The initiation of melanogenesis in the chick retinal pigment epithelium

DEVELOPMENTAL

BIOLOGY

44, 102-118

The Initiation

(1975)

of Melanogenesis Pigment JOEL

Department

of Anatomy,

Tufts University

in the Chick Retinal

Epithelium ZIMMERMAN School of Medicine,

Accepted January

Boston, Massachusetts

02111

8, 1975

Observations have been made on chick pigment retinal epithelium between 2 and 5 days of development. 2-Thiouracil has been demonstrated to be an effective agent for measuring the rate of melanin synthesis. Using [3H]thymidine and colcimid, we have found that the cells undergo a marked withdrawal from the cell cycle between 3 and 3.5 days of incubation in ouo, indicating that a majority of the population is synchronized. This withdrawal is followed, approximately 24 hr later, by a rapid rise in melanin synthesis from the basal level which first appears at approximately 3 days. 5-Bromodeoxyuridine (BUdR) has been used to determine the time at which melanin synthesis is initiated. When BUdR is administered as early as 2 days in ouo, it is incapable of blocking the appearance of basal levels of melanin even though the cells divide at least three times in the presence of this thymidine analog. However, BUdR is capable of delaying the rapid rise in the rate of melanin synthesis first observed at 4.5 days. This delay has been found to correlate, using [3H]BUdR, with a delay in the withdrawal of the cells from the division cycle. In pursuing the idea of a correlation between withdrawal and the rapid increase of melanin formation, 5-fluorodeoxyuridine (FUdR) was used. Histological and biochemical evidence suggests that those cells which have been prevented from dividing by FUdR increase their rate of melanin synthesis to the high level of the postmitotic control cells described above. Therefore, it seems that (l), in light of work done by others, the initial decision to make melanin is made prior to 2 days in ouo, and (2) the mechanism by which cells shift their synthetic capabilities to high levels of melanin production is withdrawal mediated. INTRODUCTION

In order to examine some problems in cell differentiation, it is imperative that homogeneous cell populations be looked at. Some questions that can only be answered clearly using a homogeneous system are: (1) How long before actual synthesis can a lineage of cells become committed to synthesizing those molecules peculiar to them (hereafter called luxury molecules) [Holtzer, 1968; Holtzer and Abbot, 1968]? (2) Does the cell-cycle time change as a population becomes increasingly committed to a specific terminal state? (3) Is there a unique division which must occur for a cell to begin synthesizing its luxury molecule or may there be other mechanisms, e.g., withdrawal mediation? To answer these questions, the cells

examined must have identifying characteristics, either histological or biochemical, that are in existence prior to the cells’ terminal differentiation, e.g., the cells assume specific positions before they manufacture their terminal luxury molecules. Studies in myogenesis (Bischoff and Holtzer, 1969), for example, deal with heterogeneous cell populations that are not identifiable until after terminal differentiation, while studies with red blood cells (Weintraub et al., 1971; Campbell et al., 1971) deal with homogeneous populations that are not distinguishable until hemoglobin appears. In dealing with questions about what cells do before they make their specific terminal products, it is necessary to find a system in which these cells can be identified. Some of these questions may be an-

102 Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.

JOEL ZIMMERMAN

Initiation

swered using the retinal pigment epithelium of the chick embryo between 2 and 5 days of development. This system was chosen for a variety of reasons. One is that, histologically, it is a homogeneous cell population identifiable by its position prior to the appearance of pigment (Hamilton, 1952), i.e., one may identify presumptive melanoblasts. Pigment, in addition, is easily identified in the light microscope and, if necessary, in the electron microscope and may be assayed for biochemically. Also, retinal pigment is one of the first unique cell products, along with hemoglobin and cardiac muscle myofibrillar proteins, indicating that commitment to this pigment lineage occurs early in embryonic development (Harrison, 1951). Lastly, because of its homogeneity and position, one can work in ouo in order to determine actual rather than possible modes of cell decision making. One tool for establishing at what point in a lineage a specific decision is made appears to be 5-bromodeoxyuridine (BUdR). Early work in the suppression of luxury molecule synthesis by BUdR (e.g., Wessels, 1964; Okazaki and Holtzer, 1965; Abbott and Holtzer, 1968; Clark, 1971; Turkington et al., 1971; Wilt and Anderson, 1972; Abbott et al., 1972) left it unclear as to whether the analog suppressed luxury molecule synthesis only at the time of decision making or whether suppression could occur, even after synthesis had begun, as long as division took place. More recent work seems to have clarified this point. It now appears that in erythropoietic cells the addition of BUdR to the DNA has no effect on hemoglobin synthesis if BUdR is added after hemoglobin is first manufactured (Weintraub et al., 1972). This would suggest that cellular activity, when viewed in terms of luxury molecule synthesis, falls into two classes. The first is those that have a single decision to make a product, e.g., red blood cells, and the

