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Development 121, 1227-1236 (1995) Printed in Great Britain © The Company of Biologists Limited 1995 1227 An analysis of the response to gut induction in the C. elegans embryo Bob Goldstein MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK SUMMARY Establishment of the gut founder cell (E) in C. elegans involves an interaction between the P2 and the EMS cell at the four cell stage. Here I show that the fate of only one daughter of EMS, the E cell, is affected by this induction. In the absence of the P2-EMS interaction, both E and its sister cell, MS, produce pharyngeal muscle cells and body wall muscle cells, much as MS normally does. By cell manipulations and inhibitor studies, I show first that EMS loses the competence to respond before it divides even once, but P2 presents an inducing signal for at least three cell cycles. Second, induction on one side of the EMS cell usually blocks the other side from responding to a second P2-derived signal. Third, microfilaments and micro- tubules may be required near the time of the interaction for subsequent gut differentiation. Lastly, cell manipulations in pie-1 mutant embryos, in which the P2 cell is transformed to an EMS-like fate and produces a gut cell lineage, revealed that gut fate is segregated to one of P2’s daughters cell-autonomously. The results contrast with previous results from similar experiments on the response to other inductions, and suggest that this induction may generate cell diversity by a different mechanism. INTRODUCTION induction affects only part of a single cell. The EMS cell’s response to gut induction is then characterised by determining how long the signal is presented to EMS and how long EMS is capable of responding, whether both sides of EMS can respond to inducers at the same time, whether microfilaments and microtubules are required near this time, and the potential role of induction in a mutant where a second cell develops like EMS and produces a gut cell lineage. Possible mechanisms by which gut induction may generate cell diversity are discussed. Not long ago the early nematode embryo was regarded as an archetype of mosaic development, in which cell interactions played little or no role in early development. Diverse cell types were believed to be generated almost entirely by segregating specific cell components to one daughter cell at each division (see for example, Nigon, 1965; Laufer et al., 1980; for a contrasting view see Guerrier, 1967). This view has since changed, as several cell-cell interactions have been found to play a critical role in generating cell diversity in early C. elegans development, and also because nematode embryos develop in more diverse ways than the initial evidence had suggested (Malakhov, 1994). The induction analysed here is the induction of the gut cell lineage in C. elegans. Gut specification allows a study of induction at the level of single cells; the gut (or ‘intestine’) derives from all of the progeny of a single blastomere of the eight cell stage, the E cell. During the four cell stage P2 induces EMS to produce the gut from its E lineage (Fig. 1). This interaction occurs one cell division before the gut founder cell (E) is generated (Goldstein, 1992). Although P2 normally contacts the presumptive E side of EMS, the presumptive MS side of EMS is also capable of becoming the gut founder cell, when P2 is moved to contact it instead (Goldstein, 1993). Previous results have suggested that this induction is unusual, in that it polarises a single cell (EMS), inducing gut only in the side it contacts (Goldstein, 1993). This led to interest in the mechanism by which gut induction generates cell diversity between E and MS. Here differentiation is assayed from uninduced EMS cells to determine directly if gut Key words: C. elegans, EMS cell, induction, gut differentiation, pie-1 MATERIALS AND METHODS Strains The experiments employed the wild-type strain N2 Bristol and the pie-1 strain pie-1(zu154) unc-25(e156)/qC1 [dpy-19(e1259) glp1(q339)]III under standard conditions (Wood, 1988). Embryos from mothers homozygous for maternal-effect loss of function mutations in the pie-1 gene are referred to as pie-1 mutant embryos for convenience. skn-1 and mex-1 mutant embryos are referred to similarly. Cell manipulations, culture, enzyme histochemistry and antibodies Manipulations were carried out as described previously (Goldstein, 1992, 1993). Methods for removing the eggshell and vitelline membrane and for culturing isolated cells are described in Edgar and Wood (1993). Cells were cultured in Edgar’s Growth Medium (EGM) for 12-24 hours before assaying for differentiation. Cell lineaging employed a multiple focal plane time-lapse recording system (Hird and White, 1993). In experiments where gut differentiation was assayed by both lineaging and following differentiation, lineages were traced through the time rhabditin granules were apparent. Where lineage times alone were followed to discern E and MS lineage 1228 B. Goldstein Fig. 1. Top: Four and eight cell embryos, ventral view, posterior to the right. Cell types produced by E and MS lineages are shown. The P2-EMS interaction is indicated by an arrow. Bottom: Cell lineage from fertilised egg (P0) through 26 cell stage. timings, lineages were followed in EMS for a minimum of three divisions and differentiation was followed alone in separate cases. Where EMS’s two daughters were separated from each other, this was done 4-7 minutes after EMS began to form a cleavage furrow (cases done earlier suggest the cleavage furrow is not yet complete, as the furrow generally regresses to form one cell with two nuclei). Times before or after cell divisions cited here were measured to/from the time a cytokinetic furrow was first visible in the dividing cell. The time during the cell cycle when a manipulation was carried out was determined by noting the time of the manipulation and the time of the ensuing division. The time of the preceding division was also either recorded (in most cases) or estimated as in Goldstein (1993). When P 2 and EMS are juxtaposed they stick together immediately upon contact. Their orientations are randomised, and the cells do not then rotate or move around each other to return to their original positions. This has been established first by watching for overt signs of movement in high magnification time-lapse recordings (Goldstein, 1993) and second by placing cells in contact and then looking at a marker for cell orientation, the position of the centrosomes in EMS and P2 in live cells and by anti-tubulin immunofluorescence in fixed cells (unpublished observations). Assaying for differentiation by immunofluorescence or enzyme histochemistry required fixing cells in 2% paraformaldehyde after gentle washing, as devitellinised embryos are easily disrupted. Partial embryos were washed briefly by mouth pipetting through two changes of a simplified protein-free EGM, which included only the water, stock salts, Hepes buffer, galactose, base mix stock and disodium phosphate from the EGM protocol. Paraformaldehyde was prepared in simplified EGM by first dissolving paraformaldehyde at 20% in water (at 60°C, with approx. 50 µl 5 M NaOH per 5 ml) and then diluting 1:10 in the simplified EGM. Embryos were then washed by mouth pipetting through two changes of M9 (Wood, 1988) or PBS with 0.5% Tween20 and were placed on 0.1% polylysine-treated slides. The fixed embryos stick readily to glass after washing with M9 or PBS, and hence were not allowed to sink onto glass surfaces during washes. Cell isolates were scored for differentiation of each major body tissue except neural tissue. Gut differentiation was assayed by three methods. (1) Presence or absence of birefringent rhabditin granules was determined by viewing embryos in polarised light as described previously (Goldstein, 1992, 1993; Edgar and McGhee, 1986). Except where otherwise stated, this alone was used to assay for gut differentiation. (2) Esterase histochemical staining was performed as described by Edgar and McGhee (1986), except for fixation, which is described above. (3) The monoclonal antibody 1CB4 stains the gut, and also sperm and chemosensory neurons (Okamoto and Thomson, 1985). 1CB4-positive cells were identified as gut cells by their large size. This was also confirmed by noting whether each isolate contained rhabditin granules. Pharyngeal muscle cell differentiation was assayed using the 3NB12 monoclonal antibody (Priess and Thomson, 1987). Body wall muscle differentiation was assayed using a polyclonal antibody to paramyosin kindly provided by Hiroaki Kagawa. Hypodermal differentiation was assayed using a polyclonal antibody to LIN-26 protein, which stains some descendants of isolated AB and P2 cells (unpublished observations); the antibody was kindly provided by Michel Labouesse. The 5-6 monoclonal antibody, which stains body wall muscle cells, and the 9.2.1 monoclonal antibody, which stains pharyngeal muscle cells (see Mello et al., 1992), also work on formaldehyde-fixed cultured embryos (unpublished observations). Antibodies were used by standard methods, with 1-hour incubations at 37°C for primary and secondary antibodies, and thorough washes in phosphatebuffered saline with 0.5% Tween-20, before and after each step. Immunofluorescence was observed on an MRC600 laser-scanning confocal microscope (Biorad). Photographs of confocal sections (in Fig. 2) show only a thin field of cells, roughly representative of the proportions of staining in the whole isolate. Differentiation of gut, pharyngeal muscle, body wall muscle, and hypodermis could be assayed in each isolate by photographing rhabditin granules immediately before fixation, then using a fluorescein-conjugated secondary antibody for 3NB12, and a rhodamineconjugated antibody for the paramyosin and LIN-26 antibodies. These two could be distinguished from each other as the LIN-26 antibody stains nuclei of hypodermal cells, and the paramyosin antibody stains cytoplasm of body wall muscle cells. Colchicine (Sigma) was used at 64 µg/ml for one batch, 100 µg/ml for another. Cytochalasin B (Sigma) was used at 5 µg/ml. Embryos were permeabilised by completely removing the eggshell and vitelline membrane. The concentration of each inhibitor was the lowest dose that prevented cytokinesis consistently in a dose-response assay; half the dose used did not arrest cleavage consistently. RESULTS E and MS both differentiate as MS-like lineages in the absence of gut induction Previous results have shown that gut fate is induced in the E lineage by the P2-EMS interaction. Gut differentiation does not occur from EMS cells isolated early in the four cell stage, but contact with a P2 cell will rescue gut differentiation (Goldstein, 1992, 1993). As only gut differentiation could be assayed in these experiments, it was not clear whether E is the only cell The EMS cell’s response to induction 1229 whose fate is affected by the interaction. In order to determine whether MS is also affected by the interaction, and also to determine what the