Morton DG et al. (1990) Worm Breeder's Gazette "Maternal Mutations that Alter the E Lineage"

Kemphues KJ, Morton DG

[Worm Breeder's Gazette, 1990]

In our screens for maternal effect lethal mutants that fail to differentiate intestine, we identified 4 mutants in which the embryos arrest as masses of many differentiated cells, have normal early cleavage patterns, localize P granules normally, but fail to produce gut granules. (WBG 10(2) p. 56, 1988). These mutants may identify genes that are important for specification of the intestinal cells in C. elegans development. We have found that all four of these mutants exhibit a specific alteration in the E cell lineage: Ea and Ep divide prematurely and fail to move to the interior of the embryo to begin the process of gastrulation. The daughter cells remain at the surface of the embryo. The division of Ea and Ep in the mutants occurs 6-12 minutes earlier than in wild type, and immediately follows the division of MSa and MSp. This E lineage-defective phenotype is the earliest defect that we have identified for the mutants. This early phenotype suggests that the absence of differentiated intestinal cells in late stage embryos may result from a defect in the determination of the E lineage. We hope to ascertain the fates of the E daughter cells in the mutants and to learn whether other cell lineages are affected. The mutant embryos produce body wall myosin, indicating that differentiation of body wall muscle cells, at least, is not blocked by these mutations. The four mutations, it92 V, it93 I. it103 II and it125 II, while giving very similar phenotypes, each identify a different gene. This was somewhat surprising to us, since the mutations were identified from screens of over 23,000 haploid genomes, in which we were able to identify 7 new par-4 alleles and 6 par-1 alleles. The rare mutations that give an E lineage-defective phenotype may be unusual alleles of their respective genes, or may identify genes that are unusually small or rarely mutated. We are presently mapping the mutations and doing complementation tests with lethals in these regions to distinguish between these possibilities. The fact that we obtained only one mutation in each locus also suggests that we have not saturated the genome for mutations of this kind. In fact, Jocelyn Shaw and coworkers have followed our screening procedure and identified additional mutants with different map locations that give similar E lineage alterations (C. elegans Meeting Abstracts, p. 231,1989). One mutation is allelic with it92 (J. Shaw pers. communication). We are collaborating with the Shaw lab on further genetic and phenotypic analyses of E lineage-defective mutations. We hope that our studies of these mutations will provide insights into how the embryonic intestinal cell lineage pattern and determination of cell type is controlled.

Beh CT et al. (1985) Worm Breeder's Gazette "an acid phosphatase activity localized to the gut of C elegans."

Beh CT, McGhee JD

[Worm Breeder's Gazette, 1985]

We have previously described a gut-localized esterase as a biochemical marker with which to study the early events in lineage- specific gene expression (Abstracts #14 and 86 in May, 1985, CSH; also Edgar and McGhee, Developmental Biology, in press). We wanted to find a second gut-specific hydrolase with which to compare the esterase expression and an acid phosphatase appears to be a good candidate. Adult worms are turned into extrudates by cutting in the presence of 2 g/ml of levamisole (see Lewis, et. al., Genetics 95: 905 (1980)), fixed in paraformaldehyde and stained with the following mixture: 50 l of 20 mg/ml of 1-naphthyl phosphate, 100 l of 1 M NaOH, 850 l 0.15 M sodium acetate to which is added 100 l freshly diazotized pararosaniline. Final staining pH is close to 5.0. Staining is usually rapid, intense and gut-localized. However, this staining pattern shows two important differences from the esterase pattern. Whereas esterase staining seems to occur throughout the cytoplasm of all gut cells, phosphatase staining appears localized to the edge of the gut lumen in the region of the brush border. (This same pattern is found in worms grown axenically and therefore does not result from clinging E. coli). Moreover, phosphatase staining is not found in the anterior six cells of the gut (i.e., those cells which derive from the anterior daughters of Ea(1/r)a and Ea(1/r)p cells and which show no later nuclear divisions). Since several cells at the posterior end of the gut also do not stain, there seems to be some symmetry in the phosphatase distribution but we re not yet certain how this correlates with their variable nuclear divisions. In any case, the staining pattern indicates that a spatial decision to express or activate the phosphatase is made relatively late in intestinal development. Under the usual staining conditions, the gut phosphatase appears to be the major phosphatase found in crude extracts of unsynchronized populations. Stained isoelectric focusing gels show one major band ( pI about 4.9) along with several minor bands. Unlike the esterase, the phosphatase is stable at its pI and gels can be left to stain overnight, allowing phosphatase activity to be detected in the equivalent of one worm or less. Preliminary steps in purification are consistent with the phosphatase being a membrane protein (i.e., activity is enhanced by Triton X-100) and Triton increases the apparent Molecular Weight on Sephacryl columns. Phosphatase activity in crude extracts is inhibited by tartrate but not by other phosphatase inhibitors such as alloxan, CuSO4, formaldehyde or EDTA. We will probably follow the same scheme worked out for the gut esterase, namely to purify the enzyme as a first step towards gene cloning and to induce isoelectric focusing mutants to locate the phosphatase gene on the genetic map.

