Clonal analysis of nuclear differentiation in Tetrahymena

DEVELOPMENTAL

BIOLOGY

Clonal

18,

217-231

( 1968)

Analysis of Nuclear in Tetrahymenal

Accepted

Differentiation

May 27, 1968

IKTRODUCTION

Nuclei ot the same genetic constitution in certain ciliates acquire different properties with respect to mating types and transfer these propertifas reliably to their progeny (Sonneborn, 1937, 1954, 1960). Because the cells of a differentiating clone are independent organisms. cell 1ine:age studies are easily performed, and considerable information has been accumulated on the timing and pattern of these nuclear in alteraiions. Although the mechanisms of nuclear differentiation ciliates may be of general developmental interest, some knowledge of the unique features of the ciliate life cycle is necessary for an evaluation of results available. The nuclear events during conjugation havr been repeatedly presented ( Sonneborn, 1947; Beale, 1954), but the details for Tetrahymena pyriformis (Nanney, 1964) should be rcviewed briefly. Tetrahymena pyriformis contains a compound macronucleus which controls the vegetative life of the cell and a diploid germinal or micronucleus. Conjugation triggers meiosis in the micronucleus in each of the two coconjugants. All but one of the meiotic products degenerates. while the remaining one divides mitotically. This gives rise to a migratory and a stationary pronucleus in each mate, an d reciprocal cross-fertilization follows. The diploid fusion nuclei (the syncarya) in the two conjugants are genitally identical. Each syncaryon divides twice mitotically to form four nuclei: two remain diploid and two begin rapid DNA synthesis and enlarge to form macronuclei. The old macronucleus then is resorbed and one of the two micronuclei dis1 This Institutes

investigation was supported by of Health to Dr. D. L. Nanney. 217

@ 1968 by Academic

Press Inc.

Grant

C?rI-07779

from

the

National

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integrates. At the first cell fission, the remaining micronucleus divides mitotically while the two new macronuclei are passively assorted to the daughter cells. Lineages derived from the first fission products are called ca~yonides since cells in such lineages possess macronuclei descended from a common ancestral macronucleus. Two caryonides from one exconjugant form a clone, and the four caryonides of a pair constitute a sz&one. The synclone is the unit of Mendelian inheritance; all four caryonides initially contain genitally identical nuclei. Because of macronuclear differentiation, however, the four caryonides may not express the same phenotypes. Caryonidal differences for mating types have been extensively documented. In some cases, a synclone will inherit the potentiality for an array of mating types, while the decision as to which type will be expressed occuis individually (and independently) in each macronucleus. When mating type distribution is coincident with macronuclear assortment, as in syngen (genetic species) 1 of T. pyriformis, mating type determination is said to be caryonidal. Each macronucleus usually differentiates to allow the expression of one of seven possible mating types (Nanney, 1956). A set of codominant alleles controls the occurrence of either five (the mt” series: IV-, VII-) or six (the mt” series: I-) of the seven types, so that the heterozygote may express any of the seven (Nanney et al., 1955; Nanney, 1959; Phillips, 1968). A cross between a mP/mP cell and a mt”lmt” cell results in approximately a 1: 1 ratio of the parental genotypes. Within the synclones of genotype mt”/mt”, the caryonides will be limited to five mating types, but may express any of the five. Within the remaining synclones, any of the seven may be expressed. The compounding of the macronucleus means that each genome is present in multiple copies. The question of limitation of an array of potentialities within a macronucleus is thus complicated by the problem of duplicate genomes to be differentiated. This genomic amplification may bear on theories of differentiation which suggest selective replication of genes as a possible mechanism (Brown and Dawid, 1968). Questions of macronuclear organization and macronuclear structure thus relate directly to the problem of macronuclear differentiation. Allen and Nanney (1958) studied exceptional caryonides which express mor’e than one mating type. Extensive cell lineages from such polytypic caryonides, coupled with a mathematical analysis of the

