Cell contact-dependent positioning of the D cleavage plane restricts eye development in the Ilyanassa embryo

Morgan Goulding

Section of Integrative Biology, University of Texas, Austin, Texas, 78712
Present address: Institute of Molecular Biology, University of Oregon, Eugene, OR 97403, USA

e-mail: goulding{at}darkwing.uoregon.edu

Accepted 10 December 2002


    SUMMARY
 TOP
 SUMMARY
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In embryos of the gastropod Ilyanassa obsoleta, the first-quartet micromeres of the A, B and C lineages (1a, 1b, and 1c) are each competent to form an eye in response to signaling from the 3D cell. The first-quartet micromere of the dorsal D lineage (1d) is smaller than the others, divides at a slower rate, and lacks the ability to form an eye. These properties of 1d all depend on inheritance of vegetal polar lobe cytoplasm by its mother cell D at second cleavage. I show that they depend also on the presence of cells adjacent to D during the late four-cell stage: after ablation of the A and/or C cells before this stage, 1d inherits more cytoplasm than normal, divides more rapidly, and frequently forms an eye. In non-D lineages, cleavage plane positioning and micromere division rates are relatively insensitive to cell contacts. Compressing whole embryos during third cleavage also leads to an increase in 1d volume correlated with abnormal eye formation; this suggests that the normal effect of cell contacts is to position the D cell cleavage furrow closer to the animal pole, and the enhanced division asymmetry of the D cell contributes to the suppression of eye development.

Key words: Ilyanassa, Eye, Cleavage plane, Mollusca


    INTRODUCTION
 TOP
 SUMMARY
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In gastropod mollusks, the larval head is derived entirely from the first-quartet micromeres (1a, 1b, 1c and 1d), which form at third cleavage through the asymmetric division of the A, B, C and D quadrant founder cells (Conklin, 1897Go; Wierzejski, 1905Go). Fates of individual first-quartet cells have been traced by intracellular labeling in the marine gastropod Ilyanassa obsoleta (Render, 1991Go); in this species, each first-quartet clone makes a stereotyped contribution to the larval head, as shown schematically in Fig. 1A. The 1a and 1c cells give rise, respectively, to the left and right sides of the head, including the bilaterally paired eyes and the dorsal halves of the bilateral ciliated velar lobes. The 1b cell produces a clone that spreads bilaterally to form the ventral edge of the velar lobes; the oppositely positioned 1d cell forms dorsal ectoderm as well as a small region in the left half of the head. As shown by cell ablation experiments, the normal development of an eye in each of the lateral micromere clones (1a and 1c) depends on a signal initiated by the 3D `organizer' cell between the 24-cell and 28-cell stages (Clement, 1962Go; Lambert and Nagy, 2001Go; Sweet, 1998Go). Cell grafting experiments indicate that cells of the ventral 1b lineage are unaffected by this signal as a consequence of their position in the embryo; by contrast, the dorsal 1d lineage is normally exposed to the eye-inducing signal but is not competent to respond (Sweet, 1998Go).



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Fig. 1. Clonal fate map of first-quartet micromeres (A) and origin of quadrant lineages (B) in the Ilyanassa embryo. (A) Eight-cell embryo and hatching-stage larva are seen from the animal pole with dorsal at the top; the different clonal territories are drawn schematically using the data of Render (Render, 1991Go). (B) First and second cleavages are shown in dorsal aspect with the animal pole (marked by polar bodies) at the top. PL, polar lobe.

 

The 1d cell inherits a smaller volume of cytoplasm than the other three first-quartet micromeres, and has a longer cell cycle. Both of these early features distinguishing 1d depend on the prior segregation of vegetal polar lobe cytoplasm (Fig. 1B) into the dorsal mother cell D during second cleavage (Clement, 1952Go). Inheritance of polar lobe cytoplasm by the D cell is also required for suppression of eye development in 1d (Sweet, 1998Go). These findings suggest that a vegetal pole-derived cytoplasmic factor controls positioning of the D cleavage plane, as well as both the slow cell cycle rate and the eyeless fate of the 1d micromere.

Despite strong evidence for cell-autonomous restriction of 1d fate, other experimental results imply that extracellular signals might play a role. In one type of experiment, a CD half-embryo is isolated by removing the precursor of the A and B quadrants at the two-cell stage (Fig. 2A). The CD half proceeds through subsequent cell divisions just as it would in an intact embryo [e.g. only two micromeres (1c and 1d) are formed at third cleavage (Crampton, 1896Go)]. Most CD half-embryos form a single eye, consistent with the view that an eye can arise from 1c but not 1d; however, a minority of CD halves form two oddly placed or closely apposed eyes. AD and BD half-embryos isolated at the four-cell stage develop similarly, including a low frequency of two-eyed partial larvae (Clement, 1956Go). Surprisingly, removal of the 1a, 1b or 1c micromere from AD, BD or CD half-embryos does not extinguish eye development (Sweet, 1998Go). The 1d cell is likely to be the major source of eyes in these cases, as removal of both first-quartet cells from half-embryos abolishes eye development (Sweet, 1998Go).



