The left-handed phenotype of Tetrahymena thermophila (LH) is a global mirror image of its right-handed counterpart (RH). LH cells are ‘wound’ in the opposite direction from that of RH cells with respect to the placement of all structures that are asymmetrically disposed on the cell circumference. However, the local geometry of ciliary rows, including the asymmetrically placed microtubule bands and other accessory structures, is identical in RH and LH cells. Populations of LH cells grow more slowly than those of RH cells, probably because of nutritional problems due to faulty construction of the cell mouth. LH cells, like RH cells, conjugate in a homopolar configuration, while LH cells mate with RH cells in a heteropolar union which suffices to initiate the conjugal nuclear events but is insufficient to allow survival of progeny. Subclonal analyses indicate that reversion of the LH to the RH form is relatively rare. However, the frequency of reversion is greatly increased by conditions that promote the formation of doublets by fission arrest. An analysis of intermediate doublet forms in such cultures strongly suggests that reversion takes place through a specific pathway, with LH-LH doublets regulating to LH-RH forms that then may give rise to RH singlets. The origin and fate of the LH-RH intermediate forms can be explained by applying a modified polar coordinate model of positional information with the proviso that there is a preferred direction for the intercalation of new positional values.

In the accompanying paper (Nelsen et al. 1989a), we demonstrated the non-genic nature of the ‘left-handed’ (LH) phenotype of Tetrahymena thermophila and suggested that LH cells arise through a series of doublet intermediates. Two types of doublets are found in ciliates, including Tetrahymena. The first and most common kind is the homopolar doublet with two sets of structures of similar asymmetry back to back around the cell. We designate these as RH-RH doublets. The second and much rarer kind is the mirror-image doublet with two sets of structures arranged as mirror images of each other, RH-LH doublets. Suhama (1985) produced a viable LH singlet clone by transection of an RH-LH doublet of Glaucoma, but the origin of his RH-LH doublet was unknown.

We obtained our LH singlet lines by screening for them among the progeny of regulating doublet clones. We believe the naturally occurring sequence that produces LH cells may be represented as follows: RH + RH𡊒 RH-RH𡊒RH-LH 𡊒 LH. The fusion of two RH cells yields an RH-RH doublet which, in turn, may produce RH-LH doublets, leading to LH (and RH) cells through further regulation. In this report, we first compare the RH and LH phenotypes, then explore the consequences of global reversal on growth, anatomy, development and sexual reproduction, and finally analyse the regulation from LH to RH forms via a reversal of the above pathway. In the course of this study, we confirm and extend the regulation model that we proposed previously (Nelsen & Frankel, 1986) and introduced in the accompanying paper (Nelsen et al. 1989a).

Tetrahymena thermophila was used in this study. Methods employed to obtain LH (reverse) cell lines, as well as routine procedures used for maintaining stocks, for performing genetic crosses, and for silver-staining cell samples for light microscope study, were all detailed in the accompanying report (Nelsen et al. 1989a).

The capacity of cells to form food vacuoles was assessed by suspending them in about 10 ml of medium with 2 mg of powdered carmine for 15 min with occasional agitation. Samples were fixed for Chatton-Lwoff silver impregnation to assess cell geometry. Duplicate samples were processed through the silver impregnation procedure, but OsO4 fixation was omitted. This provides permanent preparations in which the percentage of cells with carmine in food vacuoles may be accurately assessed.

SEM preparations were made of deciliated cells. Deciliation techniques used were adapated from Thompson et al. (1974), and Gaertig et al. (1988). A pellet of 4×10° cells from a log-phase culture was suspended in 20 ml of fresh 2% PPY, placed in a 100 ml beaker on a magnetic stirrer (fast stirring) and 10 mg of dibucaine, dissolved (fresh) in 1 ml of dH2O, was added. Cells were pipetted in and out of a Pasteur pipette during 3 of the 5 min they were in this solution. Then an equal volume of recovery solution [Gaertig et al. (1988): 0-1 m-sucrose and 4mm-CaCl2] was added, cells were collected by slow centrifugation and resuspended in recovery solution. After 5-10min, samples were fixed in 1% OsO4 for 2min, washed 3 times in distilled H2O, suspended in thiocarbohydrazide (Lansing et al. 1985) for 2-3 min, washed 3 times in distilled H2O, fixed again in 1% OsO4 for 2 min and prepared for SEM by the methods of Ruffolo (1974) except that cells were placed in the critical-point drying chamber in 100% ethanol instead of amyl acetate. Cell preparations were coated with gold or gold-palladium and observed on a Hitachi S-570LB scanning electron microscope.

(A) The LH phenotype

Unaltered local geometry

Global reversal of right-left asymmetry has no effect on the visible local geometry within ciliary rows. This is illustrated by comparing an LH2 cell (Fig. 2) with an RH cell (Fig. 1). At the cell’s right (viewer’s left -all directions are given as though looking out from inside the cell) of each ciliary row there is a longitudinal microtubule band (LM). A transverse microtubule band (TM) extends to the left at the anterior end of each basal body and a postciliary band (PM) extends to the posterior right from each basal body (cf. Allen, 1967). All of these structures are of exactly the same orientation in both RH and LH cells [Figs 1 and 2; LH1, LH3 and LH4 cells are the same (not shown)]. The local geometry of ciliary rows is apparently not affected by the reversal of global geometry. This is consistent with documented effects of janus mutations, which bring mirror-image global patterns to expression without altering the local geometry of the ciliary rows (Frankel et al. 1984; Frankel & Nelsen, 1986b).

Figs. 1& 2

Protargol preparations of RH and LH cells, respectively. Orientation is such that the cell’s left corresponds to the viewer’s right, and the anterior end is at the top with the oral apparatus (OA) and much of the ventral surface visible. The basal bodies and cilia are well-stained, as are the microtubule bands associated with the ciliary rows. A longitudinal microtubule band (LM) is found to the cell’s right of each ciliary row, and a transverse microtubule band (TM) extends to the left of the anterior end of each mature, ciliary-row basal body. A postciliary microtubule band (PM) arches to the posterior-right of mature ciliary-row basal bodies. The local geometry of ciliary rows is exactly the same in RH and LH cells. An oral primordium (OP) is seen at midbody in each cell. A new OA is formed from such OPs, providing a mouth for the posterior division product. The OP is at the left side of the right postoral ciliary row in the RH cell, and at the left side of the left postoral ciliary row in the LH cell. Scale bars, 5 μ.zm. cilia are visible. The cell surface around the anterior pole is deflected into ridges that are sharply compressed to the cell’s right of the OA in RH cells (Fig. 3) and to the left of the OA in LH cells (Fig. 4). The ridges progressively flatten out around the cell in a clockwise direction in the RH cell and counterclockwise in the LH cell. Each individual ridge, however, has its steepest edge to the cell’s right of the ciliary row in both RH and LH cells, an aspect of local geometry that remains constant.

