Coordination of Cellular Events That Precede Reproductive Onset in Acetabularia Acetabulum: Evidence for a ‘Loop’ in Development

Development 122, 1187-1194 (1996) 1187 Printed in Great Britain © The Company of Biologists Limited 1996 DEV0050

Coordination of cellular events that precede reproductive onset in Acetabularia acetabulum: evidence for a ‘loop’ in development

Linda L. Runft† and Dina F. Mandoli* Department of Botany 355325, University of Washington, Seattle, WA 98195-5325, USA *Author for correspondence †Present address: The University of Connecticut Health Center, Department of Physiology, Farmington, CT, USA

SUMMARY

Amputated apices from vegetative wildtype cells of the early in adult growth required more cell volume to make a uninucleate green alga Acetabularia acetabulum can differ- cap without the nucleus than did apices removed from cells entiate a reproductive structure or ‘cap’ in the absence of late in adult growth. To define the limits of the cell to reca- the nucleus (Hämmerling, J. (1932) Biologisches Zentral- pitulate development when reproduction falters, we blatt 52, 42-61). To define the limits of the ability of wild- analyzed development in cells whose caps either had been type cells to control reproductive differentiation, we deter- amputated or had spontaneously aborted. After loss of the mined when during development apices from wildtype cells first cap, cells repeated part of vegetative growth and then first acquired the ability to make a cap in the absence of made a second cap. The ability to make a second cap after the nucleus and, conversely, when cells with a nucleus lost amputation of the first one was lost 15-20 days after cap the ability to recover from the loss of their apices. To see initiation. Our data suggest that internal cues, cell age and when the apex acquired the ability to make a cap without size, are used to regulate reproductive onset in Acetabularia the nucleus, we removed apices from cells varying either acetabulum and add to our understanding of how repro- the developmental age of the cells or the cellular volume duction is coordinated in this giant cell. left with the apex. Cells must have attained the adult phase of development before the enucleate apex could survive Key words: Acetabularia acetabulum, competence, differentiation, amputation and make a cap. Apices removed from cells reproduction, gametangium

