Flagellar, cellular and organismal polarity in Volvox carteri

Volvox, like other colonial and multicellular members of the Volvocaceae, is sometimes viewed as a multicellular Chlamydomonas because of overall similarities in cell organization. Such similarities are presumed to reflect the evolution of these forms from a unicellular volvocalean ancestor (Crow, 1918; Shaw, 1919; Fritsch, 1929). Additional support for this view has been derived from observed similarities in mitosis and cytokinesis (Johnson and Porter, 1968; Coss, 1974; Triemer and Brown, 1974; Deason and Darden, 1971; Birchem and Kochert, 1979; Hoops and Floyd, 1982), cell wall structure (Roberts, 1974; Adair et al., 1987; Goodenough and Heuser, 1988; Matsuda, 1988), and of ribosomal RNA sequences (Rausch et al., 1989; Buchheim and Chapman, 1991; Larson et al., 1992). However, the unicellular and colonial members of the order also differ in a number of important respects. One such difference concerns the pattern of flagellar activity. In Chlamy domonas and other biflagellate, unicellular members of the Volvocales, the two flagella usually beat in a breaststrokelike motion, wherein the effective strokes of the flagella are oriented in opposite directions within a single plane. This propels the Chlamydomonas cell forward along its own cellular axis. However, in colonies composed of radially arranged cells, such motion would result in little or no motility. Gerisch (1959) showed that the flagella of Pleodo rina cells beat with their effective strokes angled towards the colonial posterior; such a beat pattern has now been verified by high-speed cinephotomicrography of a number of species (Hoops and Floyd, 1983; Taylor et al., 1985; Lewis et al., unpublished).

The flagellar apparatuses of the mature unicellular and colonial volvocalean algae are also organized differently. The two halves of the flagellar apparatus of the unicellular Volvocales, including Chlamydomonas, are arranged with 180° rotational symmetry (Floyd et al., 1980). This type of symmetry is of a very general occurrence; three of the four classes of advanced green algae in the classification scheme of Mattox and Stewart (1984) display this type of symmetry, as do the immature cells of colonial and multicellular Volvocales (Hoops and Floyd, 1983; Hoops, 1984; Taylor et al., 1985; Greuel and Floyd, 1985). During cleavage, the basal bodies of colonial and multicellular forms are without flagella, but flagella begin to grow from the basal bodies before hatching and while the flagellar apparatus still retains 180° rotational symmetry. Subsequently, however, the flagellar apparatus undergoes substantial reorganization before the embryo breaks free of the parent spheroid. In every case yet examined, except that of the central cells of Gonium pectorale, this reorganization results in the loss of 180° rotational symmetry (Hoops and Floyd, 1983; Hoops, 1984; Taylor et al., 1985; Greuel and Floyd, 1985). The positions of the striated fibers and microtubular rootlets suggest that the organization of the flagellar apparatus in mature cells results from an ∼90° rotation – in opposite directions – of the two halves of the embryonic flagellar apparatus. This hypothesis is supported by the morphology of cells undergoing flagellar apparatus reorganization (Hoops and Floyd, 1983; Taylor et al., 1985; Greuel and Floyd, 1985).

Since we first observed these changes in flagellar apparatus structure, we have assumed that they were correlated with the change in the orientation of the effective strokes from the opposed pattern exhibited by unicellular volvocalean algae, to the parallel one displayed by the colonial Volvocales (Hoops and Floyd, 1983). Assuming that the flagellar axonemes of the green algae are both structurally and functionally polar – as has been suggested for Chlamy domonas (Hoops and Witman, 1983; Hoops et al., 1984) and other algae (Melkonian, 1984) – the flagella would have to adopt a different rotational orientation in the colonial and multicellular species, such as Volvox, than they have in the unicellular species in order to generate effective motility. However, until now the only evidence for rotation has come from the altered position of structures external to the basal bodies and flagella, such as the striated fibers and microtubular rootlets. Thus, it has remained formally possible that the basal body/flagellum complex itself does not rotate, but that the change in direction of the effective stroke results from rearrangement of some non-axonemal component. I show here that the axonemes of mature Volvox somatic cells do indeed have an altered rotational orientation, and one that is consistent with the postulated rotation of each half of the flagellar apparatus during development, and with the observed flagellar beat patterns.