Junkersdorf B et al. (1988) Worm Breeder's Gazette "laser manipulation during early embryogenesis of Caenorhabditis elegans."

Schierenberg E, Junkersdorf B

[Worm Breeder's Gazette, 1988]

Individual blastomeres in early C. elegans embryos have been ablated using a laser microbeam coupled to a microscope. For this the nucleus of a selected cell is irradiated with several short laser pulses (386 nm). At low doses cells continue cleavage but express considerably prolonged cell cycle rhythms. For early developmental stages higher doses of irradiation are necessary to induce cleavage arrest than in more advanced stages. In the 2-cell stage we have neither been able to ablate AB nor P1 without damaging their sister cell. When the germline cell P2 is irradiated in the 4-cell stage, such that the neighboring cells do not show any visible defects, P2 does at least pass through one more cleavage. Such embryos develop into monsters with several hundred cells, expressing differentation markers like muscle twitching and gut birefringency. After irradiation of P3 (passing through one more division) a hatching worm can develop, lacking muscle cells in the central part of the body ( normally generated by D). Such a worm does not reproduce due to the missing primordial germ cell P4. After ablation of P4 (no further divisions) a sterile worm develops without any defects in somatic tissue. Ablation of the D cell results in a hatching juvenile with more or less uncoordinated movement. During postembryonic development visible defects decrease and often a normal behaving, reproducing adult emerges. The ablation of the precursor for the anterior (Ea) or the posterior half (Ep) of the intestine, results in a hatching juvenile with only half a gut (see sketch below). Nevertheless such animals have developed a functional, normal looking and pumping pharynx. They can survive several days but never reach adulthood. Our results of early cell ablations have not revealed any new cases of intercellular regulation but are in accordance with the concept of mosaic development. [See Figure 1]

Nishiwaki K et al. (1990) Worm Breeder's Gazette "On cell division sequence of C. elegans early embryogenesis"

Miwa J, Nishiwaki K

[Worm Breeder's Gazette, 1990]

The early embryogenesis of C. elegans appears to follow an invariably rigid cell division sequence. However, we do not really know how rigid this sequence is for normal embryogenesis. We report here our observation that the cell division sequence of C. elegans early embryogenesis can vary without affecting the hatching efficiency or the fertility of hatchees. We observed embryos with the same preparation method as described by Sulston et al.(1), where we used Difco purified agar (PA) for supporting embryos instead of Difco Bacto- agar. Cover glasses were sealed with vaseline. Experiments were carried out at 25 C. When we used 5% PA, we consistantly observed that AB(16) occurred 0.5-3 minutes before P(3)(2) (division of P3 to P4+D) and that MS(8) occurred 2-4.5 minutes before D(2) (5/5) in the N2 development. These cell division sequences were reversed when 2% PA was used; that is, P3(2) occurred 0.5-1 minute before AB(16) and D occurred 2-4 minutes before MS(8) (3/4). In one of the four cases, MS(8) occurred before D2 as in the 5% PA, although P3(2) occurred before AB(16). Almost all of some 50 embryos so far examined in each condition hatched normally. We tested the fertility of the worms that showed the embryonic cell division sequence typically expected for the % agar used. All of these worms grew fertile with essentially normal brood sizes. The results show that the cell division sequence of C. elegans early embryogenesis is not absolutely rigid and allows some range of variations without affecting the normal development and the fertility. We think that the observed variations in the cell division sequence resulted from difference in the pressure applied on the embryos. The embryos on the 5% PA looked flatter than those on the 2% PA under Nomarski optics. The 5% PA must have exerted more pressure on the embryos than did the 2% PA. Then, we may expect the division sequence in the 2% PA to be closer to that under no pressure Shierenberg et al observed the embryos prepared in water under a cover glass sealed with petrolatum and plastic-foam cushions (condition without pressure) to exhibit the division sequence of P3(2) -> AB(16) and D(2)-> MS8(2), which is indeed identical to that observed with the 2% PA. ---In addition to variations in the cell division sequence, we also observed variations in the cell division axes of E cells under the two different agar conditions. Both Ea and Ep divided in parallel and dorsal-ventral in the 2% PA, whereas the division axis of Ep inclined posteriorly in the 5% PA. Neither of the division axis variations resulted in any abnormal development or infertility.