INTRODUCTION actions within the cell and with its environs, not by signals in or from multicellular tissues or organs. The cellular dialog Coordination of cellular events at reproductive onset is between the nucleus, which is located in the basal rhizoid of important for the unicellular alga, Acetabularia acetabulum: the cell, and the cell apex, which is the site of differentiation, since virtually all of the contents of the cell are donated to its occurs over a long distance (3-4 cm). Second, the age of a cell progeny, if the parent cell fails to coordinate nuclear and cyto- can be assessed because the stalk is decorated with rings of plasmic events it will die without producing offspring. Thus, hairs, or ‘whorls’, that leave scars whose spacing is character- reproductive onset in A. acetabulum may be more tightly istic of the phase of development (Nishimura et al., 1992b). regulated than in organisms whose commitment to reproduc- Third, it’s giant size and ability to heal wounds (Fester et al., tion does not entail such drastic consequences for the species. 1993; Menzel, 1980) facilitates manipulations such as cell Accordingly, the cell has evolved strategies both to prevent grafting (Berger et al., 1987; Menzel, 1994) and allows the premature entry into reproduction and to reiterate reproductive developmental potential of pieces of the cell to be analyzed. development. For example, it survives winters by retracting the Finally, the cell can be stably transformed (Neuhaus et al., cytoplasm into the rhizoid (Berger and Kaever, 1992; Dao, 1986), expresses and correctly targets proteins encoded by het- 1962), it can stall development between the phases in vegeta- erologous or foreign DNA readily (Neuhaus et al., 1984), and tive growth (Mandoli, unpublished data), and the cell can is amenable to biochemical (Berger et al., 1987) and genetic execute a ‘developmental loop’ by aborting reproduction (A. manipulations (Mandoli and Larsen, 1993). Comprehensive crenulata: Puiseux-Dao, 1963; A. mediterranea: Decléve et al., knowledge of development in wildtype cells is important to 1972) if environmental conditions are not optimal or the make full use of these features. original apex is lost. The strategies used by the cell to ensure Reproductive onset is marked by an explicit shape change reproductive success are not well understood. at the cell apex, initiation of a cup-shaped structure or ‘cap’, This organism is attractive for developmental studies for and is regulated by both internal and external cues. The internal many reasons. First, in contrast to multicellular organisms, cues are the age and size of the cell (Nishimura et al., 1992b), reproductive onset in A. acetabulum is regulated only by inter- the presence of a putative population of cap-specific mRNAs 1188 L. L. Runft and D. F. Mandoli concentrated at the cell apex (Hämmerling, 1963a), and the and had 1-5 whorls consisting of clear, ephemeral hairs that branched absence of a putative, cytosolic inhibitor of the expression of 0-3 times each. ‘Early adults’ had thin stalks, were 1-3 cm tall and these mRNAs (Bannwarth et al., 1991; Beth, 1953; Li-Weber had more than 6 green whorls comprised of hairs that branched three et al., 1985; Shoeman et al., 1983; Zetsche, 1966). The external times each. ‘Late adults’ had thick robust stalks and persistent green cues include the nutrient environment in which the cell grew whorls composed of hairs that branched four times each. The ‘repro- (Hunt et al., 1995) and probably blue light (Clauss, 1970). ductive phase of development’ includes cap initiation and expansion, gametogenesis, and mating. Cells whose caps had reached their Cells reproduce only once all the internal and the external cues maximum diameters and contained gametangia were termed ‘mature’ are met. cells or caps. Vegetative development of Acetabularia acetabulum, which must be completed prior to reproductive onset, has morpho- Measurements of structures, amputation of cells, and data logically distinct juvenile and adult phases (see Materials and analysis Methods) which are temporally sequential and spatially Cap diameters were measured on a dissecting microscope fitted with stacked (Nishimura et al., 1992b). In Fig. 1, the vegetative an ocular micrometer. An isolation-fixation solution containing 4′ 6- phases are delineated on a mature or ‘late adult’ cell which is diamidino-2-phenylindole (DAPI) was used to stain nuclei (De et al., ready to make a cap. After the cap initiates, it expands laterally 1990). Plastic and glassware were sterilized with ethylene oxide (Fig. 1). During cap expansion the diploid nucleus probably (Zeller et al., 1993). All chemicals were purchased from Sigma (St. Louis, MO, USA). undergoes meiosis (Green, 1973; Koop, 1975) followed by 9- Experimental manipulations are depicted by cartoons on the 10 rounds of mitosis (Nishimura et al., 1992a). The resulting relevant figures. Before amputation, a ‘pressure-wound’ was made at haploid ‘secondary’ nuclei are transported up the stalk along the prospective cut site with a dental tool (small excavator spoon, CG- with most of the parent cell contents into the cap. After nuclear AMERICAN Model #UW A 46, GC International Corp., Scottsdale, transport, the junction or ‘septum’ between the cell stalk and AZ, USA) so as to miminize loss of cell contents. Pressure wounds the cap closes (Fig. 1, far right, center cap). The stalk is now bisected the cell contents, caused them to retract away from the wound optically clear yet retains its shape. Gametangial cell walls site, and created a region devoid of both cytoplasm and vacuole enclose each haploid nucleus with some parental cell contents (Fester et al., 1993). The cell wall was cut with a scalpel (type 21, (Fig. 1, far right, bottom cap). Gametangial formation is fairly Bard Parker, Division of Becton, Dickenson, and Company Ruther- independent in the individual partitions or gametophores of the cap (Berger et al., 1992; Shihira-Ishikawa, 1989). Temporal coordination between and interdependence of these develop- (from