of

Melanogenesis

103

second consists of those cell types that continually decide to make their product, e.g., chondrocytes and pigmented melanoma cells (Schulte-Holthausen et al., 1968; Abbott and Holtzer, 1968; Coleman et al., 1970; Silagi and Bruce, 1970; Wrathall et al., 1973). In either case, BUdR may be used to find out the latest time at which a decision is made affecting the manufacture of a given luxury molecule and will establish at what point in a lineage this decision occurs. The last question to be looked at is that of the mechanisms necessary for a cell to begin synthesizing its luxury molecule. Is there a unique division which must occur for a cell to begin synthesizing its luxury molecule or may there be other mechanisms, e.g., withdrawal mediation? In order to examine this question, the ability of the cells to manufacture increased amounts of melanin, when they were prohibited from continuing in the cell cycle, was measured. 5-Fluorodeoxyuridine (FUdR) was used as the arresting agent. It acts by blocking cells in S when all available thymidylate has been utilized or cells in the G1-S interphase where there is no thymidylate pool. If the eyes in the presence of FUdR manufacture more melanin than the controls, then it is clear that no specific division is necessary but, rather, withdrawal and melanization are linked. If, however, melanogenic levels are kept at control levels, then it is possible that a specific future division is needed. The work presented here attempts to look at these questions. First, the validity of the biochemical technique (Whittaker, 1971) for measuring the rate of melanin synthesis is discussed. Then, the normal development of the retinal pigment epithelium, both histological and biochemical, is outlined. Following this, the work with BUdR suggests in fact that (1) the decision to make pigment is made well before actual synthesis; (2) the initial decision is fol-

104

DEVELOPMENTALBIOLOGY

lowed by a series of proliferative mitotic divisions (Bischoff and Holtzer, 1969); (3) withdrawal plays a key role in the manufacture of high levels of melanin; (4) BUdR acts to disengage cells from a withdrawal program; and (5) there is another type of cell decision in which BUdR may act only to delay cells in enacting their program. Further results with FUdR indicate that melanogenesis and the cessation of division have already been tied to each other by 2.5 days of incubation and so when cells are “fooled” into a withdrawal state, they begin to make melanin at a rate not normally seen for at least another 24 hr. MATERIALS

AND

METHODS

Staging and treatment of eggs. All ages given are days from initiation of incubation of white Leghorn eggs. Eggs were incubated at 37°C. Any embryos that were obviously older or younger than expected were discarded. For the addition of coltimid (3 ml of lo- 8 M/egg), 5 ml of albumin were withdrawn from the egg. The drug, dissolved in balanced salt solution (BSS) (Weintraub and Holtzer, 1971), was then added on top of the embryo without breaking the chorion. The eggs were sealed with tape and kept in an egg incubator for the indicated length of time. Histology. Tissue to be examined was fixed with Carnoy-Lebrun fixative (Humason, 1967) for 3 hr, and postfixed in Izsaturated 70% ethanol for 5 hr. It was then stored in 70% ethanol until embedded in Paraplast or Paraplast-plus (Sherwood Medical Industries), and cut at 5 or 4 pm, respectively. Tissue was stained with Cresyl Violet. For autoradiography prior to staining, slides were dipped in Kodak NTB emuslion (diluted, two parts to one part emulsion) and allowed to develop at least 2 weeks at 4°C in Drierite-filled light-tight boxes before being developed and stained. All counting was done on sections taken through the widest part of the lens. Five sections, each five sections apart, were counted for every value.

VOLUME 44, 1975

Thiouracil assay. Several techniques for measuring the rate of melanin synthesis in cells were tried. The technique finally selected was that outlined by Whittaker (1971) using 2- [2-‘“Clthiouracil (Amersham/Searle; 50 mCi/mmole) ( [‘“CITU). The basis for the technique is that TU binds to forming eumelanin the way cysteine is thought to bind, through -SH bonds, in the creation of pheomelanin. Several studies were done to verify the validity of this technique. For 3-day or younger animals, the whole head was used, for older animals, only the eyes. The tissue was placed in the well of an organ culture dish (Falcon Plastics) that was then filled with modified Eagle’s medium (Zimmerman et al., 1974) containing [14C]TU (1 pCi/ml). The dish was incubated for the time specified (see Results) in a 5% CO,-95% air mixture at 37°C. Following this, the tissue was rinsed in BSS and placed in test tubes containing cold 12% trichloroacetic acid (TCA) for at least 1 hr. The TCA precipitate was then washed four times with 12% TCA and allowed to dry from 1 hr to overnight. Subsequently, 0.5 N NaOH (0.2 ml) was added to each tube and the tubes were kept in boiling water for 10 min. The entire sample was then placed on a glass-fiber disk (GF/A, Whatman), each sample on a separate disk, and dried prior to being counted in a liquid scintillation counter. The data are presented as rate of synthesis (cpm/4 eyes/l5 l-n). This is thought to be a truer reflection of activity since values derived using amounts of DNA or protein as a denominator fail to take into account the fact that the pigment epithelium accounts for only a small percentage of the total number of cells assayed. For measurements of protein synthesis, the treatment was the same, except that [%]leucine (280 mCi/mmole; New England Nuclear) was used. Total DNA content. After TCA washing, as described above, 0.5 N perchloric acid was added to the precipitate and the mix-

