Donahue LM et al. (1985) Worm Breeder's Gazette "molecular analysis of dosage compensation continues."

Quarantillo BA, Donahue LM, Wood WB

[Worm Breeder's Gazette, 1985]

Mutations in several unusual dpy genes appear to affect the general level of X-chromosome expression, implicating these genes in control of X chromosome dosage compensation (Hodgkin, 1983, Mol. Gen. Genet. 192:452 Meneely and Wood, 1984, C. elegans Newsletter, Vol. 8, #1, p. 6; Meneely and Wood, 1985 C. elegans Meeting Abstracts, p. 90; De Long et al., ibid., p. 99; Meyer and Champness, ibid. p. 101). For example, dpy-21 mutations appear to increase and dpy-22 mutations to decrease X expression by genetic tests. To determine whether these effects are occurring at the RNA level, we have employed a quantitative RNA dot blot procedure to compare ratios of X-linked to autosomal transcripts between males and hermaphrodites and between dpy- 21 or dpy-22 and wild-type hermaphrodites, using gene-specific probes for the X linked genes act-4 and myo-2 and the autosomal genes act-1, p. 100). We have previously reported that adult males and hermaphrodites have the same amount of act-4 mRNA relative to act-1 or unc-54 mRNA (ibid.). We have now shown that the same is true for myo-2 mRNA relative to myo-1 mRNA levels. Expression of these two genes would seem unlikely to differ for physiological reasons between males and hermaphrodites, because both code for myosins of the pharynx, which is not a sexually dimorphic structure. Therefore, the equivalence of these ratios in the two sexes supports the view that dosage compensation of these genes operates at the RNA level. Conceivably, our results could be explained by special regulatory controls on act-4 and myo-2, operating outside of, or in addition to, the general X-chromosome dosage compensation mechanism. To test this possibility, we are attempting to determine whether these genes, like other X-linked genes analyzed genetically, show dose-sensitive expression when present in extra copies because of a duplication or fewer copies because of a deficiency. We have preliminary evidence from quantitative Southern blot analysis that act-4 is covered by the duplication mnDp57 (X;I), given to us by R. Herman. If we confirm this result, we shall assay act-4 expression in mnDp57-containing strains. We are testing known duplications for extra copies of myo-2; so far we have found that this gene is not covered by mnDp10 (X;I). We previously reported that in L1 animals a dpy-21 mutation did not affect the ratio of act-4 mRNA to unc-54 mRNA (ibid.). We have now shown that in adult hermaphrodites, the dpy-21(e428) mutation increases the ratio of act-4 mRNA to unc-54 mRNA by a factor of 1.53 +- .08 relative to the same ratio in N2. We are presently testing the effect of dpy-21 in adult animals on myo-2 mRNA levels. One possible reason for the difference in results between L1's and adults with act- 4 could be that the dpy-21 gene is not active prior to the L1 stage of development. A more trivial possibility is that many of the animals in our L1 preparations were starved and could have degraded some of their RNA's by the time they were harvested. We are attempting to test these and other possible reasons for the apparent difference.

Tanner K et al. (1988) Worm Breeder's Gazette "further analysis of ct31 X, a locus affecting sex determination and dosage compensation."

Tanner K, Wood WB, Burgess SM, Trent C

[Worm Breeder's Gazette, 1988]

