DEVELOPMENTAL

BIOLOGY

45, 137-150 (19%)

DNA Synthesis

Required

Quiescent

for the Induction

Root Cortical TERRYL.

Biology Department,

University Accepted

of Differentiation

in

Parenchyma?

SHININCER of Utah, Salt Lake City, Utah 84112

February

21, 1975

Cortical parenchyma of pea roots normally does not divide nor differentiate as tracheary elements. After excision from the root these cells can be induced to undergo cell division followed by differentiation of tracheary elements in the presence of cytokinin. 5-Fluorodeoxyuridine prevents cell replication and cytodifferentiation. The thymidine analog, 5-bromodeoxyuridine, specifically prevents cytodifferentiation with little or no effect on the rate of cell replication. Thymidine can also prevent cytodifferentiation with little effect on cell replication. Thymidine reverses the effect of 5-bromodeoxyuridine (BrdU) or 5fluorodeoxyuridine (FdU) when given simultaneous with or subsequent to either BrdU or FdU. BrdU given with FdU allows up to one round of cell replication but no cytodifferentiation. Differentiation is rarely observed unless the entire cell population has undergone the equivalent of two cell generations. These results are combined with those of other investigators to present an hypothesis that implicates DNA replication but not cytokinesis in the regulation of certain types of differentiation. INTRODUCTION

It has been established by a variety of investigations using many experimental systems that the induction of certain types of development is coupled to some aspect of the cell cycle: Pancreas development (Wessells, 1964, 1968); cell fusion (Okazaki and Holtzer, 1965); mammary gland development (Stockdale and Topper, 1966); tracheary element differentiation (Fosket, 1968, 1970; Torrey and Fosket, 1970); chondroitin sulfate synthesis in presumptive chondrocytes (Abbott and Holtzer, 1968; Coleman et al., 1969; Wilcox et al., 1969). This evidence is generally based on the fact that development is usually inhibited by agents that block DNA synthesis and consequent cytokinesis (5-fluorodeoxguridine FdU]) or interfere with the synthesis of structurally normal DNA (5-bromodeoxyuridine PrdU]). The fundamental question that has been asked in these experiments has been: “Must cells undergo replication in a unique microenvironment in order for the daughter cells to be potentiated to undergo major changes in development?” Such divisions

were called “quantal” by Holtzer and Abbott (1968). The question of quanta1 mitoses is fundamental. It can be conceived in alternative ways, e.g., the transition from a totipotent into successively a multipotent, pluripotent and unipotent cell (Rutter et al., 1973). In that series the cells become progressively more restricted in their developmental potential but this restriction may not be dependent upon the cell cycle per se. The quanta1 mitosis and the totipotent to unipotent transition could be manifestations of the same type of control mechanism. Since multicellular organisms do relegate certain functions to specific cells it is obvious that for survival of the organism these functions must continue in the face of fluctuations in the cellular environment. The multicellular organism must limit the time during which cells might activate lethal information which is a part of the genome in all cells of many eukaryotic organisms. A logical time for this is a specific point in the cell cycle. This concept has been discussed previously (Stockdale and Topper, 1966; and others). This 137

Copyright All

rights

0 1975 by Academic Press, of reproduction in any form

Inc. reserved.

138

DEVELOPMENTALBIOLOGY

type of control mechanism might be unique to eukaryotic organisms and may reflect a major difference in regulation between prokaryotic and eukaryotic organisms. There is no need for such a regulatory mechanism in unicellular organisms since cells are not interdependent and there are no critical cells necessary for survival of the population. A population mistake will not be fatal since the high frequency of mutants ensures that at least one cell will survive to restore a new population. There are many observations which indicate that tracheary element differentiation could be correlated to some aspect of the cell cycle (Fosket and Torrey, 1969; Torrey and Fosket, 1970; Shininger, 1971; Dalessandro, 1973; Phillips and Torrey, 1973; Shininger and Torrey, 1973; Hepler and Fosket, 1971; and Minocha and Halperin, 1974). The only experimental evidence to provide a direct link between any aspect of the cell cycle and the induction of tracheary element differentiation was provided by Fosket (1968, 1970). He found that FdU or colchicine prevented tracheary element formation in explants of Coleus. He concluded that some aspect of a complete cell cycle is required for the auxin induction of tracheary element formation. Torrey and Fosket (1970) used the pea root cortical parenchyma system and found that incipient tracheary elements showed a higher percentage of nuclear labeling with [3H]TdR than did the cell population as a whole. These results suggest that either DNA replication or cytokinesis might be the important aspect of the cell cycle with regard to the induction of differentiation. A major problem to date with the concept that cell division is prerequisite to certain types of cell differentiation has been the lack of a clear and testable hypothesis to explain the need for the cell division. It is necessary to determine if DNA synthesis per se is an important event for the hormonal induction of cytodifferentiation. Clarification of this point will

