1 Department of Zoology, University of Washington, Seattle, WA 98195, USA
2 Friday Harbor Labs, Friday Harbor, WA 98250, USA
*Author for correspondence at address 1 (e-mail: munroem{at}u.washington.edu)
Accepted 5 October 2001
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SUMMARY |
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Key words: Ascidian, Notochord, Pattern formation, Morphogenesis
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INTRODUCTION |
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Experimental and theoretical studies have identified many possible sources of patterned cellular behavior within tissues and these fall into two classes. First, pre-existing cytoplasmic asymmetries and/or spatial pre-patterns in the expression of "morphoregulatory" genes could determine spatial patterns of intrinsic motile and adhesive behavior before any morphogenetic movements occur. For example, pre-existing gradients of adhesivity along a specific embryonic axis might be resolved by active cell movements into oriented tissue extension (Gergen et al., 1986; Irvine and Wieschaus, 1994; Mittenthal and Mazo, 1983; Nardi and Kafatos, 1976a; Nardi and Kafatos, 1976b; Wieschaus et al., 1991).
Second, interactions with surrounding tissues during morphogenesis could trigger or otherwise influence patterned cell behavior. For example, recent studies in Xenopus and Fundulus embryos suggest signals emanating from localized specific cell populations or tissue boundaries could propagate through cell populations and thereby polarize motile and adhesive behaviors within them (Domingo and Keller, 1995; Shih and Keller, 1992; Trinkaus, 1998; Trinkaus et al., 1992). Alternatively, local interactions occurring at tissue boundaries could bias the directionality of cell extension or movement, for example through contact-dependent inhibition or stabilization of protrusive behavior at planar tissue boundaries (Jacobson and Moury, 1995; Jacobson et al., 1986; Keller et al., 1989; Keller et al., 1992), or interactions with an oriented extracellular matrix at vertical tissue boundaries (Keller et al., 1991; Nakatsuji et al., 1982; Nakatsuji and Johnson, 1983; Shih and Keller, 1992).
Here, we exploit the relatively simple organization and development of the ascidian embryo to assess the relative contributions of intrinsic morphogenetic properties and tissue interactions to patterning the cell movements that drive invagination and convergent extension of the notochord. During ascidian notochord formation, morphogenetic pattern emerges rapidly within a sheet of 40 cells, in an embryo having only 500 cells. The embryos geometry remains relatively unchanged as this pattern emerges, and the simple stereotyped movements of cells within the notochord and surrounding tissues are well described (Miyamoto and Crowther, 1985; Munro and Odell, 2002; Nishida, 1987). In contrast to many other embryos wherein determination and regionalization of cell fates occurs simultaneously with morphogenesis, primary cell types and regional identities of embryonic ascidian cells are determined long before notochord cells rearrange (Kawaminani and Nishida, 1997; Nakatani and Nishida, 1994; Nishida, 1990; Nishida, 1992b; Nishida, 1993; Nishida, 1994; Nishida and Satoh, 1989; Nishikata et al., 1987; Whittaker, 1990). Moreover, it is easy to identify and manipulate notochord cells and each of the surrounding tissues at key stages.
We first document the intrinsic morphogenetic potential of notochord rudiments, isolated just after they have been determined at the 64-cell stage. We then describe the use of micromanipulation and confocal microscopy to assess the relative contributions of tissues that surround the notochord to the emergence of pattern within it.
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MATERIALS AND METHODS |
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Cell isolation and microablation techniques
Fig. 1 schematizes the micromanipulations we performed. We isolated notochord rudiments and all other embryo fragments under a stereomicroscope using hand-held glass microneedles, pulled from micropipette glass (OD: 1.0 mm; ID: 0.7 mm; Drummond Scientific, Broomell, PA) on a Flaming-Brown micropipette puller (Model P-72; Sutter Instruments, San Francisco, CA), and mounted on shortened 5" pasteur pipettes. We dipped needles in 1% agar to prevent their sticking to and lysing cells. We isolated presumptive notochord blastomeres at the 64-cell stage when notochord precursors first become lineage restricted and studied only those specimens in which all 4 notochord precursor cells remained undamaged. We performed all other fragment isolations at mid-late gastrula stages. Each isolation entailed, at most, two cuts, whose sites of initiation were well defined with respect to the stereotyped positions of identifiable blastomeres (Fig. 1).
