[
Worm Breeder's Gazette,
1983]
At the May, 1983 Worm Meetings, I described 2-nm filaments found in the pseudopods of crawling sperm. Recently, much better images of these filaments have been obtained by fixing intact crawling cells on nickel grids, positive staining with uranyl acetate, and critical- point drying. Sperm flatten their pseudopods when they crawl ( thickness ~1-2 m) so that the edges of the pseudopod of whole cells can be studied in a conventional electron microscope operated at 100 KV. These intact cells contain an impressive array of 2-nm filaments surrounded by the amorphous, granular pseudopod cytoplasm. The filaments are relatively short ( 100 nm) and are most oriented nearly parallel to the long axis of the cell. I often find several filaments of about the same length lying parallel to one another but have not seen any interconnections between them. Many of the filaments at the very edge of the pseudopod abut the cytoplasmic face of the plasma membrane. One sidelight of this work has been the continued, consistent failure to find filamentous actin. Sperm clearly lack F- actin and, therefore, must use a different mechanism to crawl. The polypeptide that makes up the 2-nm filaments has not yet been identified. Major sperm protein, described by Mike Klass, remains the leading candidate based on its abundance, its localization in the pseudopod, and its colocalization (by immunofluorescence) with filamentous structures in fibrous bodies of spermatocytes (Ward and Klass, 1982, Dev. Biol. 92: 203-208). I have been trying to immunolabel the 2-nm filaments with an antibody to MSP (we have both a polyclonal rabbit anti-MSP, obtained from Sam Ward, and a new monoclonal anti-MSP generated in our lab). This approach has been hindered by my inability to remove the granular cytoplasm surrounding the filaments without extracting the filaments as well. Recently, I have found that 10-30 min fixation in formaldehyde followed by extraction in Triton X-100 lyses the plasma membrane and removes much of the pseudopod cytoplasm. The pseudopod remnant consists of a honeycomb-like arrangement of cytoplasm which contains numerous 2-nm filaments. These filaments may be sufficiently freed of surrounding cytoplasm to label them convincingly with ferritin- or gold-conjugated antibodies.
[
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.