VOLUME 45, 1975

make it possible to set up solidly based hypotheses about the role of cell replication in the induction of cytodifferentiation. The pea root segment system (Torrey and Fosket, 1970) and the pea root cortical explant system (Phillips and Torrey, 1973) were used in the present series of experiments. In either system the cortical parenchyma is absolutely dependent upon exogenous cytokinin for cell division which is closely followed in time by tracheary element differentiation (Torrey and Fosket, 1970; Phillips and Torrey, 1973). In the segment system the pericycle will divide in the absence of cytokinin but no tracheary elements are produced (Torrey and Fosket, 1970). The induction of differentiation is not a simple consequence of the induction of cell division because both the percentage of the cellular population which differentiates and the rate of appearance of tracheary elements are functions of the exogenous cytokinin concentration (Shininger and Torrey, 1973). Tracheary element formation is therefore cytokinin dependent but is not necessarily dependent upon cell replication. Cell replication alone is clearly insufficient. It is conceivable that differentiation requires cytokinin for some critical period following either DNA synthesis or cytokinesis. The present experiments were designed to determine if tracheary element formation requires DNA synthesis. This was explored by A) utilizing inhibitors of DNA synthesis (FdU) which consequently inhibit cell division, and (B) the use of the thymidine analog BrdU which does not prevent DNA synthesis or cell division in several systems but results in the production of a structurally abnormal DNA and frequently prevents or induces certain types of development (see review, Rutter et al., 1973). MATERIALS

AND METHODS

Seeds of Pisum satiuum,

cv. Little Marvel, were surface sterilized for 20 min with 3.7% calcium hypochlorite solution (70%

TERRY L. SHININCER

Differentiation

Chlorox), rinsed four times in sterile distilled water and imbibed for 17 hr at 21°C as a monolayer of peas in large Pyrex dishes in the dark. All subsequent manipulations of seeds and tissues were performed axenically. Following inbibition, the seeds were placed on 0.7% water-agar in large Pyrex dishes for dark germination at 21°C for 72 hr. Root segments were excised at the lo-11th mm from the root tip and either cultured directly or as cortical explants (segments minus pericycle and vascular tissues; i.e., segments converted into a cylinder of cortical parenchyma (Phillips and Torrey, 1973)). Segments or explants were cultured on fully defined medium designated S2M (Torrey and Fosket, 1970) modified with the addition of kinetin, 5fluorodeoxyuridine (FdU), 5-bromodeoxyuridine (BrdU) or thymidine (TdR) in various experiments, and pH was adjusted to 6.0. Amino acids, kinetin, the vitamin components of the medium, FdU, BrdU and TdR were added by sterile filtration to the autoclaved and cooled inorganic portion of the medium. Tissues were cultured in the dark at 25°C on filter paper (Whatman #40) slants dipping into 3 ml of the medium in 25 x 150-mm culture tubes. After the required culture period, the tissues were macerated in chromic acid/HCl (Torrey and Fosket, 1970) and differential cell counts made to determine the total cell number and the number of tracheary elements. Experiments were replicated three times and the data presented are those of typical experiments. Autoradiographic determination of the sites of BrdU incorporation was done by pulsing cells for 1 hr in culture medium to which was added 2 pCi/ml of [3H]BrdU (28.5 mCi/mmole). Tissues were immediately fixed in ethanol/acetic acid (3:1, v/v) for 24 hr and rinsed in 70% ethanol. The segments were divided into halves at this point for subsequent processing. The segments were treated with pectinase (0.6 g/20 ml of acetate buffer, pH 4.5), squashed on

in Root

Cortical

Parenchyma

139

a glass slide and then one-half of the segments were treated with DNase (Worthington Biochemicals, lot #D545367, 100 pug/ml) in 10m3M MgSO,, pH 6.0, at 37°C for 1 hr. After DNase treatment the squashes were hydrolyzed for 10 min in 1 N HCl, stained with Schiff’s reagent for 1 hr, dried and dipped in nuclear track emulsion (Kodak NTB-2), exposed for 7 days and developed in cold amidol (Swift, 1955; Torrey and Fosket, 1970). RESULTS

The first experiments were to determine if blocking DNA synthesis with FdU would prevent cytodifferentiation in the presence of kinetin. Root segments were cultured 12 days on S2M medium with kinetin, 1 mg/l, and FdU and TdR as indicated (Fig. 1A). In the absence of TdR it is clear that FdU inhibited cell proliferation and that the decrease in the number of differentiated cells paralleled the decrease in cell proliferation (Fig. 1A). At 1Om6M FdU, neither proliferation nor cytodifferentiation occurred. Matthysse and Torrey (1967) showed that 10m6M FdU is just sufficient to prevent incorporation of tritiated thymidine into the nuclei of pea root cells. Thymidine (1O-4 M) also caused a decline in cell proliferation, and coupled with this was a greater than expected drop in the number of differentiated cells (Fig. 1B). Simultaneous treatment with 10m4M TdR and various concentrations of FdU resulted in both the restoration of normal proliferation and production of differentiated cells (Fig. 1B). Is 1O-6 M FdU lethal to the cells and therefore inhibiting differentiation, or is its effect reversible by subsequent TdR treatment? Segments were cultured initially on: A) control medium (S2M plus 1 mg/l kinetin), then transferred to medium containing FdU for 3 days and finally transferred to medium containing both FdU and TdR for reversal of the FdU effect or B) initially cultured on FdU-containing medium and transferred to FdU plus TdR for