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Fig. 2. (A-C) Schematic of operations used to isolate partial embryos in these experiments. Ablated cells are shaded. CD half-embryos are shown being isolated at the two-cell (A), four-cell (B) or eight-cell stage (C). In most experiments, the 1c cell was subsequently removed at the eight-cell stage, generating a CD-1c partial embryo (B,C, in box). To isolate analogous AD-1a and BD-1b partial embryos, the appropriate quadrant pairs were removed at the four-cell or eight-cell stage followed by ablation of the 1a or 1b micromere.

 

Why would the eye-forming potential of 1d be increased in partial embryos isolated at early stages? One possibility is that 1d fate depends on an eye-inhibiting intercellular signal before third cleavage, and that removing cells at an early stage disrupts this signal. Evidence is presented here that supports this view. I have found that removing quadrant founder cells during the middle of the four-cell stage permits eye development in labeled 1d clones; by contrast, removing embryo quadrants following third cleavage has little effect on the eyeless fate of 1d. In addition to promoting eye development, early cell ablations cause 1d to inherit an increased cytoplasmic volume and to divide as rapidly as the other first-quartet cells. Forcing 1d to inherit an increased cytoplasmic volume in whole embryos also results in an improved eye-forming ability, suggesting that early cell interactions restrict eye development simply by shifting the D cell division plane towards the animal pole.


    MATERIALS AND METHODS
 TOP
 SUMMARY
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals and embryo culture
Adult snails were obtained from the Marine Biological Laboratories at Woods Hole, MA in winter and early spring. Snail husbandry was as described by Collier (Collier, 1981Go). Snails were stored at 8-10°C in the dark, and batches were removed to room temperature aquaria and fed raw shrimp every other day to stimulate egg laying. Decapsulated embryos were cultured in moist chambers on the benchtop at 23±1°C in 1.5 ml 1.08x Jamarin artificial seawater (JSW) supplemented with 60-80 µg/ml streptomycin sulfate. Embryos were transferred every 1-3 days to JSW containing fresh antibiotic. Control and manipulated embryos were cultured for 7 days, after which time they were scored for ectodermal structures. Highly motile individuals were paralyzed in a solution of sodium azide and chloretone as described by Sweet (Sweet, 1998Go). Partial larvae were examined using a compound microscope; the birefringent shell, statocysts, operculum and internal mineralization were observed with the aid of crossed polarizing filters. After birefringent structures and velar cilia had been scored, the living specimens were squashed to facilitate detection of eyes and velar pigment. Experiments in which over 15% of controls developed abnormally were discarded.

Cell ablations
Cell ablations were carried out freehand with a glass needle in agarcoated dishes. Cell carcasses, which separate spontaneously from living cells within a few minutes, were plucked off or washed away by pipetting. If the sister of the ablated cell extruded any cytoplasm, the embryo was discarded. Partial embryos were raised as described by Sweet (Sweet, 1998Go), with one modification. In order to minimize the uncontrolled fragmentation often suffered by Ilyanassa embryos lacking two quadrants, the 4D yolk cell was removed from all partial embryos between 12 and 24 hours after first cleavage. Removal of 4D at the beginning of this interval did not appear to disrupt any aspect of larval development in otherwise intact embryos (n{approx}20). Eye development in half-embryos was not affected by removing 4D at any time between 10 and 24 hours after first cleavage (data not shown).

Cell labeling
The 1d cell was labeled in CD half-embryos by iontophoretic injection of 10,000 Mr lysine fixable tetramethyl-rhodamine dextran (Fluoro-Ruby; Molecular Probes). Electrodes were pulled from omega-dot fiber borosilicate capillaries (OD 1.0 mm; ID 0.75 mm) (FHC; Bowdoinham, ME) using a Flaming Brown capillary puller (Sutter Instruments) and tips were not broken off before use. Electrode tips were backfilled with 5% dextran in 150 mM KCl, 10 mM HEPES (pH 7); the electrode shaft was then filled with 3 M KCl, leaving a small air pocket between this and the dye. Current was generated using an apparatus constructed as described by Hodor and Ettensohn (Hodor and Ettensohn, 1998Go). Each 1d cell was injected with several short (1-5 seconds) pulses of current. Embryos were checked with a fluorescence microscope for successful labeling 15-30 minutes after injection and cultured in the dark.

Cleavage-stage preparations
To visualize cleavage-stage nuclei, embryos were fixed for 1 hour in 90% JSW and 2.5% paraformaldehyde supplemented with 0.1% Tween-20; washed in water and transferred briefly to methanol; rehydrated; and stained another hour or overnight with 1 µg/ml Hoechst 33528 (Polysciences) in 0.1% Tween-20. After another wash, embryos were mounted in 80% glycerol containing 4% n-propyl gallate and 20 mM Tris (pH 9) and examined by epifluorescence.

Observation of isolated cells
To isolate single micromeres, all cells of unwanted quadrants were first killed 5-10 minutes after the onset of third cleavage; 25-30 minutes later, the sister cell of the micromere' was killed and its carcass removed by pipetting. Each group of isolated micromeres was transferred to a microscope slide and fluorocarbon oil (Fluorinert FC-70; Sigma) was added under the supported coverslip to prevent evaporation. Micromeres were observed between 50 and 70 minutes after third cleavage, using a 40x objective lens. In most experiments, a video camera was used to transmit images to a monitor, and micromere outlines were traced on transparent sheets fixed to the monitor screen. Alternatively, isolated micromeres were imaged using a CCD camera and Scion Image software. The long and short axes of each cell were measured, and cell volumes were calculated using the formula for an ellipse (assuming radial symmetry about the long axis). In situ tracing and volume estimation of micromeres in CD half-embryos were carried out using the same methods.