Volvox carteri f. weismannia (UTEX no. 2180) was obtained from the Culture Collection of Algae at the University of Texas at Austin, and grown in Volvox medium as described previously (Hoops, 1984).

For the electron microscopic analysis of flagellar polarity, the cells were grown, fixed and embedded between teflon-coated slides as described previously (Hoops, 1984). Spheroids used for study had well-developed gonidia and mature, presenescent somatic cells. In addition, all spheroids analyzed were oriented in a direction that could be unambiguously determined, and were distant enough from neighboring spheroids that any given flagellar cross-section could be unambiguously assigned to the spheroid under study. These selected spheroids were mounted in known orientation on plastic blocks and sectioned onto Formvar-coated slot grids using a diamond knife, stained with uranyl acetate and lead citrate, and examined with a Zeiss 900 TEM equipped with a goniometer stage. Because the tilt necessary to obtain good flagellar crosssections influenced the magnification, magnifications given here were calculated using known axonemal dimensions rather than the nominal magnification of the microscope.

Analysis of flagellar polarity depends on the ability to correlate flagellar polarity with the orientation of the underlying cell or spheroid. Therefore, precautions were taken to assure that specimens under study were free of unattached flagella, or flagella originating from a second spheroid. Moreover, in many cases, the insertion points of flagella were determined by study of serial sections, and in every case it was as predicted from study of more distal sections.

Preparation of the sample for electron microscopy sometimes resulted in the loss of flagella. If the two flagella had different rotational orientations and one was preferentially lost, it would result in an incorrect average orientation. However, right and left flagella were found to be present in about equal numbers, even in spheroids that were missing many flagella. In addition, it was found that there is little if any difference in orientation between the right and left flagella (see Results). Therefore, it is unlikely that the average flagellar orientation observed here is skewed by overrepresentation of one class of flagella.

The analysis of the flagellar vs cellular vs spheroid orientation required us to determine the spheroid orientation at both the light microscope and ultrastructural levels. Under the growth conditions used here, spheroids usually had 8 gonidia arranged in two tiers of 4 in the posterior. Spheroids selected for study were ones in which the gonidia in a given tier were either overlapping (indicating they were viewed from the side) or oriented in a ring (viewed from the anterior or posterior). Spheroid orientation was confirmed by observing the spacing between cells; cells in the anterior hemisphere are more widely spaced than those in the the posterior.

Spheroids were placed in a drop of conditioned growth medium on a silicon-coated slide, captured by gentle suction with a micropipet held by a Leitz micromanipulator, and observed with a Zeiss Axioskop equipped with a ×40 water-immersion, phasecontrast objective. Flagellar motility was recorded on Kodak TriX 16 mm movie film at 190-290 frames per second with a Redlake Locam model 51 high-speed motion camera synchronized with a Chadwick-Helmuth power supply and strobe.

To determine the orientation of somatic cells relative to the spheroid, spheroids were fixed with 0.1% glutaraldehyde, lightly compressed between coverslips and scanned for spheroids in which all 4 gonidia from a tier were exactly in line. Such spheroids were photographed under low power to record the polarity of the spheroid and at higher power to determine the orientation of the flagellar tunnels of selected somatic cells, using an American Optical microscope equipped with DIC optics. Pairs of prints were mounted in the same orientation. Spheroid polarity was determined by drawing a line through the center of the gonidia of both tiers using the lower power micrograph. In principle, these lines are perpendicular to the anterior-posterior axis of the spheroid. If the line connecting the posterior gonidia was not exactly parallel to that connecting the anterior gonidia, the reference line was the average of the two. Cellular polarity was then determined by drawing a line between the two flagellar tunnels of a cell and measuring the angle between this line and the spheroid polarity reference line. An average cellular orientation was determined for each spheroid in which the orientation of at least 7 cells could be determined. Occasionally, cells were observed that had a very different orientation from their neighbors. Such cells were excluded from analysis if they differed from the average cellular orientation by more than 20°.