140

DEVELOPMENTALBIOLOGY

reversal. The results show that FdU treatment for three days is sufficient to block most cytokinin induced division and differentiation and both effects are fully reversible by subsequent transfer to medium 12-

A

VOLUME

45. 1975

containing both FdU and TdR (Figs. 2A and B). When is the last “differentiation-related” DNA synthesized in relation to the last DNA required for division? For this 12-

(- TdR)

e

(+ IO-4M TdR 1

4

IO-

0-

6-

6-

4-

42-

2-+.

O-

o--o

WA 0

10-e

lo-’

10-6

IFdU)M

FIG. 1. Root segments were cultured 12 days on S2M medium plus 1 mg/l of kin&in in the presence of FdU at concentrations shown in the absence (A) or presence (B) of lo-‘ MTdR. Total cell number, solid circles; total tracheary element, number, open circles; percent tracheary elements [(total tracheary elements/total cells) (loo)], solid squares. Arrows indicate day zero values of respective parameters. (A), In the absence of TdR there is a parallel decline in the number of cells and tracheary elements. (B), TdR reverses the inhibitory effects of FdU and has distinct inhibitory effects on both cell formation and differentiation. I 8-

0

A

I - 8

6-

4 x 407 5 2 2-

OL

I 0

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3

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1

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9

12

1

0

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3

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FE. 2. Cortical explants were cultured 12 days on S2M medium plus 1 mg/l of kinetin alone or with 1O-6 M FdU at various times followed by transfer to 10e6 M FdU plus lo-’ M TdR for reversal. Controls, neither FdU nor TdR treated, solid circles; FdU days O-3, FdU plus TdR days 3-12, open circles; FdU days 3-6, FdU plus TdR days 6-12, solid squares; FdU days 6-9, FdU plus TdR days 9-12, triangles; FdU days 9-12, open squares. All explants cultured on control medium (without either FdU or TdR) prior to transfer to FdU medium. (A), Cell division in cortical explants is reversibly inhibited by FdU. A normal rate of cell division is achieved after FdU treatment and transfer to FdU plus TdR. (B), Tracheary element formation is reversibly inhibited by FdU. A normal rate of tracheary element formation is achieved after FdU treatment and transfer to FdU plus TdR.

TERRY L. SHININCER

Differentiation

experiment root segments were initially cultured on standard S2M medium containing 1 mg/l of kinetin and then transerred to the same medium containing 1O-6 M FdU. Determinations of cell number and tracheary element number were done on samples taken at daily intervals. The results of a typical experiment are shown in Figs. 3A and B. Regardless of the time of the application of FdU, new cells continue to appear for 48 hr only. Tracheary elements also continue to appear for 48 hr after FdU application. If new DNA per se is required for differentiation then the last DNA synthesized for cell division is probably the last required for differentiation. Attempts to prevent an FdU inhibition of cytodifferentiation or proliferation with simultaneous uridine always failed. The FdU experiments demonstrated that prevention of DNA synthesis and, of course, events dependent upon DNA synthesis block cytodifferentiation. The next question which needed to be examined was that of whether or not normal DNA synthesis per se must occur in order for kinetin induction of cytodifferentiation to occur. One way to approach this

in Root Cortical Parenchyma

141

problem is to culture tissue in the presence of BrdU. BrdU incorporation into DNA in the place of thymidine has been shown repeatedly either to inhibit cytodifferentiation (Abbott and Holtzer, 1968; Coleman et al., 1969; Holthausen et al., 1969; Lasher and Cahn, 1969; Levitt and Dorfman, 1972; Marzullo, 1972; Mayne et al., 1971; Nameroff, 1972; Silagi and Bruce, 1970; Stockdale et al., 1964; Wessells, 1964; Wessells, 1968; Wilcox et al., 1969; Wrathall et al., 1973) or in a few cases to stimulate some form of cytodifferentiation (Koyama and Ono, 1972a,b; Schubert and Jacob, 1970). These effects occur at BrdU concentrations that have no significant effect on the rate of cell proliferation and presumably no effects on the rate of DNA synthesis. Screening for an effect of BrdU on xylem differentiation was conducted using the root segment system. Factorial experiments were done with BrdU, TdR, or both. Segments were cultured 12 days on S2M plus kinetin (1 mg/l). The results of a typical experiment show clearly that BrdU inhibited tracheary element formation to a greater extent than it inhibited cell proliferation (Figs. 4A and B). The maximum

$4w

-

32a

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FIG. 3. Segments were cultured on control medium and then transferred to medium containing 1Om6M FdU at times indicated. (A), Cell number vs time: Continuous control medium, open circles; transferred to FdU on day 4, solid triangles; transferred to FdU on day 5, solid circles; transferred to FdU on day 6, open squares; transferred on day 7, open triangles; transferred on day 8, small open square; transferred on day 9, solid large square. Cell number increases up to 48 hr after transfer to FdU medium. (B), Tracheary elements vs time: Symbols as in (A). Tracheary element number increases up to 48 hr after transfer to FdU medium.