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assortment rates of sublines pure for one mating type, led to the conclusion that the macronucleus is composed of subunits, perhaps equatable to diploid subnuclei (Nanney, 1964). Nanney and Allen (1959) examined polytypic caryonides of the mt”/mP, and mP/mt”. Each caryonide was exgenotypes mt”/mP, panded 30-fold, and the relative numbers, i.e., the output ratio of the two (or more rarely three oi four) mating types were determined. The ratios were found to vary from near equality to great eccentricity, but different output ratios appeared to be characteristic of different mating type combinations. Most of the possible mating type combinations occurred, although some mating types were more likely to be found in combination than others. From these data, certain conclusions were drawn concerning the timing and coordination of nuclear events during macronuclear differentiation. The purpose of this report is to present further work extending the above study. Cell lineage studies are employed as a means of assessing assortment patterns. Additional data extend the information on combinational tendencies. Greater expansion of the individual caryonides permits clearer definition of the frequency of a minority mating type in caryonides with eccentric outputs. This information may be important in formulating a mechanism of nuclear differentiation (Nanney, 1963, 1964). MATERIALS

AND

METHODS

Four inbred strains (families -4, A3, Dl, and F) of T. pyiformis, syngen 1, were the sources of the caryonides used. -411carry a mating type allele of the mt-’ series which permits the development of mating types (mt) I, II, III, V, and VI. Since the relative frequencies of these five mating types are similar in these four families, the mating type alleles, although of independent origin, may be identical. Accordingly. the genotypes of the caryonides will not be further considered, and all the mating type data in the next section will be pooled. The establishment of vegetative pedigrees from each caryonidc has been described (Bleyman et (II., 1966). Briefly, each caryonide was expanded to four secondary (2” ) subcaryonides, by isolation of fission products at each cell division. Each terminal culture was then ESpanded to 30 sublines. Every caryonide was thus represented by 1% sublines, of various degrees of relatedness. Sublinrs were cultured to maturitv I bv serial isolation transfers in depression slidrs. The cultural

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medium was I% proteose peptone inoculated with Aerobacter aerogenes, subsequently diluted 1: 70. At maturity, all surviving sublines (usually between 115 and 120) of each caryonide were tested for mating type. RESULTS

Macronuclear

DifJerentiation

and Coordination

Caryonidal inheritance of mating types in syngen 1 of T. pyriformis was established on the basis of an abbreviated vegetative pedigree analysis (Nanney, 1956). Exconjugants were expanded through 1” subcaryonides and a single lineage from each was carried by serial isolation transfers to maturity. In most (279/330) cases, the 1” subcaryonides were of the same mating type. Because polytypic caryonides generally occur, these exceptions were not interpreted as consequences of occasional differentiation after the first macronuclear division, but as due to assortment of mosaic macronuclei. The present study, although it involved fewer pairs, included both an extended vegetative pedigree (to 2” subcaryonides) and multiple lineages suitable for quantitative assessment. We will examine mating type distribution in both monotypic and polytypic caryonides. This information will further clarify the degree of coordination within the macronucleus at the time of differentiation, and relate the time of differentiation to the degree of compoundness of the macronucleus. A total of 54 caryonides was assessed, and of these 50 showed unequivocal caryonidal patterns. The remaining four, from a single pair, constituted an exception. The sister 2” subcaryonides were alike, but 1” subcaryonides were different as follows: II; I-VI; II; I-VI; I; III; I; III. Our first interpretation of this pair (Bleyman et al., 1966) was that it represented delayed macronuclear differentiation, i.e., subcaryonidal fixation. This interpretation, however, does not account for certain curious features of the mating type distribution pattern. Specifically, although each 1” subcaryonide is different from its sister, it is identical to its “cousin” produced by the same exconjugant. The probability of obtaining this result by random determination at the 1” subcaryonidal level is vanishingly small. Two explanations appear tenable. The first is that the lineages have been systematically mislabeled so as to switch their real relationships. While this cannot be

MACRONUCLEAR

NORMAL

POSTZYGOTIC

EXCEPTIONAL

EVENTS

PRIMARY

FIG. 1. Postulated events.

macronuclear

PAIR

SUBCARYONIDES MACRONUCLEAR DIVISION

MACRONUCLEAR ASSORTMENT normal

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DIFFERENTIATION

behavior

in exceptional

pair as compare?