above) mental events in wildtype cells is not known. cap apex expands = Our goal was to analyze the age and size requirements for initiates a cap nucleus (2n) cap initiation and to begin to assess the roles played by the divides septum nucleus and apex in regulating reproductive onset in wildtype septum cells. Our data define the spatial and temporal limits to repro- whorl closes of hairs ductive development both in intact cells and in cells regener- late adult ating from cap amputation. The ‘developmental loop’ described by these data will be a useful experimental tool in nuclei (n) analyzing how reproductive onset is regulated in Acetabularia. stalk gametangia form early adult MATERIALS AND METHODS thousands of nuclei (n) transportedthousands of nuclei nucleus (2n) Cell strain and culture juvenile Acetabularia acetabulum (L.) Silva (Chlorophyta), strain Aa0005 rhizoid (Ladenburg #17), was used in all experiments. This laboratory strain has been axenically propagated for 3 generations using all the Fig. 1. A brief life cycle of A. acetabulum. A late adult cell (left) gametangia from >200 individuals per generation to prevent loss of grows through three phases in development (from bottom to top). alleles. Gametangia-bearing caps were decontaminated (Hunt et al., The vertical arrows show the direction of growth and the relative 1992) and then stored at 10¡C in the dark until use. Following mating portions of the cell length for each phase. Vegetative morphological (Mandoli et al., 1993), the axenic zygote stock was stored in the dark differences are omitted for reasons of clarity (see Materials and at 10°C. Zygotes were grown at 1 cell/3 ml until they made the first Methods). The stalk of the cell is decorated with rings of hairs, or whorl (Zeller et al., 1993). Then, each cell was grown to reproductive ‘whorls’, which leave scars whose spacing is characteristic of the maturity in a polystyrene Petri dish with 20 ml of seawater at 21±1¡C developmental phase (Nishimura et al., 1992b). Events in the under cool-white fluorescent lights (40-70 µmol photons/m2/second) reproductive portion of the life cycle are, from left to right: at on a 14:10 hour photoperiod. One version of a novel artificial seawater reproductive onset, a vegetative cell apex differentiates a cap which (Hunt et al., 1995) which is based chemically on a derivation of then expands and is filled with haploid nuclei (center cell). Haploid Müller’s medium (Müller, 1962) as modified by Schweiger et al. nuclei which are of + or − mating type, are shown as black or white (1977) was used here except in some of the experiments involving dots and the diploid nucleus is grey. After packing of the haploid amputation (Figs 5 and 6) in which a slightly different, earlier version nuclei in the rays of the cap (repeated image shown from above at of the recipe was used. top right), subsequent changes in the cap are: partitioning of the stalk from the cap when the junction, or ‘septum’ closes, partitioning of Cell developmental age the cytoplasm with each nucleus when ‘gametangia’ form. The The phase of vegetative development was assessed for all cells mating type of the gametes in each gametangium is not indicated. (Nishimura et al., 1992b). ‘Juveniles’ were threadlike, 0.1-1.0 cm tall, These cartoons are not draw to scale. Coordinating reproduction in Acetabularia 1189 ford, NJ) perpendicular to the long axis of the stalk 5-7 seconds after RESULTS pressure-wounding. StatView¨ 4.0 was used to analyze the data (Abacus Concepts, Ability of the apex to differentiate: dependence on Inc.). Standard errors of the mean are given when possible. the nucleus Although it was shown over 60 years ago that vegetative A. acetabulum apices can differentiate a cap without a nucleus 100 (Hämmerling, 1932, 1934, 1963b), the developmental age when the apex first becomes independent of the nucleus has never been discerned. Cells used for amputation experiments by previous researchers (Beth, 1953; Hämmerling, 1932, 1934) were probably late adults (see Materials and Methods). 50 To see whether the ability of the apex to initiate a cap ? depended on the age of the cell when the rhizoid was removed, whole cells were bisected at different times after formation of the first whorl leaving all of the stalk and about 90% of the cell percent of population time contents with the apex. Fig. 2 shows that apices whose rhizoids 0 ≤ 0255075 had been amputated 20 days after the intact cells had made Survival of apices post amputation, Time, days from first whorl to amputation the first whorl of hairs had <50% probability of surviving the procedure whereas apices amputated ≥20 days after making the Fig. 2. Ability of the apex to survive without the rhizoid depends on the age of the cell. The ages of the populations (e.g. those at 25 and first whorl had an 80-100% chance of surviving. The youngest 20 days) at the time of amputation are approximate as the different apices that could make a cap without the nucleus were experimental populations were not developmentally synchronous. amputated from cells ≥35 days after formation of the first Cells whose tips were amputated at 0 to 25 days after time zero were whorl (Fig. 2, arrow), which corresponds to the early adult juveniles, at 25 to 45 days were early adults, and at 45 to 60 days portion of vegetative growth. In contrast to apices from were late adults (see definitions in Materials and Methods). Apices juvenile cells, apices from adult cells can make caps without amputated from cells at 35 days after formation of the first whorl the nucleus as shown in Fig. 3. When about 90% of the cell were the youngest to subsequently initiate caps (vertical arrow). Each point represents from 6 to 20 cells with a mean of 15.6±1.6 cells. Intact cells made caps starting 45 days and enucleate apices 55.8±0.8 days after formation of the first whorl. Cartoon to the right of the question mark indicates the terminal morphology of apices whorls post abortion that survive amputation but fail to make a cap. 3.1 ± 0.4 in Aa0006 cap abortion 4.0 ± 0.4 in Aa0005