This paper compares the orientation of axonemes relative to the direction of flagellar beat and the orientation of cells at various locations within the spheroid. This involves comparison of orientations at several levels, and potential conflicts in terminology. Therefore, it is necessary to define the terms used. The anterior-posterior axis of the spheroid is defined in the conventional manner, as the axis around which the spheroid rotates, and along which it moves. The anterior pole is defined by the direction of forward motion. In contrast, the anterior-posterior axis of each cell is conventionally defined as extending from the flagellar apparatus (anterior) through the chloroplast (posterior) and is approximately perpendicular to the surface of the spheroid. The cellular axis is seldom the same as the spheroid axis, and the angle between these axes depends on whether the cell is located in the anterior, posterior or side of the spheroid. The right and left flagella are designated as viewed from inside the cell, with the anterior pole of the spheroid upward (in other words, from the cell’s point of view). These directions are opposite those an outside observer would use when looking down on the spheroid from the outside, and are the opposite of those previously used by Coggin and Kochert (1986) for V. carteri. However, they conform to the conventions used by most anatomists to describe orientations in unicellular and multicellular organisms. All cross-sections of basal bodies and flagella, and the graph showing flagellar polarity are printed in the now-conventional manner, that is as if looking from inside the cell outward. The diagrams presented here are drawn in the opposite orientation – as they would appear to an outside observer.

In a previous study (Hoops, 1984) I described the structure of the flagellar apparatus of the somatic cells of V. carteri and showed that the orientation of the flagellar apparatus of each cell is correlated with its position in the spheroid. Here, to verify and extend that result, I analyzed basal body and rootlet orientation in neighboring cells by thin-section-TEM analysis of spheroids with known orientations. The results are summarized schematically in Fig. 1. In spheroids viewed or sectioned from the side, neighboring cells have nearly the same orientation (Fig. 1A,B). Lines passing through the basal bodies of each cell in a tier are parallel, and at approximately right angles to the anterior-posterior spheroid axis like lines of latitude on a globe. As previously described, two of the four microtubular rootlets are nearly parallel; these extend toward the spheroid posterior. The other two rootlets are nearly antiparallel, and extend in the same direction as the line connecting sister basal bodies. The distal striated connecting fiber is displaced toward the side of the basal bodies that faces the spheroid anterior. In contrast, cells in sections from near the spheroid poles are arranged differently. Cells near the anterior pole are oriented in such a way that the two inner microtubular rootlets extend outward, away from the center of the section, and the distal striated fibers lie inward, toward the center (Fig. 1C,D). On the other hand, cells near the posterior pole have opposite orientations; their inner rootlets extend towards the center, and their distal fibers lie toward the outer edge of the section being viewed (Fig. 1E,F). Despite these superficial differences in appearance, however, it is clear that cells all around the spheroid are organized according to one simple rule: the nearly parallel microtubular rootlet pairs all point to the spheroid posterior, and the distal fibers are always on the side facing the anterior pole.

Because the position of the eyespot has been extensively used as a polarity marker in Chlamydomonas, the position of the eyespot in V. carteri was determined. The eyespot is located on the side of the cell towards the posterior of the spheroid (Figs 2, 3). The 4-membered rootlet is consistently found over one edge of the eyespot while the other edge comes close to the 2-membered rootlet (Fig. 2).

High-speed cinephotomicrographic analysis reveals that the two flagella of Volvox carteri f. weismannia beat with a ciliary-type motion that resembles that of forward-swimming Chlamydomonas in many respects. It is a typical ciliarytype waveform except that, like Chlamydomonas (Ringo, 1967; Ruffner and Nultsch, 1985, 1987), there is a partial overlap between the effective and recovery strokes. Like other colonial and multicellular algae examined (Hoops and Floyd, 1983; Taylor et al., 1985), it differs from the beat pattern of Chlamydomonas in the following details: first, the effective stroke is directed predominately at right angles to the anterior-posterior axis of the cell in Volvox, rather than to the sides as in Chlamydomonas. This is presumably correlated with the fact that the basal bodies lie nearly parallel to the anterior-posterior cellular axis in Volvox, whereas they are tilted about 45° from that axis in Chlamy domonas. Secondly, the two flagella have an effective stroke in nearly the same direction in Volvox, whereas in Chlamydomonas the effective strokes of the two flagella are initially directed away from one another (Ringo, 1967; Hyams and Borisy, 1978; Ruffner and Nultsch, 1985, 1987). When viewed from the direction of the effective stroke, it is apparent that the flagella beat in nearly parallel planes (Fig. 4). This is confirmed when the flagellar beat is examined from above the spheroid. Such phase-contrast preparations are optically very poor, but in favorable sequences it is possible to determine that the direction of flagellar motion of the two flagella is approximately parallel, with both flagella beating generally towards the spheroid posterior. So far, I have been unsuccessful at precisely determining the direction of the effective stroke because of the difficulty in determining the direction of flagellar motion when filming from the side, and the poor optical quality of the phase-contrast images when viewed from above, but effective strokes directed to the cell’s left could account for the counterclockwise rotation displayed by swimming Volvox. Volvox flagella beating with a flagellartype waveform have never been observed, in spite of the relatively high light intensity used in filming. No persuasive evidence of any coordination between the flagellar beat cycles of neighboring cells, or even between the two flagella of a single cell has been observed. The ciliary-type motion is not absolutely planar but, like that of Chlamydomonas (Ruffner and Nultsch, 1985), has a small threedimensional component.