Powell-Coffman JA et al. (1995) Worm Breeder's Gazette "ama-1 antisense experiments: Embryonic transcription is required for initiation of gastrulation"

Wood WB, Powell-Coffman JA, Knight JK

[Worm Breeder's Gazette, 1995]

We are examining the requirements for maternally provided versus embryonically transcribed gene products in early developmental decisions. Past studies have shown that C. elegans embryos initiate transcription prior to gastrulation (1, 2). Indeed, the embryonic pes-10 transcript is detectably expressed by the 4-cell stage (3). Devitellinized embryos cultured in the presence of a-amanitin, which inhibits RNA polymerase II, cease dividing at approximately 100 cells. The lineage-specific timing of early cleavages in these embryos is not dramatically altered (2). a-amanitin experiments cannot address the potential role of embryonic transcription in directing gastrulation, since perturbations of the vitelline membrane disrupt gastrulation (4). To block the translation of maternally provided RNA polymerase II large subunit in otherwise intact embryos, we injected capped antisense ama-1 RNA (50 ng / ml) into the gonads of wild-type hermaphrodites. (The ama-1 cDNA clones were generously provided by David Bird.) We scored the progeny of these animals 20 hrs. to 48 hrs. postinjection, and 95% of the embryos arrested early in development (n = 850). We examined 40 of these embryos under Nomarski optics, and all arrested at 80 to 100 cells and showed no signs of morphogenesis or differentiation. To further assay embryonic transcription, we injected antisense ama-1 into a strain carrying vet-6::lacZ, which is strongly expressed by the 28-cell stage (5). The resulting arrested embryos did not detectably express the reporter gene, indicating that the antisense ama-1 RNA had effectively blocked the function of RNA polymerase II. Moreover, preliminary results show that the cleavage pattern of ama-1 antisense embryos is normal through the first four cell cycles, and P-granules are segregated appropriately; thus, these maternally-controlled events are not detectably disrupted. To control for possible artifacts caused by RNA injection, we injected hermaphrodites with RNA antisense to her-1, an embryonically transcribed sex-determining gene. Of 2609 progeny examined, 1.1% failed to hatch, and only 0.3% arrested with fewer than 300 cells. Further controls for ama-1 antisense specificity are underway. We have recorded the development of ama-1 antisense embryos past the point of cell division arrest using the 4-D microscope, and all of the embryos (n = 6) failed to gastrulate. The Ea and Ep cells and their daughters divided precociously and did not migrate into the blastocoel, reminiscent of the maternal-effect emb-16 mutant phenotype (6). These data indicate that embryonically transcribed gene products are necessary for initiating gastrulation. Further analysis of ama-1 antisense embryos will be instructive as to the role of embryonically transcribed genes in early development. 1. Schauer, I. and W. B.Wood (1990) Development 113: 1303-1317 2. Edgar, L. G., Wolf, N. and W. B. Wood (1994) Development 120: 443 - 451 3. Seydoux, G. and A. Fire (1994) Development 120: 2823 - 2834 4. Schierenberg, E. and B. Junkersdorf (1992) Roux's Arch. Dev. Biol. 202: 10-16 5. Pettitt, J. and W. B. Wood. (1993) Worm Breeder's Gazette. 13 53 6. Denich, K.T.R., Schierenberg, E., Isnenghi, E. and R. Cassada. (1984) Roux's Arch. Dev. Biol. 193: 164 - 179

Knight JK et al. (1996) Worm Breeder's Gazette "Preliminary characterization of a maternal-effect mutant defective in gastrulation initiation."