100 40 Early adult cells Aa0005 200 Aa0006 150 50 30

100 20 0 50 100

Aborted cap dia, % of mature 0 Late adult cells 10

50 0 0 50 100 150 200 Caps in aborting cell population, # Aborted final cap diameter, % of mature cap 0 Apices that made a cap, % of population Apices that made a cap, Fig. 4. Comparison of the diameters reached by aborted and mature 00.25 0.50 0.75 intact caps. Only those cells that eventually bore gametangia were used in Fraction of cell body left with apex two wild-type, laboratory strains (Aa0005: 85 out of 98 cells, Aa0006: 68 out of 121 cells). The average cap diameter at abortion Fig. 3. Apices that made a cap versus the fraction of the cell left with was 58.5± 4.1% for Aa0005 or 50.0±3.3% for Aa0006 of the mature, the apex. The percentages of cap initiation in 4 populations of intact, gametangia-bearing cap. The average diameter of the mature wild-type cells are indicated by the symbols on the right. These control gametangia-bearing cap was 5.7±0.2 mm for Aa0005 or 4.7±0.2 mm populations were grown under identical conditions and were pressure- for Aa0006. Between successive caps, cells made an average of wounded but not amputated either during the day (open circles) or 4.0±0.4 whorls for Aa0005 or 3.1±0.4 whorls for Aa0006. The inset night (solid circles). Cells amputated during the day are indicated by shows the same data analyzed in a ‘box plot’ format. The five open symbols connected with a line. Cells amputated during the night horizontal lines in each box plot represent 10, 25, 50, 75, and 90% of are indicated by solid symbols connected with a line. The mean the population. The symbols at the top and bottom of each box plot number of cells represented by data from several experiments was show ‘outliers’ or individual cells which are the 10% at the 14.9±0.1 except for the control cell populations (n = 60 cells total). population extremes. 1190 L. L. Runft and D. F. Mandoli was left with the apex, apices from late adults were 3 times more likely to initiate a cap than apices from early adults (Fig. 150 2nd cap post 3). These results suggest that for an apex to initiate a cap in amputation the absence of the nucleus, the cell from which it was taken must have reached the early adult phase of developmental (Figs intact 1st cap 2 and 3). 100 To establish how cell volume affected the ability of an enucleate apex taken from adult cells to initiate a cap, early and late adult cells were amputated at varying points along the 50 stalk (Fig. 3). Apices varied in length by increments of 1/8 of a cell. The rhizoid comprised the lower or distal 1/8 of the total cell length. Since cell height is genetically determined 0 (Nishimura et al., 1992b; Mandoli and Hunt, unpublished data), relative rather than absolute length was used. Two thirds % of mature final cap Cap diameter, 02550 of the intact controls (40 out of 60 cells) initiated caps (Fig. 3). Time, days post cap initiation The apex itself (upper quarter of the cell) was rarely capable of making a cap without the nucleus. Clearly, early adult apices Fig. 5. Rate of cap expansion for amputated and intact cells. The required more cell volume than late adult apices to make a cap linear regression for days 0-25 was y = 9.3+4.0x (R=0.98) for the without the nucleus. amputated cells (mean cells per datum for days 0-25=11.2±1.2, mean To see how cap initiation depended on the number of cells per datum point for days 25-45=2±1.2) and was y=11.9+3.6x chloroplasts in the apex, cell populations were amputated both (R=0.99) for the intact cells (mean cells per datum point = 28.8±1.3). during the day, when the chloroplasts stream continously in The average diameter of the mature gametangia-bearing cap was the stalk, and during the night, when most of the chloroplasts 5.6±0.1 mm for intact cells and 6.02±0.2 mm for the second cap of stop streaming and ‘rest’ in the rhizoid (Schweiger et al., amputated cells. Data for the amputated population at days 45 and 50 1964; Wollum, 1991). This circadian rhythm in chloroplast are missing since the