The presence of polarity markers in C. reinhardtii (Hoops and Witman, 1983) and in other unicellular green algae (reviewed by Melkonian, 1984) as well as the presence of such a marker in another species of Volvox (Melkonian, 1984) suggested that they might also be present in the axonemes of V. carteri. Reliable polarity markers were not found in the basal body, transition region or region of the flagellum immediately distal to the transition region. However, every flagellum examined distal to that point had a single doublet that lacked its outer dynein arm (Fig. 5A,B). Occasionally, the doublet lacking an outer dynein arm also possessed a two-part bridge that extended from the A-tubule of the doublet lacking the arm to the B-tubule of the adjacent doublet (Fig. 5B). Also, beak-like projections were occasionally found inside the B-tubules of three doublets (Fig. 5B). When found, these structures were present in the doublet lacking the outer dynein arm and in the two doublets directly opposite it. These three markers of flagellar polarity are the same as those found in the axonemes of Chlamydomonas (Hoops and Witman, 1983). By analogy, the doublet that lacks an outer dynein arm is identified as doublet no. 1; other doublets are numbered 2-9 in a clockwise progression as viewed from inside the cell. There is no consistent relationship between the position of the number 1 doublet and the central pair of microtubules, consistent with the hypothesis that the central pair rotates during the beat cycle, as has been shown in Chlamy domonas (Kamiya, 1982; Kamiya et al., 1982).

To answer this question, I sectioned spheroids of known polarity and examined the sections to determine the orientation of the axoneme relative to that of the spheroid and its constitutive cells. This requires that the polarity of flagella and of the spheroid be determined simultaneously. However, sections that revealed the flagellar polarity did not include the usual indicators of spheroid polarity. Therefore, the plane defined by the flagellar bases was used to determine cellular and spheroid polarity. DIC analysis of the flagellar tunnels showed that the flagellar bases were nearly at right angles to the anterior-posterior axis of the spheroid (average orientation is 5.5° counter-clockwise from the cell’s perspective; s.d. = 5.0°, n = 23 spheroids).

Dynein arms were usually not present in the axonemes of V. carteri while they were still within the flagellar tunnel, and were often absent for at least 10 sections distal to this level. Therefore, it was not possible to determine cellular and flagellar polarity of a particular cell in a single thin section. Instead, neighboring cells were used to determine the approximate cellular polarity. Axonemes having identifiable polarities were identified and photographed along with nearby cells using the same tilt and rotation settings of the goniometer. Useful data could be obtained only when the following three conditions were met: (1) the axoneme under examination showed a clear polarity; (2) the section contained at least one nearby cell with a cross-section of basal bodies or flagellar tunnels so that the axis between them could be unambiguously determined and used to define the orientation of the cell; and (3) the approximate anteriorposterior axis of the spheroid could be established by virtue of the positions of eyespots, rootlets or other flagellar apparatus structures. In addition, the same axoneme on adjacent sections was usually examined to confirm the identification of axonemal polarity.

An example of the application of these methods to determine axonemal and cellular polarity is shown in Fig. 6. The spheroid has been oriented such that the flagellar bases run horizontally (Fig. 6A). The position of the eyespot and the microtubular rootlets of the cell in Fig. 6B indicate that the posterior pole of the spheroid is down. The two flagella analyzed for polarity are seen at the top of Fig. 6B and at higher magnification, but with the same orientation, in Fig. 6C. Their position indicates that they are probably the right and left flagella of a single cell that was located slightly below this plane of section. In both of these flagella, the number 1 doublet faces the anterior of the spheroid. That this is a reproducible result can be seen in Fig. 7. Each of the flagella in this gallery has been arranged with the same orientation as the flagella in Fig. 6 (that is, with the anterior pole of the spheroid up, and the axis between flagellar bases horizontal). The number one doublets are all located on the side of the flagella toward the spheroid anterior.