Figs. 1& 2

Protargol preparations of RH and LH cells, respectively. Orientation is such that the cell’s left corresponds to the viewer’s right, and the anterior end is at the top with the oral apparatus (OA) and much of the ventral surface visible. The basal bodies and cilia are well-stained, as are the microtubule bands associated with the ciliary rows. A longitudinal microtubule band (LM) is found to the cell’s right of each ciliary row, and a transverse microtubule band (TM) extends to the left of the anterior end of each mature, ciliary-row basal body. A postciliary microtubule band (PM) arches to the posterior-right of mature ciliary-row basal bodies. The local geometry of ciliary rows is exactly the same in RH and LH cells. An oral primordium (OP) is seen at midbody in each cell. A new OA is formed from such OPs, providing a mouth for the posterior division product. The OP is at the left side of the right postoral ciliary row in the RH cell, and at the left side of the left postoral ciliary row in the LH cell. Scale bars, 5 μ.zm. cilia are visible. The cell surface around the anterior pole is deflected into ridges that are sharply compressed to the cell’s right of the OA in RH cells (Fig. 3) and to the left of the OA in LH cells (Fig. 4). The ridges progressively flatten out around the cell in a clockwise direction in the RH cell and counterclockwise in the LH cell. Each individual ridge, however, has its steepest edge to the cell’s right of the ciliary row in both RH and LH cells, an aspect of local geometry that remains constant.

Reversed global geometry

The global positions of all structures that are asymmetrically disposed with respect to each other around the circumference of the cell are reversed in LH cells, i.e. these cells are ‘wound’ in the opposite direction. This global reversal affects both the arrangement of ciliary structures and the sculpturing of the cell surface. The latter is apparent when one looks at the anterior surface of deciÚated cells. Figs 3 and 4 are oblique polar views of RH and LH cells, respectively. The sites of ciliary rows are obvious and, in some cases, stumps of

Figs. 3& 4

Scanning electron micrographs of RH and LH cells, respectively, from an oblique, ventral perspective of the anterior pole (star). Cells were deciliated with dibucaine prior to preparation for SEM. The ciliary rows (CR) are still obvious; stumps of cilia are seen in some cases, otherwise rows of ‘holes’ are seen. Two ciliary rows terminate at the OA in each cell. The cell surface around the anterior pole is deflected into ridges that are sharply compressed to the cell’s right of the OA in RH cells and to the left of the OA in LH cells. The ridges progressively flatten out around the cell clockwise in the RH cell and counterclockwise in the LH cell, demonstrating a global reversal of circumferential morphology in the LH cell. However, each individual ridge has its steepest edge just to the cell’s right of the ciliary row in both LH and RH cells, a feature of local geometry which is unaltered by the global reversal. The OA includes compound ciliary structures (membranelies) which incline to the cell’s anterior-right in the RH cell. Some of the normal differentiation of the upper right ends can be seen (large arrow, Fig. 3). The inclination is similar in the LH cell, but the lower-left ends are differentiated (large arrow, Fig. 4). The undulating membrane (UM) is on the cell’s right of the OA in the RH cell and on the left in the LH cell. The OA of the LH cell appears as a rotational permutation (180°) of that of the RH cell. Both cells’ OAs have buccal cavities and both were probably functional. A new oral primordium is present to the left of the right postoral ciliary row in the RH cell. An extended area of anterior suture is present between the OA and the anterior pole (star) in RH cells. This area is typically reduced in LH cells. Scale bars, 5 μm.

Figs. 3& 4

Scanning electron micrographs of RH and LH cells, respectively, from an oblique, ventral perspective of the anterior pole (star). Cells were deciliated with dibucaine prior to preparation for SEM. The ciliary rows (CR) are still obvious; stumps of cilia are seen in some cases, otherwise rows of ‘holes’ are seen. Two ciliary rows terminate at the OA in each cell. The cell surface around the anterior pole is deflected into ridges that are sharply compressed to the cell’s right of the OA in RH cells and to the left of the OA in LH cells. The ridges progressively flatten out around the cell clockwise in the RH cell and counterclockwise in the LH cell, demonstrating a global reversal of circumferential morphology in the LH cell. However, each individual ridge has its steepest edge just to the cell’s right of the ciliary row in both LH and RH cells, a feature of local geometry which is unaltered by the global reversal. The OA includes compound ciliary structures (membranelies) which incline to the cell’s anterior-right in the RH cell. Some of the normal differentiation of the upper right ends can be seen (large arrow, Fig. 3). The inclination is similar in the LH cell, but the lower-left ends are differentiated (large arrow, Fig. 4). The undulating membrane (UM) is on the cell’s right of the OA in the RH cell and on the left in the LH cell. The OA of the LH cell appears as a rotational permutation (180°) of that of the RH cell. Both cells’ OAs have buccal cavities and both were probably functional. A new oral primordium is present to the left of the right postoral ciliary row in the RH cell. An extended area of anterior suture is present between the OA and the anterior pole (star) in RH cells. This area is typically reduced in LH cells. Scale bars, 5 μm.

The OA is the largest and most-conspicuous structure found on the surface of Tetrahymena. It normally consists of four compound ciliary structures: three membranelies, each composed of three rows of cilia, and an undulating membrane (UM) made up of a single row of cilia. The arrangement of these structures in LH cells (as in Fig. 4) sometimes appears as a rotational permutation of that of RH cells (Fig. 3). However, OA development is complex in LH cells, in large part because the subunits of the OA have their own asymmetries which are not reversed. Figs 6, 7, 10 and 11 show the great variation of pattern found in the mature OAs of LH cells, ranging from nearly normal (Fig. 10) to a superficial mirror image of the normal (Fig. 11). This subject is treated in another report (Nelsen et al. 1989b).

The position of the developing OA also reflects the global reversal in LH cells. There are normally two postoral ciliary rows (clearly seen in Fig. 3). The oral primordium (OP) develops to the left of the right postoral row in RH cells (Figs 1 and 3) and to the left of the left postoral row in LH cells (Fig. 2). Another organelle, the cytoproct (cell anus), normally appears as a thin, silver-stained line at the posterior end of the cell just to the left of the right postoral ciliary row in RH cells. As with developing OAs, this organelle is found next to the left postoral row in LH cells (not shown). Thus, both of these structures in LH cells exhibit a mirror-image change in surface position about a plane through the anterior OA.

A basal body couplet terminates most ciliary rows at their anterior ends (McCoy, 1974). The couplets of RH cells are normally found in ciliary rows 5 through n–2 (facing the anterior pole and counting clockwise, starting with the cell’s right postoral row). The pattern of couplets is thus asymmetrically disposed with respect to the OA (Fig. 5). The pattern of placement of couplets in LH cells (Figs 6-8) is a mirror image of that in RH cells, but individual couplets retain the local geometry of the ciliary row. The OAs of the LH cells show varying degrees of pattern disorganization (Figs 6-8), but the mirror-image couplet pattern can be seen in each case. Thus, while the oral area may serve as a reference marker for establishing the couplet pattern, the couplet pattern itself is independent of the integrity of the oral pattern.

Figs. 5–8

Anterior pole views of protargol-stained cells. Fig. 5 shows an RH cell, Fig. 6-8 show LH cells. Basal body couplets can be seen terminating ciliary rows at their anterior ends. The normal pattern is seen in the RH cell: the couplets terminate every row from the 4th row to the right of the OA clockwise through the 2nd row to the left of the OA. The pattern seen in the LH cells is a mirror image of that of the RH cell, even when the OA is in disarray. Scale bars, 5 μm.

Figs. 5–8

Anterior pole views of protargol-stained cells. Fig. 5 shows an RH cell, Fig. 6-8 show LH cells. Basal body couplets can be seen terminating ciliary rows at their anterior ends. The normal pattern is seen in the RH cell: the couplets terminate every row from the 4th row to the right of the OA clockwise through the 2nd row to the left of the OA. The pattern seen in the LH cells is a mirror image of that of the RH cell, even when the OA is in disarray. Scale bars, 5 μm.