142

DEVELOPMENTAL BIOLOGY

VOLUME45, 1975

6-

!-I-0)

to-7

10-6

10-5

IO-“

(ElrdU)M

FIG. 4. Root segments were cultured 12 days on S2M medium plus 1 mg/l of kinetin in the presence of various concentrations of BrdU, TdR or both. (A), Cell number vs BrdU concentration: (TdR) = 0, solid circles; (TdR) = lo-’ M, triangles; (TdR) = lo-’ M, solid squares; (TdR) = 10e5M, open circles; (TdR) = lo-’ M, open squares. BrdU inhibits cell division in the root segment to 60% of the control at the highest concentration tested. BrdU at 1O-6M produces a distinct and fully reversible inhibition of division. (B), Symbols as in A. BrdU is extremely inhibitory to tracheary element formation. The effect is reversible by simultaneous treatment with TdR.

reduction in tracheary element formation was to one-tenth of the control value, while the maximum reduction of cell proliferation was only to four-tenths of the control. The BrdU effect on both proliferation and differentiation was reversible with simultaneous TdR treatment. The inhibition by BrdU was reversible by TdR concentrations between 10m5 and 10e7 M when the BrdU concentration was less than 10m5M. Cortical explant experiments were conducted to confirm and clarify the results obtained with root segments. Cortical explants were cultured for 12 days on S2M plus kinetin (1 mg/l) and BrdU, TdR or both. BrdU at 1O-6 A4 caused approximately a 50% reduction of the number of tracheary elements produced in the cortical explant (Table 1) in the absence of any effect on cell proliferation. Simultaneous treatment with thymidine (1O-5 M) fully reverses this effect on differentiation in the absence of any significant effect on cell proliferation (Table 1). Again, TdR alone produced a significant inhibition of tracheary element formation and also in the

TABLE 1 CORTICALEXPLANTEXPERIMENTS' BrdU(A4)

TWM) 0

0 10-S 0 IO-5

10-o

Cell number 79,850 A 6,060 83,666 * 6,660 77,083 i 20,800 74,850 +z11,800 Tracheary element number 3,800 + 160 8,206 zt 1,360 6,816 + 2,240 3,000 i 2,100

“Cortical explants were cultured 12 days on S2M plus 1 mg/l of kinetin and BrdU, TdR or both as shown. Neither BrdU nor TdR has significant inhibitory effects on cell proliferation. Both BrdU and TdR inhibit tracheary element formation. Simultaneous treatment with BrdU and TdR results in the cancellation of the effect of either BrdU or TdR. Values are: 5 * 2 (S;).

absence of any inhibition of cell division. The next set of experiments was conducted to determine if the BrdU effect was to be explained as: a) Simply a delay in the formation of tracheary elements (possibly by temporarily blocking cell division in some subpopulation of the explant) fol-

TERRY L. SHININCER

Differentiation

lowed in time by a normal rate of formation of tracheary elements, or b) normal timing of the onset of differentiation but at a subsequent low rate. A second goal was to determine if, in fact, the cells would recover the normal rate of differentiation after removal from BrdU medium. Cortical explants were cultured in S2M plus 1 mg/l of kinetin for 12 days with samples taken at 3-day intervals. Other explants were cultured in the above medium plus 1O-6 M BrdU or 10e6 A4 BrdU plus 10m5M TdR for 12 days with samples taken at 3-day intervals. Finally, explants were cultured in the above medium containing 1O-6 A4 BrdU for the first 6 days and then transferred to 1O-6 M BrdU plus 10m5 M TdR. A second version of this experiments was to culture the explants in 10e6 M BrdU for the first 9 days and then transfer to 1O-6 M BrdU plus 10e5 M TdR and continue the experiment to the 15th -

143

in Root Cortical Parenchyna

day. Controls were all run to 15 days in that case. The results from a typical experiment in which the transfer was made at day 6 show that prior to day 6 the presence of BrdU has no effect on the rate of cell proliferation but that after day 6 some experiments may show a reduction of cell proliferation (Fig. 5A). In such cases, transfer onto medium containing 10m6M BrdU plus 10m5M TdR restored the rate of cell division to normal. This return to normal rate of division may require 72 hr for expression. Continuous treatment with both BrdU and TdR always produced rates of proliferation and differentiation at least equal to the controls. Typically, the initial tracheary elements appeared at the same time in all explants regardless of treatment (Fig. 5B). The subsequent rate of formation of tracheary elements was always reduced to 25% or less of the control values by BrdU treatment.