If the rotational orientation of the flagella is correlated with cellular and spheroid polarity rather than the plane of section, then the polarity of the flagella in the anterior and posterior of the spheroid would be expected to differ from those of cells on the side, in accordance with the results summarized diagrammatically in Fig. 1D and F. Flagella of anterior cells beat away from each other, and when axonemes of such flagella are analyzed, the number one doublet faces toward the center of the section (Fig. 8A,B). In contrast, in the cells from the posterior, where the flagella beat toward one another, the number one doublets face away from the center of the section (Fig. 8C,D).

In short, cells in all parts of the spheroid have axonemes that are oriented such that the number 1 doublet is approximately perpendicular to the axis between the flagella and on the side toward the spheroid anterior. This also means, of course, that in each case the number 1 doublet is on the side of the axoneme approximately opposite the side toward which the effective stroke would have been directed in life.

To quantify axonemal polarity, I determined the position of the number 1 doublet relative to the nearest cell(s) that showed the orientation of the flagellar bases. A line was drawn through the center of the flagellum parallel to the line connecting the flagellar bases of the nearest reference cell and then the angle of the number 1 doublet was determined (to the nearest 5°) relative to the normal of the reference line.

There are two potential problems that would prevent an accurate assessment of the polarity determined in this way. First, this method uses the polarity of one cell to determine the polarity of a neighboring cell that is out of the plane of section. Because the cells are on the three-dimensional surface of the sphere, neighboring cells must have slightly different polarities. In this respect, the analysis runs into the same problem faced by map makers trying to represent the three-dimensional globe on a two-dimensional surface. To minimize these effects, the cells used to determine polarity of the basal structures were chosen to be as close as possible to those of the flagella being analyzed. Secondly, if the flagellar axoneme displayed any twist, the apparent polarity of the flagella would be different at different distances from the spheroid surface. If the axoneme was twisted severely enough for this to have an effect over the relatively short distance between the spheroid surface and the level of the flagellum cross-section analyzed, it should be apparent; no evidence of such twist was found. Nevertheless, it is conceivable that such twists might make a minor contribution to the scatter of the data.

Thirty one axonemes from the anterior, posterior and side of the spheroid were analyzed in this way (Fig. 9). Of these, 29 had the number 1 doublet at nearly right angles to the line connecting the flagellar bases, with a mean value of 5.6° clockwise from the cell’s perspective (s.d. = 18.1°). Two points lay outside of this cluster, but although they had a significant effect on the standard deviation, they did not have any effect on the mean orientation (average including all points = 5.6°; s.d. = 31.8°). The significance of these outlying points is not known. However, occasionally a few cells in a spheroid have abnormal cellular orientations; such cells probably have flagella with correspondingly altered axonemal orientations.

Because Volvox normally has a very ordered cell arrangement, it is generally possible to infer the positions of the underlying cells, and hence identify left and right flagella even when the section containing the flagella may not include any portion of the cell body from which they emanated. The orientation of flagella tentatively identified as right flagella vs left flagella was compared, to determine if the two differed significantly in their orientations (Fig. 9). The average orientation of the right flagellum (+3°; s.d. = 17.2°; n = 10) was not significantly different from that of the left flagellum (+8°; s.d. = 16.2°; n = 10).

The flagella of V. carteri contain axonemal-polarity markers similar to those of the unicellular C. reinhardtii. This is not surprising, considering the presumed close evolutionary relationship between these two species (Crow, 1918; Roberts, 1974; Adair et al., 1987; Goodenough and Heuser, 1988; Matsuda, 1988; Buchheim and Chapman, 1991; Larson et al., 1992). Somewhat more surprising is the fact that the flagella of V. carteri only rarely show the two-part bridge or beak-like projections that are regular features of the axonemes of Chlamydomonas. In view of the presence of beak-like structures in algae evolutionarily more distant from Chlamydomonas (Hoops et al., 1982), and their occasional presence in V. carteri, they might be expected to be regular features of the V. carteri axoneme. It is possible that they are regularly present in the flagella of living V. carteri, but are seldom preserved under the fixation methods used in this study. Alternatively, the difference might have a functional basis. In Chlamydomonas the beak-like projections have been postulated to be associated with the ability to execute both the ciliary-type waveform used by forward-swimming cells, and the flagellar-type waveform used by backward-swimming cells (Segal et al., 1984). Curiously, however, whereas flagella of a Chlamydomonas mutant that lacks the normal complement of beak-like projections beat only with the flagellar-type waveform, their apparent absence in Volvox is correlated with flagella that only undergo ciliary-type motion.