to

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categorically denied, the manner in which it could be accomplished is difficult to visualize. An alternative possibility (Fig. 1) suggests a cytogenetic irregularity. If for some reason the new macronuclei divided at the first postzygotic fission (instead of assorting) and assorted at the second fission (instead of dividing) the results are those predicted by a strict caryonidal determination. In our sample, monotypic caryonides were more frequent (34) than the polytypes (20) ; since polytypic caryonides contain two or more mating types, the total number of “appearances” scored for mating types in polytypes was greater than that in monotypes (41 vs. 34, Table 1). The number of caryonides studied was necessarily small, TABLE MATINS

TYPE

DISTRIBUTION

1

IN Moso-

ASU

Mating TYPO

I

II

1Monotypes

111’ (0.3’2)

7 (0.211 0 (0.00,

Polytypes

a Xumber

of appearances;

frequency

POLYTYIW

CARYONIDES

type

III

3 (0.00) 9 (0.Z 1

I

VI

N

34 (O.Oh

c0.g 41

(0”;)

in parentheses.

because of the large number of sublines examined from each. Nevertheless, differences between the mating type distributions in the two arrays are indicated. Mating type II, for example, was observed only in monotypic caryonides (7 vs. 0 cases), while mt III was more frequent in polytypic caryonides (3 vs. 9 cases). In a system of five mating types, ten different ditypic combinations are theoretically possible. Without mt II, the number of combinations is reduced to six. Among the 19 ditypic caryonides in our sample, all of these combinations were recovered at least once except for the I-V combination (Table 2). B ecause mt I and mt VI were the most common monotypes, the I-VI ditypes would be expected to predominate, and indeed half of the ditypic caryonides were of this combination. On this basis, the III-V combination should be the rarest, but this was observed twice. The V-VI combination was recovered only once, but this, like the missing I-V combination, should be an infrequent type. Only one tritypic caryonide was recovered, and this involved the three most common types (I, III, and VI 1. Because of the small number of

hfACROSUCLE.iR

223

DIFFERENTI.4TIOS

caryonides examined, little can be concluded about preferential combinations. The frequencies (excluding II) \vere generally consistent with random combinational processes. The two components of a ditypic caryonide may be designated as the majority and the minority types. Table 2 summarizes the data on the polytypic carvonides. \\‘here the sample size is Freater than two. T.WW

2

(~~~I~~sITIo~ 0~ I’OLYTYPIC~CAKYONII)ES

1

III

1.1


I

ti 1

0 4x:; (a) 0 00s (l)J

as for the I-III and the I-VI ditypes, no systematic relationship is seen between majority and minority tylles; the ditypes are either relatively balanced or indeterminate in their direction of eccentricity. Thus, not only do the mating types (excluding mt II) appear in random combinations, but there appears to be no peck order of predominance among the mating types.

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However, the data support the concept of some coordination among the macronuclear subunits during differentiation. Each macronucleus develops from a diploid unit which has the potentiality for determining five mating types. If a large number of subnuclei differentiate independently, i.e., without coordination, each caryonide would be expected to produce cells manifesting most, if not all, of the possible types. In this study, caryonides with four to live mating types were not observed; there was only one tritype, 19 ditypes (12 of which manifested less than 8% of the minority type) and 34 monotypes. If differentiation occurs at a time when many units are present, their differentiation must be coordinated. Output

Ratios and the Time of Nuclear Differentiation

The timing of subnuclear differentiation can be evaluated through an analysis of output ratios and the limits on eccentricity are related to the nature of the ciliate macronucleus. Allen and Nanney (1958) demonstrated that the “stabilization” of pure mating types from polytypic caryonides in ‘Tetrahymena could be explained by the assortment of differentiated subnuclei. Schensted (1958) showed that the mating type assortment was consistent with a model of random segregation of 91) subunits (45 after fission). Because mating types are distributed caryonidally, the macronucleus should acquire its mating type specificity prior to its first division, when the maximum number of units is approximately 90. Thus, the greatest eccentricity of output consistent with the model is 1: 89, reflecting the determination of one macronuclear subunit to manifest a minority type. If eccentricities greater than this are obtained, either the model is faulty, or some differentiations occur after the first macronuclear division, The current data permit the ascertainment of output ratios of 1: 119 or, where one of the sublines is an unresolved mixture of the two types, of 1: 239 (%: 119%). If values of this sort were to be obtained, outputs of at least this eccentricity would be implied and a reconsideration of the model would be required. The minority type outputs (Table 2) suggest that a model of 90 units is sufficient. In only one case, the tritype, did a ratio of less than 1: 59 occur. Three output ratios were 1: 59 (0.017), five were approximately 1: 39 ( 0.025) and the others were even less eccentric. Because of sampling variance, one may not exclude “real” outputs