A

IO-

6* b 6x ln i E

4_

1 0

I 3

I 6 DAYS

I 9

, 12

I

I

I

I

,

I

0

3

6

9

12

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DAYS

FIG. 5. Cortical explants cultured on S2M with 1 mg/l of kinetin: continuously treated with 10e6 M BrdU, open circles; continuously treated with lOWeM BrdU and 10e5 M TdR, open squares; or with no additives, solid circles; samples removed for cell counts at J-day intervals as indicated. Arrow indicates time of transfer of six samples from BrdU medium to the BrdU plus TdR medium (solid squares). (A), Total cells produced vs time in culture. BrdU plus TdR treatment does not reduce rate of cell proliferation. BrdU treatment alone reduces rate of cell proliferation in this experiment. The transfer of BrdU-treated cylinders from BrdU to BrdU plus TdR results in restoration of normal rate of cell proliferation after 72-hr lag. (B), Total tracheary elements produced vs time in culture. BrdU plus TdR treatment results in only a slight reduction in the rate of tracheary element formation. BrdU alone produces a large inhibition of the rate of tracheary element formation. Partial recovery from BrdU treatment (after transfer to BrdU plus TdR) occurs after a 72-hr lag. The rate of tracheary element formation does not return to normal after BrdU treatment within the time period of the experiment.

144

DEVELOPMENTALBIOLOGY

VOLUME 45, 1975

Will BrdU reverse Transfer of the explants from BrdU alone and BrdU inhibition. of cell division and, if so, to BrdU plus TdR always resulted in a FdU inhibition occur normally or not? stimulation of the rate of tracheary ele- will differentiation is, in ment formation and this usually was not If the mechanism of BrdU inhibition evident until after a 72-hr lag. The new fact, via its incorporation into DNA, then, rate was intermediate between that of the if cell division occurs in this situation (BrdU plus FdU), inhibition of differentiacontrol and the BrdU-treated explants. tion should be more complete than when Restoration of normal rate of cell division did not necessarily lead to a restoration of tissue is treated with BrdU alone. The normal rates of tracheary element formacombined BrdU plus FdU treatment would result in greater substitution of thymine tion. sites than BrdU alone. If the BrdU effect is The experiments in which the transfer into DNA, then the was made at day 9 rather than day 6 were not via incorporation BrdU plus FdU treatment performed in an attempt to determine a combined should not result in any greater inhibition possibly more rapid and more complete response to the transfer off of BrdU than BrdU treatment alone. The experimedium. This experiment confirmed the ment will only work if BrdU is as effective of day-6 transfer results. The rate of tra- as TdR in reversing the FdU inhibition DNA synthesis and cell division. cheary element formation immediately The results of a typical experiment are after transfer from BrdU to BrdU plus TdR is greater than in those explants not shown in Fig. 6. BrdU is not efficient in reversal of the FdU inhibition of cell divitransferred but less than normal. sion. Some new cells are produced with the Is BrdU incorporated into the nuclei, combined treatments of BrdU and FdU, chromosomes and DNA of pea root cortical as traparenchyma cells? To answer these ques- but none of these differentiates tions the root segments were cultured for 6 cheary elements. Figure 7 is a composite of the results of days on control medium or medium containing 1Om6M BrdU. On the sixth day the 15 various experiments in which segments were cultured for 12-15 days on control segments were transferred for 1 hr to their 1O-6 M respective fresh medium to which had been medium or medium containing added [3H]BrdU. Following the pulse the BrdU. In some of these experiments the segments were fixed in ethanol/acetic acid control and experimental samples showed and processed for autoradiography but substandard growth which was attributed to unknown variables. However, this one-half of each segment was treated with poorer than normal growth allows the comDNase prior to Feulgen staining and prior parison of samples of the same age but with to dipping in nuclear track emulsion. widely varying growth. The data are for Preliminary results from these expericontrols and BrdU-treated segments. The ments show that in these autoradiographs number of tracheary elements produced silver grains are detectable only over nuclei in any treatment, and these grains are not per segment is plotted vs the number of doublings or fractions of doublings of the present if the squash was pretreated with cell population. The number of cell populaDNase. The complete results of this experiment are being quantitated and will be tion doublings was calculated from the known number of cells present at day zero published separately. Finally, an experiment was designed to in each experiment. The results of this plot show: 1) For any given number of generadetermine if the effect of BrdU inhibition tions of cells produced there are always of cell differentiation is due to its incorpoin ration into DNA. The experiment is based fewer tracheary elements differentiated on the known TdR reversal of both FdU the BrdU treated segments than in their