The contractile vacuole pores (CVPs) are also asymmetrically disposed with respect to the OA. They are located near the posterior end of the cell, just to the left of one or more ciliary rows about one-fifth of the cell’s circumference to the cell’s right of the OA in RH cells (Fig. 9, arrow). The CVPs in LH cells are located to the left of the OA -a mirror-image position across a plane through the OA (Figs 10-11, arrows). As in RH cells, the individual CVPs are positioned immediately to the left of ciliary rows, a local geometry which is unaltered.

Figs. 9–11

Cells stained to show the relative positions of the contractile vacuole pores (CVP, marked by arrows) with respect to the OA. Fig. 9 shows an RH cell. A newly formed OA is present at midbody and a CVP is visible near the posterior end of the 4th ciliary row to the right of the OA. Fig. 10 & 11 show LH cells with the CVPs found to the left of the OAs -a mirror-image position when compared to the RH cell. The OA of the LH cell in Fig. 10 is nearly normal, while that of the LH cell in Fig. 11 is a superficial mirror image of the normal RH OA. Scale bars, 5 μm.

Figs. 9–11

Cells stained to show the relative positions of the contractile vacuole pores (CVP, marked by arrows) with respect to the OA. Fig. 9 shows an RH cell. A newly formed OA is present at midbody and a CVP is visible near the posterior end of the 4th ciliary row to the right of the OA. Fig. 10 & 11 show LH cells with the CVPs found to the left of the OAs -a mirror-image position when compared to the RH cell. The OA of the LH cell in Fig. 10 is nearly normal, while that of the LH cell in Fig. 11 is a superficial mirror image of the normal RH OA. Scale bars, 5 μm.

The global positions of CVPs in Tetrahymena have been analysed extensively by Nanney (1966a) and co-workers (Nanney et al. 1975). Nanney determined that a singlet cell places CVPs along ciliary rows within an arc of about 10% of the cell’s circumference. The number of ciliary rows with CVPs depends on the number of rows found within this arc, and hence is positively correlated with the total number of rows. Tetrahymena thermophila cells usually have about 18-21 ciliary rows, 1 to 3 of which (usually 2) have associated CVPs. Because CVPs are found just to the left of ciliary rows, Nanney scored their positions as three-fourths of the distance between rows. Using this scoring system, he found the midpoint of the CVP arc (CMP) at 0-213 of the cell’s circumference to the right of the right postoral ciliary row for RH cells of this species (Nanney, 1966a), a proportion that is roughly the same irrespective of the total number of ciliary rows. We examined the global positions of CVPs in some detail in LH clones and subclones and compared them with those of RH cells to test whether the positioning system was truly global and independent of the local (normal) geometry of the ciliary rows. In order to do this effectively, we used a scoring system slightly different from Nanney’s. The two critical departures are both described with the aid of Fig. 12. First, we score the position of the CVPs as that of the associated ciliary row. Ng (1979) has shown that CVPs develop at the posterior-left edges of basal bodies; if the ciliary row is inverted, the CVP forms on the other side of the row (Ng, 1977). Hence, it is the ciliary row that is important in the position and origin of the CVP. Second, while the right postoral ciliary row (no. 1) is the conventional origin for assessment of the position of the CVP, in LH cells we employ the left postoral ciliary row, since it is the one that bears the oral primordium and cytoproct. Applying these modified conventions, both of the CMPs shown would be 4-5-ciliary-row intervals from the oral meridian (OM). If these cells had 20 ciliary rows, this would yield 0-225 of the cell’s circumference to the right of the origin in the RH cell and to the left of the origin in the LH cell.

Fig. 12

Diagrammatic polar projection of the cell surface of RH and LH cells, respectively. Each cell has two postoral ciliary rows (l,n). The right postoral row in RH cells is called the oral meridian (OM) because new oral fields develop along it (not shown). The cell anus [cytoproct (Cyp)] is found near its posterior end. The OM is designated row no. 1; rows are counted clockwise around the cell. The contractile vacuole pores (CVP) are found next to cifiary rows near the posterior end of the cell to the cell’s right of the OM. The midpoint of the CVP set (CMP) is measured in ciliary row units from the OM. In LH cells, surface structures are found in mirror-image positions relative to those of their RH counterparts. The left postoral cifiary row is the OM; we number the ciliary rows and measure the CMP in the anticlockwise direction. See text for further explanation.

Fig. 12

Diagrammatic polar projection of the cell surface of RH and LH cells, respectively. Each cell has two postoral ciliary rows (l,n). The right postoral row in RH cells is called the oral meridian (OM) because new oral fields develop along it (not shown). The cell anus [cytoproct (Cyp)] is found near its posterior end. The OM is designated row no. 1; rows are counted clockwise around the cell. The contractile vacuole pores (CVP) are found next to cifiary rows near the posterior end of the cell to the cell’s right of the OM. The midpoint of the CVP set (CMP) is measured in ciliary row units from the OM. In LH cells, surface structures are found in mirror-image positions relative to those of their RH counterparts. The left postoral cifiary row is the OM; we number the ciliary rows and measure the CMP in the anticlockwise direction. See text for further explanation.

If Nanney’s cells (all RH) had been scored according to our system, the mean CMP distance would have been 0·26 rather than 0·213. This adjusted CMP location would be expected for both RH and LH cells if a truly global positioning system is superimposed on an unchanged local system with little interaction between the two. The data are presented in Table 1. The CMP location varies from about 0·21 to 0·23 in the LH cells, a range that also is found in various samples of RH cells (Nanney et al. 1975; Nanney, 1966a) and is not significantly different from that of the two RH clones in Table 1. The intraclonal variation seen in the subclones of LH1 is similar to the variation between the different LH clones, indicating that it is generated through sampling variations and is not a specific property of any one of the LH clones. The variance of CMP location in LH cells is greater than that in an RH control group (RH1) with rather stable numbers of ciliary rows, but similar to that of another control group (RH2) that has a wide range in numbers of ciliary rows, and (like the LH clones) was recently derived from doublet cells. A high variance in CMP location probably is inherent in all samples, whether RH or LH, which have high numbers of ciliary rows. There also is a weak, but significant, positive correlation of CVP arc width with total number of cifiary rows (e.g. the arc width reaches 4 rows as the total number of rows approaches 30). This correlation is expected on the basis of Nanney’s study of RH cells (1966a). Thus, positioning of CVPs appears to be the same in both RH and LH cells in all respects except for its direction.

Table 1

Cytogeometry

Cytogeometry
Cytogeometry

(B) Consequences of the LH phenotype

Vegetative growth and stability of the LH form

The growth of LH cells was investigated and compared to that of RH cells. Typical data are shown in Fig. 13. Under optimal growth conditions, the log-phase generation time is 2-6 h for RH cells and about 5h for LH cells. Most, if not all, of this growth differential probably relates to the cell’s capacity to feed. Figs 6 and 7 clearly show that some LH cells have defective oral structures. The capacity of cells to incorporate particulate materials into food vacuoles, therefore, was tested with carmine particle suspensions. The results are presented in Table 2. About 50% of the LH cells do not take up particulate materials. This failure is clearly related to the construction of abnormal OAs in LH cells, although any relationships of specific abnormal oral structures with their functions is inferred and not directly documented. Our results resemble those obtained by Suhama (1985) on a surgically created LH Une of Glaucoma scintillans. He has shown that some of these cells do form food vacuoles, but many others have nonfunctional oral structures which they replace, eventually leading to death in some cells. The rich medium we use for cultures of LH Tetrahymena suffices to keep RH forms alive even if they cannot form food vacuoles (Williams & Honts, 1987); it probably does the same for LH cells, allowing them a good chance of assembling a functional OA through oral replacement.

