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[
Worm Breeder's Gazette,
1993]
THE C. ELEGANS CLEAVAGE AND POLYADENYLATION SlGNAL Tom Blumenthal, Department of Biology, Indiana University. Bloomington, IN 47405; Owen White and Chris Fields, Institute For Genomic Research, Gaithersburg, MD, 20878
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[
Worm Breeder's Gazette,
1999]
For everyone who deals with the characterization of expression patterns in the nervous system, the truly impressive paper of White et al., 1986 ("Mind of the Worm") serves as the ultimate source of knowledge. While the largely invariant neural cell body positions described by Sulston et al. are an essential tool in the identification of neurons, the axon morphologies described by White et al. greatly facilitate the identification of a given neuron.
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[
Worm Breeder's Gazette,
1980]
In our laboratory, we routinely use dauer larvae to obtain large, synchronous cultures of L4's and adults for biochemical purposes. We have found that large numbers of dauer larvae can be obtained using eggwhite plates (a modification of the method of D. Baillie and R. Rosenbluth, WBG 2 (1)). Egg-white plates are prepared by stirring the white of one chicken egg with 50 ml of boiling distilled water for several seconds, homogenizing the mixture for one minute in a Waring blender, and layering 3-4 ml of the resultant liquid slurry onto a standard 100mm NGM plate containing a lawn of E . coli . After drying overnight, egg-white plates are seeded with about 1500 dauer larvae and incubated at 20 C . These dauer larvae develop into adults, but for some unknown reason a large proportion of their progeny develop into dauer larvae and become arrested at this stage . Approximately 1x10+E5 dauer larvae are usually recovered per plate after 5-7 days incubation time . Yields as high as 2x10+E5 per plate have been obtained in some instances. For purification of dauer larvae, animals washed from individual egg- white plates are incubated with 5-10 ml of 1% SDS for 30-60 minutes, collected by low speed centrifugation, resuspended in 0.5 -1 ml of buffer, and centrifuged through a 2 ml cusion of ice-cold 15% ficoll for 10 minutes at 300 xg. Intact dauer larvae pellet through the ficoll, while egg matter and worm carcasses remain at the interface . Because a small number of non-dauer animals sometimes escape this first SDS treatment, the entire procedure is usually repeated . Purified dauer larvae can be stored in Mg buffer at 16 C until use and retain good viability for 30-60 days . It has been our experience that dauer larvae obtained from egg-white plates recover much more synchronously than do dauer larvae isolated from starved E . coli plates, especially if they are used within 2 weeks of isolation. Also we have noted that the synchrony of recovering populations tends to decrease as dauer larvae are stored for longer periods of time.
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[
Worm Breeder's Gazette,
1996]
We have followed the uterine nuclei from the completion of the lineages in early L4 (Kimble and Hirsh, 1979) through the beginning of L4 lethargus. Differentiated uterine cell types have been defined by EM reconstruction (White, Southgate, and Kershaw, WBG 11(3) p.75); they are the toroidal ut cells that make structural epithelium and specialized utse and uv cells that make the connection between the uterus and the vulva. Comparison of Nomarski and EM data enabled us to correlate differentiated fate with lineal position. The 60 uterine cells are produced by the dorsal uterine (DU) and ventral uterine (W) precursor cells. The anchor cell (AC) induces adjacent W granddaughters (which are intermediate precursor cells) to adopt the fate rather than the ground state p (Newman, White, and Sternberg, Development 1995). For convenience, we have assigned names to the DU and W great grandprogeny: Dl-8 (from anterior to posterior) and Vl-12 (see diagram). The cells that contribute to each of the differentiated cell types are as follows:
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[
Worm Breeder's Gazette,
1981]
High population density influences dauer larva formation as demonstrated by growth of nematodes to high density either in liquid medium, on enriched agar plates or on egg white plates. Dauer larvae are produced under these conditions by wild-type animals prior to exhaustion of the food supply. Our recent work, based on the observations of Cassada and Russell (Develop. Biol. 46:326,175), has shown that the medium from such cultures contains a stable, nematode- produced substance of low molecular weight which both promotes dauer larva formation and inhibits exit from the dauer stage. We have named the substance DRIF, for dauer recovery inhibition factor. DRIF can be detected in starved or non-starved liquid culture media, and it can be extracted from worms washed off of non-starved NGM plates. A