Embryo compression
For compression during third cleavage, late four-cell stage embryos were placed in a 70 µl drop of JSW supplemented with 200 µg/ml swine skin gelatin (Sigma) on a microscope slide resting on a level surface. A 22x22 mm coverslip (no.1 thickness) was gently placed on the drop. A moment later (70 to 75 minutes after second cleavage), 15 µl of medium was removed gradually from the side of the coverslip, thereby compressing the embryos. After a 10 minute period in which the D cell divided in most embryos, the coverslip was floated off by adding JSW around its edges. Embryos were flattened to varying degrees, depending on their location under the coverslip. In `over-compressed' embryos (identifiable during compression by their girth and translucency, and afterwards by their wrinkled surfaces) 1d tended to form either abortively or not at all. Embryos that stably formed an abnormally large 1d cell were otherwise normal in appearance, except for their initially flattened shape. Cytokinesis in the A, B and C cells seemed less sensitive to compression, as it was invariably completed on schedule.


    RESULTS
 TOP
 SUMMARY
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
1d can form an eye in half-embryos, independently of the neighboring first-quartet micromere clone
The development of larval tissues in CD half-embryos has been investigated by Clement (Clement, 1956Go) and Sweet (Sweet, 1998Go). In the earlier work, 50% of all CD halves developed a single eye and another one-third developed two eyes; in the more recent study, a single eye developed in every case examined. I repeated these experiments by removing either the AB blastomere at the two-cell stage or its daughters A and B at the four-cell stage (Fig. 2A,B, excluding the box). Each ablation was made 35-50 minutes after the last cell division (25-40 minutes prior to second or third cleavage). After 7 days of development, one eye had formed in about 70% of CD half-embryos, and two eyes in about 20%, regardless of the stage of isolation (Table 1). In a few experiments, partial larvae were observed for 5-7 days more; among these, none was found to form any additional eyes.


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Table 1. Eye development in CD half-embryos isolated at different stages before third cleavage

 

Sweet (Sweet, 1998Go) also generated CD-1c partial embryos by isolating CD halves at the four-cell stage and then killing 1c after third cleavage, leaving 1d as the only first-quartet micromere. Surprisingly, CD-1c embryos were able to form an eye. To verify this, I repeated the CD-1c isolation. The A and B cells were ablated during the mid four-cell stage (35-45 minutes after second cleavage onset), and the 1c cell was ablated 30-40 minutes after third cleavage (Fig. 2B, box). Removing 1c any earlier usually caused damage to its sister cell 1C. Eleven CD-1c partial embryos (46%) formed an eye by the seventh day of development (Table 2) compared with a frequency of 29% reported by Sweet.


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Table 2. Isolation of partial embryos before or after third cleavage: effect on eye-forming potential of 1d

 

Sweet concluded that the eyes in CD-1c partial larvae were formed by the 1d cell, as removal of both first-quartet micromeres from half-embryos abolished eye development. Another explanation is that 1d can induce other cells to form an eye, while being unable to form any itself. To resolve this ambiguity, I labeled the 1d cell by iontophoretic injection of fluorescent dextran in CD-1c partial embryos. Four such embryos were successfully labeled. After 7 days of development, three out of these four had formed a single eye. In each case the pigmented retina was closely surrounded by fluorescent label and lay close to a region of labeled epidermis (Fig. 3). This confirms that after removal of cells at the four-cell stage, the 1d lineage can deviate from its fate by forming an eye. It is assumed here that eyes developing in analogous partial embryos consisting of different quadrant pairs (AD-1a; BD-1b) also arise from 1d.



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Fig. 3. A representative CD-1c partial larva in which the labeled 1d cell formed an eye (arrows). Scale bar: 50 µm.

 

The effect of neighboring first-quartet micromeres on 1d potential was tested by repeating the above lineage-tracing experiment in CD halves without removing 1c. If the 1c cell is capable of inhibiting eye development in 1d strongly, one would predict eyes to be unlabeled in these experiments. The 1d cell was successfully labeled in ten CD halves, all of which formed at least one eye (Fig. 4). In every case, one or two patches of epidermis were labeled; if an eye was labeled, the labeled epidermis was found nearby. Four out of 14 eyes were labeled; the 10 unlabeled eyes (formed in eight half-embryos) were presumably derived from the normal eye precursor 1c. The observed rates of labeled and unlabeled eye formation in these CD halves resemble the frequencies of eye development previously reported for CD partial embryos that, respectively, lack either 1c or 1d (Sweet, 1998Go). Consistent with the results of other cell ablation experiments (Clement, 1967Go; Sweet, 1998Go), these data suggest that the decision of each first-quartet micromere to form an eye is not strongly influenced by neighboring first-quartet clones.



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Fig. 4. Results of 1d labeling experiments in CD half-embryos. Each oval represents an individual with the stated number of labeled and unlabeled eyes.

