Rosenbluth RE et al. (1987) Worm Breeder's Gazette "an autosomal X-linked duplication."

Johnsen RC, Rosenbluth RE, Baillie DL

[Worm Breeder's Gazette, 1987]

The duplication sDp30(V:X), on the X chromosome, carries wild type copies of dpy-11(V) and surrounding LGV genes. It arose in a strain carrying a gamma ray induced deficiency, sDf30(V), and presumably was a transposition of most (but not all) the genes deleted by the deficiency. [See Figure 1] The duplication was discovered when conflicting results were obtained from reciprocal complementation tests between the following individuals: dpy-18/dpy-18(III):unc-46 crossed to dpy-18/dpy-18(III);(unc-46) /eT1(V), - [where let-x(sy) was an allele of one of the genes in zones 11B-15 and, in some cases, was linked to unc-46]. Adult Dpy were produced when sDf30 and sDP30 were carried by the hermaphrodite parent, but no such males were produced from the reciprocal crosses. Suppression of the lethal phenotypes by the duplication was not uniform in F1 hermaphrodites having the genotype dpy-18:unc-46 sDf30/( unc-46) let-x(sv);sDp30/+(X). At 15 C most of these hermaphrodites reached adulthood, but not all were fertile (Table), even though the stock strains, maintained at 15 C with the same let gene dosage [dpy- 18/eT1:(unc-46) let-x/eT1], were fertile. This suggests that some genes were under-expressed in the duplication. [See Figure 2] The under-expression of some of the genes may be related to the phenomenon of X-dosage compensation, known to occur in C. elegans ( Meneely & Wood, Genetics 117: 25,1987), which involves the equalizing of X-linked mRNA transcripts in the two sexes (Meyer and Casson, Cell 47: 871,1986). Whether compensation is achieved by elevating expression in the single X chromosomes of males or by reducing expression in the two X chromosomes of hermaphrodites or by a combination of these two mechanisms is not yet clear. Based on data of Meneely & Wood (1987), using dpy-21 and dpy-26, and those of Meyer & Casson (1986), using dpy-27 and dpy-28, reduction of X chromosome expression in hermaphrodites appears to be part of the mechanism. We may, therefore, speculate that in our experiments (1) reduction of X chromosome expression occurred in hermaphrodites, (2) this reduction spread to the linked autosomal genes on sDP30 and (3) either the genes were variably affected by the spreading effect or the mutant phenotypes were variably sensitive to reduced doses of wild type product. To test whether the apparent under-expression of certain sDp30 genes was due to the effects of X-dosage compensation, we plan to study sDp30's properties in the background of dpy-21 which is known to effect dosage compensation (Meneely & Wood 1987). In addition, we hope to acquire new duplications that carry the same genes as sDP30 but are transposed to an autosome. Supported by grants from NSERC and MDA of Canada to DLB.

Fabio Piano et al. (2001) Worm Breeder's Gazette "On the reproducibility of large-scale - RNAi screens"

Aaron Schetter, Fabio Piano, Ken Kemphues, Kris Gunsalus, Valerie Reinke, Stuart Kim, Diane Morton

[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.

Sternberg PW et al. (1980) Worm Breeder's Gazette "postembryonic gonadal cell lineages of Panagrellus redivivus."

Horvitz HR, Sternberg PW

[Worm Breeder's Gazette, 1980]

Anatomical differences between the gonads of the P. redivivus female and the C. elegans hermaphrodite suggested that these structures might differ in cell lineage. The female of P. redivivus is monodelphic (has one ovary) with a single reflexed gonadal arm. A uterus, spermatheca, oviduct, and ovary are directed anteriorly from the vulva; a post-vulval sac of unknown function is directed posteriorly from the vulva. The male gonads of P. redivivus and C. elegans are similar in shape but differ in the internal structure of the vas deferens. We have determined most of the postembryonic cell lineages of the somatic gonad (Z1, Z4) of the P. redivivus male and female. Early in the development of the P. redivivus female gonad Z4. pp dies; this cell is lineally equivalent to the posterior distal tip cell (dtc) of the C. elegans hermaphrodite. In C. elegans, the laser ablation of a dtc prevents growth of the relevant gonadal arm (J. Kimble, Newsletter, January 1980). Thus, the monodelphy of P. redivivus probably results from the programmed death of Z4.pp. P. redivivus may have evolved from a didelphic (two-ovaried) species. The cell divisions in the Z1 and Z4 lineages occur in two distinct periods--the early period extending from the late L1 through the mid- L2, and the late period from early L3 to mid-L4. Early period divisions are shown in the Figure along with those of C. elegans for comparison. The early lineages are similar in both sexes and both species, whereas later lineages differ as do the structures of the gonads. The early lineages of the C. elegans hermaphrodites and P. redivivus female are identical except for the cell death and the timing of divisions (in the female, Z4 divides substantially later than Z1; this first indication of an anterior-posterior asymmetry may be related to the death of Z4.pp and/or to the late period lineage differences between the cell labelled in the Figure as SA and PS). The obviously homologous structures are generated by lineally equivalent cells. Z4.app and Z4.pa give rise to the posterior sheath and spermatheca in C. elegans and the post-vulval sac in P. redivivus, which suggests that the sac is homologous to the sheath and spermatheca. The post-vulval sac may be an evolutionary vestige. In the C. elegans male, Z1.a migrates posteriorly to the distal end of the developing gonad. In the P. redivivus male, the apparently homologous cell is Z1.p, which is generated in the correct position to produce the ordered (cloacal-proximal to cloacal-distal) set of structures in the gonad. The only other difference between the two male early lineages is an extra division of Z1.p and Z4.p in P. redivivus. The linker cell (lc) and the anchor cell (ac), which appear to be homologous, define the proximal end of the gonad in the male and female/ hermaphrodite respectively. The dtc defines the distal end of the gonad. Given these homologies, the early lineage differences between the P. redivivus male and female appear to involve a rotation of Z1, whereas the differences between the C. elegans male and hermaphrodite appear to involve a rotation of Z1.p. Homologies between the structures of the two sexes remain elusive. [See Figure 1] Comparison of the early lineages of the somatic gonad in both sexes of C. elegans and P. redivivus. The lineages up to the time of the L2 molt are shown schematically. C. elegans lineages have been adapted from Kimble and Hirsh (Develop. Biol. 70: 396, 1979). The lineage tree and directions of mitosis are as defined in Sulston and Horvitz (Develop. Biol. 56: 110, 1977); anterior is drawn to the left and posterior is drawn to the right except where otherwise indicated. We do not know at present whether or not P. redivivus exhibits variability in cell fates, for example as in the case of Z1. ppp and Z4.aaa in the C. elegans hermaphrodite. Terminal cell fates are noted in lower case. Cells that divide further are indicated in capitals according to the structures they predominantly generate; a particular structure may be derived from different lineages. Alternate fates are as shown. Cell death is indicated by an 'X'. A dashed line indicates that a migration occurs. Abbreviations: dtc, distal tip cell; ac, anchor cell; lc, linker cell; sh, sheath-like cell equispaced around the developing testis (Z1.pp in P. redivivus might become a dtc in some animals, or might have dtc function although it does not reside at the distal tip); SA, anterior sheath and spermatheca (includes the oviduct in P. redivivus); DU, dorsal uterus; VU, ventral uterus; SP, posterior sheath and spermatheca; PS, post-vulval sac; VD, vas deferens; SV, seminal vesicle; ED, ejaculatory duct (proximal portion of vas deferens); DS, vas deferens and seminal vesicle.