Differentiation

TERRY L. SHININGER

20

in Root Cortical Purenchyma

a

I6 10 bx

FdU IO-%l,BrdU

I I 0

3

6

9

l4-

10-4M I2

I5

DAYS

FIG. 6. Root segments were cultured up to 15 days on S2M medium plus 1 mg/l of kinetin alone or with: lo-’ MFdU;10-6MFdUplus10-“MTdR;10~‘MTdR;10-6MFdUplus10-‘MBrdU;10-BMFdUplus10-5M BrdU; lo-* M BrdU or 10e5 M BrdU, as indicated. Samples were taken after 6,9 or 15 days of culture. (A), FdU inhibition of cell division is fully reversed by TdR and only very slightly reversed by BrdU. BrdU ( 10e5 M) is less inhibitory than BrdU (lo-’ M) during the early part of the culture period and a normal rate of cell division is recovered. BrdU (10e4 M) continues to be inhibitory to cell division. TdR (10-l M) is not inhibitory during the first part of culture but eventually reduces the rate of cell division. (B), FdU inhibition of tracheary element formation is fully reversed by simultaneous TdR treatment but not by simultaneous BrdU (10m5 or lo-’ M). Either BrdU or TdR alone greatly reduce the rate of tracheary element formation. *, This line also represents the following treatments: FdU, 1Om6M, BrdU, 10e5 M; FdU, 10e6 M, BrdU, lo-’ M, BrdU, lo-’ M.

.

controls, and 2) tracheary elements do not appear in the segments until two doublings of the population have occurred. DISCUSSION

FIG. 7. The number of tracheary elements produced in 15 separate experiments in which root segments were cultured on control medium or medium containing lo-’ M BrdU for either 12 or 15 days plotted vs the number of cell generations (multiples of the day-zero cell number, calculated for each individual experiment). Both plots intersect abscissa at two generations.

The results of these studies demonstrate that either the prevention of DNA synthesis or the synthesis of an abnormal DNA will prevent cytokinin induction of tracheary element differentiation. FdU totally prevents the induction of tracheary element differentiation along with inhibiting DNA synthesis and cell division. The time course of FdU inhibition of tracheary element differentiation parallels the inhibition of cell division. The time course of the recovery of tracheary element differentiation from inhibition by FdU also parallels the recovery of cell division. This result could indicate a requirement for DNA synthesis or for cytokinesis, or both. This

146

DEVELOPMENTAL BIOLOGY

is in agreement with the work of Fosket (1968, 1970) which showed that auxin induction of vessel element differentiation in Coleus was blocked by FdU. Similar results have been reported for a number of other investigations in animal development (Wessells, 1964, 1968; Coleman et al., 1969; Lasher and Cahn, 1969; Levitt and Dorfman, 1972; Stockdale et al., 1964; Wilcos, 1969). Most of these investigations have not shown that FdU inhibition is reversible by subsequent TdR treatment. This is crucial if FdU effects are not to be attributed to irreversible side effects. Fosket (1968) further attacked the question of DNA synthesis vs cytokinesis by using 0.04% (w/v) colchicine to block cytokinesis in Coleus explants while presumably allowing normal DNA synthesis. He found that colchicine blocked most auxininduced vessel-element differentiation. The conclusion was that differentiation probably required cytokinesis rather than DNA synthesis. In the pea cortical parenchyma system, cells which replicate their DNA in the presence of BrdU have a reduced ability to respond to cytokinin in terms of differentiation, yet most metabolism related to DNA synthesis and cell division must be normal. In this system both normal activation of cell division and subsequently normal rates of replication occur in the presence of differentiation-inhibiting concentrations of BrdU. BrdU reduced the rate of differentiation to 25% of the control rate while having little or no effect on the rate of cell replication. Transfer from BrdU to BrdU plus TdR allowed the restoration of a normal rate of cell proliferation. In contrast, it was never possible to obtain a normal rate of tracheary element formation following a BrdU treatment. These results suggest that BrdU is incorporated into some component of the nucleus which turns over at a low rate and further that differentiation is more sensitive to the presence of BrdU than is cell replication. This conclusion is supported by autoradio-

VOLUME45, 1975

graphic evidence of DNase-sensitive nuclear incorporation of [SH]BrdU. It is generally thought that BrdU effects on differentiation are due to incorporation into DNA (Coleman et al., 1969; Hill et al., 1974; Lasher and Cahn, 1969; Levitt and Dorfman, 1972; Lin and Riggs, 1972; Silagi and Bruce, 1970; Stockdale et al., 1964; Wessells, 1964, 1968; Wilcox et al., 1969; Wrathall et al., 1973). DNA replication and cell replication can proceed normally in these cells and it is the new daughter cells without BrdU in their DNA that are capable of differentiation. Mayne et al. (1973) showed that normal BrdU inhibition of development in chick chondrocytes occurs only when the cells replicate in the presence of BrdU. Levitt and Dorfman (1972) found that differentiation in embryonic limb tissue can be permanently repressed by fairly short exposure to BrdU. They also found that the nuclei of 95% of these cells showed incorporation of [3H]BrdU. Schubert and Jacob (1970) however, came to the opposite conclusion based on their work showing BrdU induction of neurite development. In their work, the inhibition of DNA synthesis during BrdU treatment did not prevent the BrdU effect. Differentiation in their system is not dependent upon DNA synthesis. Attempts to determine directly that BrdU effects in the pea root system are or are not due to BrdU incorporation into DNA are not conclusive. The failure of BrdU to reverse the FdU inhibition of cell division activity might not be due to a failure of BrdU effectively to replace thymine in DNA. Under these conditions total substitution of thymine sites should occur and so the inhibitory effects of BrdU substitution on cell replication should be more pronounced than for the same concentration of BrdU given alone. This is exactly the result obtained. A consequence of this is that differentiation should be even more suppressed by combined BrdU and FdU treatment. Again, this result is obtained. The third conclusion is that non-

TERRY

L.