Table 2

Carmine uptake by cells

Carmine uptake by cells
Carmine uptake by cells
Fig. 13

Typical growth curves for populations of RH and LH cells grown at 29°C in PPYGFe medium. Cultures of RH cells consistently grow with approximately the generation time (gt) shown. LH cultures show considerable variability. The curves shown are among those with the fastest gts found. Some other LH cultures grow much more slowly.

Fig. 13

Typical growth curves for populations of RH and LH cells grown at 29°C in PPYGFe medium. Cultures of RH cells consistently grow with approximately the generation time (gt) shown. LH cultures show considerable variability. The curves shown are among those with the fastest gts found. Some other LH cultures grow much more slowly.

Subcloning experiments were used to test the viability and stability of LH cells. Healthy, growing clones which, by inspection, appeared to consist entirely of LH cells were subcloned by random selection of cells. Results assessed after about 15 generations are presented in Table 3. 90 out of 105 subclones survived, indicating a likelihood that most nonfeeding cells either themselves become feeders through oral replacement or produce feeding cells by fission. The character of the subclones indicates that each was likely begun by an LH cell. The stability of the LH phenotype is shown by the fact that no RH cells were found in 74 out of 90 subclones, and only one cell in 50 was RH in an additional 10 subclones. The positive growth differential shown for RH cells in Fig. 13 is sufficient to change an LH:RH ratio of 1:1 to l:2x109 in one week of optimal growth. This great potential for overgrowth by RH cells reinforces the conclusion of relative stability of the LH phenotype. This stability has allowed us to keep cultures of LH cells growing for years by weekly subcloning with selection.

Table 3

Stability of the LH phenotype

Stability of the LH phenotype
Stability of the LH phenotype

Deficient regulation of numbers of ciliary rows

RH Tetrahymena thermophila have a ‘stability range’ of about 18 to 21 ciliary rows (Frankel, 1980; Nanney, 1966b), the basis of which is probably genic as in other ciliates (Heckmann & Frankel, 1968). A developmental mechanism that causes the insertion of a new row may be triggered in cells with ciliary row numbers below this normal range (Nelsen & Frankel, 1979). Cells with row numbers above the normal range regularly lose rows by unknown mechanisms (Frankel, 1980; Nanney, 1966b). Indeed, doublets with 28 or more rows rapidly lose the ‘extra’ rows when they become singlets.

LH cells often have numbers of ciliary rows in the mid-20s (Table 1), as though they were not properly regulating downward toward the 18- to 21-row range. Studies of subclones from cultures of LH cells with about 22 to 26 ciliary rows have not shown the expected trend toward lower row numbers observed in RH subclones (Nanney, 1966b; Frankel, 1980); rather a mid-20s range was usually reestablished (data not shown). The failure of LH cells to regulate row numbers downward might be explained by recurring cleavage arrest in which doublets were frequently being formed and then regulating back to singlets. Observations made on samples of these cultures revealed very few cells undergoing apparent fission arrest, making this hypothesis unlikely. The failure of downward regulation in these LH clones suggests that the successful regulation in RH clones is caused by an active system for downward regulation and not simply a growth differential in favour of cells with lower numbers of ciliary rows. Therefore, it seems more likely that in RH cells the global system interacts with the local system in some unknown way to regulate row numbers, and that a normal local geometry interacting with the reversed global geometry of LH cells somehow fails to carry out this process. Thus one might predict that LH Tetrahymena would fail to trigger the ciliary row insertion process if row numbers fell below the stability centre. The LH form of Glaucoma generated surgically by Suhama (1985) had fewer ciliary rows than RH Glaucoma cells. These cells also failed to regulate to the normal number of rows, perhaps for a similar reason.

Anomalies in conjugation

Conjugation in Tetrahymena thermophila is preceded by two distinct preparatory stages. The first, initiation, is a response to starvation conditions and is independent of cell-cell contact (Bruns & Brussard, 1974). The second, costimulation, requires cell-cell interaction (Bruns & Palestine, 1975) and leads to the elaboration of a conjugal-bonding surface just anterior to the OA (Wolfe & Grimes, 1979; Suganuma et al. 1984). RH cells pair in a mouth-to-mouth homopolar position; fusion then proceeds in the differentiated area anterior to the OAs (Fig. 14). The preoral area in LH cells usually is considerably smaller than that of RH cells (compare Fig. 3 with Fig. 4, also Fig. 5 with Figs 6, 7, 8). LH cells do, however, form conjugal bonds with RH cells, but they join in a heteropolar orientation (Figs 15, 16). This strongly suggests a right-left asymmetry within the conjugal-bonding surface which is important for bonding (Nanney, 1977) and which is reversed in LH cells; any anterior-posterior asymmetry within this surface cannot be important for bonding per se. LH ×RH crosses with heteropolar bonding have not yielded viable progeny in tests which should have selected for even rare cases of conjugal success (Nelsen et al. 1989a). The heteropolar bond in LH-RH pairs suffices to trigger meiosis (Fig. 15), and conjugation proceeds through late macronuclear-anlagen stages (Fig. 16), but death apparently ensues. Isolated pairs always die or else prove to have aborted conjugation (data not shown). We have postulated possible damage to micronuclei during transfer (Nelsen et al. 1989a) but the cause of death is unknown. The formation of new macronuclear anlagen before death seems to be common in lethal conjugal situations (Doerder & Shabatura, 1980; Kaney, 1985; Gaertig & Kaczanowski, 1987). Gaertig & Kaczanowski (1987) have shown a suggestive correlation between longer nuclear transfer times and death after macronuclear anlagen formation in a Tetrahymena cross. Slow or difficult nuclear transfer would not be surprising in LH×RH crosses because such cells form pairs slowly and asynchronously, and swimming in the heteropolar position often separates them. Perhaps disturbances of the normal temporal sequence of integration of new nuclei with the old cell body can lead to dysfunction and death.

Figs. 14–17

Conjugating, protargol-stained cells. Fig. 14. Typical homopolar configuration seen in RH cell x RH cell unions. These cells face each other and fuse just anterior to their juxtaposed oral areas (OA). A late stage of conjugation is shown with macronuclear anlage (MA) visible. Fig. 15 & 16. Cells displaying the heteropolar union found in RH cell x LH cell unions. These cells also fuse just anterior to the OAs but their cell bodies are turned away from each other. Fig. 15 shows an early stage in which meiotic prophase of the micronucleus (mic) is seen. The old macronucleus (mac) is still present. Fig. 16 shows a later stage with MA present. LH × RH matings are apparently lethal (see text). Fig. 17 shows the typical homopolar configuration found in LH cell x LH cell unions. As in Fig. 15, the micronuclei (mic) are in meiotic prophase and the old macronucleus (mac) is still present. Scale bars, 10 μm.