uninduced state of the E lineage is, differentiation was assayed from the progeny of isolated EMS cells. The results are described briefly here; the complete results are shown in Fig. 2. EMS cells isolated before gut induction had occurred (1013 minutes before cytokinesis began in EMS) divided to form two daughters, both of which produced body wall muscle and pharyngeal muscle, but no gut or hypodermis, suggesting that both daughters differentiate as MS-like lineages, as MS normally produces this suite of cell types. These findings confirm previous suggestions made on the basis of the timing of cell divisions: in the absence of gut induction, E loses its distinctive cell lineage timing and takes on an early lineage timing of an MS-like lineage (Goldstein, 1992, 1993). EMS loses the competence to respond before it divides; P2 continues signalling The time when the P2-EMS interaction normally specifies gut in E is known. This occurs 5-6 minutes into the four cell stage (this is 9-10 minutes before EMS cleaves, as the EMS cell cycle is 15 minutes long); isolating EMS later than this usually will not prevent gut specification (Goldstein, 1992). If P2 and EMS are not in contact at this time, placing them in contact later might still rescue gut differentiation. Here I determine when contact between the two cells can no longer induce gut in an uninduced EMS. EMS and P2 blastomeres were isolated before gut fate had Fig. 2. Differentiation from the progeny of induced (A-E) and uninduced (F-I) EMS cells. (A) Gut-specific rhabditin granules. (B,F) Pharyngeal muscle marker 3NB12 antibody (green) and body wall muscle marker anti-paramyosin antibody (red). (C,G) Gutspecific esterase histochemical stain (red), only in progeny of the induced EMS. (D,E,H,I) Differentiation from the progeny of each of the two daughters of an EMS cell. D and E derive from the two daughters of an induced EMS cell, (D) from one daughter, (E) from the other. H and I derive similarly from the two daughters of an uninduced EMS cell. (D) Gut-specific rhabditin granules. All cells in this isolate produced rhabditin granules. This isolate produced no pharyngeal muscle or body wall muscle (not shown). (E,H,I) Pharyngeal muscle marker 3NB12 antibody (green) and body wall muscle marker anti-paramyosin antibody (red). These isolates produced no rhabditin granules (not shown). (J) Table showing that in the absence of gut induction, both daughters of EMS differentiate as MS-like lineages. gut is gut differentiation, as assessed by three assays (gran, rhabditin granules; ab, 1CB4 antibody staining; est, gut esterase histochemical stain); bwm, body wall muscle; phm, pharyngeal muscle; hyp, hypodermis (see Materials and Methods); induced, EMSs isolated after gut was induced were isolated 0-7 minutes before cytokinesis began in each EMS cell; uninduced, those isolated before gut was induced were isolated 10-13 minutes before cytokinesis began in each EMS cell. Both parts show differentiation from isolated EMS cells (labelled EMS), and from EMS’s two daughter cells separated from each other (labelled ‘E/MS’), to find which daughter(s) produce the tissue indicated. The daughter that produced gut cells (when there was one) is shown on the left of each pair. Each + or − represents 5-16 cases; each case resulted invariably as indicated, except when EMS was isolated 0-7 minutes before dividing, in which EMS occasionally (8/40 cases) produced only bwm and phm and no gut or hyp, suggesting that gut has not yet been induced in these cases, as this occurs when EMS is isolated before gut is induced. been induced, and were kept separate for a time, after which they were placed back in contact, cultured, and later assayed for gut differentiation. When P2 and EMS were isolated before gut fate had been induced and were placed back in contact 8 minutes after EMS cleaved (this would be during the eight cell stage in an intact embryo), gut fate could no longer be rescued in EMS. In order to rescue gut fate in an early-isolated EMS, P2 had to be placed back in contact with EMS 3 minutes before EMS cleaved or earlier (Fig. 3). 1230 B. Goldstein To determine whether this time represents the time that EMS loses the competence to respond to P2’s signal, or the time that P2 stops signalling, cells from embryos of differing ages were combined. First, uninduced EMS blastomeres were tested to see if they can respond after this time. An EMS blastomere was isolated before gut fate had been induced (10-13 minutes before EMS cleaved), was left in isolation at least 10 minutes, and then was placed in contact with a younger P2 blastomere (from an embryo in the first half of its four cell stage). In this way EMS blastomeres of varying ages could be tested to see if they can still respond to a signal. This experiment was carried out six times; 0/6 produced gut, suggesting that EMS loses the competence to respond 3 minutes before it cleaves. Second, P2 blastomeres were tested to see if they can induce later than this time. Each P2 was isolated, was left until after EMS had cleaved, and was placed in contact with another EMS that was isolated from a younger embryo, before gut had been specified. Some of the P2 cells had cleaved once or twice before being placed in contact with EMS. Eight cases of this experiment were assembled; 5/8 produced gut cells. In some of these cases, P2 had cleaved twice before being placed in contact with the uninduced EMS, and yet gut differentiation still occurred. The results show that P2 and some or all of its descendants