SHININCER

Differentiation

halogenated nucleosides may have the same differentiation-inhibiting effects as generally attributed to only the halogenated nucleosides. Most BrdU experiments have, with some exceptions (Coleman et al., 1969; Koyama and Ono, 1972a,b; Lasher and Cahn, 1969), failed to incorporate TdR controls. This is undoubtedly due to the original report by Stockdale et al. (1964) in which TdR at ten times the concentration of BrdU failed to have an inhibitory effect on differentiation. It does not seem reasonable to attribute a DNAbased mechanism for the TdR inhibition of tracheary element formation because: a) TdR is normally incorporated into DNA, and b) TdR inhibition of DNA synthesis (Cleaver, 1967) should act essentially to reduce cell proliferation and differentiation in parallel as would any inhibitor of DNA synthesis. These BrdU and TdR effects might be explained in either of two simple and obvious ways: The first possibility is a mixed-mode system. BrdU would be inhibitory because of its incorporation into DNA and consequent formation of a structurally abnormal DNA which cannot be properly expressed possibly because of steric hindrance. Simultaneous TdR treatment would reverse this effect by competition for sites in the DNA molecule. TdR, in the absence of BrdU, might inhibit differentiation by end-product inhibition (thymine triphosphate, TTP) of enzymes crucial to differentiation. Such TTP inhibition is known in the case of TdR kinase (see discussion in Cleaver, 1966). BrdU reversal of TdR inhibition could then be explained on the basis of competition for attachment to a specific site on the enzyme resulting in reactivation of the pathway. BrdU attachment to the enzyme would have to result in active enzyme systems and effectively reduce BrdU availability such that insignificant amounts were available for incorporation into DNA. These effects could be similar to allosteric effects (Monod et al., 1963) but clearly different from the original

in Root Cortical Parenchyma

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concept of allosteric effects. The second possible explanation is that BrdU incorporation into DNA is not involved, but the effect is only manifest when DNA synthesis has occurred. In this case BrdU and TdR would both be allosteric effecters for one or more enzymes involved in differentiation. This could involve an alteration of sugar nucleotide metabolism as suggested by Schubert and Jacob (1970) or be an effect on transport systems. The results of the present experiments along with the published literature make possible the suggestion of a system for the regulation of certain types of development that is consistent with the concept of quanta1 mitoses and with the concept of the change from totipotency to unipotency. The following are the critical observations which must be taken in account: 1) Only a few tracheary elements differentiate in the pea root cortical parenchyma in segments that have not undergone two or more complete cell population doublings; 2) most newly formed tracheary elements have synthesized DNA (Torrey and Fosket, 1970); but 3) ploidy increases which occur (Libbenga and Torrey, 1973) are probably not important to differentiation (Phillips and Torrey, 1974); 4) FdU blocks both cell replication and tracheary element differentiation (see papers cited above); 5) BrdU blocks tracheary element formation or other forms of differentiation while not blocking cytokinesis in this and other systems (see papers cited above); 6) BrdU can block differentiation after incorporation into only one strand of DNA, “unifilar dominance” (Bischoff and Holtzer, 1970); 7) colchicine can block differentiation (Fosket, 1968; Hepler and Fosket, 1973). The hypothesis suggested by these observations is that normal differentiation of cells involves repression of certain blocks of genes via a repressing factor which is attached to or integrated into a strand of DNA. The factor could be synthesized continuously or discontinuously. Basically, the system would operate in a fashion

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similar to regulation in the operon as described by Jacob and Monod (1961). The major difference would be that the repression factor would not be available for binding with inducer (possibly cytokinin) until freed from the DNA molecule during the process of DNA replication. The induction of differentiation within one generation would be unlikely since there are undoubtedly many genes involved in the differentiation of any given eukaryotic cell and these genes may be sequentially derepressed using as inducer the cytokinin and a product of the early transcribed genes. This would ensure that a new pathway of differentiation could not generally be induced in differentiated cells without two rounds of DNA replication and the usual accompanying cytokinesis. This explanation fits the concept of quanta1 mitoses. The progressive repression of totipotent cells toward the unipotent state could be via the acquisition of repressed genes which become repressed by the activity of specific gene products in combination with the milieu of the cell. The quanta1 mitosis is then a mitosis or mitoses that lead to a specific differentiated state from any point in the spectrum of “potency.” The result of the scheme is that preventing DNA synthesis would block changes in differentiation because of the inability of cells to form new nonrepressed DNA. Blocking cytokinesis should not prevent changes in the state of differentiation. Colchicine did prevent cytokinesis and differentiation in Coleus explants (Fosket, 1968; Hepler and Fosket, 1973). However, the chromatin in colchicine-treated cells of Coleus appears condensed (D. E. Fosket, personal communication). Colchicine effects may also be explained by interference with DNA synthesis (Fitzgerald and Brehaut, 1970). The repressing factors should be present in very small quantity and be available for interaction with cytokinin (if it is an inducer) only in a dividing population of