JOEL ZIMMERMAN

Initiation

ture placed in a boiling water bath for 20 min. The supernatant was then assayed by the diphenylamine method of Dische as revised by Schneider (1957). Thiouracil incorporation into nucleic acid. After incubating the tissue in medium containing [“C]TU, washing and precipitating as described above, 0.5 N perchloric acid was added and the mixture kept in boiling water for 20 min. The supernatant was then decanted and counted and the precipitate hydrolyzed in NaOH and counted as above. Deoxyadenosine incorporation into nucleic acid. [3H]Deoxyadenosine [3H]AdR) incorporation into DNA was measured by liquid scintillation counting. Tissue that had been treated in ouo with [3H]AdR (50 PCi) (New England Nuclear; 15 Ci/mmole) was washed four times in 12% TCA. The precipitate was then treated with 0.2 N KOH at 37°C for 90 min to hydrolyze the RNA. Cold 12% TCA and carrier medium (Zimmerman et al., 1974) were then added to precipitate the labeled DNA. The precipitate was then washed twice with 12% TCA and hydrolyzed in 0.5 N NaOH (0.2 ml) for 10 min in a boiling water bath. The hydrolyzate was then dried on glass fiber disks and counted. BUdR and FUdR. Drugs were administered as previously described for colcimid. The following doses were given per egg: BUdR, 24 or 36 hr, 600 pg (unless otherwise noted); BUdR, 48 hr, 300 pg; [3H]BUdR, 50 PCi (New England Nuclear; 28 Ci/mmole); FUdR, 24 hr, 1 ml of 1O-6 M/egg; FUdR, 36 hr, 3 ml of 1O-7 M/egg. The points on all graphs of synthetic rates are the averages of at least three separate experiments, each done in duplicate.

105

of Melnnogenesis

sufficient level (0.5 mMJ to stop melanin synthesis (Lerner et al., 1950; Whittaker, 1966; Whittaker, 1971). The results are presented in Fig. 1. In the presence of PTU (which has been shown by Whittaker [1971] not to effect viability), incorporation of label is extremely reduced over the entire 25 hr, being kept to a very low constant level. Incorporation without PTU increases gradually for 10 hr and then begins to rise more rapidly. This increased rise is thought to reflect a change in the synthetic activity which occurs at approximately 4.5 days (see below). In any case, it is clear that [14C]TU is not limiting. On the basis of these data, an incubation of 15 hr was thought adequate. To insure that during these 15 hr of incubation the tissue remained viable, [‘%]leucine-incorporation studies were done (1 pCi/ml). These data (Fig. 2) demonstrate that during the entire incubation time the tissue remains viable, as judged by its ability to produce protein at a constant rate. The possibility had to be checked that the [l%]TU was being incorporated into nucleic acid. Three-day heads, after 15 hr of incubation with [14C]TU and the appropriate TCA washings, were treated with perchloric acid to extract the nucleic acids. The extracted material contained less than the level of PTU-resistant activity. There-

RESULTS

Thiouracil

Assay

To test the kinetics of [l*C]TU incorporation into melanin, 4-day embryo eyes were incubated in medium containing the labeled compound for up to 25 hr with and without phenylthiourea (PTU) present at a

0

5

IO Time

15

25

(hrs)

FIG. 1. The rate of [“Clthiouracil incorporation into pigment was measured over 25 hr with and without the presence of FTU which inhibits melanin syn. thesis.

106

DEVELOPMENTALBIOLOGY

VOLUME 44, 1975

incubated with [14C]TU also confirms this point. It is clear from the above data that [14C]TU incorporation is an accurate and specific measure of the rate of melanin synthesis and may be used as such in this system without any qualifications.

Normal Development of the Retinal Pigment Epithelium

FIG. 2. Protein synthesis, as measured by [14C]leutine incorporation, was examined over 25 hr to check the viability of the eyes in the incubation system

fore, by subtracting the background levels present (see next section), the problem of label incorporated into nucleic acid is resolved. Whittaker’s work (1971) indicated that other dihydroxyphenylalanine-containing tissues also demonstrate the ability to incorporate TU. This point is examined in Table 1. It is clear that any tissue having dihydroxyphenylalanine (whether adrenergic or melanogenic) shows some level of [14C]TU incorporation above background. In addition, the thyroid, which makes a sulphenyl protein known to bind TU (Jirousek, 1968), also incorporates label. However, in agreement with Whittaker, tissues without tyrosine hydroxylase activity, e.g., kidney, show little [‘%]TU incorporation beyond what is attributable to incorporation into nucleic acid. As the whole eye is assayed, and the adult neural retina is known to have tyrosine hydroxylase activity, some idea of the activity attributable to this in the embryonic system was necessary. Therefore, eyes were dissected into (1) neural retina and lens and (2) pigment epithelium with a small amount of attached choroid. The results are presented in Table 2. They show without a doubt that the major portion of counts detectable are those coming from the specific incorporation of [14C]TU into pigment cells. Radioautography of tissues