dauer-recovery bioassay for DRIF has been developed which allows its detection at a 1/120 dilution from starved liquid culture media. DRIF's effect on both dauer larva formation and recovery is in competition with the food supply and can be overridden by an abundance of food. E. coli cells in a concentration greater than 2x10+E9/ml overcome inhibition of dauer recovery by DRIF at starved media concentration. Since the bacteria do not degrade DRIF, we conclude that bacteria and DRIF provide competitive chemosensory signals. DRIF is produced by C. elegans var. Bristol and Bergerac, C. briggsae, and all of the dauer-defective mutants assayed so far. It cannot be found in liquid culture media which has not been inoculated with worms, nor can it be found in starved liquid cultures of Panagrellus redivivus. (Panagrellus does not make dauers.) DRIF has a molecular weight of less than 1000 daltons and has a net negative charge at pH7. It is nonvolatile and cannot be extracted with ether or chloroform:methanol (2:1). It is stable to various treatments including autoclaving, treatment with acid or base, and digestion with protease, DNAase, RNAase, and phosphodiesterase. We are in the process of purifying DRIF so that its structural and biological properties may be more thoroughly studied. Our hypothesis is that DRIF may be the environmental cue which triggers dauer formation in crowded cultures. Our liquid cultures reach a density more than 10-fold greater than that reached by populations on NGM plates. As reported by Peg Swanson in the last Newsletter, dauer-defective mutant phenotypes suggest that the response to starvation is at least partially distinct from the response to high population density. Some mutants, e.g. CB1377
daf-6, do not form dauers in liquid or on plates, while other dauer- defective mutants, e.g. CB1376 (
daf-3), form many dauers when grown to starvation in liquid. Thus, CB1377 is blocked in both responses, while CB1376 is blocked in the starvation response, but nearly normal in the response to high population density. It is conceivable that different sensory neurons could mediate the responses to different stimuli. Several laboratories have observed independently that large numbers of dauers form in cultures grown on egg-white plates. Interestingly, worms grown on such plates 'phenocopy' dauer-constitutive mutants in that adults retain eggs, and the dauers formed are large (well-fed). Since chicken egg-white itself does not contain DRIF activity, we presume that dauer formation on egg plates is due to nematode DRIF production, perhaps combined with a declining food supply. It is difficult to determine when an egg white plate is 'starved' because the bacterial food is not visible in all that mess. The worms will not grow on sterile egg white plates.
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[
Worm Breeder's Gazette,
2002]
The nervous system of C.elegans displays significant patterns of bilateral symmetry (Hobert et al., 2002). Deviations from this symmetry are usually highly stereotyped. For example, the unilateral RIS neuron is always located on the left side of the animal, but never on the right side (White et al., 1986; this observation, based on a limited number of EM-reconstructed animals, has now been confirmed with gfp reporters). One major exception to this stereotyped laterality is represented by the expression of the putative odorant receptor gene
str-2, which occurs stochastically in either the left or right AWC neuron (the distribution is about 50:50; Troemel, et al., 1999). Are there other examples of stochastic laterality in the nervous system? In their EM reconstruction work, White et al. (1986) noted that the unilateral RID motor neuron (see Fig.), whose cell body is located on the dorsal midline, sends its axon along the right side of the nerve ring in one reconstructed animal, but along the left side in another reconstructed animal. Could this observation reflect a stochastic choice of the growth cone when encountering the dorsal side of the nerve ring? The sample size of two obviously did not allow one to derive such a statement, but the advent of gfp reporter technology allows us to address this question. Several previously described gfp reporter strains show RID expression, yet the presence of GFP in the axons of many other neurons precluded the tracing of the RID axon. A promoter fragment from a putative serotonin-like receptor gene that we study in the lab drives expression of gfp in RID and only one additional pair of neurons, allowing us to trace the axon of RID. Taking advantage of this, we found that in 56 out of 58 animals, the axon traveled along the left side of the nerve ring, while in 2/58 animals, the axon migrated on the right side. In another genetic background in which a gfp marker is ectopically expressed in RID (Is[
unc-119::
ttx-3;
ttx-3::gfp]), we found a similar distribution; namely 46 out of 48 animals have their RID axon on the left side.