 

Cell contacts during the late four-cell stage suppress eye development in 1d
Why is 1d able to form an eye in half-embryos isolated at an early stage? One possibility is that the eye-forming potential of 1d is normally inhibited by intercellular signals shortly before or concurrent with third cleavage. To test this idea, CD halves were isolated by removing all cells of the A and B quadrants immediately after third cleavage (Fig. 2C); the 1c micromere was then removed as before, leaving CD-1c partial embryos in which normal cell contacts had existed at the four-cell stage. Strikingly, only 5% of these partial embryos formed an eye (Table 2). Other markers of the larval head (velar pigment and long velar cilia), which are frequently observed in CD-1c partial larvae operated at the four-cell stage, were lacking in nearly all of these specimens; by contrast, markers of the larval trunk ectoderm (shell, statocysts, operculum and internal birefringent masses) appeared at about the same frequencies regardless of the timing of operation (not shown). AD-1a and BD-1b partial embryos were also isolated by first removing two quadrants either before or after third cleavage, and then removing the 1a or 1b micromere. The results (Table 2) were essentially the same as in CD-1c partial embryos: regardless of which half of the embryo remained, the frequency of eye development was greatly reduced when quadrants were removed from the early eight-cell rather than the middle four-cell stage. These results suggest that during the late four-cell stage, the A, B and/or C cells interact with the D cell in a way that prevents 1d from forming an eye or other head structures.

The next question asked was whether A, B and C each have an equal inhibitory effect on eye development in 1d. To address this question, the A, B or C cell was removed singly during the middle four-cell stage (35-45 minutes after second cleavage). To facilitate comparison with the first set of experiments, an additional quadrant was removed from these embryos immediately after third cleavage; AD, BD and CD half-embryos were thus generated that, during the late four-cell stage, had lacked only one cell (A, B or C). The remaining non-1d micromere in these half-embryos (1a, 1b or 1c) was also removed 30-40 minutes after third cleavage, again leaving 1d as the only eye precursor. After removal of either A or C at the four-cell stage, eyes developed at rates comparable with those seen after removal of two cells (Table 3). Removal of the B cell alone did not lead to a significant increase in eye development. These results suggest that the combination of A and C cells contacting the D cell is necessary and sufficient to suppress eye development in 1d.


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Table 3. Single cells removed at the middle four-cell stage: effect on the eye-forming potential of 1d, as assayed in different partial embryos

 

To characterize the time course of this interaction, a series of BD-1b partial embryos was made by removing A and C at different intervals during the middle to late four-cell stage. The 1b micromere was killed at the eight-cell stage as before, and partial embryos were scored for eye development at 7 days. The results are charted in Fig. 5. Based on X2 tests, no significant difference was found in the rates of eye development after A+C ablation at timepoints between 35 and 55 minutes after second cleavage; likewise, removing A and C at 65-75 minutes after second cleavage (0-15 minutes before micromere formation) did not yield significantly different results compared with removing the A and C quadrants in the early eight-cell stage. Between the two pooled groups, however (35-55 minutes versus 65 minutes to early eight cell), a significant difference was found ({chi}2=5.79, P<0.05). Thus, the eye-inhibiting action of the A and C cells seems to be completed between 55 and 65 minutes into the third cell cycle, which corresponds to the transition from prometaphase to metaphase in the D cell.



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Fig. 5. Frequencies of eye development in BD-1b partial embryos generated by removing the A and C quadrants during different time windows (x-axes). Each time point represents samples from at least three experiments (seven clutches of eggs used in all). Phases of mitosis (shown at the top) were determined by chromatin staining of samples fixed in parallel during two experiments.

 

Cell contacts act to position the D cell division plane
The earliest visible difference between 1d and the other first-quartet micromeres is their cytoplasmic volume: whereas 1a, 1b and 1c are all approximately the same size, 1d is normally only two thirds as big. After isolation of half-embryos in the middle four-cell stage, the 1d cell consistently inherited an abnormally large volume of cytoplasm. This point was made by measuring the size of micromeres isolated either from whole embryos or from CD halves which had themselves been isolated in the middle of the four-cell stage (35-50 minutes after second cleavage). Ablation of A and B during this time interval was found to affect the volume of cytoplasm inherited by both 1c and 1d, but to different degrees. The mean volume of 1c was increased by 28% in CD halves; in the same embryos, the 1d cell experienced a proportionally greater (70%) mean volume increase, inheriting, on average, a volume about equal to the normal size of 1c. Increases in mean 1d size were also observed in 1d cells isolated from AD and BD halves and from D quarters; ablation of the A, B or C cells alone resulted in smaller but significant increases in 1d size (Fig. 6).



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Fig. 6. Histograms showing first-quartet micromere volumes in normal embryos or partial embryos isolated at the four-cell stage. Micromeres were measured after isolation from whole or partial embryos. Quarter embryos, half embryos and three-quarter embryos were isolated by puncturing unwanted blastomeres between 35 and 45 minutes after second cleavage (30 to 40 minutes before micromere formation). One micromere was isolated from each whole or partial embryo; this was achieved by isolating the desired quadrant (either during the four-cell stage or immediately after third cleavage) and then ablating the sister cell of the desired micromere 30-40 minutes after third cleavage.

 

To examine more generally how cell contacts in the late four-cell stage influence positioning of third cleavage planes, the A, B and C cells were each isolated by killing the three other cells 35-45 minutes after second cleavage onset. The first-quartet micromeres formed by isolated cells were in turn isolated and measured. The results of these experiments (Fig. 6) indicate that cell contacts in the four-cell stage have a relatively large effect on cleavage plane positioning in the D cell compared with the three other cells. The variability of cleavage plane positioning is obviously increased in isolated D cells, suggesting that cell contacts may normally act to `fine-tune' the position of the cleavage furrow.