continue to present a gut-inducing signal for some time after the four cell stage. EMS loses the ability to respond to gut induction 3 minutes before it divides. Induction on one side of EMS interferes with induction on the other side Previous results showed that either side of EMS can respond to gut induction (Goldstein, 1993). Here it is determined whether a single EMS cell can respond to inductions on both sides. This was tested in two ways: by presenting a single P2 cell sequentially to two sites on an EMS cell, and by sandwiching an EMS cell between two P2 cells. Presenting P2 sequentially to two sites on EMS P2 was moved to a random orientation after gut specification had initially occurred (Fig. 4A). To do this, EMS cells were isolated after gut fate had already been induced, 5-9 minutes before EMS cleaved. P2 was then placed onto EMS. This randomises the orientation of cell contact (see Materials and methods). After EMS cleaved, whether one or both of EMS’s daughters produced gut was determined by two methods. In 12 cases, lineage times were recorded from EMS. In 10/12 cases, only one of EMS’s daughters took on E-like lineage times, and the other took on MS-like lineage timing. In the other 2/12 cases, neither cell took an E-like lineage timing. In an additional 13 cases, the two daughters of EMS were separated and cultured independently. In all 13 of these, gut cells differentiated from only one of the daughters. By both assays, when a gut lineage was established it was always established in only one daughter of EMS. Despite P2 being presented to two sites with a timing that should allow two inductions to occur, in no case did both of EMS’s daughters produce the gut lineage. Which daughter of EMS gave rise to the gut lineage is of interest because it can reveal for how long the site of gut specification, once established, is movable. As P2 was moved to a random site on EMS, in approximately half of the cases P2 will have been placed on a region that normally would have become time (minutes) Fig. 3. P2-EMS contact can rescue gut specification up to 3 minutes before EMS cleaves. A timeline is shown of the four to eight cell stage; the time that EMS cleaves is at zero. Each bar represents one case where P2 and EMS were isolated before gut was specified (more than 10 minutes before EMS cleaved) and were placed back in contact at some time later, except the top bar, which shows that isolating EMS 10 or more minutes before EMS cleaved prevents gut differentiation (Goldstein, 1992). The bars below show that rescuing gut differentiation requires placing P2 back onto EMS at least 3 minutes before EMS cleaved. In these cases it was additionally confirmed that gut differentiation occurred in the daughter of EMS which derived from the side of EMS secondarily contacting P2 (11/11 cases by following differentiation of rhabditin granules after isolating EMS’s two daughters, 4/4 by following the lineage timing of EMS’s two daughters). Additional results described in text show that EMS loses the ability to respond 3 minutes before dividing. P2 signals throughout its cleavage cycle and passes the ability to signal to at least some of its descendants for the next two rounds of cell division. The possibility that only some of P2’s descendants inherit the ability to signal might explain why only some (5/8 cases) of the heterochronic combinations using P2’s daughters or granddaughters were able to induce gut fate in previously uninduced EMS cells. MS. When the manipulation was carried out at 5 or 6 minutes before EMS divides, the gut lineage derived from the side P2 was moved to in half of the cases (2/4 cases at 5 minutes, 2/4 at 6 minutes). In contrast, when the manipulation was carried out earlier the gut lineage nearly always derived from the side to which P2 was moved (6/7 cases at 7 minutes, 8/8 at 8-9 minutes). These results suggest that although gut is specified by 9 minutes before EMS divides (Goldstein, 1992, 1993), this site can be moved by moving the inducer 7-9 minutes before EMS divides. During this 3-minute period, removing P2 no longer prevents gut differentiation, however, moving P2 moves the site of gut specification. Sandwiching EMS between two P2 cells To present inducers to both sides of EMS simultaneously, EMS blastomeres were isolated before gut had been specified and The EMS cell’s response to induction 1231 each EMS was sandwiched between two P2 cells (Fig. 4B). In The results show that although both sides of EMS are ten cases gut differentiation was then assayed in each of EMS’s competent to respond to the inducing signal (Goldstein, 1993), two daughters, isolated after EMS divided. Three additional when presented with signals on both sides, only one side cases were assembled differently in order to record which side generally forms a gut lineage. of EMS was the original presumptive E side: an extra P2 was placed onto an intact but devitellinised four cell stage, on the presumptive MS side of EMS, in the first 4 minutes of the four cell stage. EMS was then lineaged to determine lineage timings and which daughter(s) produced gut. Gut differentiation occurred in 10/13 cases. In nine of these the gut cell lineage derived from only one of EMS’s two daughters, and in one case both daughters produced complete gut cell lineages. The finding that only one daughter usually produced a gut cell lineage was surprising, as either side of EMS can consistently respond to P2 (Goldstein, 1993), however it appears they are generally unable to do so at the same time. In the cases