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To ablate identified neural plate or endoderm precursors we first lysed them at the 64-cell stage using sharpened tungsten needles under a stereo dissecting microscope, then allowed dead cells to separate from the remaining embryo through one cell division round, and finally cleaned them away using a hand held microneedle prepared as described above. In all cases we conclusively verified the specific ablation of the desired cell population (see Results for details).
Time lapse analysis of dorsal anterior quadrants
To visualize cell shape changes over time in dorsal anterior quadrants, we used the fluorochrome Bodipy (488 nm excitation; Molecular Probes, Eugene Ore) which diffuses freely into living embryos and concentrates into yolk granules within the cytoplasm yielding a "negative" image of cell outlines in confocal thin sections (Cooper and Kimmel, 1998). We pre-incubated dorsal anterior quadrants for 60 minutes in a 100 µM solution of Bodipy in FSW, transferred them through several FSW rinses, and then mounted them between two coverslips within a custom temperature-controlled chamber, as described in the accompanying paper (Munro and Odell, 2002), on the stage of a Biorad MRC 600 LSCM on a Nikon upright microscope. We imaged embryos using the 488 nm line of a krypton-argon laser and a 25x Plan Neofluor multi-immersion lens (Carl Zeiss) adjusted for water immersion. We collected images at 10-minute intervals from each of several identified embryo fragments within a field. We used the minimal exposure required to obtain usable images. Under these conditions, the embryo fragments developed to the same extent as either Bodipy-labeled or unlabelled embryos cultured without fluorescent excitation in Petri dishes.
Histological analysis
The methods we used to fix embryo fragments, stain them with Bodipy-phalloidin and analyze them with confocal microscopy are essentially the same as those described elsewhere (Munro and Odell, 2002). For morphometric measurements, we mounted embryos in a mixture of 30% glycerol in PBS to avoid the shrinkage and distortion that alcohol dehydration causes.
Notochord extension index
To characterize the extent to which notochord rudiments formed normal notochords under different experimental conditions, we devised a notochord extension index (NEI) which varies between 0 and 1, and reflects both convergent extension within the notochord plate, and overall extension of the notochord plate along the AP axis. Notochord rudiments that did not change their aspect ratio at all received an NEI score of 0. Rudiments that exhibited the degree of extension seen in normal embryos at the end of stage II when formation of the cylindrical intermediate is completed, but in which no cell had come to span the entire circumference, received a score of 0.5. The NEI score then increased in direct proportion to the fraction of cells within the rudiment that had completed rearrangement (i.e. rearranged to span the entire circumference of the notochord rudiment); thus a rudiment in which half the cells had completed rearrangement received a score of 0.75, and one in which all cells had completed rearrangement received a score of 1.0. In no case have we ever observed a notochord rudiment in which some cells had completed rearrangement, but which had not extended to the degree seen at the end of stage II. Thus our NEI is well defined.
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RESULTS |
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To determine how much of their normal developmental program ascidian notochord cells can express in the absence of surrounding tissues, we isolated primary notochord blastomeres at the 64 cell stage as a 4-cell rudiment consisting of blastomeres A7.3 and A7.7 and their bilateral partners (Fig. 1C), cultured them in filtered sea water (FSW) on non-adhesive coverslips, and monitored their development intermittently through a stereo microscope (n>100) or using time-lapse microscopy (n=8).
In normal embryos, cells of the primary notochord lineage divide three times: once just before the onset of gastrulation to form an arc of 8 cells; once at mid-gastrula stage along the AP axis; and again at the beginning of neurulation along the AP axis (Munro and Odell, 2002; Nishida, 1986). Just after the final notochord cell division, notochord cells within whole embryos become motile, and the notochord plate begins to converge and extend along, and invaginate about, the AP axis. Within six hours, they have achieved the stack of coins configuration.
Fig. 2 summarizes our observations of isolated notochord rudiments. In nearly all cases examined, notochord cells within isolated rudiments executed all three of their final divisions, with roughly normal timing relative to whole embryos cultured at the same temperature, giving rise to a full complement of 32 cells. Occasionally, one or two individual blastomeres began to divide at the appropriate time and then failed to complete their divisions. In some cases (<50%), non-adjacent blastomeres adhered to one another, disrupting normal neighbor relations within the notochord rudiment, and it was difficult to document the relative orientations of the two final notochord cell cleavages. But in every remaining case, normal neighbor relations were preserved, and the final two notochord cell cleavages occurred perpendicular to the axis of the first, as they do in situ. Thus by the end of the final notochord cell division, these rudiments had assumed proportions and a monolayer organization similar to that in normal intact embryos at the same time.