MACRONUCLEAR

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more eccentric than 1:90, but the data are obviously more consistent with a system of perhaps 30 to 60 subunits. The data on outputs can indicate not only the limit on the number of subunits, but also the number of subunits present at the time of fixation. If differentiation regularly occurs, for example, at the 32-unit stage, the minority output fractions should form a series l/32, 2/32, 3132, etc., or 0.03, 0.06, 0.09, etc. At the 16-unit stage, the series would be 0.06, 0.125, 0.19, etc. If, on the other hand, the time of fixation is variable, highly eccentric outputs could represent late differentiations with 32 units; moderately eccentric ratios, differentiations with 16 units; and balanced ratios, differentiations when only two to eight units are present. Differentiations occurring when only one unit is present could account for many of the monotypic caryonides. Nine caryonides with highly eccentric ratios (one to three minority sublines) constitute the largest class observed, except for the monotypes. The simplest interpretation of this group is that one subunit differentiated to an alternate mating type at the %-unit stage, producing a class with a modal value of 0.03. The two caryonides with minority outputs of 0.042 and 0.050 could belong either to this class or to the two subunit class with a modal value of 0.06, with possibly four examples. The two examples with minority outputs of 0.183 indicate either a 6/32 or 3/16 subunit differentiation. The number of caryonides with more balanced outputs is too small to provide strong evidence for or against uniform differentiation at any particular stage.

Assortmmt Patterns and hlacronuclear Events (Szlbnuclemr Behavior) The Schensted ( 1958) model for subnuclear assortment postulates that macronuclear division yields products within which the subnuclei are equal with respect to number and random with respect to kind. The number of subunits is then estimated on the basis of the assortment rate (Allen and Nanney, 1958). If the numbers of subnuclei are markedly unequal after division, assortment will occur more rapidly and the estimated number of subunits will be too small, The number will also be in error if distribution after division is nonrandom, the direction of error depending on the distribution pattern. If sister subnuclei (produced by replication during the preceding fission cycle) tend to be separated at division-perhaps by moving away from each other after replication -the number of assorting units will be over-

226

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oc?ooo

d’OOIu

coooc~c

ICOO@?

m

c

30

53 2

3

cj

.c

I

MACRONUCLEAR

DIFFERENTIATIOX

227

estimated. A tendency for sister subnuclei to remain together would reduce the number of “effective assorting units” and lead to more rapid segregation and an estimate of units lower than the actual number. The data provided by vegetative pedigrees permit the randomness of distribution to be directly assessed, particularly for the first two macronuclear divisions. If assortment is random, the number of sublines of minority mating type produced in sister 1” subcaryonides (a cs. b column m in Table 3) should be similar. No evidence of imbalance is apparent in the most highly eccentric caryonides 1, 2, and 3 with a: b ratios of 0: 9. 1: 1, l:l, but the next five (4-S) show some clumping. In three of these five, all three of the minority type sublines are found in the same 1” subcaryonide. A similar asymmetry is seen among the moderately eccentric caryonides (9-12) ; the a:b ratios are 3: 2, 6: 0, 1: 6, and 1: 7.5. At least five of the eight balanced caryonides ( 13-20) manifest significant differences between 1” subcaryonides. Therefore, the first macronuclear division is probably not entirely random with respect to subnuclear assortment. A similar asymmetry is seen for the second macronuclear division (al cs. a2 and bl US. b2). When two minority sublines were involved. the ratios were 1: 1, 0:2, O:& 0:2 (lb; 4a; 6b; 9b). Imbalance is more striking where more sublines are involved. Where a 1” subcaryonide produced three minority sublines the ratios were 0:3 or 3: 0 (two CRSCSeach: 5b; 8a; 7a; 9a). Three 1” subcaryonides (lOa, llb, and 14a) contained six minority sublines, and these were distributed 6:0. 3::3, and 6:0. The only 1” subcaryonide ( 17a) with eight minority sublines had them distributed 0: 8 and the one ( 13b ) with 10 minorit? sublines had them distributed 0: 10. These distribution patterns do not indicate a randomness of assortmcnt. DISCUSSION