where gut cells were produced from one daughter of EMS, the two sides of EMS appeared equivalent in their potential to become the E lineage: which daughter produced the gut could not be consistently predicted by the age of the two P2s, the order in which they were placed on EMS, which P2 was from the same embryo as the EMS used, nor which side of EMS would normally have given rise to the E lineage. One case is shown in Fig. 4C-K, where a P2 was added to an intact four cell stage on the presumptive MS side of EMS, and the cell which Fig. 4. Induction on one side of EMS generally inhibits the other side of EMS from responding to a normally would have been E took second signal. (A,B) The diagram shows two experiments designed to test if EMS can respond to on MS-like lineage timing. The inducers on both sides, sequentially in A and simultaneously in B. EMS cells are shaded. The timeline cell that normally would have represents the EMS cell cycle (between the two vertical bars on the ends), which lasts 15 minutes. been MS took on E-like lineage Each time division marked is 5 minutes. Only one daughter generally produced gut cells. (C-K) An timing and produced the gut. EMS cell sandwiched between two P2 cells in an otherwise intact embryo. To do this, a P2 cell from One potential caveat to these another embryo was added to the presumptive MS side of EMS. In the case shown the presumptive MS experiments is that secondary lineage (labelled ‘MS’) produced the gut cell lineage. Which P2 is the extra one was determined by interactions between EMS’s noting slight asymmetries in the embryoid immediately after the manipulation, and was confirmed by daughters might affect results, for watching P2 cell cycle times. (C) Ventral view after EMS divided. (D) Lower focal level at same time, example by lateral inhibition as showing the four daughters of AB, on which the embryoid is resting. (E) ‘E’ and ‘MS’ after dividing. This next cell cycle lasted 37 minutes in the two ‘E’ cells, 54-56 minutes in the two ‘MS’ cells; normal occurs between developing vulval cell cycle times appear switched. (F) The presumptive E lineage after its second division. (G) Lower cells (Sternberg, 1988). This pos- focal level at the same time, showing two presumptive MS cells. (H) Approximately 12 hours later. sibility is unlikely, since isolating (I) Same view, under polarised light, showing gut-specific rhabditin granules. (J,K) are as (H,I), EMS’s daughters from each other showing a different focal plane. The gut cells derived from the entire presumptive MS lineage. Note after EMS divided did not affect gut cells are larger than other cells; this is also the case in the E lineage of normal embryos, as the E lineage goes through relatively few cell divisions. Bar, 10 µm. results (see above). 1232 B. Goldstein Microfilaments and microtubules Microfilaments and microtubules appear to play a crucial role in cytoplasmic localisation in many systems including C. elegans (see Sardet, 1994 for review). To investigate the role of the cytoskeleton in the response to gut induction, it was determined whether microfilaments or microtubules are required during the four cell stage for gut to differentiate. Although cell divisions do not occur, cleavage-arrested cells of a variety of embryos commonly continue to differentiate (Whittaker, 1973). The role of microfilaments has been tested previously with varying results (Laufer et al., 1980; Cowan and McIntosh, 1985; Edgar and McGhee, 1986). Embryos were devitellinised at various stages, placed in microfilament- or microtubule-depolymerising agents (cytochalasin or colchicine, respectively), and were cultured through the time that gut cells would normally differentiate. Whereas depolymerising microfilaments in eight to twenty cell stage embryos had no effect on subsequent gut differentiation, treating four cell stage embryos nearly always prevented gut differentiation (Fig. 5A). Embryos treated during the six cell stage, which represents the last 3 minutes of the EMS cell cycle, produced gut in about half of the cases. The results suggest that microfilaments may be required during the four cell stage for gut differentiation to occur, and that they may no longer be required soon after this time. Results with microtubule-depolymerising agents were similar (Fig. 5B), suggesting that microtubules also may be required during the four cell stage. Gut specification in pie-1 mutant embryos EMS fate has been proposed to be specified by the activity of the SKN-1 protein (Bowerman et al., 1992a; 1993). The pie-1 gene product is believed to repress SKN-1 activity in the P2 cell, preventing P2 from developing as an EMS-like cell. In pie-1 mutant embryos, P2 generally develops as EMS normally does (Mello et al., 1992). Thus pie-1 mutant embryos produce two EMS-like lineages, one from EMS and another from the cell that would normally be P2. Two gut cell lineages result, one from a daughter of EMS and one from a daughter of P2. As there is no P2-like blastomere in pie-1 mutant embryos, it was of interest to examine whether the two gut lineages produced in these embryos are specified via induction. The simplest apparent explanation was that an EMS blastomere might normally possess the ability to induce gut but cannot induce gut in itself, perhaps because its signal must be presented on an apposing membrane. If EMS normally presents a gut-inducing signal, then the formation of two gut lineages in pie-1 mutant embryos could be explained by both of the EMS-like blastomeres inducing gut in each