Figs. 14–17

Conjugating, protargol-stained cells. Fig. 14. Typical homopolar configuration seen in RH cell x RH cell unions. These cells face each other and fuse just anterior to their juxtaposed oral areas (OA). A late stage of conjugation is shown with macronuclear anlage (MA) visible. Fig. 15 & 16. Cells displaying the heteropolar union found in RH cell x LH cell unions. These cells also fuse just anterior to the OAs but their cell bodies are turned away from each other. Fig. 15 shows an early stage in which meiotic prophase of the micronucleus (mic) is seen. The old macronucleus (mac) is still present. Fig. 16 shows a later stage with MA present. LH × RH matings are apparently lethal (see text). Fig. 17 shows the typical homopolar configuration found in LH cell x LH cell unions. As in Fig. 15, the micronuclei (mic) are in meiotic prophase and the old macronucleus (mac) is still present. Scale bars, 10 μm.

One would expect homopolar bonding during conjugation in LH×LH crosses, since both cells would have a reversal of the right-left asymmetry of the bonding surface. This is precisely what is found in cells that are properly stimulated for conjugation (Fig. 17). The preoral region, reduced in LH cells, appears to be required for the cell-cell interaction of costimulation; LH ×LH crosses will yield homopolar pairs and viable progeny in the presence of RH cells of a third mating type which can costimulate (stimulate) both of the LH mating types (Nelsen et al. 1989a).

(C) Reversion to the RH phenotype

The importance of cleavage block

The LH phenotype, as shown above (Table 3), is relatively stable. Experience has shown, however, that RH cells will occasionally be encountered and should be expected in any LH culture which is maintained by loop or drop transfer. One reason for this is the strong selective advantage of RH cells conferred by their more rapid multiplication rate. However, RH cells must first appear before they can selectively multiply. Our experience suggested the existence of a developmental pathway that, under certain circumstances, can commonly produce a change of cellular handedness.

Based on previous studies (Nelsen & Frankel, 1986; Frankel & Nelsen, 1986a), we strongly suspected that cells with the LH phenotype are produced when RH-RH doublets regulate back to the singlet state. We therefore predicted that the RH singlets found in some of our LH cultures were produced by LH-LH doublets regulating back to the singlet state. RH forms often appear in LH cultures that are growing poorly; cells with cleavage difficulties are frequently seen in such cultures (data not shown). A cleavage block would be expected to produce doublets; this, in fact, was the technique used to produce the RH-RH doublet culture from which clone LH1 was derived. The LH1 cells are homozygous for a temperature-sensitive mutation (cdaAT) which causes cleavage arrest in virtually every growing cell within a few hours at a restrictive temperature (Frankel et al. 1980) and also tends to do so in cells entering stationary growth phase at nonrestrictive temperatures (unpublished observations). To test the relationship of cleavage arrest to the appearance of cells with the RH phenotype, LH1 (cdaAl/cdaAT) daughter cells were separated after cleavage. One sister of each pair was subjected to a restrictive temperature regimen of 39 °C for 6h and then returned to a nonrestrictive temperature (30°C) for growth. The other sister was retained, and grown, at the original transfer temperature (26°C) to avoid any possible temperature-shift effects. LH2 (cdaA+/ cdaA+) sister cells were used as controls. The heat-treated LH2 sister cells were left at 39 °C overnight and monitored to be sure each had divided at that temperature before shifting them to 30°C, the temperature at which their nontreated sisters were maintained continuously. The data are presented in Table 4. The clones derived from the LH1 sister cells subjected to the restrictive temperature regimen had RH cells in 18 out of 23 clones, whereas their untreated sister clones had RH cells in only 4 out of 23 clones (chi-square = 14·72, P < 0 001). The heat treatment of LH2 controls was deliberately excessive but even this had little effect in producing clones with RH cells (chi-square =0-21, 0·5<P<0-9). The controls show that the heat per se has little if any effect on RH cell production; rather, it is the cleavage block promoted by heat in LH1 cells that is associated with production of RH cells.

Table 4

LH stability in sister cells

LH stability in sister cells
LH stability in sister cells

The reversion pathway

The pathway from LH cells to RH cells can be predicted from our previous experience with regulation of RH-RH doublets (Nelsen & Frankel, 1986; Frankel & Nelsen, 1986b). We would expect LH-LH doublets formed after cleavage block to regulate back to the singlet state, producing intermediate compound forms as well as LH and RH singlets. Intermediate forms were seen in some of the LH clones noted above, and such forms are almost always found in any extensive search of cultures that have both LH and RH singlet cells (data not shown). The LH1 clone from the above experiment which showed the largest numbers of intermediate forms was selected for more detailed study to catalogue the various forms and to discern possible direct relationships between them. The survey was continued until 100 cells of other than singlet LH and RH forms (and astomatous forms) were scored. The data are presented in Fig. 18, excluding 45 astomes. The frequency of each form is given in the centre of the circle in which a schematic polar projection of the relevant morphology is shown. These numbers may be read as percentages exclusive of the RH and LH singlet cases (a and j). Arrows mark transitions occasionally seen at cleavage. The four double-headed arrows relating pairs b-c, e-i, f-j, and g-k each show observed changes in morphology which are accomplished by adding or subtracting the expression of one LH oral meridian. When more than one oral meridian is present, as in janus mutants which produce mirror-image doublets (Jerka-Dziadosz & Frankel, 1979) and in RH-RH doublets which produce LH oral meridians during regulation (Nelsen & Frankel, 1986), the LH meridian sometimes is not expressed. The reasons for this are unknown, but the above-noted pairs are consistent with such intermittent expression. The f-j pair is likely to be exceptional in that Fig. 18j represents the morphology of a normal RH cell. Many of these RH cells probably are free of any ‘hidden’ LH meridian.

Fig. 18

Polar projections showing the OA and CVP geometries observed in a detailed study of one of the clones of Table 4 which showed regulating intermediate forms. Oral areas are designated by an RH or an LH to indicate their global asymmetry, while CVP sets are indicated by circles. Mature cells were scored; astomatous cells are not included. Scoring was continued until 100 cells were tabulated exclusive of the LH singlet (a) and RH singlet (j) forms (and astomatous cells). The central numbers record the frequency of a given form (this is also the percentage for the intermediate forms). Arrows mark transitions seen in dividing cells: for double-headed arrows, transitions were seen in both directions, while a single-headed arrow marks the apparently directional e to f transition. See text for further explanation.

Fig. 18

Polar projections showing the OA and CVP geometries observed in a detailed study of one of the clones of Table 4 which showed regulating intermediate forms. Oral areas are designated by an RH or an LH to indicate their global asymmetry, while CVP sets are indicated by circles. Mature cells were scored; astomatous cells are not included. Scoring was continued until 100 cells were tabulated exclusive of the LH singlet (a) and RH singlet (j) forms (and astomatous cells). The central numbers record the frequency of a given form (this is also the percentage for the intermediate forms). Arrows mark transitions seen in dividing cells: for double-headed arrows, transitions were seen in both directions, while a single-headed arrow marks the apparently directional e to f transition. See text for further explanation.

The forms shown in Fig. 18e and f are connected by a single-headed arrow. This transition appears to be directional, toward the coalescence of 2 CVP sets into one. The spacing between CVP sets in form e is quite variable, but the midpoint of the two CVP sets is generally close to halfway between the two oral meridians (data not shown). Our observations suggest that this form first appears with widely separated CVP sets. Regulation in successive cleavage cycles brings them closer together until they fuse into one set. This fusion is accomplished at cleavage by development of a continuous array of new CVPs at midbody in the anterior division product while two separate CVP sets remain at the posterior end of the cell and are retained by the posterior division product. In a growing culture, such posterior fission products would soon be lost by dilution. The reverse process, the broadening of one CVP set into two, has not been seen in any transitional cells; cultures have been followed in which 2-CVP-set forms disappeared and only the single-CVP-set form remained (data not shown). These observations are consistent with our previous findings in regulating RH-RH doublets (Frankel & Nelsen, 1986a); however, in the earlier studies, forms e and i appeared to regulate to the RH singlet form (j) without the appearance of form f.