In beginning to examine any system, it is important to know what the general cell population is doing during the time of investigation (in this case, 2-5 days of embryogenesis). One thing to know is the size of the dividing-cell population and how it changes over time. To learn this, embryos were treated with 3 ml of 10m6M colcimid in ovo for 24 hr. Specimens were then fixed and sectioned, and mitotic arrest nuclei were counted (Fig. 3). It is clear from these results that approximately 55% of all cells present between 2 TABLE

1

THE INCORPORATIONOF [%]THIOURACIL INTO VARIOUS TISSUES AT 4, 8, AND 17 DAYS OF INCUBATION OVER 15 HR Organ

Brain Limb Heart Liver Kidney Thyroid Skin Eye

Day 4 (cpm/organ)

Day 8 (cpm/mg DNA)

Day 17

18.7 4.6

1,160 -

2,550 -

27.9 42.3

550 680 -

231 730 146 4,600 1,460 1,560

1,060 TABLE

2

THE INCORPORATIONOF [“CITHIOURACIL INTO THE 8- AND IT-DAY WHOLE EYE AND ITS PARTS OVER 15 HR Part of eye

Retina and lens Pigment epithelium Whole eye

Day 8 (cpdmg DNA) 820 127,900 2,200

Day 17

(cpmhg DNA)

455 2,150 810

JOEL ZIMMERMAN 70

size of the starting dividing-cell population (50%), and the number of cells labeled by the end of a given time period (90%), it is possible to calculate the number of divisions needed in that time period to generate that number of labeled cells, and, so, the average cell cycle time. This is done theoretically below:

1

6Ot

107

Initiation of Melunogenesis

t

starting cells labeled cells/total cells 50/100 -------+ 100/150 --------+ 200/250 ------+ 400/450 80% 50% 66% 89% I .o

2.0

3.0

4.0

5.0

DAYS OLD AT ADDITION

FIG. 3. The percentage of cells in the pigment epithelium arrested by colcimid over 24 hr.

and 3 days are in the dividing-cell population. This number changes markedly between 3 and 3.5 days of development when the number of dividing cells decreases by approximately 40%. This is then followed by a slow, but steady decline, until by 5 days, only 8-9% of the cells present are in the dividing cell pool. To obtain a qualitative idea of the generation of new cells over the same time period, [3H]thymidine (50 pCi/egg; New England Nuclear; 47 Ci/mmole) autoradiographs were used. One finds a similar result (Fig. 4). Between 2 and 4 days, 90-95% of all cells are generated within any given 24-hr period. Following this, there is a precipitious drop of almost 70% in new cells generated and this drop is followed by a steady decline out to the 5-6day labeling period. Since, between 3 and 4 days, 90% of all cells are labeled, and between 3.5 and 4.5 days only 23% are labeled, it is obvious that between 3 and 3.5 days of incubation in ouo most of the observed withdrawal activity must have taken place. This rapid shift in cells from the dividing to the nondividing cell population is indicative of a well synchronized cell population. The data presented above may be used to calculate cell-cycle times. Knowing the

2.5 days ________ -- _._______ 3 days ___.-.-- ________ --3.5 days

In the above example it takes three divisions in 24 hr to generate 89% labeled cells from 50% originally dividing cells. This would suggest a maximum cell-cycle time of approximately 8 hr. However, when colcimid is added at 3 days, only 50% of the cells are seen to be arrested, whereas one would expect that between 66 and 80% of the cells would be arrested. This indicates a continual withdrawal of cells so that at any given time only 50% are capable of dividing. Taking this into account should yield a more accurate picture of the number of divisions, as illustrated below: 50/100 ---50%

(75 divide) +100/150--------

-----------+

66%

(112) 1751225 -----+287/337-----+ 77% 85%

(169) 456f506 90%

2.5 days__..__________.____ 3 days._________________ 3.5 days This example, in which only 50% of the cells divide each time, indicates four cell divisions within 24 hr, each of 6-hr duration. Actual cell-cycle time calculations are presented in Table 3. The cell-cycle time for 3-day cells is given as the same as those preceding it; it is thought that two to three cell divisions occur between day 3 and 3.5, followed by one or two divisions afterwards, by some cells. Cell-cycle times for days 3.5 onwards are calculated without taking into account the continual with-

108

DEVELOPMENTALBIOLOGY

drawal of cells (and are thus maximal values), this method being used only to calculate the short cycle times of the very earliest cells. With the cell cycle kinetics of the pigment epithelial cells known between 2 and 5 days of development, it now becomes of interest to see what the melanin synthesizing capability of these cells is over the same period. Accordingly, heads were incubated in [‘“CITU, as described in the text, and the data collected (Fig. 5). It should be noted here that there is no detectable melanogenic ability before 3 days in ovo, i.e., incorporation levels without PTU and those with PTU are equal, so 100 f

