Zaidel-Bar, Ronen (2012) Worm Breeder's Gazette "New lab announcement: Ronen Zaidel-Bar at National University of Singapore"

Zaidel-Bar, Ronen

[Worm Breeder's Gazette, 2012]

Sundaram MV et al. (2000) Worm Breeder's Gazette "Possible perils pertaining to the lin-31 promoter and Pn.p cells"

Dudley NR, Sundaram MV

[Worm Breeder's Gazette, 2000]

Studies of vulval development would be greatly aided by the availability of promoters that drive gene expression specifically in vulval precursor cells (VPCs). One not quite VPC-specific but still useful promoter is that of the lin-31 gene (Tan et al., 1998). P. Tan and S. Kim generously provided us with the PB255 version of the lin-31 promoter, which drives heterologous gene expression in Pn.p cells (including the VPCs) and a limited number of additional cells (mostly neurons). We have been using this promoter ("plin-31") to drive the vulval expression of various ksr-1 variants (*), and as a control, we have also analyzed a plin-31::GFP transgene. When injected into N2 at concentrations of 50-100 ng/l, these transgenes dont have many obvious detrimental effects. However, we find that integrated arrays of our experimental and control transgenes sometimes cause a bar-1-like phenotype (Eisenmann et al., 1998) in which some VPCs never divide but rather adopt a "quaternary" or "fused" fate. The severity of this phenotype varies from line to line, but can be quite strong in some cases (e.g. one plin-31::KSR-1* line had 88% abnormal animals, n=36). Since we also see a weak bar-1 like phenotype in a control plin-31::GFP line (9% abnormal animals, n=22), we suspect that this phenotype could have less to do with ksr-1 than with high levels of lin-31 regulatory sequences (perhaps titrating out other factors such as BAR-1 or LIN-39). We advise other users to be cautious in interpreting similar defects caused by other plin-31 constructs.

Froehli E et al. (1998) Worm Breeder's Gazette "The APC-related gene apr-1 is required for specification of the vulval equivalence group"

Kim SK, Eisenmann DM, Hajnal AF, Froehli E

[Worm Breeder's Gazette, 1998]

The six vulval precursor cells (VPCs) P3.p through P8.p are equivalent in their potential to adopt one of three vulval cell fates that are specified by the anchor cell signal. VPCs are connected to each other and to hyp7 through adherens junctions that stain with the MH27 antibody, while Pn.p cells that do not belong to the vulval equivalence group loose their adherens junctions and fuse with hyp7 during the first larval stage. We are interested in the role of the apr-1 gene in vulval development. apr-1 encodes a protein similar to the human Adenomatous Polyposis Colon tumor suppressor APC. APR-1 co-localizes with the MH27 antigen at the adherens junctions of the VPCs, the seam cells and the lateral hypodermis of the embryo. Since RNAi experiments have indicated that loss of apr-1 function causes an embryonic lethal phenotype (C. Rocheleau et al., Cell 90:707-716 (1997)), we have specifically down regulated APR-1 expression in the VPCs by expressing antisense apr-1 cDNA under control of the Pn.p cell-specific lin-31 promoter. VPCs expressing antisense apr-1 RNA exhibit no detectable APR-1 staining, lack adherens junctions in 53% of the cases (n=114) and adopt an undivided, non-vulval cell fate in 70% of the cases (n=30), resulting in an 80% penetrant vulvaless phenotype. Mutations in the beta-catenin/armadillo related gene bar-1 and in the HOX gene lin-39 cause a similar phenotype, and APR-1 specifically interacts with BAR-1 in a yeast two-hybrid assay. These observations have suggested two possible models: (1) APR-1 might act together with BAR-1 to specify the vulval equivalence group by inducing lin-39 expression in the VPCs, or (2) APR-1 might be required to maintain the adherens junctions and thus prevent the VPCs from fusing with hyp7 before the vulva is induced.

Diana McCulloch et al. (2002) Worm Breeder's Gazette "Evolution of sex differences in lifespan in nematodes"

David Gems, Diana McCulloch

[Worm Breeder's Gazette, 2002]