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Notochord cells within isolated rudiments express normal motility with normal timing
To assess the possibility that isolated notochord cells failed to express normal motile behaviors, we stained isolated notochord rudiments with Bodipy-phalloidin and used 3D confocal microscopy to characterize the occurrence, distribution and orientations of their F-actin-rich protrusions at selected stages. At mid-gastrula stage, F-actin organization was indistinguishable from that of notochord rudiments within intact embryos at the same stage, with no sign of the broader flattened protrusions characteristic of notochord cells during active rearrangement in situ (Fig. 3A). By 1 hour after the final notochord cell division (1 hour PFD), when cells in situ have begun to extend and rearrange, there was a pronounced general increase in the intensity of F-actin staining at interior edges and along many edges we could detect flattened F-actin rich protrusions, resembling those seen in intact embryos at the same stage (Fig. 3B). By 3 hours PFD, most interior notochord cell edges bore F-actin rich protrusions (Fig. 3C). These tended to be longer on average than those seen at 1 hour PFD as is the case for protrusions seen in situ at later stages. However, we could detect no obvious bias in the orientation of protrusions within the rudiment, in contrast to the in situ situation (Munro and Odell, 2002). These results suggest that cells in isolated notochord rudiments autonomously express the basic motile and adhesive machinery necessary to produce cell shape changes, but absent interactions with bounding tissues, these correct local behaviors produce no globally coherent result.
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Posterior muscle and lateral mesenchyme are not necessary for notochord formation
To remove the influence of presumptive muscle, we ablated the entire posterior third of the embryo, leaving the notochord plate intact (Fig. 1D; n=54). The resulting anterior half embryos extended small tails consisting of a central notochord, axial neural tissue, and a somewhat disorganized epidermis (Fig. 4A). To characterize the overall extent to which a normal notochord formed, we used a simple notochord extension index (NEI) (see Materials and Methods). For ventral half embryos, the NEI was 0.79±0.13, with an average 18/32 notochord cells spanning the entire notochord circumference. The best cases formed entirely normal notochords and the worst resembled the elongated cylindrical intermediates seen at late neurula stages (Munro and Odell, 2002). In general, notochords were most normal anteriorly and became more disorganized posteriorly.
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To look more closely at notochord formation in the absence of contact with mesoderm, we examined intermediate stages in living dorsal anterior quadrants using confocal microscopy with unconjugated Bodipy as a fluorescent label, to enhance contrast (Cooper and Kimmel, 1998). Mediolateral intercalation occurred with a time course similar to that seen in whole embryos (Fig. 5A-D). Cell shape changes tended to appear first in the anterior of the notochord plate and then spread posteriorly across the plate row by row (Fig. 6). We have observed this effect in some normal embryos (Munro and Odell, 2002), but its much more robust here. Only the posteriormost notochord cells, which normally contact posterior muscle, failed to converge and extend or invaginate. These cells eventually lost contact with the overlying neural plate as the notochord rudiment extended and formed a disorganized mass of cells at the posterior of the extended notochord rod (see Fig. 4B).
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Contact with neural plate alone is sufficient for the emergence of global polarity and notochord extension
To assess whether contact with neural plate alone is sufficient for the expression of normally polarized convergent extension and invagination, we co-isolated just the notochord rudiment and the overlying neural plate (Fig. 1F; n=40). In the resulting coisolates, notochords invaginated to form cylindrical intermediates (data not shown) and showed pronounced AP extension. However, rearrangement was less complete than in dorsal anterior quadrants (NEI=0.58±0.105), with no discernible difference in the degree of organization between anterior and posterior (Fig. 4C). In addition, at mid-neurula stages, both planar length/width ratios for individual cells, and AP extension of whole rudiments were significantly greater for dorsal anterior quadrants than for notochord/neural plate co-isolates (see Table 1). Thus, contact with neural plate alone is sufficient for both convergent extension and invagination, but contact with anterior endoderm may speed the emergence of planar polarity within the notochord plate, particularly at its anterior end (Fig. 6).