“x--F

L

Y i $I 5o 3 z IO 2-

5

DAYS OLD AT ADDITION

FIG. 4. The percentage of cells in the pigment epithelium labeled by [3H]TdR over 24 hr. TABLE

3

INDICES OF ARREST ANDLABELING, WITH [8H]T~R AND [*H]BUDR FOR 24 HR, AND THE CALCULATED CELL-CYCLE TIME Day of addition of label

% Arrest

2 2.5 3 3.5 4 4.5 5

59.0 49.0 55.0 17.4 18.6 13.4 8.5

% Cell[SH]- cycle TdR t&r; r 95.0 89.5 90.5 22.9 29.5 15.2 8.0

4.8 6.0 6.2 55.8 28.2 136.4 -

% [9H]tf4JfR r

Cellcycle time (hr)

90 [3H]BUdR 36hP

91.5 94.1 94.4 47.9 37.8 37.5 12.0

8.0 6.0 6.4 11.2 17.3 12.3 48.9

91.0 82.5 55.0 57.5 -

a The labeling index when BUdR is present for 36 hr but label is only present for the last 24; values are given for the days label is present.

VOLUME44, 19% 750 1

I

I,

AGE OF EMBRYOS (DAYS)

FIG. 5. The changing rate of melanin between 2 and 5 days of development.

synthesis

the value of 2.5 days is always taken as background. At 3 days, slight levels of incorporation are detected and these rise steadily over the next 24 hr. Then, sometime between day 4 and 4.5, the rate of melanin synthesis markedly increases and continues to climb very rapidly. This increase occurs approximately 24 hr after the cells have withdrawn from the cell cycle. It is important to state, however, that some synthesis occurs while the cells are still dividing rapidly, at day 3, and that metaphase cells have been observed to contain melanin. It is clear then that cell division and some levels of synthesis are compatible. BUdR Treatment In order to determine the appropriate dosage of BUdR, 3.5-day eggs were given, for 24 hr, various amounts of BUdR, up to 600 pg, and the eyes were then assayed for melanogenic activity (Fig. 6) by measuring [14C]TU incorporation. There is no apparent decrease in synthetic activity when 200 pg or less of BUdR is added; however, there is a sharp difference when the dosage is increased only slightly (by 50 pg) and this suppression is maintained at increasingly higher doses. Therefore, 600 pg was chosen as the highest effective tested dose which did not kill the embryos. (For 48-hr treat-

JOEL ZIMMERMAN 3kp-

5

F 8

01

4k2 DAYS TREATMENT

100

200

300

Initiation

WITH BUdR

400

500

600 I

BUdR CONCENTRATION (pg/vgg)

FIG. 6. The effect on the rate of melanin synthesis of treatment of 3.5.day eyes with increasing amounts of BUdR.

ment times, this dose was reduced to 300 pg in order to maintain the embryo’s life.) Using [3H]BUdR (50 PCi in 600 pg), the number of cells incorporating the analog during any 24-hr period was determined (Fig. 7). As was seen before for [3H]TdR, the percentage of labeled cells is in the 90-95s range when the drug is added between 2 and 3 days. This drops off, although not as sharply as with [3H]TdR, by 3.5 days and steadily declines thereafter. The maximum values for cell-cycle times, calculated according to the method previously described are shown in Table 3. They are contrasted against the values for [3H]TdR-treated cells. It should be noted that the maximum cycle time for cells that are still dividing at 3.5 days or later is less in BUdR-treated cells than in the controls. This could be due to either a more rapid cell cycle and/or a failure of cells to withdraw from the cell cycle on schedule. If the cell-cycle time were shortened in the presence of BUdR, one would expect to see a more significant difference between the two populations earlier than 3.5 days. Other data, suggesting in fact that the cells in BUdR do fail to withdraw on schedule, are presented in the last column of Table 3. Eggs were treated for 36 hr with BUdR, but label ( [3H]BUdR) was present only for the last 24 hr. This was done to see what effect the pretreatment would have on the number of cells that divide and incorporate label. It is clear that treatment from 2.5-4 days (label from 3-4 days) in BUdR has no

of

109

Melanogenesis

significant effect in terms of the number of cells generated in the population, i.e., they are not dividing faster. The other labeling indices illustrate very clearly that there is a large increase over both controls and 24-hr BUdR-treated eggs. As there does not seem to be an effect on cell cycle time, this may only be attributed to a failure to withdraw on schedule, after 3.5 days, as was suggested above. How BUdR affects the rate of melanin synthesis is illustrated in Fig. 8. There is no apparent effect on [14C]TU incorporation if BUdR treatment begins by day 3 and ends by day 4, even though at least 90% of all cells have incorporated the analog. There is also no effect in the 4-5 day treatment period. When the BUdR is present between 3.5 and 4.5 days in ovo, however, there is a marked effect on the ability of the cells to synthesize melanin. Rather than increase their rate of synthesis as the normal cells do, they seem to stall at a level in accord with their slow synthetic phase. This stalling seems to occur when BUdR is present during or just after the cells begin to withdraw, i.e., at 3.5 days. The next step was to expose the eyes to BUdR for 36 hr. The results are shown in Fig. 9. Treatment with BUdR from 2-3.5 days shows no apparent BUdR effect. The

2-

5

DAYS OLD AT ADDITION

FIG. 7. The percentage of cells in the pigment epithelium labeled by [3H]BUdR (solid line) or [3H]TdR (dashed line) over 24 hr.