The data considered here bring to 70 the number of ditypic caryonides examined in various genotypes of syngen 1, and modify somewhat the previous interpretations concerning the polarity of eccentricities and the possible systematic relationships of majority and minority types suggested by the observations of Nanney and Allen ( 1959 ) . The following conclusions were supported by data from both

228

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studies: (a) mt II was unique in appearing primarily as a monotype or as the majority type in highly eccentric ditypes; (b) the seven I-III ditypes were either relatively balanced or indeterminate in their direction of eccentricity; (c) mt VI was the majority type in all the III-VI ditypes (eight examples). The current study differs from the earlier one in results for the following combinations: (a) the I-VI combination, rather than being primarily eccentric in the direction of mt I (H/12 cases in the earlier study) now had mt VI as the majority type in half the caryonides (5110); (b) the V-VI combination in which the degree and the direction of eccentricity differed in limited examples from each study. Sufficient data to support generalizations regarding rarer combinations such as III-V, I-V, and I-VII are unavailable. With the possible exception of the III-VI combination, which shows consistent polarity toward VI, no evidence is available for systematic numerical predominance in dtiypic caryonides involving types I, III, IV, V, VI, and VII (all the types but II, which appears to be unique), In addition, the majority type is not necessarily one which is common as a monotype, but may be one of the rarer types (such as III or V). In the earlier study on caryonidal composition only 30 sublines were analyzed. The most eccentric output ratio ordinarily encountered therefore would be 1:29, when all but one of the sublines were of one pure type, or %:29% (1:59) w h en the one exceptional line was a selfer (an unresolved mixture of the two types). Among the 51 ditypic caryonides previously analyzed, four had output ratios of I:29 and three had ratios of 1: 59. Any smaller minority fraction could not be ascertained because of the limits of sampling. Thus, the earlier data could place no useful limit on the output ratio; the current data, however, permit the limit to be extended to approximately 1: 119 (or 1:239 with unresolved sublines). The data from this experiment have clarified this problem. The output ratios obtained are consistent with a subunit number of 30 to 60, which encompasses the number of subunits (45) calculated on the basis of assortment rates by Allen and Nanney (1958). Variations in minority fractions might reflect variable numbers of units of that type in macronuclei differentiating at the same time (same level of compoundness) or constant numbers of units in macronuclei differentiating at different times (at different levels of

MACRONUCLEAR

DIFFERENTIATIOS

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compoundness). Both variables might be relevant and are difficult to discriminate with limited data. The present data are not sufficiently extensive to resolve this issue. Differentiation evidently occurs as late as the 32-unit stage (or as late as one replication cycle prior to the first macronuclear division) in some macronuclei, but probably no later than this in any macronucleus, and possibly not this late in some macronuclei. If the differentiation occurs at the same time in all macronuclei, the most likely time for this differentiation is the period when approximately 32 units are present. A surprising consequence of the pedigree analysis was the finding that the assortment of subnuclei was nonrandom, at least for the first two macronuclear divisions. If similar nonrandomness characterizes subsequent fissions, the number of assorting units may be considerably underestimated. However, events of these first two macronuclear divisions, which involve nuclear differentiation and growth. mav be significantly different than those in later divisions. In developing from a single diploid unit at fertilization to perhaps 90 units at the first macronuclear division, a total of at least seven replications must occur for some subnuclear lineages--rcitkout an intervening assortment. Subsequently assortment follows each replication, and the “shuffling” of subunits might be more thorough. Possibly only six replications occur and the final subnuclear limit of 90 units may not be achieved by the first division. Therefore, some caution must be exercised in extrapolating from the results of these divisions to those occurring later in the life cycle. The necessity of similar studies on vegetative lines before any general conclusions can be drawn on random assortment of subnuclei is apparent. The uniqueness of the early divisions of the macronucleus is emphasized by the exceptional pair described in the Results section. We assumed that the pair could be explained by a cytogenetic irregularity. An assessment of this possibility must take into account not only the assortment pattern of mating types, but also that for H-serotypes which was explored in this same pair ( Bleyman et al., 1966). Although mating types were assorted at the 1” subcaryonidal level, i.e., at the second fission, H-serotypes were apparently assorted at the first fission as they were in other pairs of this genotype. However. H-serotypes in