N2 males live ~20% longer than hermaphrodites when maintained in isolation to prevent deleterious interactions with other worms (1). We have found that this is also true of 9/12 other C. elegans wild isolates tested. Increased male lifespan is therefore typical of C. elegans as a species, and not unique to N2. Why might this male longevity bias have evolved? One possibility is that it is a consequence of protandrous hermaphroditism, which is likely to lead to a skew in the male reproductive probability distribution to later ages. The evolutionary theory of aging suggests that this would result in the evolution of greater longevity in males. To test this idea, we measured the lifespans of both sexes of four dioecious and three more hermaphroditic terrestrial nematode species. However, males proved to be significantly longer-lived than hermaphrodites/females in all other nematode species tested, bar one. This disproved our hypothesis, and suggested that a male longevity bias is typical of free-living nematodes. The exception was C. briggsae, since hermaphrodites were significantly longer-lived than males in the three isolates tested, G16, HK104 and VT847. This could be an evolutionary consequence of the fact that following mating in this species, oocytes are preferentially fertilised by X-bearing sperm, so that outcross progeny are initially mainly hermaphrodites (2). Given that the frequency of males among progeny of selfing hermaphrodites is comparable to that in C. elegans (3), it seems likely that C. briggsae males are exceptionally rare in the wild. Potentially, this rarity results in reduced selection against deleterious mutations with male-specific effects, and the evolution of reduced male longevity. Interestingly, lifespans of males of dioecious species as a whole were greater than those of males of hermaphroditic species (p < 0.001); females were not longer-lived overall than hermaphrodites. Thus, the rarity of males in hermaphroditic species may lead to the evolution of reduced male lifespan.

Goldstein B (1992) Worm Breeder's Gazette "The Timing of Gut Induction, and a Hint of the Mechanics Involved"

Goldstein B

[Worm Breeder's Gazette, 1992]

Previous work has shown that induction plays a role in gut specification in C. elegans: Isolating the EMS cell early in the four cell stage prevents gut from differentiating, and gut differentiation can be rescued by recombining EMS with P2 ,but not by recombining EMS with ABa and/or ABp (1). The time when induction specifies gut was defined by determining how early EMS must be isolated in order to block gut differentiation. In order to determine at what point the induction is no longer occurring, an EMS cell was isolated early in the four cell stage (more than ten minutes before it cleaved - gut will not have been specified by this time). After waiting some time, EMS (or its daughter cells) was recombined with P2 (or its daughter cells). If the induction was still occurring at this time, P2 would still be able to rescue gut differentiation in EMS. If, however, the induction was no longer occurring, P2 would have no rescuing effect. By varying the length of time that EMS is isolated from P2 ,the time at which the induction ends can be determined. Preliminary results may have revealed something about the mechanics of gut induction. In the figure shown below, the first bar represents a summary of the isolation experiments (when EMS is isolated more than ten minutes before it cleaves, gut doesn't differentiate; note that the EMS cell cycle takes 15-20 minutes in culture.) Each of the 10 bars below represents one case where an EMS was isolated early in the four cell stage, was left in isolation for some time, and then was placed back in contact with P2 .These cases define the time at which the induction ends as a few minutes before EMS cleaves (2). In addition, there is no overlap in the timing of positive and negative cases, suggesting that this time is fairly reproducible from one embryo to another. That P2 must contact EMS before EMS cleaves suggests that the induction may effect a cleavage stage specific event, such as the segregation of particular EMS contents into E or MS. Experiments are being done now to determine whether this time represents the time that P2 no longer induces or the time that EMS is no longer competent to respond to the induction. [See Figure 1]

Marie Delattre et al. (2001) Worm Breeder's Gazette "Microevolution of vulval cell lineages within two nematode genera : Caenorhabditis and Oscheius."

Marie-Anne FELIX, Marie Delattre

[Worm Breeder's Gazette, 2001]