Unlike the case in Chlamydomonas, the number 1 doublets of the two axonemes do not face each other in mature V. carteri cells; rather, both point in the same direction, and perpendicular to a line connecting them. This implies that the two axonemes have each been rotated by about 90° but in opposite directions during development.

The Chlamydomonas cell, like other unicellular volvocalean algae, at first glance appears to be symmetrical. However, detailed analysis of the flagellar apparatus, eyespot, contractile vacuoles, mating structure and mitotic poles reveals a fundamental cellular asymmetry that appears to be intimately associated with behavior of the basal bodies during mitosis (Holmes and Dutcher, 1989). This cellular asymmetry is demonstrated by the positions of cellular structures associated with particular microtubular rootlets (notably the eyespot and mating structure; Goodenough and Weiss, 1978; Huang et al., 1982) and by developmental (Huang et al., 1982; Melkonian et al., 1987) and functional differences in the basal body and flagellum (Kamiya and Witman, 1984; Ruffner and Nultsch, 1985, 1987). In Volvox, as in other colonial and multicellular members of the order, the cellular asymmetry is much more prominent (Pocock, 1933; Gerisch, 1959; Coggin and Kochert, 1986). Volvox contains the same markers of cellular asymmetry as the unicellular forms, but their positions are changed during maturation of the flagellar apparatus. The demonstration that the basal bodies do indeed rotate confirms our suggestion that the flagellar apparatus of the colonial and multicellular Volvocales results from rotation of each half of the flagellar apparatus. It is therefore necessary to take rotation of the basal bodies and associated structures into account before they can be reliably used to compare cellular polarity in these two forms.

On the basis of the observation that the eyespot and the mating structures are found in specific locations with respect to particular flagellar rootlets (Goodenough and Weiss, 1978; Holmes and Dutcher, 1989), Holmes and Dutcher, (1989) have suggested that the cells of Chlamy domonas can be divided by a plane perpendicular to that connecting the basal bodies into cis and trans halves, and by a second plane passing through the basal bodies into syn and anti halves. These names are assigned with respect to the position of the eyespot, which is always located in the syn/cis quadrant (Fig. 10A). Despite the differences in basal-apparatus morphology that results from rotation, it is possible to apply these same axes to the Volvox cell, which has secondarily lost the superficial symmetry shown by Chlamydomonas. In Chlamydomonas and other algae, the eyespot lies on one side of one of the larger (4-membered) microtubular rootlets (Gruber and Rosario, 1974; Moestrup, 1978; Goodenough and Weiss, 1978; Melkonian and Robenek, 1984; Holmes and Dutcher, 1989). In mature somatic cells of Volvox, the eyespot is located between one 4-membered and one 2-membered microtubular rootlet. Presumably, during reorganization of the basal apparatus, the eyespot and the 4-membered microtubule rootlet with which it is associated have rotated in the counter-clockwise direction (from the cell’s perspective) while one 2-membered microtubule rootlet emanating from the other basal body was rotating clockwise, until it came to lie near the other edge of the eyespot. The V. carteri somatic cell can also be divided into syn/anti and cis/trans halves by planes passing through and between the basal bodies, respectively (Fig. 10). However, because of this rotational displacement, the eyespot is bisected by the cis/trans plane and does not, by itself, define the cis/trans plane as it does in Chlamy domonas and related unicellular algae (Huang et al., 1982; Holmes and Dutcher, 1989). I propose that the terms syn and trans may still be used to indicate cell polarity in cases where the original position of the eyespot can be inferred. In V. carteri somatic cells the syn/cis quadrant can be defined not by the eyespot per se, but by the 4-membered microtubular rootlet associated with it (Fig. 10C).