Knight JK, Wood WB

[Worm Breeder's Gazette, 1996]

The genetic control of gastrulation is not well understood in C. elegans. We have isolated a temperature-sensitive mutation, ct226ts, which results in failure to initiate gastrulation. The mutation, tentatively defining a gene which we propose to call gad-1 (gastrulation defective), exhibits a strict maternal effect. At 16C, homozygous worms grow slightly slower than wild-type, are variably defective in egg-laying and gonad placement, and lay 24% dead eggs. At 25C, 100% of embryos laid by a homozygous hermaphrodite arrest with a clear gastrulation defect: the E blastomeres do not migrate into the embryo, and the embryo does not undergo gastrulation. Analysis of ct226 mutant embryos with the 4D microscope has shown that the 2E4 division is premature and in the a-p rather than the d-v direction. The 4E8 division is also early and in an aberrant plane, and all 8 E cells remain on the ventral surface of the embryo. Gastrulation initiation appears to be completely blocked; there is no delayed migration of the gut precursors, nor do the mesoderm or germ-line precursors migrate into the interior of the embryo. Despite lack of morphogenesis, terminally arrested defective embryos have differentiated (though misplaced) muscle and hypodermal cells, as assayed with monoclonal antibodies MHCA (5.6.1) and MH27, respectively. Gut granules are also present, although clearly mislocalized in a cluster at the very posterior of the embryo. We have mapped ct226 to the center of LG V, between unc-83 and dpy-11. This region is uncovered by the deficiency nDf18, but not by the deficiency sDf26. Embryos laid by ct226/nDf18 hermaphrodites at 16C are inviable about 90% of the time, a substantially higher penetrance than for embryos from ct226 homozygotes, suggesting that ct226 is a hypomorphic allele. The Gad phenotypes of the inviable embryos from ct226/nDf18 and ct226/ct226 hermaphrodites are similar. None of the lethals in this immediate region, (let-439, let-473, and let-337) fails to complement ct226. Thanks in part to deficiency endpoint mapping of nDf18 by C. Malone and A. McGee in the Han lab, we have narrowed the location of ct226 on the physical map to a small region between the left endpoint of nDf18 and dpy-11. We are currently injecting appropriate cosmids in an attempt to rescue ct226 homozygous mutant animals. Previously described mutations in at least seven other genes cause gastrulation defects; five of these show maternal effects (emb-5, emb-13, emb-16, emb-23, and emb-31) and two do not [zen-1 (Ferguson et. al WBG 13(5):66) and end-1(Zhu et. al 1995 Worm Mtg. abstract 33)]. It is noteworthy that the non-maternal-effect mutants isolated to date have delayed E cell migration, but are able to complete gastrulation in most cases, while all the maternal-effect mutants arrest with mislocalized gut granules and only partial or no gastrulation (1, reviewed in 2). emb-16(g19ts) and ct226 display the most severe phenotype, with very early Ea and Ep divisions and no E cell migration. Interestingly, these two characteristics are also shared by embryos in which embryonic transcription has been blocked by ama-1 antisense RNA (Powell-Coffman, Knight and Wood WBG 14(1):70). We intend to combine our characterization of ct226 with a search for embryonically transcribed genes required for gastrulation in the hopes of understanding the functional relationship between maternally and embryonically expressed genes in the control of gastrulation. 1. Denich, K.T.R., Schierenberg, E., Isnenghi, E., and Cassada, R. (1984). Rouxs Arch. Dev. Biol. 193, 164-179. 2. Bucher, E.A. and Seydoux, G. (1994). Seminars Dev. Biol. 5, 121-130.