cells had formed gametangia and so could not make a second cap. Other developmental events which occur during movement results in a gross asymmetry of organelles so that this time frame are shown in Fig. 10. amputation of cells at night yields apices with few chloroplasts and rhizoids which are highly chloroplast enriched. Conversely, amputation of cells during the day yields cell Note that we could not tell how much the aborted cap expanded pieces with fairly equal chloroplast densities. Apices after growth of the stalk recommenced because we can’t amputated from early adult cells during the day needed more predict when and in which cells cap abortion will occur. The cell body to make a cap than those amputated at night (Fig. 3, diameters of the aborted caps varied: the mean diameter of the top) whereas apices amputated from late adult cells needed aborted caps was about 50% of the mature, gametangia-bearing less than half of the cell body to initiate a cap regardless of caps with 3-4 whorls made between successive caps (details in time of day at which amputation occurred (Fig. 3, bottom). Fig. 4 legend). Although cap diameter at abortion varied Hence, the ability of an enucleate apex to make a cap was not widely, all cells of a strain made the same number of whorls strictly dependent on the number of chloroplasts in the apex before reproduction was attempted again. (Fig. 3). To estimate the time of abortion, the final diameter of the aborted caps (Fig. 4) was correlated with the cap diameter in The ability to make more than 1 cap: a ‘loop’ in cells that developed normally. For this estimate to be valid, reproduction cap expansion must be linear and similar regardless of Cells can repeat the transition from vegetative to reproductive whether a cell is making a first or second cap. First, as Fig. 5 development: a cell will cease expansion of a cap, produce illustrates, caps expanded at a linear rate until they reach their more stalk, and then initiate a new cap as diagrammed in Fig. final mature diameter about 25 days after initiation. Second, 4. The cell contents are withdrawn from the ‘aborted’ cap back the rate of cap expansion is similar for intact cells and the into the stalk so that the aborted cap eventually appears white. second cap of cells whose first cap had been removed (Fig. 5: This ‘loop’ in development is repeated until the youngest (i.e., intact, 3.6%; amputated, 3.9% of the final cap diameter per uppermost) cap formed at the cell apex completes gametogen- day). Note that although 13.3% (10 out of 75) of the mature esis. A cell that has aborted resembles a stack of 2 or more gametangia-bearing caps were smaller in diameter than the umbrellas which are fused top to bottom (Fig. 4, top right). immature caps that had been aborted by the same cell (Fig. 4), Abortion occurs both in the ocean and in the laboratory. this does not affect the calculation of the rate of cap expansion Cells with aborted caps were isolated from wildtype labora- since the data were normalized. These data predict that cap tory strains in which they had spontaneously appeared. Cap abortion occurred about 15 days after a cap initiated: at 3.6% abortion occurred in about 5% of a population (6 out of 130 expansion of the final cap diameter per day, caps reach 55.4% cells). In two cell lines, only 56.2 to 86.7% of the cells that of the mature diameter 15.4 days post cap initiation (Fig. 4, had aborted a cap made gametangia (see Fig. 4 legend). Cells inset box plots). that failed to make gametangia died and were excluded from this analysis. The diameters of aborted caps were measured and Triggering the loop in reproduction with cap then normalized to the diameter reached by the mature amputation gametangia-bearing cap eventually made by the same cell in To determine the limits of cells to reiterate reproductive onset, order to nullify genetic variation in cap diameter (Koop, 1977). apices of reproductive cells were amputated until all cells in Coordinating reproduction in Acetabularia 1191 Whorls, # made post amputation