Regardless of the position of the somatic cells within the spheroid, the anti side always faces the anterior pole of the spheroid and the syn side faces the posterior pole. In addition the trans side is on the cell’s right and the cis side is on its left.

In Volvox, the plane of flagellar motion and the orientations of the two axonemes are both rotated by a similar amount relative to their orientations in Chlamydomonas. This suggests that there is an intrinsic structural polarity of the axoneme that is related to the functional polarity of the flagellar beat in both Chlamydomonas and Volvox. Presumably the need for effective swimming has supplied the selective pressure that has resulted in the rather dramatic reorganization of the flagellar basal structures in the colonial and multicellular Volvocales. This also suggests that the flagellar apparatus modifications probably occurred early in the evolution of the colonial green flagellates.

The axonemal orientations reported here agree with the earlier analyses of flagellar development in V. carteri and V. rousseletii (Hoops, 1984), Platydorina caudata (Taylor et al., 1985), Astrephomene gubernaculifera (Hoops and Floyd, 1983) and Gonium pectorale (Greuel and Floyd, 1985). In each of these cases, the flagellar apparatus of the cells during and immediately after cleavage was observed to be similar to that of Chlamydomonas. In all of these cases (save the four central-most cells of Gonium), however, the flagellar apparatus lost 180° rotational symmetry as the cells differentiated and the flagellar apparatus matured. This structural modification has been interpreted to be the result of rotation of each half of the basal apparatus. The present demonstration that the number 1 doublets face the same direction in Volvox, rather than opposite directions as in algae such as Chlamydomonas, strongly supports this hypothesis. The altered axonemal orientation suggests that the cis half of the flagellar apparatus rotates counterclockwise (from the cell’s perspective) while the trans half rotates clockwise. The cellular mechanism that causes this rotation is completely unknown.

The sites at which the microtubular rootlets and the distal fiber are attached to the basal bodies also rotate during the reorientation of the basal body/flagellum complex. This suggests that these appendages are always attached to a particular set of basal body triplets. We had previously suggested that these fibers may set up the proper rotational positioning of the basal body/flagellar complex by binding to particular points on the surface of the basal body in Chlamydomonas (Hoops and Witman, 1983; Hoops et al., 1984). The flagellar apparatus of cleaving volvocalean cells is very similar to that of Chlamydomonas, so similar mechanisms may be involved in forming the immature flagellar apparatus of Volvox. Because rotation of the basal bodies during flagellar apparatus maturation occurs after this stage, it requires a reorganization of the entire flagellar apparatus.

Basal body rotation is, however, not unique to colonial and multicellular algae such as Volvox. In all biflagellate green algae, basal bodies are replicated and transmitted semi-conservatively. Thus, after division a new flagellum grows from a basal body that matured during the last division cycle and each pair of basal bodies includes one new and one old one (reviewed by Melkonian et al., 1987; Segaar and Gerritsen, 1989). If the absolute orientation of the flagellar apparatus is to be preserved, then it is essential that in every division cycle there be rotation of the old basal body with respect to the newer one (O’Kelly and Floyd, 1984). Such rotation has been observed in Chlamy domonas by Holmes and Dutcher (1989) and in the related unicell Brachiomonas by Segaar and Gerritsen (1989). If the schemes proposed by Holmes and Dutcher, and Segaar and Gerritsen, are correct for the division stages of Volvox – and there is no reason to think that they are not – the older (trans) basal body of each pair must rotate counterclockwise (as viewed from the cell’s perspective) in each division cycle. Thus, this basal body rotates in opposite directions in the two episodes of basal body rotation. Segaar and Gerritsen (1989) suggested that sliding of oppositely oriented 4-membered rootlets may be responsible for the rotation of the basal bodies in Brachiomonas after mitotic division. In Volvox, such a mechanism might be involved in the first episode of rotation (to set up the Chlamy domonas-type flagellar apparatus typical of immature cells), but it cannot be responsible for the later rotation, because the two 4-membered rootlets in the flagellar apparatus do not overlap one another during this period.

I have shown that both flagella of the V. carteri cell beat in nearly parallel planes toward the posterior pole. This would account for the forward motion of the spheroid, but not for its rotation. However, effective strokes directed to the cell’s left (the spheroid’s right as viewed from the exterior) would result in the observed (‘left-hand screw’) rotation. For technical reasons, I was unable to measure the precise angle between the flagellar effective strokes and the anterior-posterior axis of the spheroid using phase-contrast microscopy, and so cannot confirm this assumption. We are now using DIC optical sectioning of flagellar beat as viewed from above to answer this question.

