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]

Goldstein B (1994) Worm Breeder's Gazette "What Happens to E When It Doesn't Make Gut?"

Goldstein B

[Worm Breeder's Gazette, 1994]

The E lineage in C. elegans is induced to form gut by an interaction between P(2) and EMS early in the four-cell stage(1). Normally P(2) contacts the side of EMS which gives rise to the E lineage, however the other side of EMS also is competent to respond to gut induction: moving P2 to the opposite side of EMS causes gut to form from what normally would have been the MS lineage. (2) Here I discuss recent results concerning how E and MS differentiate in the absence of gut induction, to address what the "uninduced state" of EMS is, and to determine if MS is also affected by the P(2)-EMS interaction. EMSs were isolated at various times in the four-cell stage and were cultured overnight. Isolates were examined for birefringent rhabditin granules, and then were processed for either an esterase histochemical stain or for immunohistochemistry. The progeny of induced EMSs (isolated late) formed gut as assayed by rhabditin granules, esterase stain and ICB4 antibodies, body wall muscle as assayed by antibodies to paramyosin, and pharyngeal muscle as assayed by 3NB12 antibodies. No hypodermis was formed, as assayed by antibodies to LIN-26 .This suite of cell types is consistent with EMSs normal fate,(3) E forming gut, and MS forming mostly pharyngeal muscle and body wall muscle. The progeny of uninduced EMSs (isolated early) formed about twice as much body wall muscle and pharyngeal muscle as induced EMSs, but no gut and no hypodermis. This suite of cell types suggests that both daughters of EMS are differentiating as MS lineages, and is consistent with previous results concerning the lineage timing of uninduced EMSs.(l) Each daughter of EMS was examined similarly to see if each do in fact form body wall muscle and pharyngeal muscle in the absence of gut induction. Again EMSs were isolated at various times in the four-cell stage, only this time after EMS divided once, the two daughters were separated and were cultured independently. lnduced EMSs (isolated late) formed all gut from one daughter, and body wall muscle and pharyngeal muscle but no gut nor hypodermis from the other daughter, consistent with one acting as an E lineage and the other as an MS lineage. Uninduced EMSs (isolated early) formed two daughters both of which formed body wall muscle and pharyngeal muscle, and no gut nor hypodermis, consistent with both acting as MS lineages. These results and the experiment in which P(2) was moved to the other side of EMS imply that induction by P(2) is the cue that males one side of EMS differentiate differently than the other side. The results also suggest that this induction affects only E and not MS; the uninduced state of EMS is essentially as an "MSMS" cell. It is interesting then that the induction occurs before EMS divides to form E and MS; gut induction appears to affect only part of a single responding cell. Additionally the results lend further support to the notion that the MS suite of cell types form independently of cell interactions with for example the gut lineage or with ABa or ABp derivatives.(4)

Goldstein B (1994) Worm Breeder's Gazette "Cell-Cell Contacts Controlling Early Cell Division Axes."

Goldstein B

[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.

Goldstein B (1992) Worm Breeder's Gazette "Identification of the Cues Used to Generate Blastomere Diversity:The Role of the P2-EMS Interaction in Positioning Gut Fate Into E"

Goldstein B

[Worm Breeder's Gazette, 1992]

The gut of C. elegans derives from all the progeny of the E cell, a cell of the eight cell stage (Sulston et al. 1983 Dev. Biol. 100:64.) In order for E to form gut, EMS must contact P2 during the four cell stage (Goldstein 1992 Nature 357:255; [See Figure].) P2 'sposition on the "E" side of EMS raises the possibility that the P2 -EMSinteraction is the cue that makes E different from MS. Alternatively, the E and MS sides may be different from each other before the P2 -EMSinteraction, such that only the E side can form gut in response to the interaction. In order to determine which is the case, I tried to move P2 to the MS side of EMS, to see if MS then forms gut. This is a particularly frustrating manipulation. A more feasible way to address the question turned out to be isolating P2 and EMS, and then placing the two cells back in contact in a random orientation. If any side of EMS can form gut in response to the interaction, then gut should always form from the daughter of EMS that contacts P2 .If however only the E side can form gut, then in some cases either gut will form from the daughter away from P2 or gut will not differentiate. Fortunately, P2 and EMS stick immediately upon contact and they do not rotate around each other when placed back together, precluding the possibility that P2 moves back to its original position. Which daughter of EMS formed gut was then determined by one of two methods: In five cases cell division times were watched to see which daughter took on E-like cell division timings, and in another five cases the daughters of EMS were separated from each other and cultured individually to see which produced gut granules. Gut differentiated from the side of EMS on which P2 was placed in all ten cases. This result indicates that any side of EMS can form gut in response to the induction, and hence that the interaction with P2 is what makes E different from MS. Current work is aimed at identifying cellular mechanisms by which the induction makes E different than MS, and determining whether cell interactions are required at other stages for gut fate to be segregated into E.