The plethora of forms shown in Fig. 18 can be ordered into arrays of related forms and regulation pathways by reference to the observed forms and pathways established by our previous studies of RH-RH doublet regulation (Nelsen & Frankel, 1986; Frankel & Nelsen 1986a). The previously observed forms are ordered into probable sequences of intermediate and regulating forms in Fig. 19, pathways A and B; their predicted mirror-image counterparts are shown in pathways C and D. Singlet cells, shown in the centre of Fig. 19, have one set of cortical structures asymmetrically arrayed around the cell. When two cells fuse (Fig. 19, fusion arrows), the resulting doublet has 2 sets of structures arrayed back to back. We use the index (placed in the centre of each icon of Fig. 19) to keep track of these sets; we arbitrarily designate an RH system as +1 and an LH system as −1. The index is an integer that is the algebraic sum of the number of complete cortical-organelle systems found in any given cell (doublets with two tandem systems have an index of +2 or −2; juxtaposed partial + and–systems would yield an index of 0). The doublets resulting from fusion are probably balanced initially (i.e. corresponding parts are 180° apart), but most tend to become unbalanced. This condition is dynamic. As doublets grow and divide, balanced doublets can become unbalanced and vice versa. Unbalanced RH-RH doublets (Fig. 19A) often insert a partial set of reverse (LH) structures in place of part of the normal set, leaving only one complete set (+1) of RH structures. The remaining complementary partial sets may be lost, returning the cell to the starting RH singlet (+1) condition. Intermediates in pathway A from doublet to singlet have often been observed (Nelsen & Frankel, 1986). Pathway B of Fig. 19 shows the regulation sequence deduced for balanced doublets. We focus on pathway B because it leads to the possible production of the alternative LH form. As shown in pathway B, one of the two RH oral meridians disappears and an LH meridian appears on the other side of the corresponding CVP set (loss of an RH meridian without the appearance of an LH meridian might also occur). This form in turn is presumed to regulate back to the RH singlet (+1) or the alternate LH singlet (−1).

Fig. 19

Regulation pathways relating intermediate forms to each other and to the doublet and singlet forms found in cultures in which doublets are regulating to the singlet state. The central number in each icon is the index, which is the algebraic sum of the number of complete RH (+) and LH (−) systems. Arrows show the direction of observed or deduced change. The forms on the right were observed during previously published studies of RH-RH doublets as they regulated to the singlet state. Pathway A was observed in unbalanced doublets (i.e. with their OAs not directly opposite to each other) and pathway B in balanced doublets. The corresponding pathways C and D were predicted for LH-LH doublets. In all cases doublets are obtained by fusion of singlets (dark arrows). The letters at the lower left of the icons denote the forms found in the survey shown in Fig. 18. Asterisks denote symmetry reversals. See text for details.

Fig. 19

Regulation pathways relating intermediate forms to each other and to the doublet and singlet forms found in cultures in which doublets are regulating to the singlet state. The central number in each icon is the index, which is the algebraic sum of the number of complete RH (+) and LH (−) systems. Arrows show the direction of observed or deduced change. The forms on the right were observed during previously published studies of RH-RH doublets as they regulated to the singlet state. Pathway A was observed in unbalanced doublets (i.e. with their OAs not directly opposite to each other) and pathway B in balanced doublets. The corresponding pathways C and D were predicted for LH-LH doublets. In all cases doublets are obtained by fusion of singlets (dark arrows). The letters at the lower left of the icons denote the forms found in the survey shown in Fig. 18. Asterisks denote symmetry reversals. See text for details.

We had elucidated pathways A and B by studying clones of RH-RH doublets (Nelsen & Frankel, 1986; Frankel & Nelsen, 1986a). RH-RH doublets are sufficiently stable to allow selective subcloning for extended periods. This is apparently not true for LH-LH doublets. One can usually find them in regulating cultures, but all attempts at propagating such doublets in subclones have failed. They appear to lose expression of one LH meridian (converting from the form of Fig. 18b to that of 18c) or otherwise regulate soon after formation. The forms predicted in Fig. 19C and D, corresponding to A and B, respectively, were found in the survey shown in Fig. 18, suggesting that LH-LH doublets undergo regulation through these pathways.

However, in regulation of LH cells, pathway D appears to be the common one, with pathway C much less frequent. This is in contrast to regulation of RH cells, in which pathway A, the RH equivalent of C, was prevalent. This difference may be related to differences between the mode of regulation of numbers of ciliary rows in RH cells and that of LH cells. Pathway B appears to be followed only by RH-RH doublets which remain balanced and have reduced their numbers of ciliary rows to a ‘threshold’ of about 29. Imbalance leading to pathway A is likely before both conditions are met. LH singlets do not regulate numbers of ciliary rows properly (shown above). Perhaps LH-LH doublets have similar difficulties in assessing their ciliary-row complement, allowing them to take pathway D without meeting the presumed conditions noted above for RH-RH doublets. Indeed, the RH-LH doublets found in our previous study mostly had 29 ciliary rows, while those of the present study varied widely with a range of 27 to 39 rows (data not shown). This is probably a simple result of the latter having been generated with more rows, which in turn may allow them to reach the somewhat stable, mirror-image doublet form (f) before regulating further to the singlet state.

The letters at the lower left of the schematic diagrams of Fig. 19 correspond to those of the forms found in the survey (Fig. 18); letters in parentheses correspond to the survey forms obtained when LH meridians are sometimes not expressed. The regulation pathways C and D of Fig. 19 thus account for all of the forms found in the Fig. 18 survey except d, m, g and k. These will be discussed in the context of the model presented below for conversion of global asymmetry.

Conversion through intercalation

How are all of the forms shown in Figs 18 and 19 generated? To account for such forms and relate them to regulation pathways, we earlier proposed a reverse intercalation model modified from the shortest-intercalation rules of the polar coordinate model of French et al. 1976 (Nelsen & Frankel, 1986; Frankel, 1989). Positional values are assigned as displayed around the circumference of the singlet cells shown in Fig. 20. We use an arbitrary set, 1-10, ordered clockwise for RH singlets and counterclockwise for LH singlets. Homopolar doublets are assigned two sets of values. Positional values determine which structures will form at a given site, e.g. 5 designates an OA and 7 a CVP. The rules for shortest-route intercalation include the following postulates. (1) Continuity is maintained everywhere around the cell, as in a clock face. Ten is equivalent to zero. (2) Positional values can be ordered in either direction. (3) There normally is a uniform spacing of positional values; if a sufficient disparity in spacing develops, the system tends to regulate to a new state of uniformity following a route that involves a minimum number of newly generated positional values. (4) The positional coordinates may be propagated longitudinally into daughter cells (cf. Nelsen & Frankel, 1986).