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In normal embryos, muscle cells rearrange during the first 3 hours PFD from a cap of cells 6 high and 6 wide to form an arc of cells 3 high and 12 wide that extends anteriorly along either side of the presumptive midline (Munro and Odell, 2002). Thereafter, all muscle cells elongate as the entire tail extends. In isolated posterior caps (without notochord), muscle cells rearranged normally during the first several hours, and the caps increased in length by a factor of 2.1±0.15, n=17), but the muscle cells did not subsequently elongate, so that at late tailbud stage the posterior cap remained short and stubby relative to normal tails (Fig. 11A). In notochord/posterior cap recombinants, notochord rudiments showed little shape change during the first several hours as the muscle cells rearranged and the caps elongated (data not shown). But thereafter, notochord rudiments extended along the AP axis defined by the posterior cap regardless of their initial orientation (NEI=0.66±0.1; n=24), accompanied by AP elongation of muscle cells and an increase in overall length of recombinant embryoids by a factor of 3.25 (±0.48; n=24). In the best cases, they formed recognizable tails with a central notochord flanked by parallel rows of extended muscle cells (Fig. 11B). While we did not directly confirm that notochords formed by convergent extension and invagination, cross-sectional views of recombinant embryos at tailbud stage revealed the typical cylindrical morphology and pizza-slice shaped cells seen in normal embryos during later phases of extension (not shown).
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DISCUSSION |
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Convergent extension and invagination are robust properties of the notochord plate, expressed whenever minimal conditions are met
To our surprise, we find that none of the tissues normally bounding the notochord are required for it to form an extended invaginated rod. Basal contact with neural plate OR posterior muscle OR anterior endoderm/lateral mesenchyme is sufficient. Planar contact with anterior endoderm, while not alone sufficient, enhances the response to neural plate alone. Furthermore, each of these interactions can determine a different axis of invagination/convergent extension relative to the original AP axis of the embryo (Fig. 12): in normal contact with neural plate with or without endoderm, the notochord extends AP as in normal embryos. In embryos lacking neural plate, the notochord plate invaginates and extends perpendicular to the embryonic AP axis. In notochord/muscle recombinants, the notochord extends along the axis defined by the posterior muscle, regardless of its original axis.
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What are the minimal sufficient requirements for invagination and convergent extension to occur? One may be the polarizing cue discussed above, although we cannot rule out the possibility that the notochord plate has a weak intrinsic polarity, which external cues override. The other is almost certainly basal contact with an external tissue substrate. Each of the sufficient interactions listed above meets this condition, and notochord cells that lose basal contact with an underlying substrate lose (or never gain) planar and epithelial polarity, even when they retain planar contact with adjacent notochord cell neighbors (see e.g. Fig. 4B and Fig. 5A-D). However, planar contact with neural plate or anterior endoderm, in the absence of any other basal contact, does not induce or support globally organized convergent extension and invagination of notochord rudiments (data not shown), even though basal contacts with the same tissues do, and even though planar contact with anterior endoderm alone can induce locally polarized convergent extension (data not shown). Thus basal contact with an external tissue is almost certainly a necessary condition, and basal contact plus a polarizing cue is probably sufficient. Presumably basal contact with neural plate, posterior muscle, and anterior endoderm/lateral mesenchyme are each sufficient because they supply basal support and a polarizing cue. Whether basal contact with any adhesive substratum (e.g. an artificial one), coupled to a polarizing cue, would suffice remains to be seen.
Mechanisms for the establishment of global polarity
How basal contacts and planar polarization cues interact with notochord cells to produce invagination and convergent extension is unclear. However, it seems likely that they act by aiming autonomously expressed basolateral extension within the plane of the notochord plate and along its apical-to-basal axis in a particular direction so as to produce convergent extension and invagination respectively. In the companion paper (Munro and Odell, 2002), we hypothesize that local attempts of basolateral notochord cell edges to extend are opposed by cortical forces that attempt to contract the cell boundary about a constant cytoplasmic volume. This results in a tug-of-war in which basolateral edges compete for a limited cell volume, with winning edges extending and losers retracting. In this context, any number of mechanisms could act either locally or globally to bias the outcome of this competition. Basal contact with an external tissue could provide the apico-basal bias either indirectly by providing an external cue necessary for the maintenance of general epithelial polarity and the polarized deployment of force-generating cytoskeletal machinery, or more directly by providing an adhesive contact that retards active contraction of basal notochord cell surfaces relative to apical ones. Alternatively, it may operate in more subtle ways to allow the coordination and or spread of planar polarity within the notochord monolayer.