Edgar LG et al. (1992) Worm Breeder's Gazette "Embryonic Functions of the pal-1 Gene"

Wood WB, Carr SH, Edgar LG

[Worm Breeder's Gazette, 1992]

We are investigating embryonic functions of the pal-1 homeobox gene (Waring and Kenyon, Cell 69:123; Nature 350:712). The two psoralen-induced alleles ct224 and ct281 produce hatching L1 swith a a severely deformed posterior, which we call a Nob (no back end) phenotype. The earliest detectable abnormality is a misarrangement of the gut cells at the 100-cell (16 E cell) stage. Neither allele complements pal-1 (e2091 )and we presume that they represent null alleles of this gene. 1. Using Southern analysis, we have determined that both of these alleles are deletions covering the 3' end of pal-1 .ct224 ,Approximately 4.2 kb, lies entirely within the smallest pal-1 rescuing plasmid, the 6 kb Kpn I/ Pst-1 restriction fragment pWPK6 (a gift from Waring and Kenyon), beginning in the homeobox and deleting the rest of the 3' coding sequence. ct281 is approximately 4.7 kb, beginning slightly 3' of ct224 and deleting into the next 3' Kpn 1 fragment; it may include a small part of the homeobox. Northerns show a doublet of 1.25 and 1.45 kb (the appropriate size for pal-1 )when probed with any of the pal-1 coding fragments from pWPK6 .The 3' 3 kb Kpn I fragment encodes a transcript of 800 bp which is presumably affected in ct281 but should not be affected in ct224 unless its untranscribed sequence extends beyond the Kpn I site. 2. We have injected pWPK6 ,the larger plasmids pWK9 and pWK14 ,and the cosmid WO5E6 (all gifts from Waring and Kenyon), together with rol-6 DNA, into ct224 and ct281 ,and now have integrated or transmitting lines from each possible combination. In all cases, we recover Unc Dpy animals as well as the heterozygotes from the balanced strains mut Unc Dpy/qC1 carrying an array. However, the Unc Dpy animals produce only nonhatching embryos with a severe Nob phenotype. Although one explanation for this maternal effect is that a second, maternal effect mutation is closely linked to the deletion mutations, both alleles and the heterozygotes (ct224 unc-32 dpy-18 /ct281dpy-18 unc-25 )show this effect when rescued, even with the cosmid. Thus we suspect that pal-1 may also have a maternal function which is not rescued possibly because this transforming DNA is not expressed in the germ line. 3. To investigate expression patterns of the wild type pal-1 gene, we have injected a construct containing approximately 5 kb of pal-1 upstream genomic sequence and about half the coding sequence fused to the reporter bacterial lacZ gene. The earliest expression observed is in the 4, then 8, C cells in 70-80 cell embryos. This disappears, and at approximately 300 cells Earpp, Ealpp, and 4 cells lateral to the pharyngeal primordium stain. These cells migrate posteriorly, and are probably Z1 ,Z4 ,M, and mu int. By lima bean stage, several additional ventral midline cells and 2 cells at the forming rectum, probably U and K, begin to stain, continuing through hatching [See Figure 1 &2]. Earpp and Ealpp form int-5 at the center of the gut, where Ea and Ep descendants interdigitate, and like Z1 and Z4 ,contact Z2 and Z3 during embryogenesis. U and K form the intestino-rectal junction. Thus all the staining cells lie posterior, at positions where malfunction might well give the Nob phenotype of the mutants. Isolated and cultured P1 ,EMS, and P2 blastomeres appear cell-autonomous for pal-1 expression; isolated AB blastomeres give no blue cells. Since U and K are AB descendants, inductive interactions may be required for their expression of pal-1 .