We described earlier the isolation of ct31 as a gamma-ray-induced, semi-dominant, feminizing mutation that suppresses the masculinization of XX animals caused by the her-1(gf) allele n695, and reported that ct31 increases the penetrance of the X-linked hypomorph lin-15(n765), suggesting that ct31 might cause a decrease in X-chromosome expression (Burgess et al., WBG May, 1986). ct31 XX animals are Dpy, Egl hermaphrodites; XO animals are Dpy, nonfertile, partially feminized males. Here we present evidence that ct31 lowers expression of the X- linked act-4 gene and that it is probably a gain-of-function mutation involving duplication of a region of the X chromosome. Assay of act-4 mRNA in ct31 and N2 L1 hermaphrodites using an RNA dot blot assay ( Donahue et al., PNAS 84:7600,1987) showed that the level of this transcript in ct31 animals is only 46% + 4% of the level in N2 animals. This finding supports the genetic evidence cited above that ct31 causes a decrease in expression of X-linked genes. Three-factor mapping data places ct31 in the lin-2 - unc-9 interval on X. Deficiencies of this region have not been obtained, but it is covered by the duplication mnDp10. XX hermaphrodites of genotype mnDp10/+; ct31 lin15, which have two mutant and one wild-type copy of the ct31 locus, are more severely Dpy and Egl and generally sicker and more slow-growing than ct31 homozygotes. When the former animals are allowed to self-fertilize, one quarter of the progeny arrest as L1 larvae. We surmise that these arrested larvae are the class carrying two copies of mnDp10 (ct31/ct31/+/+). Since the ct31 phenotype appears to be enhanced by additional copies of the wildtype locus, these results are consistent with ct31 being a gain-of-function mutation. However, given the large size of mnDp10, and the fact that it carries at least one other locus known to affect X expression (sdc- 1), more definitive evidence on this point must await isolation and characterization of deficiencies in the region. The high reversion rate of ct31 described earlier (Burgess et al., 1986) suggested that it might involve an unstable chromosomal rearrangement. In an attempt to test this suggestion, we used a probe derived from the cloned myo-2 gene, which is located on the physical map at a position that should be near the ct31 locus, to carry out quantitative Southern blot analyses. The copy number of the myo-2 gene in the ct31 strain is clearly higher than in N2 or in the her-1(n695) parent strain of ct31. The elevated copy number of myo-2 appears identical to that measured in a strain homozygous for mnDp10. Seven independent nonDpy ct31 revertants do not show the elevated myo-2 copy number, correlating the visible ct31 phenotype and the apparent duplication. Preliminary experiments using as probes cosmid clones from the myo-2 contig ( obtained from A. Coulson and J. Sulston) indicate that at least 40 kb including and to the left of the myo-2 gene is duplicated in the ct31 mutant. Experiments are in progress to characterize further the extent and nature of the rearrangement.

(1982) Worm Breeder's Gazette "Goettingen worm group activities 1981 and prospects 1982"

[Worm Breeder's Gazette, 1982]

After Gunter von Ehrenstein's death of a heart attack December 26, 1980, the 6 directors of the Max-Planck-Institute for Experimental Medicine decided to close the Department of Molecular Biology as of the end of 1982. We are continuing work to finish up and publish work in progress with gradual attrition as people find new positions elsewhere and with correspondingly diminishing budget. A list of our recent papers and manuscripts is given below. Eddi Isnenghi completed his paper on the phenotypes of the Gottingen emb mutants (including TSP's and maternal tests) and is trying to microinject cloned DNA to rescue worm mutants. He is also trying to transform yeast mutants with worm DNA. Randy Cassada is still trying to map embs on LG III with deficiencies and duplications and is looking for new strains of C. elegans in the wild. Eddi has received a 6-months' extension of his DFG (= NSF) fellowship until July. Ken Denich is writing up his work on emb mutant lineages in Gottingen for a paper and as PhD Thesis for the Department of Zoology, University of Manitoba and will be returning there this summer to finish up PhD requirements. Einhard Schierenberg has continued with laser-induced cell fusion experiments. If the membrane between 2 cells is disrupted at the right time, the division of both nuclei results in 4 more or less normal cells. Development continues to a twitching lima bean monster. Fusing cells in later stages results in uncoordinated animals. All treated embryos show timing defects in the E-cell line similar to emb mutants, perhaps due to remixing of cytoplasm. A copy of Einhard's film on embryogenesis is available (with commentary) from Scott Emmons for those who want to show it to students. Einhard is going to Bill Wood's lab this fall. Khosro Radnia has left for a job in industry. Ulli Certa and Franz Scharfenberg are characterizing histone H2A variants in emb mutants. They have also identified several histone H4 DNA clones in a C. elegans genomic library using a sea urchin probe obtained from Mike Grunstein. Ulli has finished his PhD and plans to go to Grunstein's lab at UCLA this spring for a post- doc on yeast histones. Ulli and the other gene cloners have been working successfully with similar minded members of the Institute's Department of Chemistry. Franz hopes to do his PhD in that department. Shahid Siddiqui has just joined Joe Culotti at Northwestern. Tom Cole is very happy with his new Zeiss EM 109 electron microscope. Our computer-aided reconstruction program with color display now works for LM and EM input. We are trying to transfer the programs and data to the MRC, so they will still be available after our department has closed. Chris Carlson has gone to the EMBL in Heidelberg as of Jan 1 to work on computer graphics of molecules. Ursula Reuter also has a position at EMBL as of Jan 1 as an EM technician. Ed Hedgecock visited for 3 weeks this fall to show us microinjection, antibody staining (with anti-actin from Mary Osborne and Klaus Weber), post- embryonic lineage techniques and the like. It was a very useful and pleasant visit. We hope to have some of you as visitors in 1982!