The 1d division pattern is altered in partial embryos isolated before third cleavage
In normal development, each first-quartet micromere divides asymmetrically to produce a large apical cell (denoted as 1m1) and a small turret cell (1m2). The turret cells in all four quadrants have small, condensed nuclei and generally do not divide for at least 11 hours (M. G., unpublished). The apical cells shortly undergo another asymmetric division, forming a secondary apical cell (1m11) and a basal cell (1m12). In other gastropod species, all four 1m1 cells contribute some of their progeny to the definitive head ectoderm, with the largest contributions made by the lateral (1a12 and 1c12) and ventral (1b12) basal cells. In Ilyanassa, the 1d cell immediately exhibits a unique division pattern (Fig. 7A,B), dividing about 30 minutes later than the three other first-quartet cells. The apical daughter cell (1d1) divides asymmetrically to form a secondary apical cell (1d11) that is larger than its basal sister cell (1d12); this asymmetry is reversed with respect to the other three quadrants, in which the basal cell is slightly larger than its sister (Clement, 1952Go). The 1d12 cell is very small, has a highly condensed nucleus (Crampton, 1896Go), and arrests for a long period while 1a12, 1b12 and 1c12 rapidly proliferate (M. G., unpublished). Thus, the relative contributions of first-quartet micromere derivatives to head ectoderm are presaged by differences in early cell division rate.



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Fig. 7. Effect of cell ablation before third cleavage on 1d division pattern. (A) Normal division chronology of first-quartet micromeres. Minutes after first cleavage are indicated on vertical axis; horizontal gray lines indicate the ages of embryos shown in B, F and G (bfg) and in C, D and E (cde). (B-G) Camera lucida drawings of nuclei in whole and partial embryos, shown at the same scale; first-quartet micromere derivatives are shaded. (B) Normal embryo, 500 minutes after first cleavage. The 1b12 nucleus is in prometaphase; the small 1d12 nucleus is in interphase. (C) Nuclei in a representative D quarter embryo isolated in the mid four-cell stage and fixed at 375 minutes after first cleavage; 1d1 is precociously in metaphase. (D) Another D quarter isolated in the mid four-cell stage, and fixed at the same stage as the specimen in C; both 1d1 and 1d2 are in metaphase. (E) Nuclei in a D quarter embryo isolated in the early eight-cell stage and fixed at 375 minutes after first cleavage. Interphase 1d1 and 1d2 nuclei exhibit their normal size difference. (F) Nuclei in a BD half-embryo isolated in the mid four-cell stage and fixed at 500 minutes after first cleavage, showing two abnormal pairs of nuclei apparently derived from 1d. The 1b lineage has a normal appearance, including one small nucleus (1b2) and two larger ones (1b11 and 1b12); the 1b12 nucleus is in metaphase. (G) Nuclei in a BD half-embryo isolated in the early eight-cell stage and fixed at the same stage as the embryo in F. The 1d cell has produced the normal complement of three nuclei. Uncharacteristically, the two daughters of 1b1 are dividing synchronously. In both F and G, the 3b nucleus has entered prophase earlier than normal, probably as a result of abnormal exposure to 3D signaling. 4d is in metaphase; 4D is not shown.

 

After removal of the polar lobe, 1d divides with the same rate and pattern as the other first quartet micromeres (Clement, 1952Go) and readily forms an eye when transplanted into a normal embryo (Sweet, 1998Go). To find out if a transformation of the 1d division pattern is likewise correlated with eye development following early cell ablation, I fixed D, BD and CD partial embryos during late cleavage stages and examined them after staining for nuclei. In D quarters isolated in the middle four-cell stage and fixed between 360 and 420 minutes after first cleavage, the apical cell 1d1 was invariably seen to divide earlier than normal, roughly on schedule with the 1abc1 cells in controls (Fig. 7C). Surprisingly, the turret cell 1d2 also divided during this period in at least eight out of fifteen D quarters (Fig. 7D). In most of the D quarters examined here, the nuclei of 1d2 and its daughter cells were as large or nearly as large as the nuclei of 1d1 and its daughters, suggesting that the first division of 1d was abnormally symmetric with respect to both size and cell cycle rate of sister cells.

Similar effects on the 1d cell division pattern were observed following isolation of BD and CD half-embryos at the four-cell stage. In early-isolated BD halves fixed 500 minutes after first cleavage, four 1d lineage cells with interphase nuclei were observed (2/3 cases), rather than the three normally present at this stage (1d11, 1d12, 1d2); the positions and sizes of these nuclei suggested that they were formed by equal division of both 1d1 and 1d2 (Fig. 7F). In CD halves fixed 560 minutes after first cleavage, the same profile of two apparent cell pairs was found (2/3 cases); in another specimen 1d2 was small and undivided as normal, but both daughters of 1d1 (1d11 and 1d12) were prematurely in mitosis.