Fig. 20

An intercalation model relating the RH and LH OAs and the CVP sets in singlets, doublets and intermediate forms, as seen in polar projections. The central number in each icon, the index, is the sum of the number of complete sets of positional values ordered in a clockwise (+) or anticlockwise (−) direction. Anticlockwise sequences are indicated by stippling. Left: The assigned positional values for RH and LH singlets. The direction of ordering is clockwise for RH cells and anticlockwise for LH cells. Position 5 designates an OA, while the direction of ordering determines the LH or RH character. Middle: Unbalanced RH-RH and LH-LH doublets (upper icons) with their two complete sets of unequally spaced positional values. The line across each circle denotes the proposed path of reversed intercalation to obtain the forms seen in the bottom icons. The numbers along each Une replace those of the arc that it subtends. These forms correspond to those of pathways A and C of Fig. 19. Right: Balanced RH-RH and LH-LH doublets (upper icons) with their two complete sets of equally spaced positional values. Here two lines across each circle represent two reversed-intercalation events required to give the respective forms seen in the bottom icons. These forms correspond to those seen in pathways B and D of Fig. 19.

Fig. 20

An intercalation model relating the RH and LH OAs and the CVP sets in singlets, doublets and intermediate forms, as seen in polar projections. The central number in each icon, the index, is the sum of the number of complete sets of positional values ordered in a clockwise (+) or anticlockwise (−) direction. Anticlockwise sequences are indicated by stippling. Left: The assigned positional values for RH and LH singlets. The direction of ordering is clockwise for RH cells and anticlockwise for LH cells. Position 5 designates an OA, while the direction of ordering determines the LH or RH character. Middle: Unbalanced RH-RH and LH-LH doublets (upper icons) with their two complete sets of unequally spaced positional values. The line across each circle denotes the proposed path of reversed intercalation to obtain the forms seen in the bottom icons. The numbers along each Une replace those of the arc that it subtends. These forms correspond to those of pathways A and C of Fig. 19. Right: Balanced RH-RH and LH-LH doublets (upper icons) with their two complete sets of equally spaced positional values. Here two lines across each circle represent two reversed-intercalation events required to give the respective forms seen in the bottom icons. These forms correspond to those seen in pathways B and D of Fig. 19.

Applying this system first to unbalanced doublet pathways (Fig. 19A and C), we note the lack of uniform spacing of positional values shown in the unbalanced doublets of Fig. 20. In such cells, a third oral area often appears with reversed asymmetry (LH in RH-RH doublets and RH in LH-LH doublets). We interpret this as a replacement of some of the nonuniform positional values with a shorter set of reversed values (shortest route reversed intercalation, indicated with an asterisk beside the arrows between icons), maintaining continuity of positional values and including the value for a new oral apparatus in a uniform reverse sequence of values (Fig. 20, unbalanced doublets; the numbers that cut across the circle in the upper icons are shown in place around the circle in the lower icons). Only one complete set of positional values remains (hence an index of +1 or −1). The partial + and − sets may gradually be eliminated incrementally along a fine of symmetry between them (cf. Nelsen & Frankel, 1986). This results in the elimination of two oral meridians as RH and LH OAs fuse, generating the singlet form again (Fig. 19A and C). We presented evidence of such fusion in the regulation of RH-RH doublets (Nelsen & Frankel, 1986) and we think it also occurs in LH-LH doublets, although perhaps less frequently.

The balanced doublet pathways (Fig. 19B and D) require double intercalation events to yield the observed forms, one to eliminate an OA position and another to generate a new one with the reverse order. These are shown in Fig. 20. The bottom icons show the mirror-image doublets that result. The return to LH or RH forms would require elimination of either the LH or RH partial set of values and completion of the remaining set.

The, as yet unaddressed, patterns found in the Fig. 18 survey are forms d and m, plus the interconvertible forms g and k. Form d is predicted and expected from reverse intercalation in balanced doublets. The single intercalation Une across the cell between 7 and 1 in balanced LH-LH doublets need only be moved to between 6 and 0 with the reverse insertion of 7, 8 and 9 to result in positional information requiring 3 CVP sets. These forms should be transient, regulating to the singlet form by incremental elimination of values across Unes of symmetry between CVPs. The three-CVP form has seldom been seen in regulating RH-RH doublet cultures, but is not uncommon in the survey culture (Fig. 18d). Perhaps the LH forms regulate more slowly due to disharmony between global and local systems.

The rare form shown in Fig. 48m is very unusual. It appears to be a left-over relic after regulation in which the oral meridian was moved in the direction of the CVP during development of the new OA at midbody. A form similar to m was found dividing in the survey culture after the survey shown in Fig. 18 was completed. The new OA at midbody was placed directly posterior to the anterior OA, the old CVP at the posterior end was next to the oral meridian, but the new CVP at midbody was placed at the ‘proper’ angle to the right of the OA meridian, yielding a normal RH singlet anteriorly and form m posteriorly. Thus the CVP in form m appears to be out of its normal position but without influence on the positioning of new CVPs. Form m should soon be lost by dilution in a growing culture, but it provides evidence that the posterior division product does not resorb and reposition its CVPs. We will return to the remaining forms, g and k, at the end of the next section.

Directional intercalation

Although several intermediates of regulation shown in Fig. 18 were predicted by the intercalation model and others are consistent with it, our current observations suggest an extension of the model. Intercalation of reverse sequences of positional values necessarily produces two meridians of symmetry about which further incremental loss of values in sequence is predicted. For example, in the bottom icons of Fig. 20, such meridians are found at the borders of the stippled reverse segments, at positional values 1 and 3 in the forms derived from balanced doublets and at positional values 6 and 2 in those from unbalanced doublets. In every case, we have seen evidence of loss at only one of the two possible positions. In the balanced doublet sequences of Fig. 20, evidence for loss was only found between CVPs (position 1), leading to eventual fusion of CVP sets. In the unbalanced doublet sequence, evidence for loss of values was found only between an LH oral meridian and the RH meridian to its left (position 6). In a previous study of regulating janus doublets (with four oral meridians, alternating LH and RH), we predicted loss of values at the longitude of symmetry between each RH and LH OA, but found convincing evidence for such loss only between LH OAs and the RH OAs to their left (Frankel & Nelsen, 1987).

There is no compelling theoretical reason, based on the intercalation model presented, why positional values should be eliminated only on the CVP side of mirror-image doublets, just to the left of LH OAs found in unbalanced doublets, or just to the left of the LH OAs in janus doublets. A new, supplementary postulate would account for these observations. If we treat each positional value as a vector pointing in the direction of decreasing values (picture small arrows on each number pointing toward the next lower integer), then all observations are consistent with a higher probability of incremental elimination of values only where arrows face away from each other. If incremental insertion of values were possible, they would be expected in positions where the arrowheads face toward each other.

Theoretically, the mirror-image forms (Fig. 18e and f) might be relatively stable if uniformity of positional values made new intercalation events unlikely. But form 18f may convert to form j, apparently by failure of expression of the LH meridian, only to reappear occasionally many generations later. All cultures followed, however, have eventually shown only the RH singlet form, perhaps due to overgrowth of RH forms ‘free’ of a cryptic LH system. Mirror-image doublet forms are rather stable in some ciliates (Suhama, 1982; Tchang et al. 1964) and have been maintained for years by selective subcloning (Suhama, 1982). The apparent failure to develop OAs along LH meridians in many cases and the lack of really distinctive characteristics of living RH-LH cells makes selective subcloning difficult in Tetrahymena. Therefore, we cannot make a final judgement concerning the stability of RH-LH mirror-image doublets in Tetrahymena, though our experience suggests that they are less stable than mirror-image doublets in certain other ciliates.