The analysis of axonemal orientation did not give evidence for effective strokes directed at an angle to the spheroid axis. The mean values for the tilt of the basal body pair (5.5° counterclockwise, from the cell’s perspective) and the orientation of the number one doublet (5.6° clockwise), would result in the number 1 doublets facing directly toward the spheroid anterior. If these values are correct, and if the flagella beat directly away from the number one doublet and towards doublets number 5 and 6, as we proposed for Chlamydomonas (Hoops and Witman, 1983), then flagellar orientation does not account for the observed spheroid rotation. It is possible that the number 1 doublet of one or both axonemes is actually rotated clockwise from the mean values obtained in this study. However, it is more likely that the direction of the effective stroke may not be exactly towards the number 5 and 6 doublets in Volvox. We had originally determined that the number 1 doublet of each Chlamydomonas axoneme directly faced the second, and the two flagella are known to beat away from one another at the start of the effective stroke (Ringo, 1967; Hyams and Borisy, 1978; Ruffner and Nultsch, 1985, 1987). Our analysis of flagellar orientation and motility assumed that the two flagella beat precisely in the plane of the basal bodies. However, observations by Ruffner and Nultsch (1985) suggest that this might not be correct. Therefore, it is possible that the flagella do not beat directly towards the number 5 and 6 doublets even in Chlamydomonas.

If the direction of the effective strokes are structurally determined, and if the flagellar orientation is specified as a result of the cell’s structural organization and polarity, then the orientation of the cell within the spheroid will determine the orientation of flagellar motion. Analysis of cellular orientations indicates that the spheroid possesses rotational symmetry. In the context of the Volvox spheroid, rotational symmetry can be operationally defined by the ability to rotate any sector from the plane perpendicular to the anterior-posterior axis around a central point with resultant overlap of homologous structures. Because each somatic cell has a cis side and a trans side, overlap of cellular structures can only be produced by rotation and not by reflection. This excludes the use of the term ‘radial symmetry’ which, in biology, usually describes objects as having multiple lines of mirror image symmetry. That the spheroid exhibits rotational symmetry is very obvious in the anterior and posterior tiers (see Fig. 1), but this symmetry is also characteristic of the other tiers. Rotational symmetry is the simplest cellular arrangement that results in spheroid rotation during forward swimming. Rotational symmetry of the flagellated cells within the spheroid will ensure that any lateral component of flagellar motion will contribute to rotation of the spheroid, as long as all cells in the spheroid behave similarly (e.g., that a component of the effective stroke of one or both flagella is directed to each cell’s left).

This symmetry is probably formed during the first cleavages. After three cleavages, the embryo of another forma of V. carteri (Starr, 1969; Green and Kirk, 1981, 1982) like many other colonial and multicellular volvocalean algae (Pocock, 1953; Stein, 1958; Gerisch, 1959; Goldstein, 1964; Nozaki, 1986) form the ‘Volvox cross’ (Gerisch, 1959) with a prominent 90° rotational symmetry. The rotational symmetry of cell positions is preserved through subsequent cleavage events, as illustrated with exceptional clarity in the SEM images of developing V. carteri (Green and Kirk, 1981, 1982). However, the development and maintenance of the cellular orientation must be a little more complex. Huskey (1979) has isolated a number of mutants in which the cells have random cellular orientations as determined by the position of the flagellar tunnels and the eyespot. As one might expect, these spheroids do not display effective motility. However, the cells are found in their normal position in the spheroid. This organization reflects a precise set of cleavages in the Volvox embryo (Starr, 1969; Green and Kirk, 1981, 1982). It would seem unlikely that these cleavages could be maintained without the correct polarity of the embryonic cells. This suggests that the cells developed their random orientation after the completion of cleavage, but before the individual cell walls and their associated flagellar tunnels were laid down. The mechanism by which the cells develop their proper orientation within the colony is unknown, but the process may require functional flagella (Huskey, 1979).

I thank Kim Feathers for her assistance in high-speed cinephotomicrography and David Kirk for comments on the manuscript. This work was supported by NSF grants 895078 and BSR-9020013.