With regard to planar polarization, diffusible signals arising at junctions between different tissues could influence the frequency or strength of local protrusions (Domingo and Keller, 1995; Shih and Keller, 1992). Local contact inhibition of protrusive activity or adhesive stabilization of local cell extensions at tissue boundaries could favor protrusive extension away from those boundaries (Jacobson and Moury, 1995; Jacobson et al., 1986; Keller et al., 1989; Shih and Keller, 1992). Cell extensions might be selectively stabilized along a particular axis through basal contact with an oriented extracellular matrix (Nakatsuji et al., 1982; Nakatsuji and Johnson, 1983; Weiss, 1945) or through interaction with an adhesive gradient (Poole and Steinberg, 1982). Finally, our own modeling studies (E. M. M. and G. O., unpublished) suggest a number of mechanical and geometric effects, mediated through adhesive contacts with surrounding tissues, that could provide the necessary cues.
Mechanical integration of local polarity cues within the notochord rudiment
Regardless of what these cues are, their combined effect must be determined by how they are integrated through local cell-cell interactions within the notochord. In a close packed tissue, any interior edge, in order to extend, must displace neighboring cells. Neighboring cell edges attempting to extend at right angles to one another will compete for the same tissue volume while those attempting to extend in parallel will synergize in their efforts. This simple mechanical observation has several important implications: First, locally established polarity will tend to propagate across a monolayer (Weliky et al., 1991) consistent with what we have observed in dorsal anterior quadrants (Fig. 5) and with what others have described elsewhere (Domingo and Keller, 1995; Shih and Keller, 1992). Secondly, competing orientation biases arising at different local positions, either within the notochord or along its boundary, will be resolved at any given point in a field of cells as a compromise orientation, weighted by the relative strengths of those biases (Elsdale and Bard, 1972).
Resolving forces and cell volume conservation globally provides a general mechanism that converts orientation biases arising at different locations and by different mechanisms into a common currency, integrated across the notochord to produce a global result. This provides a satisfying explanation for the robustness with which the correct orientation emerges, since, in this common currency, one cue can readily substitute for another, and adding additional cues only enhances the overall reliability of the process. Indeed, from this perspective, it is not surprising that many independent cues might have arisen during evolution.
Regulative mechanisms of notochord formation
We were surprised to observe a completely different mode of notochord formation in embryos lacking neural plates. In this case, invagination and convergent extension along an axis perpendicular to the normal axis drives the formation of a U-shaped rudiment which must subsequently regulate to form a normally oriented cylindrical rod; and this regulation fails to occur when posterior muscle is absent. To complete formation of a cylindrical rod, both free edges of each lateral arm must meet and anneal at the midline both dorsally and ventrally, but this is exactly what happens ventrally, in normal embryos. Since notochord cells clearly have a mutual adhesive affinity, we would expect these edges to fuse if brought into sufficiently close contact. From this perspective, the contribution of posterior muscle may be: (a) to provide a narrow channel which forces lateral arms together as they coextend into it; and (b) to provide an external substrate that coextends with the notochord rudiment providing the basal support necessary for notochord cells to maintain planar and epithelial polarity. In the absence of this support, notochord cells would lose basal contact as the lateral arms extended and become disorganized.
Another regulative aspect of notochord formation revealed by our experiments is an intrinsic tendency for the notochord rudiment to adopt a uniformly circular cross section despite any differences in its initial shape and the shapes and mechanical properties of the tissues it contacts. These observations imply that the forces that generate uniform circularity reside within the notochord plate itself. The simplest explanation, consistent with our observations and the mechanical framework outlined above (and in Munro and Odell, 2002) is this. The same cortical contractile forces that cause a single cell to round up, or make all cells in a sheet isodiametric, will force a flat sheet of cells to form a circular disk, and a solid mass of cells or a hollow epithelial cyst to become spherical mass. At the same time, active convergent extension forces drive extension of the notochord rudiment along a single axis and thus the preferred shape becomes a circular cylinder.
Evolutionary implications
These observations have important implications for the evolution of notochord formation and function within the chordate phylum. The cylindrical rod form of an ascidian embryos notochord is a characteristic intermediate stage in all chordate embryos examined thus far (Bancroft and Bellairs, 1976; Brun and Garson, 1984; Conklin, 1928; Keller et al., 1989; Lofberg, 1974; Sulik et al., 1994; Wood and Thorogood, 1994). However the way notochord cell precursors collectively arrive at this stage, the embryonic context in which they do so, and the subsequent elaboration and ultimate fate of the notochord rudiment, differs considerably from one organism to the next. If the intrinsic robustness with which this structure forms in ascidians were a conserved characteristic of the notochord lineage, it would help to explain its persistence in the face of divergent developmental origins and fates.
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ACKNOWLEDGMENTS |
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