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other allelic combinations appear to assort at the second or third fission as well as the first, while this pair appears to be the only exception to first fission mating type assortment. The results of the H-serotype analysis are not, however, unambiguously interpretable, primarily because the assessment depends on quantitative not qualitative distinctions among sublines. In addition, the analysis of the time of differentiation is based on the assumption that subunit assortment is random (Nanney et al., 1964). The nonrandom assortment of subunits indicated in this report could be the explanation for the variability which occurred, at times even within one H-allelic combination. Thus, regardless of precisely how the peculiarities of this pair are to be interpreted, the evidence provides no substantial support for a delayed mating type determination. Consequently the data for this pair (number 13, Table 3) have been included with that from the other ditypic caryonides. SUMMARY

Nuclear differentiation is responsible for mating type determination in many ciliates. In Tetrahymenu pyriformis, syngen 1, the focus of differentiation is the macronuclear subunit. We have analyzed mating type distribution within caryonides of these strains, in order to derive some insight into nuclear events. Cell lineages were established for the first two macronuclear divisions following conjugation. The resulting secondary subcaryonides were expanded 30-fold, and each subline was tested for mating type. Two kinds of caryonides were observed: monotypic caryonides yielded sublines all of the same mating type; polytypic caryonides produced sublines of different mating types. Most polytypic caryonides were ditypes, with one mating type (the majority type) in excess over the other (the minority type). No evidence was found for any systematic relationship between the mating types in a ditypic caryonide, except that mating type II appears primarily as a monotype. Thus, the mating type combinations and some aspects of the quantitative patterns involve indeterminate or random nuclear events. The nuclear events are not entirely random, however. Coordination among subunits being differentiated is indicated by the large numbers of monotypic caryonides, the rarity of high polytypes and the eccentricity of outputs in ditypes. The output ratios suggest that the most likely time for subunit differentiation is the 3%unit stage.

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The distribution of mating types among sublines indicates that subunit assortment is nonrandom for the first two macronuclear divisions. Finally, reexamination of an aberrant pair which might have indicated an exception to early macronuclear differentiation provides no nnc,cpivocal evidence for an exception to the r111eof car\,onidal inheritance in this svngcn of T. prp?formi.~. REFERENCES

1). L., and ALLES, s. I,. ( 1959). Intranr~clear cool-tlination ill Tetrah\wen;l. Phl/~SiOZ.zooz. 32, 221-229. NAYSKY, Il. L;, &WHEY, I’. X., and TEFASKJIAN, A. (1955). The genetic control of mating type potentialities ill Tctmh!ynw?u pyriformis. Gotetics 40, 668-680. SSSSISY, D. l,., BAGEL, hl. J., and Tocrxru~~mu, R. u’. (1964). The timing of II alltigrrlic differentiation in Trtrahymena. J. Eq~tZ. Zoo/. 155, 25-42. PHII.I~II~X, R. R. ( 1968). llating-type alleles in Illinois strains of Tctrahynrma pyriforrliis, svngen 1. Gefzel. Hes. 11, 21 I-214. SC:HL’:NS.I CD, I. i’. ( 19%). klotlcl of srll)~ll~clear segregation iI1 the rn;iCronllClells of ciliatrs. .,lltr. Naturcllist 92, 161-170. SOS YEl1011\) T. 11. ( 1937). Sex, sex inheritance and sex drtrrmination in Parcltwcirrrt~ cum&~. Proc. Not/. Acad. Sci. U. S. 23, 37X-39,5. SONP*EIN)R\, T. hl. ( 1947). Recent advances in the, g;encatics of Paranlrcillm and Enplotes. A~IXLII. Genet. 1, 26:1-358. SONNEISOHS, T. hl. ( 1954). Patterns of nrlcleoc~toplasmic integration in Paramrcium. Proc. 9th Intern. Congr. Geuet. Caryologia 6, Suppl., 307-325. S<~~EIIOR~, T. hl. ( 1960 ), The g ene and cell differentiation. Proc. Nat!. Acad. Sri. I’. S. 46, 149-165. SA\.SE~,