other. To test this hypothesis EMS cells were isolated from wild-type embryos in the first 4 minutes of the four cell stage, and were placed back in contact in pairs. In none of five sets of EMS pairs did gut differentiation occur, suggesting that EMS does not in fact present a gut-inducing signal. Blastomeres were then isolated from pie-1 mutant embryos, to determine directly if EMS and the EMS-like P2 cell require cell-cell interactions to produce gut cells. EMS blastomeres isolated in the first 5 minutes of the four cell stage did not produce gut cells (0/7 cases), whereas P2 blastomeres isolated at the same time did (7/7 cases). The gut cells occupied roughly half the volume of each P 2 isolate (Fig. 6). The results demon- Fig. 5. Microfilaments and microtubules may be required near the time of the induction for subsequent gut differentiation. Embryos were treated with cytochalasin (A), colchicine (B), starting from the stage indicated and continuing until gut differentiation was assayed. Positions on the Y axis represent the percentage of the cleavagearrested embryos at each stage in which gut differentiation occurred. The timeline begins at the beginning of the two cell stage. The transition from the four to six cell stage (when ABa and ABp divide) occurs near when EMS normally loses the ability to respond (3 minutes before cleaving), and is used to separate embryos placed in inhibitor in the first 12 minutes of the EMS cell cycle from those placed in inhibitor in the last 3 minutes of the EMS cell cycle. 97 embryos were used in A; gut differentiation occurred in embryos treated from the four cell stage in 2/29 cases, and in those treated from the six cell stage in 11/17 cases. 96 embryos were used in B; gut differentiation occurred in embryos treated from the four cell stage in 0/17 cases, and in those treated from the six cell stage in 4/12 cases. strate that in pie-1 mutant embryos gut is still induced in EMS, presumably by ‘P2’, however P2 produces gut cells independent of interactions with other cells. DISCUSSION This work has characterised some aspects of an unusual induction. Gut induction generates cell diversity between the two daughter cells of a single responding blastomere. Uninduced EMS cells produced two MS-like lineages, suggesting that the induction generates polarity in EMS before it divides. Heterochronic cell combinations showed that EMS can only respond to gut induction in a single cell cycle, whereas P2 can signal over at least three cell cycles. Induction on one side of EMS inhibited the other side of EMS from responding to a second P2-derived signal. Microfilaments and The EMS cell’s response to induction 1233 Fig. 6. pie-1 mutant embryos: gut fate is established cell-autonomously in one daughter of the P2 cell. (A,B) EMS cells from pie-1 mutant embryos isolated in the last 7 minutes (A) or the first 5 minutes (B) of the EMS cell cycle, which lasts approximately 15 minutes. Isolating EMS early prevents gut induction. (C,D) P2 cells from pie-1 mutant embryos isolated at the same times as above. Isolating P2 early did not prevent gut specification in P2. Bar, 10 µm. (E) Model for E and MS specification in wild-type embryos, mutants, and manipulations, from results presented by Mello et al. (1992), by Bowerman et al. (1992a, 1993), and here. Results from intact embryos are shown at left, from isolated cells at right. Shading summarises how cells differentiate in each situation. MS-like fate is indicated by light shading, E fate (gut) by dark shading. Unshaded cells differentiate as other lineages. Note that all of the cells with shading are the ones believed to have active SKN-1 protein (Bowerman, 1993). In wild-type embryos (WT), gut fate is induced in the posterior side of EMS. In pie-1 mutant embryos, both P2 and EMS have EMS-like fate, although occasionally both of P2’s daughters produce gut lineages (Mello et al., 1992). In mex-1 mutant embryos, ABa and ABp each have SKN-1 protein and produce pairs of cells with MS-like fates (Mello et al., 1992; Bowerman et al., 1993). Results presented here suggest that in wild-type embryos and in pie-1 mutants the P2-EMS interaction causes a segregation event in the EMS, and in pie-1 mutants gut fate is segregated autonomously in P2. In mex-1 mutants, ABa and ABp might not form E-like lineages because they may lack the ability to segregate gut fate. microtubules appeared to be required near the time of the induction. Lastly, in pie-1 mutant embryos, where the P2 cell has an EMS-like fate, gut fate is segregated to one daughter cell-autonomously. Implications of results Two interactions serve to elaborate the anterior-posterior axis of the C. elegans embryo: on the ventral side of the embryo, E and MS develop distinctly because of the P2-EMS interaction. On the dorsal side, ABa and ABp develop distinctly because of the P2-ABp interaction (Bowerman et al., 1992b; Mello et al., 1994; Hutter and Schnabel, 1994; Mango et al., 1994a; Moskowitz et al., 1994). Hence P2 is the source of two inductions during the four cell stage. Mango et al. (1994a) have suggested that P2 may present two distinct signals to EMS and ABp. Gut induction and the mechanism of generating cell diversity Previous work on inductions in many embryos has elucidated a general mechanism by which many inductions generate cell diversity: of the cells that are competent to respond to an induction, only some encounter the inducing molecule(s) (see Slack, 1991 for review). As a result, these cells differentiate along a different pathway than the