Control partial embryos (D, BD and CD) isolated after third cleavage showed no evidence of abnormal cell division timing through the stages examined. In most (13/14) late-isolated D quarters, the 1d1 and 1d2 nuclei exhibited their normal size difference (Fig. 7E). Among late-isolated BD and CD halves, the 1d2 cell likewise had a very small undivided nucleus in 6/9 cases (Fig. 7G); in the other cases, however, all three 1d-derived cells had nuclei of the same intermediate size, suggesting that 1d division asymmetry might also be impaired in late-isolated half-embryos. In most half-embryos isolated either before or after third cleavage, the subsequent division of 1d1 was also abnormally symmetric with respect to the sizes of sister nuclei.

In all half-embryos examined, cells of the 1b and 1c lineages appeared normal with respect to number, positioning and nuclear size (n=15). A normal cell pattern was also observed in C quarter embryos isolated in the mid four-cell stage, examined at 390 or 420 minutes after first cleavage (n=4).

These observations do not indicate a transformation of the 1d division pattern to the 1abc pattern in partial embryos. Although the overall division rate in the 1d lineage is increased, the geometry of cell divisions in early-isolated partial embryos is unpredictable; this suggests a deficiency of regulation, rather than a switch between two regulatory states. Normal execution of the 1d division pattern seems to depend on extrinsic factors acting both before and after third cleavage. It is possible that the geometry of corresponding (and superficially similar) divisions is regulated differently in D and non-D lineages, as the 1b and 1c cells always divided with their normal asymmetry in partial embryos. Precocious division seems to be linked to increased cell size in both 1d and 1d2, suggesting a general effect of cell size on division rate.

Increasing the inherited size of 1d promotes eye development
The increase in both 1d size and eye-forming potential in partial embryos suggested that cell contacts at the four-cell stage might normally restrict 1d eye development by shifting the D cell division plane towards the animal pole. If so, then forcing D to divide further from the animal pole in an intact embryo should allow 1d to form an eye.

I found that compressing embryos under a coverslip during third cleavage caused some 1d cells to inherit more cytoplasm than normal, without perceptibly disturbing the angle of cleavage planes. To investigate the effects of compression on 1d size and division timing, a total of thirty-seven 1d cells were isolated from whole embryos compressed in four experiments. Cells were photographed at high magnification (2.7-3.2 pixels/µm) for size measurement, then cultured under a sealed, supported coverslip and photographed every thirty seconds for eight hours. Size measurements indicated a subtle effect on D cleavage plane positioning: mean 1d size was not significantly different from normal (29 pl), and most cells were within the normal range. Seven cells (19%), from three of the four experiments, fell within the size range of the normal eye precursors (35-57 pl), indicating that this method can in some cases force the D cleavage plane away from the animal pole. Surprisingly, the size of isolated 1d cells was not significantly correlated with the length of their cell cycles (r=–0.27).

In another set of sixteen experiments, the eye-forming potential of measured 1d cells was assayed after compression at third cleavage. To facilitate measurement of 1d volume, CD half-embryos were isolated by puncturing the incompletely formed 1A and 1B cells immediately after embryos were released from compression. CD half-embryos were quickly transferred to slides and the 1d cell was traced or photographed. The 1c cell was subsequently killed, and CD-1c partial embryos were raised as before in separate dishes. Forty partial embryos were scored for eyes at 7 days; results are shown in Fig. 8A. In ten cases, 1d volume fell within the normal range of 1a and 1c; four of these developed a single eye. In another partial embryo, with an especially large 1d cell (72 pl volume), a pair of closely spaced eyes developed. The remaining 29 partial embryos, in which 1d was smaller than the normal eye precursors, did not develop any eyes. Logistic regression analysis showed a strong relationship between 1d size and eye development ({chi}2=13.75, P=0.0002). This result suggests that the small size of 1d limits its eye-forming potential, and that A and C cell contacts inhibit 1d eye development by positioning the D cell division plane close to the animal pole.



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Fig. 8. Ectopic eye development correlated with displacement of the D cell division plane. (A) Histograms showing 1d volume in embryos that were subsequently assayed for eye development. 1d cells were measured in situ following compression at third cleavage (compressed embryos) or ablation of neighboring cells during the mid four-cell stage (partial embryos). Black boxes represent individuals that developed one or more eyes; light-gray boxes represent eyeless individuals. Normal micromere volumes (data from Fig. 6; dark-gray boxes) are shown on the same scale for comparison. (B) A three-eyed larva that developed after compression during third cleavage. Scale bar: 100 µm.

 

To test this model in another way, 1d cells were measured in CD half-embryos after ablation of either A or both A and B during the four-cell stage. As before, the 1c cell was removed from CD halves, and partial embryos were scored for eye development (Fig. 8A). Among these partial embryos, 1d exceeded the normal eye precursors' upper size limit of 57 pl in five cases, three of which formed an eye. Among nine cases where 1d volume fell within the upper 90th percentile of normal eye precursor cell volumes (40-57 pl), five formed an eye. At 1d volumes of 32-40 pl (a range straddling the lower limit of eye precursor size), an eye formed in one case (1d volume 37 pl) out of five. Although not enough small 1d cells were obtained in these experiments to test the relationship of cell size and eye development formally, pooling these data with the results of compression experiments strengthened the logistic model fit (P<0.0001)

While the above results argue that 1d eye-forming potential is restricted by D cleavage plane positioning, they leave room for doubt as to whether this parameter is important in the intact, normally developing embryo. In six of the compression experiments described above, this question was addressed by culturing embryos intact after compression. Among 169 such whole embryos, 103 developed into approximately normal larvae, with the rest suffering from a variety of trunk deformities. Twelve larvae that were otherwise normal in appearance developed an ectopic eye; in every one of these cases the third eye was on the left side of the head, either just dorsomedial to the normal left eye (Fig. 8B) or just to the left of the midline. The 1d clone normally contributes mainly to left-sided and mid-dorsal regions of the head (Render, 1991Go) (M. G., unpublished), suggesting that these ectopic eyes were derived from 1d. Together with the size increase observed in some isolated 1d cells following compression, this result suggests that the enhanced asymmetry of normal third cleavage in the D cell does in fact constrain the pattern of eye formation in the intact embryo.