110

DEVELOPMENTALBIOLOGY 24 HR BWR TREATMENT

900 BOO

5 8~ a F f k rd

e-a

CONTROL

6OOC

4

500-

400

-

300

:

2001

VOLUME~~, 1975

Table 4, we see that while labeling in the first 24 hr in BUdR was high, little additional labeling was noted by extending the period 12 more hr. It is as though many of the cells had pulled out of the division cycle. Associated with this withdrawal is an increased rate of melanin synthesis (Figs. 8 and 9). The effect of BUdR on the rate of melanin synthesis after a 48-hr treatment period was examined (Fig. 10). Addition of BUdR at, or later than, 3.5 days has no effect on [‘4C]TU-incorporation levels. While treatment from 3-5 days seems to be

I

H) M i

1

CONTROL BUdR

AGE OF EMBRYOS(DAYS) FIG. 8. The rate of melanin synthesis after the embryos have been treated with BUdR for the preceding 24 hr.

addition of BUdR from 2.5-4 days displays no significant depression of melanogenic activity. (This is true when the 4-day value for all the data are examined rather than solely for this series. It appears that the 4-day value, in this set, is slightly elevated due, perhaps, to the use of slightly older embryos.) From the 3-4.5 day data, however, it is obvious that BUdR-treated eyes fail to initiate the high synthetic activity levels that the controls do (Fig. 9). Looking at the 3.5-5-day treatment period, it is clear that high melanogenicactivity levels have begun in cells that were initially restrained from achieving these levels. In other words, BUdR action in this instance seems to have a delaying effect upon the cells rather than a suppressive effect. To test the idea further that withdrawal may be important in initiating high activity levels, labeled BUdR (or TdR) was added to eggs at 3.5 days for 24 or 36 hr. In

7

6

AGE OF EMBRYOS(DAYS) FIG. 9. The rate of melanin synthesis after the embryos have been treated with BUdR for the preceding 36 hr. TABLE 4 LABELING INDICES OBTAINED BY TREATING 3.5.DAY EGGS WITH TDR OR BUDR FOR 24 OR 36 HR Time (hr)

TdR (W)

BUdR (%)

24 36

22.9 35.0

47.9 55.0

JOEL ZIMMERMAN

Initiation of Melnnogenesis

4ooc

48

HR BUdR

W 3000

TREATMENT

CONTROL 4

BUdR

z u

fl

z 2ooc !F 5 t; B I+ d 1000

200

.: AGE

3 OF

4 EMBRYOS

111

24-hr FUdR-treated embryo are shown (Figs. 11 and 12). The treated eye appears to contain fewer cells and to be at a much younger stage than the control eye. To test the effect of FUdR on DNA synthesis, the following experiments were performed. FUdR was added to 4-day eggs. Two hours later, [3H]AdR was added and, after 22 hr, the eyes were removed and treated as described in Materials and Methods. The results for treated and control (22 hr with [3H]AdR) eyes are shown in Table 5. They demonstrate a 60% inhibition of [3H]AdR incorporation into the DNA. As this is a measure of the whole eye and not just the pigment epithelium, and in order to determine whether the activity present was due to cells going through S and dividing or simply to the utilization of the available thymidylate pools, autoradio-

(DAYS)

FIG. 10. The rate of melanin synthesis after the embryos have been treated with BUdR for the preceding 48 hr.

reducing the rate of melanin synthesis, it is clear that the eyes have moved into the high activity phase. Once again though, measurement at 4.5 days (2.5-4.5-day treatment) shows an effect in that the eyes do not begin manufacturing melanin at high rates. The eyes treated with BUdR from 2-4 days show a modest amount of melanin synthesis probably due to the first few melanogenic cells having gone through their decision-making division prior to the addition of BUdR at 2 days. FUdR Treatment The first question that arises is the efficiency of the FUdR dose given. After 24 hr in FUdR, the embryos appear retarded in development; i.e., at a younger stage than they should be, but the heart continues to beat. The eyes generally appear smaller and blacker than controls. Sections through the whole eye of a control and

FIG. 11. A section through control eye. 44 x

the center of a 4-day

112

DEVELOPMENTAL

BIOLOGY

FIG. 12. A section through the center of an eye treated with FUdR from 3-4 days. The pigment epithelium (p), neural retina (n) and optic vesicle (0) are clearly seen. 88 x TABLE