25 100 15 20 2nd cap 15 10 50 time

population 10 5 5 whorls 0 Cells that made a new cap, % of cap, Cells that made a new 0 0 0 5 10 15 20 25 0102030 Successive apex amputations, number per cell Time, days from amputation to next cap from amputation to next days Time, Time, days from cap initiation to amputation

Fig. 6. Cells could make a cap again despite repeated amputation of Fig. 8. Time of second cap initiation and number of intervening the reproductive apex. All cells made a cap after the first amputation whorls made between amputation of the first cap and initiation of the (first datum) but none made a cap following the 23rd amputation (n = second. Note that the number of cells represented by each datum point 30 cells total). Caps were amputated on the day of initiation. Note decreased as the cells failed to make a new cap or failed to heal. For that the number of cells represented by each point decreases as the clarity, only one-sided standard errors for both parameters are drawn. cells either failed to make a new cap or to heal. Cell population size per datum point was 8.14±1.56 for time and 5.14±1.1 for whorls. The interval between the 2nd and 3rd cap from Figs 5 and 6 is replotted here for comparison (solid triangle). the population stopped initiating caps. Apices were amputated on the day of cap initation and were discarded. All cells initiated a second and then a third cap following amputation of These data show that A. acetabulum has a remarkable capacity the first and second caps respectively (Fig. 6). The probability to recover from wounds and can repeat reproductive onset if that a cell would initiate 4 or more caps was inversely related the apex is lost. to the number of apices that had been previously amputated. We wondered whether the cells were returning to the same On average, cells made 9.0±1.0 caps after successive apex earlier point in development regardless of how the cap was lost. amputations before losing the ability to reiterate reproductive That is, was the loop in development the same in aborted and onset. One cell initiated 23 caps in succession, failing to make amputated cells? We measured both the time from loss of the a new cap only after the 23rd apex amputation. Fig. 7 shows cap to initiation of a new one and the cell structure of the stalk that new caps were initiated an average of 11 days after ampu- made during this developmental loop. A quantitative temporal tation of the previous cap. Although the intervals needed to comparison of the abortion and amputation data (Figs 4 vs. 6) reinitiate a cap following amputation of caps 3 through 23 was not possible since we could not induce cap abortion but ranged from 7 to 17 days there was no pattern in the time only see that a cap had aborted retrospectively. Data in Fig. 8 required: one cell might take 17 days to make the 10th cap but show that cells whose apices were removed on the day of cap only 7 days for the 11th suggesting that the amount of time initiation made a new cap 5 days post amputation with 1 whorl required to initiate a new cap was not a function of the ampu- between the amputation site and the new cap. Cells whose tation history of the cell but of that particular amputation. apices were amputated 5-20 days post cap initiation took longer to make a new cap and made more whorls than those amputated on the day of cap initiation (Fig. 8). From 0-20 days these increases are nearly linear. When apices were removed >20 days post cap initiation, most of the cells could not make 20 a new cap but the few that could did so 8-11 days after amputation. The time required for healing the wounds was not assessed. These data suggest that a cell can repeat reproductive development at almost any time during cap expansion if the cell returns to a point in vegetative growth at least 5 days 10 prior to cap initiation and suggests that the amount of vegetative growth after cap amputation depends strongly on the R = 0.999 amount of cell contents that were removed. Point of no return: loss of ability to repeat 0 reproductive onset 0 100 200 Successive cap initiations, # per cell cap initiations, Successive Time, cumulative days since first cap initiation If there existed a point when a cell had to finish reproduction Fig. 7. Rate of cap initiations during repeated amputation of the with the last cap made and could no longer repeat reproduc- reproductive apex. The rate of cap initiation (y=11.0x−4.4) was tive onset, then cells whose apices were amputated after this derived from the previous experiment (Fig. 5). Caps were amputated would not be able to form a new cap. As shown in Fig. 9, cell on the day of initiation. Standard errors of the mean time between populations were amputated at several times post cap initiation successive caps are indicated. to see if such a point exists. All cells that were amputated ≤15 1192 L. L. Runft and D. F. Mandoli

cap initiation (Fig. 10) after cap expansion was complete (Fig. 100 5). All cells ﬁnished septum closure about 41 days post cap initiation (Fig. 10). Gametangia ﬁrst appeared in cells at 30 days and 50% of the population of cells contained gametangia at about 75 40 days post cap initiation. All of the cells completed gametangia formation about 55 days post cap initiation (Fig. 10). Cells completed cap expansion, septum closure and gametangial 50 formation about 1 week apart (Figs 5, 10).