Seydoux GC et al. (2000) Worm Breeder's Gazette "Reversal of Anterior/Posterior Polarity in 1-Cell Embryos Arrested in Meiosis - Implications for How Embryonic Polarity Is Established"

Seydoux GC, Wallenfang MR

[Worm Breeder's Gazette, 2000]

In C. elegans, A/P polarity is established after fertilization and is thought to be determined by the site of sperm entry, which defines the posterior pole of the embryo (Goldstein and Hird, 1996). The nature of the cue brought in by the sperm is not known. Using a collection of 1-cell arrested mutants, we have obtained evidence that the cue may be provided by microtubules emanating from the sperm asters.

Wiegner O et al. (1996) Worm Breeder's Gazette "Cephalobus spec. shows a different pattern of gut induction and differentiation"

Schierenberg E, Wiegner O

[Worm Breeder's Gazette, 1996]

Gut induction is a well known example for cellular interaction in the early C. elegans embryo (Schierenberg, 1987; Goldstein, 1992). Contact between the germline cell P2 and the gut precursor cell EMS in the early 4-cell stage is necessary to specify the gut fate. As a consequence of the interaction, EMS divides into an anterior MS cell and a posterior E cell and the gut primordium is formed by the progeny of the latter.

Badrinath AS et al. (1998) Worm Breeder's Gazette "Different patterns of mitochondrial redistribution in early C. elegans and Acrobeloides sp. PS1146 embryos."

White JG, Badrinath AS

[Worm Breeder's Gazette, 1998]

The redistribution of mitochondria in the period following fertilization through the first cell division was followed in the early embryos of two related nematodes, Caenorhabditis elegans and Acrobeloides sp. PS1146, using a combination of vital staining with a mitochondrial-specific dye Rhodamine 6G, and confocal laser-scanning microscopy. In C. elegans embryos mitochondria are part of the same bulk flow directed toward the sperm pronucleus that has been implicated in generating the initial A-P axis asymmetry (Strome, S. And Wood, W.B. Cell 35, 15-25 (1983); Hird, S.N. And White, J.G. J. Cell Biology 121, 1343 1355 (1993)). Concomitant with these flows, during the period of cytoplasmic rearrangement known as !pseudocleavage!, most of the cortical mitochondria are found only in the posterior, and following the first cell division the posterior blastomere P1 inherits roughly twenty per cent more mitochondria than its anterior sister AB. This redistribution does not require the presence of either an anterior actin cap or a pseudocleavage since the same pattern was observed in nop-1 mutant embryos in which these structures are missing. In contrast, in Acrobeloides sp. PS1146 embryos the sperm pronucleus does not direct any cytoplasmic rearrangements, and the P-granules are segregated differently. During pronuclear migration they do not become asymmetrically distributed. Most are on the surface of the pronuclei, and the rest are loosely distributed throughout the cytoplasm. During metaphase most of the P-granules are situated around the chromosomes, and by anaphase they redistribute primarily around one of the two microtubule asters (Goldstein, B. et al., Current Biology 8(5): 303. 1998 Feb 26). We found that during pronuclear migration the mitochondria are either around the pronuclei or distributed throughout the cytoplasm, and following the formation of the first spindle they are seen to translocate in radial paths toward the aster centers. This probably indicates transport along astral microtubules mediated by minus-end directed motors. Comparing the two species studied we find that the pattern of redistribution of mitochondria correlates closely with that of the P-granules.

Goldstein P (1982) Worm Breeder's Gazette "nondisjunction and knobby chromosomes."

