[
Worm Breeder's Gazette,
1994]
Update on deficiency screens Joohong Ahnn and Andrew Fire Carnegie Institution of Washington, Department of Embryology, Baltimore, MD 21210 We have been screening available chromosomal deficiencies in order to identify genetic loci whose zygotic expression is required for early events in embryogenesis including formation of body wall muscle cellsl. A surprising result of deficiency screens (oursl and others2 3) has been the extensive tissue differentiation that occurs in many of the deficiency homozygote embryos. In our initial analysis we found only one region (on the left tip of LGV) which was required for accumulation of body-wall myosin. This gazette article reports updates on our deficiency analysis. Analysis of additional characterized deficiencies: Embryos homozygous for nDf3 (II)4 and nDf4 (Jl)4 arrest elongation before comma stage (although a small fraction develop to the 1-112 fold stage). These embryos form pharyngeal structures, have gut granules and stain with antibodies to
unc-54 and
myo-3 produsts. Deficiency sDf23 (IV)s homozygous embryos arrest before comma stage, have gut granules but no pharyngeal structures; these embryos do not show any movement but stain with both anti-myosin antibodies. nDf42 (V)6 homozygous embryos show an early arrest with no movement, no gut granules, and no pharyngeal structures. Homozygous embryos from nDfl 7 (m)4 and nDfl 9 (X)7 both arrest at 2-fold stage, show twitching movement, have gut granules, form pharynx and stain with myosin antibodies (n.b. mapping data on ACEDB suggests nDfl 7 and nDfl 9 could be compound deficiencies). Producing a bank of Dfs of VL: In order to isolate small deficiencies in the region of LG VL which appears to be essential for myogenesis, gamma-ray induced mutagenesis has been conducted. Mutagenized N2 males were crossed into
unc-34 dpy-l l animals, and rare Unc Non- dpy F1 progeny were cloned. Seven deficiencies have been isolated among 17,000 haploid chromosomes screened so far. Further mapping of these deficiencies by physical and genetic means should allow us to narrow down the myogenic locus (or loci) in the region. A very early zygotic requirement ? In a more general screen for early arrest mutants, several very early lethals linked on LG m have been isolated. These segregate a high fraction of dead embryos, suggesting a chromosomal break or rearrangement. About 1/4 of the embryos from these strains show a characteristic very early terminal phenotypes. These embryos have 200-2S0 nuclei, with no overt tissue differentiation. The embryos fail to stain with myosin antibodies and do not have gut granules. Embryos from two of these strains were observed with 4-D microscopy to look for defects during early embryogenesis. The early cleavage pattern of early-arrest embryos look normal, but these embryos fail to complete last 1-2 cell divisions. In some cases, nuclear divisions appear to complete without subsequent cytokinesis. It is interesting to compare these terminal phenotypes with those of
emb-29 (V).