    DISCUSSION
 TOP
 SUMMARY
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
From previous work it is known that inheritance of polar lobe cytoplasm by the D cell is required for specifying the reduced size and division rate of 1d (Clement, 1952Go), and for suppressing eye development in the 1d clone (Sweet, 1998Go). As shown here, these properties of 1d depend also on contact between D and neighboring cells during the middle-to-late four-cell stage. The D cell appears to be more sensitive than A, B or C to the influence of cell contacts on third cleavage plane positioning. Thus, it seems that, in addition to specifying the inductive potential and endomesodermal fate of 3D (Clement, 1962Go), the polar lobe cytoplasm acts to sensitize the D cell cytoskeleton to an early extracellular stimulus. The induced poleward displacement of the D cleavage plane apparently prevents 1d from forming an eye.

As in vertebrate and insect embryos, the dorsoventral axis in Ilyanassa is patterned largely by intercellular signaling originating from a polar organizer (Clement, 1952Go; Clement, 1962Go; Sweet, 1998Go; Lambert and Nagy, 2001Go). In the embryos of mollusks and a number of other phyla, the early formation of fate-restricted cells places a relatively large informational burden on the behavior of each individual cell, and it might be expected that variation in the geometry of an early cell division would have an impact on later development. This study demonstrates one case where lineage-specific division geometry contributes to ectodermal pattern formation. Exactly how cell contacts cause a shift in the division plane, and how this shift in turn restricts eye-forming potential, are matters for further investigation.

Control of cell division geometry by intercellular contacts
Several examples are known in which cell contacts help to position the mitotic apparatus. One way for cell contacts to act is by constraining the shape of a dividing cell. In studies on pulmonate gastropod embryos, Mescheryakov (Mescheryakov, 1976Go; Mescheryakov, 1978Go) found that prior to third and fourth cleavages, macromere spindles rotate between prophase and metaphase to orient approximately parallel to the longest axis of the cell, which is also parallel to zones of cell contact. Reducing or eliminating cell contacts before this stage blocks spindle rotation, an effect attributed to loss of the constraint imposed by neighboring cells on blastomere shape. Consistent with this view, mechanical deformation of mammalian epithelial cells has recently been shown to reorient spindles parallel to the longest axis of the cell (O'Connell and Wang, 2000Go). Surface deformation can also play a role in generating division asymmetry, as shown for the CD cell of leech embryos (Symes and Weisblat, 1992Go).

Another way for cell contacts to affect division geometry is by changing the way that part of the cell cortex interacts with the mitotic apparatus. In early embryos of the nematode Caenorhabditis (Goldstein, 1995Go) and the annelid Tubifex (Takahashi and Shimizu, 1997Go), spindle poles have been shown to migrate toward zones of cell contact. The opposite effect is seen in embryos of the crustacean Sicyonia; in this case, spindle poles are repelled by cell contact zones, apparently independent of cell shape (Wang et al., 1997Go). In early mouse embryos, cell contacts have been shown to reduce the accumulation of cortical myosin locally (Sobel, 1983Go), suggesting a possible molecular mechanism for cleavage plane positioning in Ilyanassa.

Control of multicellular pattern formation by cleavage geometry
Lineage-specific variation in third cleavage geometry might affect eye development by mediating differential inheritance of an `eye determinant' by the first-quartet micromeres. Such a determinant, for example, might be localized in all four cells at a level just below the presumptive cleavage plane of the D cell; it would then be inherited by 1a, 1b and 1c, but not 1d. A related possibility, not necessarily involving localized factors, is that the developmental potential of the micromere is determined by its cytoplasmic volume. By virtue of its smaller size, the 1d cell inherits larger ratios of surface area to volume, and DNA to both surface and cytoplasm, etc., compared with the other first-quartet cells. Enlarging the cell could effectively dilute a component that acts concentration dependently to specify 1d fate; such a factor might exist in all four quadrants or could be enriched in D. In a number of systems, developmental transitions are triggered by changes either in absolute cell volume or the ratio of cell volume to DNA content (Edgar et al., 1986Go; Kane and Kimmel, 1993Go; Kirk et al., 1993Go; Mita, 1983Go; Newport and Kirschner, 1982Go; Yamashiki and Kawamura, 1986Go). It is reasonable to suspect that this type of mechanism might commonly influence the fates of cells produced by asymmetric division.