The cell lineage of Caenorhabditis elegans and some other nematodes is mostly invariant. Given this invariance, one can wonder how a cell lineage can evolve. We have started a microevolutionary approach by observing lineage variations of vulva precursor cells (VPCs) in different natural nematode populations : 13 strains of the C. elegans species and 5 different species in the Caenorhabditis genus, as well as 32 strains belonging to 3 different species of the Oscheius/Dolichorhabditis genus. Our results show that, within both genera, lineage variations between species and even between strains of the same species occur mostly for VPCs that do not participate to the vulval invagination (VPCs with a 3deg non-vulval fate). These cells are probably not under strong selection pressure and this could explain the large variations observed at a small evolutionary timescale. In the reference strain C. elegans N2, the lineage of the P3.p cell is not invariant (Sulston and Horvitz, 1977). In 50% of N2 animals, P3.p is a true VPC : it is competent to form the vulva and divides once before fusing with the surrounding hypodermis. In the other 50%, P3.p is not competent, does not divide and fuses with the hypodermis. The percentage of occurrence of P3.p division varies between strains of the C. elegans species (from 10% to 60%) and between species of the Caenorhabditis genus (from 0% to 100%). In the C. elegans N2 strain, P3.p division is correlated with the competence of the cell. However, the correlation does not hold in the C. elegans strain CB4857, where P3.p divides in only 15% of the animals whereas it is competent in about 60% (as determined by ablation of P(4-8).p in the L1 stage). P3.p competence varies between Caenorhabditis species, suggesting that the size of the competence group (controlled by lin-39 / bar-1 / lin-22 in C. elegans N2) has evolved between closely related species, but that the program of cell division can be affected even between strains of the same species. Species of the Oscheius genus can be easily found in soil samples from around the world (much more easily than Caenorhabditis species). Thus, we have been able to compare a large variety of strains in this group. Variations mostly concern P4.p and P8.p lineages : these cells divide twice, once, or not at all, but are always competent to replace the vulval cells P(5-7).p. Variations observed between strains of the same species are larger in Oscheius sp.1 than in C. elegans . Therefore, we have undertaken a genetic analysis between two pairs of strains of Oscheius sp. 1 that show distinct P4.p and P8.p lineages. For each pair, at least three loci are involved in the phenotypic differences. We also find that variation at one locus has a relatively strong effect on the phenotype (instead of small additive effects of many genes). Within both Caenorhabditis and Oscheius , a similar range of variations is found between strains of the same species and between closely related species of the same genus. Thus, cell lineage variations observed between species could be due to fixation of variants that segregate within species.

Cleavinger PJ et al. (1988) Worm Breeder's Gazette "analysis of transcription in early Ascaris embryos."

Cleavinger PJ, Bennett KL

[Worm Breeder's Gazette, 1988]

We have exploited some of the unique features of embryos of the parasitic roundworm, Ascaris des to observe transcriptional patterns in nematode early embryogenesis. In contrast to the free-living nematode Caenorhabditis ble to obtain large synchronous populations of Ascaris embryos at various stages of development. It has been reported in C. elegans that transcription is detectable at approximately the 100-cell stage. [Hecht, et al., (1981). Dev. Biol. 83:374-379]. More recent work in C. elegans suggests that transcription is occurring at or before the 30-cell stage. [Schauer, et al., WBG 10:1, 72-73.]. Our laboratory has recently reported that Ascaris embryos in the 5-6th cleavage stage are actively transcribing. [Dalley, et al., WBG 10:1, 76.]. From staged embryos we have isolated nuclei to determine run-on transcription characteristics. Initial studies performed on nuclei isolated by the method of Dixon et al. [WBG 9:3, 73-74.] resulted in contamination of the embryonic preparations with mitochondria, as seen by microscopic examination after diamidinophenylindole (DAPI) staining, as well as a strong hybridization signal using a mitochondrial probe. Subsequent purification of nuclei on 50% Percoll gradients has yielded about a 100-fold visual reduction in mitochondria, although a hybridization signal can still be detected. Run-on transcription assays were performed on nuclei isolated from staged embryos of 4-8 cell, 24-30 cell, ~60 cell, and 10 day embryos (about 600 cells). Parallel assays were carried out with and without alpha-amanitin present at 1 g/ml a level which inhibits RNA polymerase II activity. The results are shown on Fig. 1 as counts of [3H]-UTP incorporated per 10 l reaction. Total levels of incorporation were typically 10-35 fold over background, which has been subtracted from each of the values shown. The darkened portion of each bar represents the alpha-amanitin sensitive (RNA polymerase II) fraction of total transcription. We consistently detect RNA polymerase II activity as early as the 4-8 cell stage and by the 30 cell stage active mRNA transcription is occurring. When DNA content of preparations was measured by fluorimetric assay the highest level of incorporation per pg DNA was at the 30 cell stage. Similar transcription reactions were carried out with [32P]-UTP, with the labelled RNAs used to probe duplicate dot blots containing cold DNA probes. Results from the 60-cell stage showed no hybridization to the SP6 vector or the Ascaris sperm- specific MSP cDNA. Ascaris ribosomal and actin probes showed significant hybridization (Fig. 2). When the labelling was carried out in the presence of 1 I/ml alpha-amanitin, the actin signal was eliminated, while the ribosomal signal was retained as expected. This shows that we are able to detect specific mRNA transcription occurring in staged embryos. Similar studies are being carried out with each of the mentioned cell stages to establish if specific messages, i.e., actin for now, can be detected at the 4-8 cell stage. We propose that differential library screening using staged early vs. Iate RNA populations will yield transcripts unique to the early embryo. The following two references have been valuable for run-on transcriptions: J. R. Nevins, Methods in Enzymology, (1987), 172:235-241. M. E. Greenberg, Current Protocols in Molecular Biology, (1987), 4. 10.1-4.10. 7