Avery L (1993) Worm Breeder's Gazette "Quantitative Measurement of the Currents Associated With PharyngealExcitation and Relaxation"

Avery L

[Worm Breeder's Gazette, 1993]

When a worm's head is sucked into a pipet and the inside and outside of the pipet connected to a high-impedance voltage amplifier, voltage transients are measured between the inside and outside of the pipet every time the worm pumps (Raizen and Avery, WBG 12(5): 62). Although the time-course of these transients is quite reproducible from pump to pump and from worm to worm, their size varies from less than a few hundred V to several mV, even in the course of a few minutes' recording from a single worm. We suspected that this variability was an artifact of our recording arrangement; that in fact each transient in the electropharyngeogram (EPG) carried roughly the same current in every pump, and that the variable voltage we measured resulted when this current flowed across the variable resistance of the seal between the pipet mouth and the cuticle of the worm's head. To test this hypothesis, I connected the pipet to a low-impedance current-measuring amplifier (an Axopatch 1D patchclamp amplifier operated in voltage-clamp mode). As hoped, the transients were now constant in size, not only from pump to pump, but even among worms of the same age and size. A typical record of a single pump is shown below (left), accompanied by a typical voltage trace for comparison (right). Positive currents represent current flowing out of the worm's mouth. All the usual features can be recognized. What is the origin of this current? When pharyngeal muscle is excited, the voltage changes across the basal membrane, which separates the muscle cell cytoplasm from the pseudocoelom. The total voltage difference between the muscle cytoplasm and pseudocoelom must be the same alone any path that connects them, including the long path that goes across the apical membrane of the muscle cell into the pharyngeal lumen, out the mouth, through the surrounding liquid, across the cuticle and hypodermis, and back into the pseudocoelom (see diagram above). Most of the voltage drop along this path should appear across the membranes, since cytoplasm and pseudocoelom have low resistance. Biological membranes are capacitors: any voltage difference between the two faces of a membrane is accompanied by a proportional difference in charge. Thus, when pharyngeal muscle is excited, current must flow to charge the apical membranes and hypodermis. If we assume that all the charging current flows out of the mouth, and that most of the voltage drop appears across the apical muscle membrane instead of the hypodermis (equivalent to assuming the capacitance of the apical membrane to be much smaller, reasonable since capacitance is proportional to area), we can estimate roughly the voltage change associated with each transient. This analysis was done using the R1 spike, since it is large and well-isolated, and since we understand it best. The R1 spike results from the repolarization of the corpus muscle that takes place at the end of an action potential. The total charge (measured as the area of the trough) transferred during the R1 spike of a healthy adult N2 hermaphrodite was close to 1 F/cm(2). From the pictures in Albertson and Thomson's reconstruction of the pharynx (Phil Trans Roy Soc 275B: 299-325), the area of the apical membrane of the corpus can be very roughly estimated at 2000 m(2). The capacitance of biological membranes is usually close to 1 F/cm(2). These numbers give an estimated voltage change of 50 mV. Byerly and Masuda (J Physiol 288: 263-284) measured a voltage change of 100 mV when Ascaris pharyngeal muscle repolarizes. Considering the uncertainties in the calculation, this agreement is remarkable, and suggests that much of the current that flows out of the mouth arises as described. In the course of this analysis, I noticed a correlation between the length of the pump (i.e., the interval from E2 to R1 spike) and the depth of the R1 spike. The figure below shows pump length and R1 depth for 12 N2 adults (a -1) and 3 egl-30 adults (ea - ec; egl-30 worms tend to have longer pumps). The correlation could also be seen among the pumps recorded from a single worm. This correlation may be explained by the properties of the Ascaris negative spike channel described by Byerly and Masuda (ibid) and believed to mediate the repolarization at the end of the action potential. This channel is inactivated at resting potential, and the inactivation is removed by depolarization with a time constant in the range of hundreds of msec. (The precise time constant depends on membrane potential.) Thus, the longer the muscle cell remains excited, the more negative spike channels will be available to open when the action potential ends, and the larger the peak current will be. The increased current is expected to speed up the repolarization of the membrane, but should have little effect on the total voltage change. In fact, the deeper troughs tended to be narrower so that the total charge transferred (which, as described above, should be proportional to the change in membrane potential) didn't vary as much as the peak current. In summary, the currents that flow out of the mouth due to pharyngeal activity are of reproducible amplitude. The total charge that flows into the mouth during corpus repolarization is of about the order of magnitude predicted from simple electrical considerations. Finally, the peak current during corpus repolarization is correlated with the length of the action potential, a result that can be explained by the hypothesis that the negative spike channel of Byerly and Masuda passes this current.