cells that do not encounter the inducing molecule. In vertebrate embryos, inducing molecules are commonly growth factors which diffuse many cell diameters. After some time cells lose their competence to respond, and only the cells that have encountered the inducer before this time are affected by the induction. Unlike gut induction in C. elegans, these inductions commonly involve large numbers of cells, which can respond through several cell cycles (Gurdon et al., 1985; Dale et al., 1985; Jones and Woodland, 1987). In invertebrate embryos the inducer is commonly presented on cell surfaces, and only the cells competent to respond that contact the inducing cell(s) are affected. Other cells are also competent to respond, but do not contact the inducing cell(s) (see Slack, 1991 for review). Gut induction is peculiar in that the area which is competent to respond is a single cell (EMS), and only one side of this cell is affected by the induction. One way of explaining how gut induction generates cell diversity is via the type of mechanism described above, in which only part of EMS encounters an inducing molecule and develops differently than the other part, on this basis. Alternatively, the inducer might cause a directed movement of particular components, in the cytoplasm or the membrane, to one side of the responding cell, causing one daughter to differentiate differently than the other (Fig. 7). Several of the experiments presented here address these models. The two models are not mutually exclusive, and many variations on the models are possible; the key point addressed here is whether or not the induction causes a directed movement of cell components to one side of the responding cell, and by doing so generates cell diversity. Either side of EMS is competent to respond to gut induction (Goldstein, 1993). If P2 causes the segregation of particular components to one side in EMS, this might interfere with the other side’s ability to respond to a second signal. This was found to be the case, in two types of experiments. First, moving the P2 cell after gut had been specified moved the site of gut specification for the first 3 minutes (6-9 minutes before EMS 1234 B. Goldstein divided), and no longer had an effect if moved during the next 3 minutes (3-6 minutes before EMS divided). Throughout this 6 minute interval, an uninduced EMS can respond to gut induction, however moving P2 did not establish a second site of gut specification (Fig. 7A). Second, when EMS was sandwiched between two P2 cells, either of EMS’s daughters could form the gut founder cell, however only one generally did. The findings show that as well as inducing gut fate in the side of EMS it contacts, P 2 has a detectable effect on the other side of EMS, inhibiting it from responding to a second gut inducing signal. In other inductions (where many cells can respond to the inducer) presenting the inducer to more of the area competent to respond results in more of the induced tissue, in both vertebrate embryos (Jacobson, 1963) and invertebrate embryos (for example, van Vactor et al., 1991). Both microfilaments and microtubules were found to be required at the four cell stage for subsequent gut differentiation. Depolymerising microfilaments or microtubules later than this time, during the eight cell stage or later, had little effect on gut differentiation. Although the timing of the requirement for microtubules and microfilaments is suggestive, this result is difficult to interpret. Microfilaments and microtubules, rather than functioning to localise a cell component, could have a non-motile function, for example to stabilise the localisation of a cell component. Also, previous experiments testing the role of microfilaments in gut specification have had varying results (Laufer et al., 1980; Cowan and McIntosh, 1985; Edgar and McGhee, 1986), most roughly consistent with those presented here, except for one in which nearly every embryo produced gut after cytochalasin treatment beginning in the four cell stage (Cowan and McIntosh, 1985). An interesting finding in some of these studies is that cytochalasin treatment beginning at the two cell stage prevents gut differentiation less effectively than treatment beginning at the four cell stage. This might be explained if an inhibitor of gut specification were normally segregated into MS, and it was too diluted to be effective in the large P1 cell of the two cell stage, however other explanations are equally plausible. The role of cell-cell interactions was examined in specifying gut in pie-1 mutants, where the P2 cell has EMS-like fate and also produces a gut lineage from one daughter cell. Results showed that in the EMS-like P2 cell, gut fate is segregated to one daughter independently of cell contact, yet the normal EMS cell still requires a cell-cell interaction to establish gut fate in one daughter. The possibility remains that an isolated pie-1 P2 cell produces a signal on its future P3 side and induces its own future P3 side to produce gut cells; this appears unlikely, however, since in several experiments no evidence has been found for a signal produced only on part of P2; P2 appears to present a signal on all sides (Goldstein 1992, 1993, and unpublished observations). The result, that in pie-1 mutants gut fate appears to be segregated cell-autonomously to one daughter of the P2 cell, lends credence to the notion that the induction normally causes a segregation of cell components in EMS. The results of experiments thus far are consistent with the hypothesis that