emb-29 produces embryos with comparable numbers of nuclei, but these embryos exhibit extensive differentiation8. This suggests that the putative chromosome m defect is not simply due to blockage or failure of cell divisions. WBG 12: 2, 106 Genetics (137:483-498) 2. F. Storfer-Glazer & W. Wood Genetics (137: 499-508) '93 C. elegans Meeting (Abt. 497) 4. I. Greenwald & R. Horvitz unpublished 6. M. Hengartner & R. Horvitz unpublished 7. V. Ambros & R. Horvitz unpublished 8. R. Hecht et. al J. of Cell Science (87:305- 314)
[
Worm Breeder's Gazette,
2001]
RNAi is being used routinely to determine loss-of-function phenotypes and recently large-scale RNAi analyses have been reported (1,2,3). Although there is no question about the value of this approach in functional genomics, there has been little opportunity to evaluate reproducibility of these results. We are engaged in RNAi analysis of a set of 762 genes that are differentially expressed in the germline as compared to the soma (4 -- "Germline"), and have reached a point in our analysis that allows us to look at the issue of reproducibility. We have compared the RNAi results of genes in our set that were also analyzed by either Fraser et al. (1 -- Chromosome 1 set "C1") or Gonczy et al. (2 -- Chromosome 3 set "C3"). In making the comparison we have taken into account the different operational definition of "embryonic lethal" used by the three groups. In the C3 study, lethal was scored only if there were fewer than 10 surviving larva on the test plate, or roughly 90% lethal. In our screen and the C1 screen the percent survival was determined for each test. To minimize the contribution of false positives from our set, in our comparison with the C1 set we defined our genes as "embryonic lethal" if at least 30% of the embryos did not hatch, but included all lethals defined by Fraser et al. (> 10%). For our comparison with the C3 set, we used a more restrictive definition of "embryonic lethal" that required that 90% of the embryos did not hatch. (This means that in Table 1, five genes from our screen that gave lethality between 30-90% were included in the not lethal category; one of these was scored as lethal by Gonczy et al.). We have analyzed 149 genes from the germline set that overlap with the C1 set and 132 genes that overlap with the C3 set. The table below shows the number of genes scored as embryonic lethal (EL) or not embryonic lethal (NL) in each study. (Note that these comparisons do not include data from our published collection of ovary-expressed cDNAs.) Table 1. Comparing RNAi analysis of the same genes in different studies. Germline Chromosome 1 Germline Chromosome 3 NL (117) EL (32) NL (97) EL (35) NL (104) 100 4 NL (89) 87 2 EL (45) 17 28 EL (43) 10 33 Overall, the degree of reproducibility is high. The concordance between our results and the published results was 86% with C1 (128/149 genes) and 90% with C3 (120/132). However, we scored a larger number of genes as giving rise to embryonic lethal phenotypes than the other studies did. What does this mean? One possibility is that we are generating a large number of false positives (God forbid!). The other interpretation is that there is a fairly high frequency of false negatives in each screen (4-8% in our screen (2/45; 4/49); 22% in the C3 screen (10/45); and 35% (17/49) in the C1 screen). It is no surprise that the different methods used by the three groups resulted in slightly different outcomes and we can only speculate on which methodological variation contributed most. In comparing our methods to those used in the C3 study we note that our two groups used different primer pairs for each gene; that we tested genes individually while they tested genes in pairs; and that the operational definition of "embryonic lethal" differed. Considering the latter two differences, we speculate that even with pools of two, the competition noted by Gonczy et al. in dsRNA pools could reduce levels of lethality below the 90% cutoff. The major difference between our approach and the C1 approach is feeding vs. injection, raising the possibility that for some genes feeding may be a less effective means of administering dsRNA. Whatever the basis for the difference, these comparisons indicate that genes scored as "non-lethal" in any single study may show an embryonic lethal RNAi phenotype when reanalyzed. It therefore seems useful to have more than one pass at analyzing C. elegans genes via RNAi. We are indebted to P. Gonczy for very useful comments. Fraser, A. G., Kamath, R. S., Zipperlen, P., Martinez-Campos, M., Sohrmann, M. and Ahringer, J. (2000). Functional genomic analysis of C. elegans chromosome I by systematic RNA interference. Nature 408 , 325-330. Gonczy, P., Echeverri, G., Oegema, K., Coulson, A., Jones, S. J., Copley, R. R., Duperon, J., Oegema, J., Brehm, M., Cassin, E. et al. (2000). Functional genomic analysis of cell division in C. elegans using RNAi of genes on chromosome III. Nature 408 , 331-336. Piano, F., Schetter, A. J., Mangone, M., Stein, L. and Kemphues, K. J. (2000). RNAi analysis of genes expressed in the ovary of Caenorhabditis elegans. Curr Biol 10 , 1619-1622. Reinke, V., Smith, H. E., Nance, J., Wang, J., Van Doren, C., Begley, R., Jones, S. J., Davis, E. B., Scherer, S., Ward, S. et al. (2000). A global profile of germline gene expression in C. elegans. Mol Cell 6 , 605-616.