VOLUME 44. 1975

no cells have divided. This, coupled with the fact that the embryo’s general appearance after 24 hr in FUdR is of an embryo approximately 1 day younger suggests that FUdR is almost totally effective in preventing cell division. (As to whether it is causing cell death, the viability of the embryos, as judged by heartbeat, suggests that this is not the case. While this is not the best criterion, others, such as leucine incorporation, have their own drawbacks and therefore this is considered sufficient to suggest that cell death is not significantly involved in the results.) As the general appearance of those embryos treated with FUdR for 36 hr is the same as for a 24-hr treatment, it is assumed that treatment is equally effective, even though the dosage is lower, in preventing mitotic activity, as judged by the retardation of size and the developmental stage. Figure 13 shows the rates of synthesis of melanin (as judged by [14C]TU incorporation) of control and 24-hr, FUdR-treated M

5

COUNTS PER MINUTE OF [3H]AnR INCORPORATED INTO THE DNA OF A I-DAY EYE IN THE PRESENCE OF FUDR

CONTROL 8 I

x

FUdR CORRECTED VALUE ASSUMING 100%

AND A ~-DAY CONTROL EYE Specimen

Cpm

% Inhibition

Control FUdR

12,800 5,100

60

graphs were prepared. Use of [3H]TdR has shown that approximately 30% of the pigmented epithelial cells incorporate label between 4 and 5 days and that 19% of the cells are arrestable in metaphase with colcimid. When FUdR and [3H]AdR are present, however, only 9% of the cells are seen to be labeled (a 67% decrease from the total possible number of labeled cells). Thus, while one-third of all possible cells appear to incorporate some label, the fact that the number is less than the colcimidarrested value suggests in fact that almost

FIG. 13. The rate of melanin synthesis after the embryos have been treated with FUdR for the preceding 24 hr. The corrected value adjusts the rate of synthesis for the effect of FUdR in decreasing the population size.

JOEL ZIMMERMAN

Initiation

embryos. As the rate-of-synthesis data are presented here in terms of cpm per 4 eyes and since FUdR effectively reduces the number of cells present, it is clear that this is not a fair comparison of activity per cell and that some compensation will have to be made for a reduced cell number. Assuming that FUdR is 100% effective (see above) and knowing the number of dividing cells and the number of cells generated, one can determine the factor by which the rate of melanin synthesis in the presence of FUdR must be adjusted for an equivalent population size. The factor is equal to (1 metaphase arrest index)/(l ~ labeling index). (This is equivalent to 1 plus the number of generated cells divided by the original number of cells.) The factor is then multiplied by the rate of synthesis of the FUdR-treated embryos. The corrected values shown here in Fig. 14 are the rates after just such an adjustment has been made. What is clear is that eyes treated between 2.5 and 3.5 and between 3 and 4 days exhibit at least control levels of activity which are well within the range of the high synthetic phase (i.e., greater than approximately 175 cpm as judged from the general pattern of all control data generated). This activity can only be considered a precocious (up to 24 hr) appearance of high levels of melanogenesis. Looking at the effect of FUdR after 36 hr (Fig. 14), it is clear that the same pattern of results exists. What should be noted, however, is that the correction factors used here are the same as those for 24 hr and, therefore, underestimate the actual factor by which the rate should be multiplied to approximate the same numbers of control cells.

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of kfehogenesis

use the pigment epithelium of the developing chick eye, a homogeneous group of cells, which may be identified by their position. The activities of these cells have been characterized, a summary of which is shown in Fig. 15, and it is seen that they go through a rapidly dividing phase, until 3-3.5 days of development. At that point most of the cells, seemingly synchronously 2500

CORRECTED VALUE x x r -coNTRoL

Ii FudR i

AGE

OF w3R~os

(DAYS)

14. The rate of melanin synthesis and the corrected rate after the embryos have been treated with FUdR for the preceding 36 hr. FIG.

DISCUSSION

In order to examine cells prior to the appearance of their terminal state, it is necessary to use some other marker, which itself may be a differentiated characteristic of the cell. Accordingly, I have chosen to

i AGE OF EMBRYOS IDAYSI

FIG. 15. A time line summarizing impel *tant points in the initiation of melanin synthesis.

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DEVELOPMENTAL

BIOLOGY

(i.e., * 6 hr), withdraw from the cell cycle, while the cells that continue to divide do so, but at a much slower rate (i.e., 24 hr). Very shortly before withdrawal occurs, the cells first begin synthesizing melanin. It is not know at present whether all cells begin manufacture at the same time (this would have to be determined at the electron microscopic level). However, the consistent slow and steady rise in the rate and the fact that this follows the steady increase in the number of cells in the epithelium are suggestive of the idea that all cells do not initiate melanogenesis at the same time. The rate of initial melanin synthesis is relatively small but increases steadily during the next 24 hr (probably as the number of cells has been increasing). After this, the level of melanogenic activity rises very rapidly (Fig. 5). This increase does not appear to be due to an increased number of cells present, as most cells have dropped out of the cell M x--x