contact with P2 causes a cytoplasmic segregation event in EMS. No cell components however have been seen to segregate in EMS. Following granule movements in EMS in normal embryos and in P2-EMS combinations, as has been done for the segregation event occurring before first cleavage (Hird and White, 1993), showed no apparent directed granule movements (unpublished observations). Granules of the size class found in C. elegans embryos might not however move in cells as small as EMS (S. Hird, personal communication). Gut induction might cause the segregation of a gut determinant, distributed throughout EMS below putative threshold levels, toward the site of contact with P2. Alternatively an inhibitor of gut fate might be distributed throughout EMS, and then segregate away from P2. Although the results in pie-1 mutants point to a cytoplasmic segregation event, the results are also compatible with segregation via capping of membrane components. Both cytoplasmic segregation and capping occur in some cells as a response to an external cue (Sardet et al., 1994; Taylor et al., 1971). The Numb protein in Drosophila is segregated prior to cell division and acts as a determinant (Rhyu et al., 1994); whether a cell-cell interaction is required to segregate Numb in a particular direction is not known. It will be interesting to see whether any genes required for gut specification encode gene products which are segregated in EMS, toward or away from the site of contact with P2. Mutations in genes involved in distinguishing the fates of E and MS have recently been identified (B. Bowerman, C. Mello, J. Priess, and B. Draper, personal communication); molecular characterisation of these may shed light on the mechanism of gut induction. Some other inductions in invertebrates may also affect only a part of a single responding cell. One such example is the induction of the mesentoblast in equally-cleaving molluscan embryos. In this case as in C. elegans gut induction, only one daughter of the cell which receives the induction has its fate altered by the induction (van den Biggelaar and Guerrier, 1979; see Davidson, 1986 for review). Fig. 7. (A) Summary of events during the EMS cell cycle. (B) Model for generation of cell diversity by gut induction: the P2-EMS interaction may cause a segregation of cytoplasmic components (arrows), making the presumptive E side of EMS (dark shading) different from the presumptive MS side (light shading). The experiments here do not address whether the proposed segregation event would concentrate a gut-specifying factor into the presumptive E side (as depicted) or a gut-inhibiting factor away from the presumptive E side. The EMS cell’s response to induction 1235 Gut specification and the role of SKN-1 The skn-1 gene product has been proposed to act as a determinant for EMS fate, as it is required for EMS-like fate, and can alter the fate of other cells when mislocalised (Bowerman et al., 1992a, 1993; Mello et al, 1992). The SKN-1 protein is normally found in the nucleus of EMS, and also in the nucleus of P2 (Bowerman et al., 1993). SKN-1 has no apparent normal function in P2; its activity is believed to be repressed in P2 by the pie-1 gene product, as mutations in pie-1 can confer an EMS-like fate on the P2 cell (Mello et al., 1992). P2 also appears to retain some characteristics of a normal P2 cell in pie-1 mutants: results presented here suggest that P2 retains its ability to induce gut in EMS. P2 also inherits P granules, as it does in wild-type embryos (Strome and Wood, 1983; Mello et al., 1992). The phenotype of mex-1 mutant embryos (Mello et al., 1992) is puzzling, as mex-1 mutant embryos have SKN-1 protein in the nuclei of the other two cells of the four cell stage (ABa and ABp) but rather than producing EMS-like lineages, these cells each produce two MS-like lineages. It appears as though SKN1 activity in the EMS or the P2 cell may promote EMS-like fate, yet in ABa and ABp only pairs of MS-like cells result. Mello et al. (1992) have indicated that an additional mechanism may need to operate for a cell with SKN-1 activity to produce a gut cell lineage. Results presented here suggest what the additional mechanism may be: EMS may segregate gut fate to one daughter cell via an interaction with P2, and P2 (in pie-1 mutant embryos) may segregate gut fate autonomously; ABa and ABp (in mex-1 mutant embryos) may be unable to segregate gut fate to one daughter cell, and hence develop like uninduced EMS cells do, producing pairs of MSlike daughters. In support, the ABa and ABp cells probably lack the ability to segregate cell components, as their daughters develop differently from each other by virtue of their positions, as a result of later cell interactions (Wood, 1991; Mello et al., 1994; Hutter and Schnabel, 1994; Mango et al., 1994b; Gendreau et al., 1994). ABa and ABp in mex-1 mutant embryos alternatively might lack the competence to respond to P2’s gut induction signal. Both of these interpretations (diagrammed in Fig. 6E) imply that SKN-1 activity, rather than simply specifying EMS fate, specifies the uninduced EMS fate, which produces pairs of MS-like daughters. Segregation of gut fate could then occur in EMS via the interaction with P2, and in P2 (in pie-1 mutants) cell-autonomously. I thank Gary Freeman, in whose laboratory I began these experiments, for suggestions and support. I thank S. Hird, B. Podbilewicz, P. Lawrence, S. Hoppler, J. Gurdon, J. White, S. Gendreau, J. Rothman, S. Mango, C. Mello, B. 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