Cassada RC et al. (1976) Worm Breeder's Gazette "studies on development by the Goettingen C e-group (GCE)."

Von Ehrenstein G, Cole TS, Cassada RC, Deppe U, Schmitt D, Schierenberg E, Krieg C, Yoder B

[Worm Breeder's Gazette, 1976]

We are continuing our study on the development of C. elegans, concentration on embryogenesis. Cell lineage on living eggs has been followed past the 170-cell stage, using Nomarski optics and recordings on video tape. Selected cell types have been followed further, including the gut cells taking advantage of their large size and characteristic autofluorescence. We were able to identify cells uniquely by recording cell division, position of nuclei and cell size. Mitoses proceed in waves from anterior to posterior. From the 16-cell stage onwards groups of cells are clearly distinguishable. The members of each group form a clone. Again, these groups of cells divide largly sequentially from anterior to posterior. Within each group, cell division is essentially synchronous. Every complete wave starts with division of the AB descendants (classically designated Ectoderm), strictly alternating with the mitoses of the P1 descendants (classically called EMST, C and D). After four rounds of cell division a new cycle is initiated before the previous one has been completed. The length of the cell cycle in early cleavage is 15 to 20 minutes (at 22 C). The cycle lengthens gradually to 30 to 40 minutes at the 170-cell stage. We did not observe any cell death in the stages investigated. A coeloblastula with 2-3 very small cavities has been formed at the 24- cell stage. Cleavage and the formation of the three germ layers are not clearly separated in time. Gastrulation starts after the 24-cell stage when the two precursor cells of the endoderm begin to slide into the center. This movement is complete at the 51-cell stage. This type of gastrulation, known as morula delamination, is different from the types of gastrulation so far known in nematodes, as listed in Chitwood. Morula delamination also occurs in some Cnidaria (Clava Laomedea etc.) and also in some insects (e.g. some Collembola). Between the 24- and the 170-cell stage, bilateral symmetry as has been described for Ascaris is not apparent. In the fertilized egg the male and the female pronuclei meet in that part of the egg which at the 2- cell stage becomes the smaller P1-cell (situated at the posterior end). This allows early assignment of the anterior-posterior axis of the egg. The dorso-ventral assignment is possible at this stage by the asymmetry of the membrane of the first cell division. This membrane begins to form dorsally and the final hole in this 'asymmetric iris' closes well in the ventral part of the egg. So far 14 eggs in various stages have been serially sectioned for EM and are being reconstructed by hand or by computer for correlation with and extension of the Nomarski results. The fixation-embedding procedure works on all stages of embryogenesis, including prehatching. Computer capabilities have been increased this year by major hardware additions and by the installation of a new operating system ( RSX). Special attention is being given to computer-aided reconstruction of the embryo from EM series, from video film of light microscopic data, and to the correlation of the EM and LM data. Major enhancement in computer capabilities are in process along two lines. On the one hand the mechanical details associated with the recording process will be made as simple as possible for the user. On the other hand the analysis of the data involving abstraction and simplification will become much more efficient by organizing and storing the data in a 'tree-structured' data base. This will facilitate study not only of the cellular anatomy but also of subcellular structures. For example, we have begun to analyze the distribution of mitochondria in eggs and early cleavage stages. One ultimate goal is to display the important events in embryogenesis in a highly schematized movie on the screen of the graphics display. Some 10,000 EMS mutagenized clones have been tested for temperature sensitive mutants by methods similar to those of Hirsh and Vanderslice (1976), except that about half of the clones came from mutagenized hatchees. Over 100 ts mutants have been selected (designated ts G1, ts G2 etc. according to David Hirsh's nomenclature proposal, where G denotes Gottingen), including some 20% embryological arrest. Our first interest in these mutants is microscopic characterization. Dr. Johji Miwa joined the group at the end of the summer.