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[
Worm Breeder's Gazette,
1984]
The anatomy of the nervous system of the large parasitic nematode Ascaris des is similar in many respects to the nervous system of C. elegans. An adult Ascaris can be as large as 30 cm and weigh 10 grams while a typical C. elegans may be 1 mm long and weigh a few micrograms. Yet, both worms have about the same number of neurons. In addition, the nerve cells are arranged in the same basic pattern throughout the animals and many of the neurons have similar shapes in the two worms. It has been known for some time, for example, that motorneurons in the two animals have identical shapes and make similar synaptic connections. On the other hand, homologies between interneurons in the two species have been less clear. In order to compare different classes of neurons more fully, we have examined the anatomy of the retrovesicular ganglion (RVG) of Ascaris, which contains both interneurons and motorneurons. We find that each RVG cell in Ascaris appears to have a homolog in the RVG of C. elegans. The homologies are based on the unpublished reconstructions of head neurons by John White. [see Figure 1]
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[
Worm Breeder's Gazette,
1994]
Cell-cell contacts controlling early cell division axes Bob Goldstein, MRC, Cambridge, England Cell division axes are often controlled precisely in embryos, by intracellular cues (for example, Hyman and White, 1987; Dan, 1979), or by extracellular cues. Extracellular cues have been found to work either by mechanical deformation of cell shape (Symes and Weisblat, 1992) or by induction, as changes in cell fate are often followed by changes in cell division patterns (Sternberg and Horvitz, 1986). I have found evidence that the division axes of some early cells, EMS and E, are controlled by specific cell-cell contacts with their posterior neighbors (EMS P2 or E-P3 contact). Altering the orientation of contact between these cellsl alters the orientation in which the EMS or E cell divides. I have been following this up with timelapse videomicroscopy of centrosome movements and anti-tubulin immunofluorescence to visualise asters and centrosomes. These have been done primarily in the EMS cell, in both intact embryos and isolated cell pairs. Contact-dependent mitotic spindle orientation appears to work by establishing a site of the type described by Hyman and White (1987) and Hyman (1989) in the cortex of the responding cell: one centrosome moves toward the site of cell-cell contact during rotation, both in intact embryos and re-oriented cell pairs.2 The effect is especially apparent when two donor cells are placed on one side of the responding cell. Both centrosomes are "captured", pulling the nucleus to one side of the cell. No centrosome rotation occurs in the absence of cell-cell contact, nor in nocodazole-treated cell pairs. The relationship between gut induction and spindle orientation in EMS is being examined, as both require contact between P2 and EMS. When P2 and EMS are isolated in the first five minutes of the EMS cell cycle, neither effect occurs. Placing P2 onto EMS'soon after this time still rescues gut induction, but can no longer rescue the spindle orientation effect. In these cell pairs EMS divides in various orientations, yet gut differentiation generally occurs, suggesting that proper spindle orientation is not necessary for gut induction. There is however one spindle orientation which appears to be incompatible with gut induction: when the EMS cleavage furrow forms directly through the site of cell-cell contact, gut differentiation does not occur (0/10 cases, compared to 14/14 of other orientations). The results suggest that some of the cortical sites described by Hyman are established cell-autonomously (in P1, P2, and P3), and some are established by cell cell contact (in EMS and E). Contact- dependent mitotic spindle orientation appears to play a role in ensuring that developmental information received via induction is partitioned between daughter cells. It might also play a role later in morphogenesis, generating lines of cells in the embryo. Notes: 1. That orientation of cell pairs was altered has been confirmed by seeing that no whole cell rotation occurs in high magnification timelapse recordings of cell pairs, and by noting in live and fixed cells the random position of the centrosomes after cells are apposed. 2. The direction of rotation in EMS is at odds with Waddle's finding that actin- capping protein localises to the anterior side of EMS during rotation (Waddle et al., 1994). Schierenberg previously noted that the nucleus in these cells moves posteriorly to the site of cell-cell contact, and is dependent on contact with P2 in EMS (Schierenberg, 1987). References: Dan (1979) Dev Growth Diff 21(6):527-535. Hyman and White (1987) J Cell Biol 105:2123-2135. Hyman (1989) J Cell Biol 109:1185-1193. Schierenberg (1987) Dev Biol 122:452-163. Sternberg and Horvitz (1986) Cell 44:761-772. Symes and Weisblat (1992) Dev Biol 150:203-218. Waddle et al. (1994) Development 120:2317- 2328.
