect (M,M: mutant animals produce no viable embryos after mating with N2 males), linked to dpy-10 II or unc-4 II, and carried as balanced heterozygotes were roughly mapped relative to the linked morphological marker and tested for suppressibility by sup-7(st5) X. The five alleles, all recovered following EMS mutagenesis, were ct1 and ct5 (isolated by John Laufer) and mn40, mn202, and mn203, obtained from R. Herman. Three of them, ct1 (2% to the right of dpy-10), ct10 (2% to the left of unc-4), and mn203 (about 2% from unc-4) were not suppressed; crosses of the corresponding balanced heterozygotes with sup-7 males gave no higher frequency of fertile morphologically marked F2 animals than expected from recombination. However, a balanced heterozygote carrying mn40 (1% to the left of unc-4) in similar crosses gave about 50% fertile F2 Unc progeny (1 to 50 viable embryos; unc-4mn40 homozygotes normally produce many defective embryos with <0. 02% viability). A balanced heterozygote carrying mn202 (22% to the right of unc-4) in crosses with sup-7 males carrying the suppressible allele dpy-18(e364) III gave about 40% fertile F2 Unc progeny as expected from recombination. Suppression was demonstrated by picking semiDpy unc-4mn202/+;dpy-18/dpy-18;sup-7/+ F2 hermaphrodites and showing that their nonDpy Unc progeny produced viable embryos, whereas their Dpy did not. Some semiDpy Unc progeny also produced viable embryos; the frequency was uncertain due to the difficulty of unequivocally distinguishing semiDpy from nonDpy and Dpy animals. These results show that both mn40 and mn202 are suppressible and suggest that both are suppressed marginally by one copy of sup-7 and better by two copies. Suppressed lethals (unc-4mn;sup-7), easily distinguishable from recombinants by their reduced fertility and slower development, were isolated from each of these crosses and shown by backcrossing to carry the lethal mn allele. The suppressed lethals can be maintained near 20 C but not at lower or higher temperatures; at 16 C sup-7 is lethal, and at 25 C it suppresses the mn alleles poorly. The genes defined by mn40 and mn202 must be transcribed during oogenesis. Neither mutant homozygote produces viable embryos following mating with sup-7 males, consistent with the possibility that the corresponding messages are also translated prior to fertilization. We are attempting to determine the translation period by temperature-shift experiments, taking advantage of the temperature- sensitive properties of sup-7. We are also using these mutants in attempts to detect transformation of worms with cloned suppressor genes introduced by microinjection.

Wood WB (1976) Worm Breeder's Gazette "maternal effect and linkage of the ts lesion in the wild strain of C elegans Bergerac."

Wood WB

[Worm Breeder's Gazette, 1976]

Some French strains of C. elegans exhibit a zygote-defective phenotype at 25 C. Crosses of English (N2) males with French ( Bergerac) hermaphrodites (obtained from Dr. R. Stokstad, Univ. of Calif., Berkeley, through Carl Johnson) yield progeny that are fertile at 25 C, although some F2 segregants show reduced fertility. When A and B tests for maternal effect (see preceding report) were carried out using F1 hermaphrodites from such a cross and wild type Bergerac hermaphrodites, respectively, the French strain scored M,M. Therefore, the ts lesion in this strain is recessive, and the gene(s) that it defines must be expressed in the parent for zygote survival. Linkage of the ts allele(s) was tested by crossing English males heterozygous for known dpy markers with French hermaphrodites and scoring F2 dpy progeny for the ts phenotype. The ts character is linked to dpy-10 in linkage group II. The N2 ts zyg mutant B1 characterized by Vanderslice and Hirsh exhibits the same maternal effects, and linkage as the Bergerac strain, and displays a lethal phenotype at 25 C very similar to that of the French strain studied by Nigon. However, the two alleles complement; a tsB1/ts Bergerac heterozygote was constructed and found to be fertile.

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.

Wood WB (1994) Worm Breeder's Gazette "Progress Towards Cloning nob-1 and a Rapid Method For Cloning Genomic Sequence Flanking Tc1 Insertions Kimberly Van Auken, Mark Yandell, Lois Edgar and William"

Wood WB

[Worm Breeder's Gazette, 1994]

As part of our efforts to understand posterior patterning in C. elegans embryos, we are cloning and characterizing one of several loci defined by mutations that result in the Nob (no back end) phenotype. nob-1 ,the first locus thus identified, is defined by two psoralen-induced mutations: ct223 ,which results in 100% L1 lethality and the Nob phenotype and ct230 ,a weaker allele which results in only 15% lethality and production of viable, fertile animals with variable tail abnormalities. Thus far, our cloning endeavor has been directed towards correlating the genetic and physical maps on the right arm of LGIII, where nob-1 resides approximately 2 map units right of spe-6 and 1 map unit left of pie-1 .In this region, no genes have been cloned so far, and ambiguities have been found in the cosmid map. Therefore, we constructed a congenic strain to identify and map Bergerac-specific Tc1 elements that might be useful in correlating the genetic and physical maps. We identified two Tc1 elements that map approximately 2.5 map units left of nob-1 . When attempts to clone the flanking genomic sequences by inverse PCR met with difficulties, we tried a different strategy that in our hands has proved to be more efficient. Using size-selected, EcoRI-digested genomic DNA, we first performed a primer extension using a Tc1 -specificprimer that was directed outward. This resulted in double-stranded, blunt-ended molecules to which we ligated a linker-adaptor molecule that contained a unique NotI restriction site. We then performed two rounds of PCR with nested primers specific to sequences in Tc1 and the linker adaptor. After the second round of PCR, we observed strong signals from each of the expected Tc1 elements in our size-selected genomic fractions. These PCR products were digested with NotI and EcoRV (which cuts at a site in the 54 bp terminal repeat of Tc1 )and cloned efficiently into a plasmid vector. Subsequent probing of Southern blots with these cloned genomic fragments have confirmed the identity of the PCR products, which fortunately appear to contain unique sequence. We can now use these fragments as probes to assay for presence of the nob-1 region in members of a set of ten YAC clones identified by genomic DNA flanking a mutator allele of pie-1 (Craig Mello and Alan Coulson, personal communications and thank you's) and thus proceed towards transformation rescue of nob-1 .