25 ? DISCUSSION percent of population 0 time Our data suggest that for A. acetabulum to reproduce it must Cells that made a new cap, Cells that made a new be old and/or big enough and establishes the limits of the cell’s 02040 Time, days from first cap initiation to amputation ability to reproduce when the reproductive apex aborts or is lost. Based primarily on our data, Fig. 11 summarizes the Fig. 9. Ability to make a new cap declined as time from cap relative timing of the developmental events or interactions that initiation to apex amputation increased beyond 15 days. Mean cells surround reproductive onset in A. acetabulum. This is the first per datum point was 15.1± 0.2. The linear regression of the data from − time that events preceding and during reproductive develop- day 15-45 was y=151.9 3.6x (R=0.987). ment have been quantified with the amputations feasible in A. acetabulum. days after the first cap was initiated were able to make a second Regulation of cap initiation: putative mRNAs and a cap. When amputation occurred >15 days after cap initiation putative inhibitor the probability that a cell would initiate a second cap decreased The ability of the apex to make a cap without the nucleus has by 3.6% per day. When amputation occurred ≥40 days after been attributed to the presence of a putative population of stable cap initiation, no cells in the population could initiate a new mRNAs (Berger et al., 1987; Beth, 1953; Hämmerling, 1963a). cap. Fifteen days post cap initiation corresponds to 54% cap This ability is largely confined to the apex since isolated mid- expansion, roughly the calculated mean time of abortion (Fig. sections of the cell rarely initiate caps (Hämmerling, 1932, 9 vs. Fig. 5). Also, when the first cap was removed 15 days 1934). Furthermore, Beth (1953) reasoned that differentiation after initiation, the time required to make a second cap nearly of the apex was inhibited by the nucleus until the cell was ready doubled (Fig. 8). These data suggest that cells 0-15 days post to reproduce since the enucleate apex differentiated sooner than cap initiation can enter the loop in reproduction and that loss it would have in the presence of the nucleus. This hypothesis of the ability to make a new cap occurs as early as 15 days post has been supported by comparison of the kinetics of cap cap initiation. initiation and the activity of developmentally regulated enzymes between intact and enucleate Acetabularia cells Nuclear division and post meiotic events during cap (Bannwarth et al., 1977, 1982; de Groot and Schweiger, 1983, expansion 1985). The existence of the putative mRNAs and the role and Nuclei were stained with 4′,6-diamidino-2-phenylindole identity of the putative cytosolic inhibitor of cap initiation (DAPI) at various times during development to determine if meiosis in the diploid nucleus in the basal rhizoid of the cell was correlated with a particular point in cap expansion. Unfor- septum closure tunately, meiotic tetrads were not easily seen since the nucleus 100 seems to reside near the center of the fist-like rhizoid. However, nuclei being transported could be visualized with DAPI (De et al., 1990; Shihira-Ishikawa, 1984; Shihira-Ishikawa et al., 75 1984). Of the cells stained with DAPI, 0% of the vegetative gametangial cells (n=92) and 24% of reproductive cells (n=296) had visible 50 formation nuclei in the stalk. Multiple nuclei were detected in cells at any >1 nucleus in stalk time post cap initiation until the end of cap expansion (25 days, 25 Fig. 5). The highest percentage of cells with nuclei being transported up the stalk occurred 15 days post cap initiation as shown in Fig. 10 about when the cell population begins to lose the 0

capacity to enter the loop in reproduction (Fig. 9). These data percent of population Late events, 0204060 argue against the idea that the loss of ability to enter the loop Time, days from cap initiation in development and meiosis are concomitant (Figs 7-9). Fig. 10. Timing of three nuclear events which occur post cap The last two developmental events that the cell undergoes prior initiation. Percentage of the population which had closed the septum to gametogenesis are septum closure and gametangial formation. or formed gametangia was plotted as a function of the time in days Septum closure marks the end of nuclear transport (Fig. 1). post cap initiation (mean cells per datum point was 10 and 15.1±0.2 Septum closure ﬁrst occurred about 24 days after cap initiation respectively). DAPI-stained nuclei of vegetative cells were early and 50% of the cells completed septum closure at 32 days post adults and never had more than one nucleus (black square). Coordinating reproduction in Acetabularia 1193

late adult on a morphological basis (Nishimura et al., 1992b). The portion of vegetative growth which is repeated is invariant within a given strain. Three possible causes for cap abortion are: unfavorable environmental conditions (Fester et al., 1993), an error in coordination between the nucleus and apex, or the presence of a lethal mutation. Short days may induce cap