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[
Worm Breeder's Gazette,
1997]
Cell interactions, fusions and migrations are processes involved in the formation of organs. In C. elegans these have been widely reported to occur in the formation of the hypodermis, uterus, excretory gland cells, pharynx and vulva. We have been studying the formation of the vulva in C. elegans by characterizing the pattern of cell recognitions, migrations and fusions by using immunofluorescent techniques and confocal microscopic reconstructions. Vulval sections at different times during the developmental process were stained for MH27, an adherens junction protein (1). Events have been followed from the time the fates of the six vulval precursor cells (VPCs) have been specified (27 hours; times are in hours after hatching), the generation of the VPC daughters (30 hours), VPC grand-daughters (32 hours; late L3), which results in 22 epithelial cells of the vulval lineage (34 hours) to the construction of a tube that consists of seven toroidal rings stacked together. These rings are designated as: vula, vulb1, vulb2, vulc, vuld, vule and vulf (2). These studies have revealed a definite pattern and order of cell migrations and fusions which allow us to propose a pathway for vulval morphogenesis, with the following intermediates being identified till now In the first stage a vulval primordium consisting of a longitudinal row of 12 cells (after two rounds of division), shows no signs of differentiation (Fig.1A) followed by a stage where the six central cells are polarized towards the center of symmetry before the last round of division (Fig.1B). During the third and final round of divisions six cells divide transversely, two cells do not divide and four divide longitudinally (3), (Fig.1C). This intermediate shows a central ring precursor containing four cells (vulf), four more that form the next ring (vule) and a third ring (vuld) with two cells. The next two cells (!c!) on each side have divided and are sending processes towards the centre of symmetry of the vulva surrounding vuld. The last schematic intermediates (Figs. 1D-H) show the seven rings that have completed the migrations and are in different stages of cell fusions starting with an intermediate where all seven rings are unfused followed by several intermediates: Fig.1D-rings vula and vulc are partially fusedand contain two binucleate cells each, Fig.1E-vuld has fused, Fig.1F-vula and vulc have fused, Fig.1G-vulf has fused, Fig.1H-vule has fused. These events studied in time thus reveal a pathway for vulva formation in C. elegans. 1. Podbilewicz, B. and White, J.G. (1994). Dev.Biol. 161, 408-424. 2. White, J.G. Personal Communication. 3. Sulston, J.E. and Horvitz, H.R. (1977). Dev. Biol. 56, 110-156.
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[
Worm Breeder's Gazette,
1992]
I have assembled an image archive system based in principle on John White's '4D microscope' (now marketed by BioRad) but differing in that images are stored digitally as opposed to the analog LaserDisk storage used in the White/BioRad system. Message: internal system error : Malloc error at 10041a8c length 256: end overwritten with 345678 Message: internal system error : Malloc error at 10041a8c length 256: end overwritten with 345678 Our archive system is based on a relatively inexpensive computer (a 'clone' of an IBM type PC). The computer controls a focusing motor, which cycles the microscope through different focal planes. For each focal plane, an image is collected from a video camera, digitized, and stored (in a somewhat compressed format) on a high capacity magnetic disk drive. In a typical experiment lineaging a single embryo, images would be taken in each of 25 consecutive focal planes in a cycle, with each cycle lasting 30 seconds. The current system (with 2.5 gigabytes of disk storage) can accommodate approximately 18,000 full frame images: six hours of continuous recording at maximum resolution. Once recorded, images stored on the disk can be 'played back' in any order. Generally a series from a single focal plane is played consecutively in either forward or reverse time. The focal plane can be controlled during playback (using keyboard or joystick), allowing individual cells to be followed as they move between focal planes. Theoretical playback rates are as fast as 60 full frame images per second, but currently the system can only run at a fraction of this rate (3-5 full frame images per second). Although rapid playback is useful for general impressions of developmental events, precise lineaging generally involves using the keypad to step through time and focal planes to follow single cells. Image series can be archived by copying information from the magnetic disks onto digital tape. An image series stored on tape can be restored at any time by recopying the data onto the hard disk of the system. Each digital tape holds about 40,000 full frame images, costs $5 and can be rewritten multiple times. A disadvantage of digital tape archiving is a waiting time of 1-3 hours for an archived image series to be copied from tape back to disk. When compared to the magnetic tapes that we are using, the analog LaserDisks used to store images in the White/Biorad system have some advantages and disadvantages: LaserDisks can be readily interchanged, provide rapid access to 43,500 images per side, and provide faster playback rates. Unfortunately the LaserDisks cannot be erased or rewritten and are costly (around $200 each; the laserdisk recorder is also considerably more expensive than magnetic disk and tape drives). Unprocessed image quality should be comparable for analog and digital storage systems. The digital system allows for more sophisticated image enhancement and analysis techniques, but at present we are using only very simple image averaging and contrast enhancement. This gives screen images almost equivalent to viewing specimens directly through the microscope. While early lineages can easily be followed with the system as it is now working, later lineages (i.e. the last two cell divisions) are more difficult to follow reliably. Current efforts aimed at improving image resolution and data compression should facilitate later lineaging. A second type of experiment for which the setup has been used is to follow large groups (10-30) of embryos at lower magnification (nomarski observation with a 20 or 40x objective; generally 8-12 focal planes recorded for 12-14 hr series). At this resolution one can observe early cleavages, cell movements, morphogenesis and embryonic muscle function. Being able to follow embryos in retrospect (e.g. only those embryos that fail to hatch) facilitates analyses of the earliest defects and course of development of zygotic lethal mutations and deficiencies. A parts list and the software driving the system are available. Because our system is pieced together and includes some older equipment from the Carnegie attic, duplication would require some mechanical tinkering and likely some programming. (Thanks to John White, Joe Vokroy and Jim Priess for their help)