The concept of directional intercalation might account, in part, for the putative instability of mirror-image doublets in Tetrahymena, and at the same time account for the strange interconvertible forms g and k of the Fig. 18 survey. In form g, the CVP was located at a position nearly directly opposite the midpoint between the LH and RH OAs. The high incidence of these forms with dorsal CVPs, coupled with the fact that we never observed dividing cells with a return of expression of a second LH meridian to the left of the RH meridian, suggests that such a meridian is absent and form g therefore is unrelated to form h. Rather, form g would occur as a natural consequence of continued directional incremental intercalation in form f. The possible sequence is shown in Fig. 21. During downward regulation of ciliary row numbers, form e would give rise to form f by incremental loss of positional values between the CVP sets. Further directional intercalation can only lead to loss of the CVP from the position shown in form f. However, a new CVP (7) could appear on the other side of the cell by insertion of positional value 7 (forms that did not insert the CVP value 7 across the cell would presumably die from failure to osmoregulate). Directional intercalation should make the reappearance of form f from form g unlikely. In fact, directional intercalation should continue, eventually leading to fusion and loss of the LH-RH OA pair, yielding an astomatous cell (Fig. 21). Thus, indefinite continuation of directional intercalation would trap the cell in an inviable geometry. 45 astomes in various states of disorganization were encountered during the survey in Fig. 18.

Fig. 21

Proposed pathway of directional intercalation, relating forms e, f and g of the Fig. 18 survey, and the astomatous forms also found in that survey. The illustrative conventions are the same as in Fig. 20. Assume that each positional value is vector-like, with its direction oriented toward the next smaller value (but 0 = 10). If new positional values can be incrementally intercalated only at positions in which a smaller value lies between two larger values (i.e. where arrowheads face toward each other) and eliminated only at positions in which a larger value lies between two smaller values (where arrowheads face away from each other), then the sequence shown might occur. The RH-LH doublet with two CVP sets (form e) would become an RH-LH doublet with one CVP set (form f). The CVP set next to the RH-LH OAs would disappear as a new CVP set appeared across the cell (form g). Continued loss of values would lead to fusion and elimination of the RH-LH OAs, and an astome would result.

Fig. 21

Proposed pathway of directional intercalation, relating forms e, f and g of the Fig. 18 survey, and the astomatous forms also found in that survey. The illustrative conventions are the same as in Fig. 20. Assume that each positional value is vector-like, with its direction oriented toward the next smaller value (but 0 = 10). If new positional values can be incrementally intercalated only at positions in which a smaller value lies between two larger values (i.e. where arrowheads face toward each other) and eliminated only at positions in which a larger value lies between two smaller values (where arrowheads face away from each other), then the sequence shown might occur. The RH-LH doublet with two CVP sets (form e) would become an RH-LH doublet with one CVP set (form f). The CVP set next to the RH-LH OAs would disappear as a new CVP set appeared across the cell (form g). Continued loss of values would lead to fusion and elimination of the RH-LH OAs, and an astome would result.

Forms g and k were not found in our studies of RH-RH doublet regulation (Nelsen & Frankel, 1986; Frankel & Nelsen, 1986a) and astomatous cells were seldom seen. However, in those studies mirror-image doublets were only found with about 29 ciliary rows. RH-RH doublets normally regulate to singlets at about this threshold number of ciliary rows (Nanney et al. 1975). Such a threshold of regulation may also apply to the RH-LH mirror-image doublets of the present study. The unusual forms (g, k and astomes) may have resulted from incremental intercalary regulation before such a threshold was attained.

The parallels of our observations to regulation forms and pathways seen in LH Glaucoma cultures are striking. Suhama (1985) reported that RH singlets appeared only in cultures in which doublets were also found. He also reported (Suhama, 1984), without presenting details, that in regulating cultures, mirror-image doublets are found with two CVP sets between the OAs (our form e of Fig. 18), but that when the distance between OAs is somewhat less, only one CVP set is present (our form f). Sometimes he observed a CVP on the dorsal surface when there was still a CVP between the OAs on the ventral surface (Suhama, 1984, 1985). As the distance between OAs was even further reduced, only the dorsal CVP was present (our form g). This is precisely the sequence that directional intercalation, as noted above, would predict. Further, it would be expected that the cells with only dorsal CVPs would seldom produce progeny with ventral CVPs, and in fact they should produce astomatous cells.

Directional intercalation also occurs in regulating multicellular systems. Muneoka et al. (1986) demonstrated directional circumferential intercalation in axolotl limb stumps with certain dorsal-ventral confrontations. They found that, of the two discontinuities generated, only one was normally resolved by addition of new cells and tissues, the other remained unresolved. This could be rationalized with a polar coordinate model (French et al. 1976) by assuming that cells with new positional values could be added only in one direction. Kiehle & Schubiger (1985) showed that pattern regulation in imaginai leg discs of Drosophila involved differential directional cell growth after wound healing. While these examples do not necessitate homologous processes of pattern regulation in Tetrahymena and multicellular systems, they do suggest that such homologies might exist and that some aspects of pattern development and regulation could transcend cellularity.

In the accompanying paper (Nelsen et al. 1989a) we demonstrated that the LH condition could not have arisen from a mutation of the expected kind, namely a macronuclear mutation. However, that demonstration was not totally conclusive because it could not rule out macronuclear mutations affecting the processing of new macronuclei during their development, nor the unprecedented possibility of an expressed dominant micronuclear mutation. The genetic proofs and arguments offered for a non-genic basis of the LH phenotype were primarily negative in character. The present study addresses these concerns from a positive direction. In this work, we not only characterize and document the LH phenotype, but also show that an unrelated temperature-sensitive lesion which causes arrest of cleavage, brings with it a strong tendency of the LH cells to revert to the RH phenotype following heat treatment. Heat-treated LH cells without such a lesion, and non-heat-treated sister cells of cells with this genetic lesion, do not show this trend. The change of handedness thus appears to be associated with the doublet condition that is often the consequence of arrest of cleavage. A genic hypothesis for the LH condition would have to postulate that a heat treatment can induce mass genic reversion in a specific background genotype. It is vastly simpler to suppose that the ‘reversion’ is a direct consequence of the doublet phenotype provoked by the fission-arrest per se.

In support of the idea of direct conversion, we have demonstrated and documented a series of compound intermediate forms between the LH and RH forms (Fig. 19). Most of these forms in the proposed pathway of LH to RH transition were predicted from earlier studies of regulation of RH-RH doublets (Nelsen & Frankel, 1986; Frankel & Nelsen, 1986a). The actual appearance of these predicted forms in cultures undergoing changes in cellular handedness, in turn reinforces our original conclusions of such a pathway. Thus, the postulate of genes specifying cellular handedness is not only virtually excluded by the experimental evidence, but is also unnecessary to explain the phenotypic shift. LH and RH phenotypes are alternate, selfpropagating cortical arrangements that are possible for any given genotype, even though they are sufficiently different from each other as to suggest separate species.

A major remaining challenge is to explain why transitions between the RH and LH forms should occur in regulating structurally compound cells. Our observations are in accord with, indeed were largely predicted by, a previously proposed model (Nelsen & Frankel, 1986; Frankel & Nelsen, 1986a) which was inspired by the polar coordinate model of French et al. (1976). The challenge for the future is to find a mechanistic basis for the dynamic behaviour revealed by our observations and formalized by our model.

The authors thank Dr N. E. Williams, Dr M. Suhama and Mr E. C. Cole for their helpful comments. This work was supported by grant HD 08485 from the U.S. National Institutes of Health.

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