mRNA expression? abortion (Puiseux-Dao, 1963). Abortion in cells that failed to cap initiation bear gametangia (see Fig. 4, legend) suggests the presence of ÷ nuclear & transport, septum closure lethal mutations in some cells. Acetabularia cells that have light? cap expansion gametogenesis aborted superficially resemble mutants in yeast (cdc4 and 34: -10 0 10 20 30 days Fong et al., 1986; Goebl et al., 1988), Aspergillus nidulans (brlADb: Miller, 1993) and possibly Drosophila (polycomb: abortion loop Lonie et al., 1994): in each case reproductive structures are competence reiterated several times, stacked one above another. Whether differentiation point of no return spontaneous abortion in A. acetabulum also has a genetic basis is not known. Fig. 11. Summary of the reproductive phase of development in Although apex loss via abortion and amputation both trigger Acetabularia acetabulum. Times indicate when an event was first initiation of a new cap, the developmental pattern that ensues seen in a population. Cap initiation, the first identifiable event in the is not the same. During abortion the pattern of development reproductive phase of development, is defined as day zero on the bar that shows time. Measurable developmental events in reproduction does not vary, there is no wound to heal, and all the cytoplasm are above and conceptual terms are below the time bar. Events is recovered from the old cap. In contrast, when a cap is phys- followed by question marks are included for the sake of ically removed, cells make an increasing number of whorls as completeness but were not measured here. the interval between cap initiation and cap amputation increased (Fig. 8) and later amputations remove an increasing proportion of the cell body as more of the parental cell contents (Bannwarth et al., 1991; Beth, 1953; Li-Weber et al., 1985; are moved into the expanding cap. (Note that the apparently Shoeman et al., 1983; Zetsche, 1966) remains hypothetical. faster cap initiation and fewer whorls made in this experiment (Fig. 8) should not be overinterpreted because the population Coordinating linear developmental between the was small by this time.) In sum, although cap abortion or nucleus and apex amputation both trigger cells to repeat reproductive onset, it is Our results show that the isolated apex of a cell was competent not clear how these phenomena are related. to differentiate a cap without the nucleus only after the cell had reached the adult phase of development and only if there was Point of no return enough of the cell body associated with it (Figs 2, 3). Clearly, these are not just requirements for a certain number of chloro- Cells cannot enter the loop in reproductive development after the plasts since apices from adult cells make caps when relatively cap reaches a certain age, when it must finish reproduction or die achlorotic (Fig. 3). Apices from juvenile cells could fail to (Fig. 9). We avoid the term ‘commitment’ because our assay is make a cap because (1) they do not survive the physical trauma a loss of function, inability to make a new cap post amputation, of amputation well, (2) they were not yet competent to form a rather than a gain of function or commitment to a defined event cap without the nucleus-containing rhizoid (i.e. they lack the such as the beginning of cell division, called START (Hartwell putative cap-specific mRNAs or are not yet independent of the et al., 1989). Chromosome behavior visualized with DAPI nucleus for basic vegetative, autotrophic growth), or (3) staining in A. calyculus suggests that premeiotic chromosome because they take too long to make a cap (e.g. exceed the half- condensation started 3 days after cap initiation and finished in the life of the putative, cap-specific mRNAs estimated at 20 days; cell population when the cap was about three quarters of its final Kloppstech et al., 1982). Whether competence of the apex to diameter (Shihira-Ishikawa, 1984) but these events have not been make a cap without the nucleus, acquired during early adult documented well in A. acetabulum. The relationship between growth (Fig. 3), can be correlated with accumulation of specific meiosis and the point of no return is not obvious. mRNAs in the apex will be interesting to determine. These data contribute to our understanding of which cellular Once initiated, the cap expands radially at a steady rate (A. activities characterize the different developmental ages of acetabulum: Fig. 4 and Zetsche, 1966; A. calyculus: Shihira- Acetabularia acetabulum and describe the limits of the ability Ishikawa, 1984). Cap expansion was 3 times slower in A. of the organism to control reproductive onset. How the apex acetabulum than in A. calyculus (24 versus 8 days respectively: and the nucleus of this organism work together to coordinate Fig. 5 vs. Fig. 18 in Shihira-Ishikawa, 1984) although caps of progress through the phases in vegetative development and to the two species had similar final diameters. Cap diameter correct errors in the onset of reproduction will be intriquing depends on both genetic (Koop, 1977) and physiological puzzles to unravel. factors (Schweiger et al., 1977). The authors contributed equally to this work. This research was Repetition of reproductive development: abortion supported by a Hughes Undergraduate Research Fellowship (L. L. R.), in part by a Project R/B-8 of the Washington Sea Grant Program versus amputation with funding from Grant no. NA89AA-D-SG022 from the National To make a new cap after abortion a cell repeats part of veg- Oceanic and Atmospheric Administration, U.S. Department of etative growth (Figs 4-6) that has been previously defined as Commerce (D. F. M.), and in part by the National Science Founda- 1194 L. L. Runft and D. F. Mandoli tion #IBN-9305473 (D. F. M.). We thank Peter Ray, Paul Green, Lee rapid, high cell density culture of Acetabularia acetabulum without a change Hartwell, and Frank Russo for stimulating discussions and Susan in medium. J. Phycol. (in press). Singer and Winslow Briggs for comments on the manuscript. Kloppstech, K. and Schweiger, H.-G. (1982). Stability of poly(A)+ RNA in nucleate and anucleate cells of Acetabularia. 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