Alternatively, the inherited cytoplasmic volume of a cell might affect development by determining properties of clonally derived tissue at a later stage. There is some evidence that in the gastropod head, a minimum threshold mass of first quartet-derived ectoderm is required to form an eye. In normal development, eye precursor cells delaminate from paired regions of proliferating ectoderm called cephalic plates, which give rise to the definitive head ectoderm and are initially separated by an apical band of non-proliferating larval epithelium (Conklin, 1897Go; Tomlinson, 1987Go) (N. H. Verdonk, PhD Thesis, Rijksuniversiteie, Utrecht, 1965). In embryos of the pulmonate snail Lymnaea, early micromere pattern defects that reduce the size of a cephalic plate typically abolish eye development there; conversely, cephalic plates that are larger than normal seldom lack an eye, and frequently develop two or more. In cases where a deficiency of apical larval cells allows the two cephalic plates to fuse, a single eye or a closely spaced set of two or three eyes is reported to form in the center of the fused ectoderm (Arnolds et al., 1983Go) (N. H. Verdonk, PhD Thesis, Rijksuniversiteie, Utrecht, 1965). As noted by Arnolds et al. (Arnolds et al., 1983Go), abnormal eye patterns do not necessarily correspond to loss or gain of specific lineage elements. All of these results are more consistent with eyes being specified by a tissue size-dependent field than by a strictly lineage-dependent mechanism.

The effect of the polar lobe on 1d fate
Removal of the polar lobe at first cleavage seems to transform the 1d cell into a duplicate of 1a, 1b and 1c in terms of inherited size and early division pattern (Clement, 1952Go), as well as eye-forming potential (Sweet, 1998Go). One way to account for this is with a simple `cleavage plane positioning model', whereby the polar lobe potentiates an asymmetry-enhancing effect of cell contacts on the D cell, and the consequent increase in D division asymmetry determines the slow cell cycles and eyeless fate of 1d. The observed lack of correlation between size and division timing in isolated 1d cells argues against this model; however, further study is needed to determine whether micromere size controls division rate in the context of the whole embryo. More fundamentally, the cleavage plane positioning model is contradicted by two differences between the effects of polar lobe removal and early cell ablation on 1d behavior. These differences can be reconciled only by postulating additional mechanisms.

The first difference is related to eye development. In this study, eyes formed in 10 of the 23 CD-1c isolates in which 1d was as big as an eye precursor. According to Sweet (Sweet, 1998Go), half-embryos lacking the 1d cell (AD-1d, BD-1d, CD-1d) formed an eye in 62/73 cases. Considering that partial embryos in the present study generally formed eyes at a higher rate than the same isolates made by Sweet, the above comparison argues that removing early blastomeres does not sufficiently elevate 1d eye-forming potential to match the normal eye precursor cells. By contrast, 1d cells transplanted from embryos lacking the polar lobe developed an eye just as frequently as 1a, 1b and 1c cells transplanted to the same position (Sweet, 1998Go). Thus, it is reasonable to think that 1d eye development is restricted by a polar lobe-derived factor that does not affect cleavage plane positioning.

Second, this study showed that the 1d cell often divides symmetrically in partial embryos; by contrast, symmetric divisions do not follow polar lobe removal (Clement, 1952Go) (data not shown). In quadrants that naturally lack the polar lobe, early isolation does not make the first quartet micromere divide symmetrically, as 1c always divided normally in C quarter embryos isolated at the four-cell stage. These observations suggest that cell contacts enhance 1d division asymmetry by antagonizing an effect of the polar lobe. This relationship could be explained if a polar lobe-derived factor acts to position the cleavage plane of 1d with reference to the apical pole, resulting in an equal division only when 1d size is increased by loss of A and C cell contacts (Fig. 9).



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Fig. 9. Hypothesis to explain the difference in 1d division asymmetry when the polar lobe or the A/C cells are removed. Micromeres are shown with their apical pole towards the top; broken lines indicate the plane of their first division. (A) The normal 1a, 1b and 1c micromeres, which are presumably equivalent to the 1d micromere formed after polar lobe ablation. (B) In the normal 1d cell, a polar lobe-derived factor repositions the division plane with reference to the apical pole. (C) In partial embryos, the polar lobe-derived factor still positions the 1d division plane, but as the cell is bigger, division occurs proportionally higher on the axis of the cell. In this diagram, the resulting volume of the apical cell is intermediate between the normal volumes of 1d1 and 1a1/1b1/1c1.

 

One way to account for the differential effects of removing the polar lobe or the A/C cells on eye development is to speculate that the size of the apical daughter cell of a first-quartet micromere, rather than the size of the micromere itself, determines the eye-forming potential of the clone. The apical cells 1a1 and 1c1 are required for eye development in Ilyanassa (H. C. Sweet, PhD Thesis, University of Texas at Austin, 1996), suggesting that their sisters 1a2 and 1c2 cannot form eyes. Assuming that this is also true of a bigger than normal 1d2 cell, then the relatively poor 1d eye development in partial embryos versus polar lobe-deficient embryos could be attributed to loss of cytoplasm from the 1d1 lineage during an abnormally symmetric division.


    ACKNOWLEDGMENTS
 
This work was supported by NSF grant IBN-9982025 to Gary Freeman. I thank Dr Freeman for his support, advice and comments on the manuscript. I also thank Bruce Bowerman for his support during the completion of this work. Thanks are also due to Justin D. Lambert, David Parichy, John Pezzullo, Marty Shankland, Hyla Sweet and Peter Vize for sharing ideas, methods and/or results; to John Morrill for sharing his translation of Wierjezski's paper; and to Bobby Blakeway for construction of the iontophoresis box.


    REFERENCES
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