Goldstein P

[Worm Breeder's Gazette, 1982]

Why don't chromosomes at meiotic prophase fall apart (or undergo disjunction) when they are supposed to? Part of the answer lies in peculiar knobs attached to the synaptonemal complexes (SC) in C. elegans hermaphrodites and males. It has recently been reported that these 'SC knobs' may represent active regions of DNA, as opposed to the more compact chromatin usually found at pachytene (Goldstein and Slatan, 1981). Also, the number and distribution of these SCKs vary, such that the wild-type hermaphrodite has 6 SCKs (and shows 0.01% nondisjunction) while the him8 mutant (with 37% nondisjunction) has no such knobs (Goldstein, 1981). The him4 mutant, which shows 16% nondisjunction and represents an intermediate situation, has 3 SCKs. Thus, it was suggested that the presence or absence of SCKs may mediate the nondisjunction process, such that the product of the active DNA region inhibits nondisjunction if present (6 SCKs in wild- type-0.01%) and if this product is not present, then nondisjunction occurs (no SCKs in him8, 37% nondisjunction). Recently, further evidence has been obtained to support this theory. There are two SCKs in wild-type young males (4 days) this represents a true sex- difference) however, in older males (7-9 days) no such SCKs are present. The report by Rose and Baillie (1979) that nondisjunction increases with age (as revealed by increase in male progeny) correlates with the absence of SCKs in older males.

Richards JP et al. (1991) Worm Breeder's Gazette "The Cloning of a Putative C. elegans Cyclin B Gene Using the Polymerase Chain Reaction."

Bennett KL, Richards JP, Temenak JJ

[Worm Breeder's Gazette, 1991]

Cyclin proteins are intimately involved in cell cycle regulation. Two types of cyclin proteins have been identified, cyclin A and cyclin B. The cyclin A protein has been shown to be critical to embryonic development in Drosophila; homozygous mutant embryos lacking cyclin A die when zygotic transcription fails to provide cyclin A. The presence of cyclin B protein cannot rescue these mutants (C.F. Lehner and P.H. O'Farrell, Cell 1990. 61:535-547). No mutants in cyclin B have been reported in Drosophila. Cyclin B protein has been shown to be involved in meiotic maturation in clams (Westendorf et al., J. Cell Biol. 1989. 108:1431-1444). Perhaps surprisingly, in Drosophila the mRNA for cyclin B localizes by in situ hybridization to the Drosophila pole cells, the fly equivalents of the nematode P germline cell lineage (Whitfield et al., Nature 1989. 338:337340). The Bennett laboratory is interested in the nematode germline lineage and in identifying the components of the germline P granules ( Strome and Wood, Cell 1983. 35:15-25 and K. Bennett, WBG 9:1 55). We plan to determine if nematode cyclin B RNA is similarly localized to the nematode germline precursor cells, and would like to address what possible role the sequestration of cyclin B RNA plays in the determination of the germline. Using primers to conserved cyclin B regions, we have isolated, cloned and sequenced a PCR product from genomic Caenorhabditis DNA which is a putative cyclin B. As illustrated below, this fragment has 72% exact match at the amino acid level to the corresponding region of the clam or sea urchin cyclin B genes; this is exactly the same match ( 41/57 aa) that sea urchin and clam show to each other in this region. Southern blot analysis using this fragment as a probe with C. elegans DNA indicates a single copy gene. The PCR fragment recognizes a single 1.4 kb message on Northern analysis using polyA+ RNA from mixed stage worms; this size is large enough to accommodate the coding region of the cyclin B protein. We have recently screened R. Barstead's lambda ZAP mixed stage cDNA library. Thirty putative cyclin B cDNAs were detected on duplicate lifts when 120,000 phage were screened. [See Figure 1]

Miller LM et al. (1998) Worm Breeder's Gazette "The Effects of Benzo[a]pyrene (cough cough!) on C. elegans"

Miller LM, Hartman PS

[Worm Breeder's Gazette, 1998]

The polycyclic aromatic hydrocarbon (PAH) benzo[a]pyrene (B[a]P) is a major carcinogenic component of cigarette smoke and many other combustibles. Wild-type (N2) and four radiation hypersensitive mutants (rad-1, rad-2, rad-3 and rad-7) were exposed to different B[a]P concentrations for either one or 24 hours. Survivorship curves were generated for both embryos and L1s. In addition, hermaphrodites were exposed to B[a]P and the effects on brood sizes were measured. Relative to most studies, very high B[a]P concentrations were required to significantly reduce survival or brood sizes in C. elegans. All strains showed greater sensitivity with longer exposure. Surprisingly, the rad mutants were no more sensitive than wild type. Since no recessive lethal mutations were detected in the 500 plates screened using the eT1 balancer system, B[a]P does not readily induce mutations in C. elegans. Finally, using a specific and sensitive RIA, no adducts were detected in C. elegans DNA extracted from animals exposed to high B[a]P concentrations. The extreme resistance of C. elegans is likely due to the failure of this nematode to metabolize B[a]P into more toxic and mutagenic compounds.