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cells. Proof of this concept requires knowledge of the site of cytokinin action. Is cytokinin active in induction of terminal differentiation in differentiated cells when it interacts with the cell surface, or must the cytokinin enter the cytoplasm or nucleus? The hypothesis predicts that the nucleus of a young tracheary element will always be doubly labeled if a synchronously dividing population of cells is treated with [l%]TdR during the first round of DNA replication and with [3H]TdR during the second. Also, differentiation will be blocked by allowing only one round of DNA replication or two rounds of DNA replication with cytokinin present during only one round. Efforts are currently directed toward synchronizing the pea cortical parenchyma system in order to test this. Previous results with the unsynchronized system suggest cytokinin must be present for more than one round of DNA replication (Shininger and Torrey, 1973). The few tracheary elements which can be detected prior to two cell population doublings in a few of the present experiments and in other experiments (Torrey and Fosket, 1970; Phillips and Torrey, 1973) might be derived from a population of cells that divide more rapidly than the general population or were incompletely repressed during development. These may be those few new tracheary elements which do not become labeled with [3H]TdR (Torrey and Fosket, 1970). I thank Mrs. Lissa Elliot for technical assistance, Dr. Donald E. Fosket for critical reading of the manuscript and Dr. E. W. Hanly and Dr. K. G. Lark for valuable discussions during the course of this work. This work was supported by the National Science Foundation Grant No. GB 36948. REFERENCES ABBOIT, J., and HOLTZER, H. (1968). The loss of phenotypic traits by differentiated cells. I. The effect of 5-bromodeoxyuridine on cloned chondrocytes. Proc. Nat. Acad. Sci. USA 59,1144-1151. BISCHOFF R., and HOLTZER, H. (1970). Inhibition of myoblast fusion after one round of DNA synthesis

TERRY L. SHININGER

Differentiation in Root Cortical Purenchymu

in 5-Bromodeoxyuridine. J. Cell Biol. 44, 134-150. CLEAVER, J. E. (1967). “Thymidine Metabolism and Cell Kinetics,” p. 93. North-Holland, Amsterdam. COLEMAN, J. R., COLEMAN, A. W., and HARTLINE, E. J. H. (1969). A clonal study of the reversible inhibition of muscle differentiation by the halogenated thymidine analog 5-bromodeoxyuridine. Deuelop. Biol. 19, 527-548. DALESSANDRO,G. (1973). Interaction of auxin, cytokinin and gibberellin on cell division and xylem differentiation in cultured explants of Jerusalem artichoke. Plant Cell Physiol. 14, 1167-1176. FITZGERALD,P. H., and BREHAUT, L. A. (1970). Depression of DNA synthesis and mitotic index by colchitine in cultured human lymphocytes. Exp. Cell Res. 59, 27-31. FOSKET, D. E. (1968). Cell division and the differentiation of wound-vessel members in cultured stem segments of Coleus. Proc. Nut. Acad. Sci. USA 59, 1089-1096. FOSKET, D. E. (1970). The time course of xylem differentiation and its relation to deoxyribonucleic acid synthesis in cultured Coleus stem segments.

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acids. In “The Nucleic Acids” (E. Chargaff and Davidson, J. N., eds.). Academic Press, New York. TORREY, J. G., and FOSKET, D. E. (1970). Cell division in relation to cytodifferentiation in cultured pea mot segments. Amer. J. Bot. 57, 1072-1060. WESSELLS, N. K. (1964). DNA synthesis, mitosis and differentiation in pancreas acinar cells in vitro. J. Cell Biol. 20, 415-433. WESSELLS, N. K. (1968). Problems in the analysis of determination, mitosis and differentiation. In “Epithelial-Mesenchymal Interactions” (ed. R. Fleischmajer and R. E. Billingham eds.), pp. 132-151. Williams and Wilkins, Baltimore. WILCOX, C., SANGER, J., and ABBOTT, J. (1969). Quantal mitosis and the induction of vertebral cartilage. J. Cell Biol. 43, 157a. WRATHALL, J. R., OLIVER, C., SILAGI, S., AND ESSNER, E. (1973). Suppression of pigmentation in mouse melanoma cells by 5-bromodeoxyuridine, effects on tyrosine activity and melanosome formation. J. Cell Biol. 57, 406-423.