VOLUME 44, 1975

cycle already (see also Coulombre, 1955). Rather, the increase is probably due to an actual change in the rate of synthesis in the pigment cells already present. BUdR and FUdR have been used to examine the mechanisms behind this rate of change. In addition, BUdR has been used to examine the question of when cells decide to make any melanin. The results, presented above, show the effect of the continued presence of BUdR. They are summarized in Figs. 16-18, composites of the graphs (Figs. 8-10) presented in Results. Beginning at 2 days (Fig. 16B), it is clear that over the next 48 hr some melanogenic activity commences in the presence of BUdR. Treatment begins fully 24 hr before any pre-melanosomes are noticed (Zimmerman et al., 1974). It is probable, then, that for this to occur the decision associated with the making of melanin must be occurring before 2 days in ouo in some cells and that, after this decision, cells may incorporate BUdR into their

CONTROL BUdR 2’12 DAY ADDITION M x---x

CONTRDL BUdR

:

392 DAY ADDITION

DAY ADDITION

M X---X

0

CONTROL BUdR

2 DAY ADDITION 100 50 B”

0

NX

2

3

4

AGE

OF EMBRYOS (DAYS)

FIG. 16

2

5 AGE

OF

EMBRYOS (DAYS) FIG. 17

3

4

5

6

AGE OF EMBRYOS (DAYS) FIG. 18

FIGS. 16-18. Composites of the data from 24, 36 and 48 hr of BUdR treatment. The change in the rate of melanin synthesis versus the normal change in rate is presented when BUdR (BU with arrow) is added at 2 days (16B), 2.5 days (17), 3 days (16A) and 3.5 days (18) and the eyes are assayed over the next 48 hr.

JOEL ZIMMERMAN

Initiation

DNA without affecting the initiation of low levels of melanin synthesis. These data give one of the few pieces of evidence, in vertebrates, that somatic cells are finally committed to a given end-product well before that end is realized and that the final step in determination of a line may be made many divisions before the differentiation of the cells (Hadorn, 1965; Gehring, 1968). Another region of non-effect occurs after 4 days in ovo, when the addition of BUdR fails to halt the high levels of melanin synthesis within the following 48 hr, even though more than 58% of the cells must incorporate BUdR (see also last column, Table 3). The data give additional credence to the suggestion (Holtzer, 1968) that for any given end-product there are at least two kinds of divisions, ones that affect its production and are involved in decision making, i.e., BUdR-sensitive, and ones that have no affect on the product syntheeven if the sis, i.e., BUdR-insensitive, product is not yet being made. A similar noninterference has been observed in the red blood cell system after hemoglobin synthesis is initiated (Weintraub et al., 1972). It is also clear that these divisions, in which BUdR is incorporated without effect, are examples of proliferative mitotic divisions, i.e., those divisions which act to increase the number of cells without changing the type of cells (Bischoff and Holtzer, 1969). This is opposed to decision-making divisions, here defined as those divisions that both increase the number of cells and change the cell type, e.g., presumptive myoblast to myoblast. However, one must realize that they are only proliferative divisions for the product being measured. In other words, one measures cell type using a specific product and thus judges whether a change has occurred. If, however, one used a different product, it might be found that a different set of proliferative and decision-making divisions existed. Thus, the proliferative divisions of one product (e.g., melanin) might, in fact,

of Melanogenesis

115

in some cells be the decision-making divisions of another product (e.g., that which causes withdrawal from the cell cycle). Looking at the curve of activity when BUdR is added at 2.5 days (Fig. 17), it is clear that initial activity levels, i.e., up to 4 days, are within the range of the control values. Only when comparing 4.5-day values does it seem that BUdR has acted to prevent the rise in the rate of synthesis that normally occurs and has instead allowed the cells to increase their activity only at the initial rate, as judged by the slope of the curve of BUdR-treated cells. (A similar delay in maturation as an effect of BUdR has been observed by Coleman et al., 1970). The addition of BUdR at 3 or 3.5 days (Figs. 16A and 18) suggests that what is occurring is a 12-hr delay in the increased rate of melanogenesis. Instead of increasing at 4.5 days, it now increases at 5 days. This delay appears to be linked to a delay in the withdrawal of the cells from the division cycle, so when this withdrawal occurs, melanogenic activity rises. It is possible that this prevention-ofwithdrawal mechanism is present in other BUdR-treated systems, without the delaying effect. In skeletal myogenesis, for example, BUdR prevents fusion and the synthesis of myofibrillar proteins, but that BUdR may do this simply by keeping the cells in the cell cycle, i.e., by acting as a growth factor (Coleman et al., 1970), rather than by disturbing the “myogenic program”, has been proposed (Holtzer et al., 1973) and deserves a reexamination in light of the data presented here. Zimmerman et al. (1974) have shown in cultured somite cells that BUdR affects the “melanogenic program”, as have Wrathall et al. (1973) for cultured melanoma cells. But it is obvious that BUdR does not, at any time examined, affect the “melanogenie program” of the pigment epithelium in ovo. This difference may be due to two factors. One is that those other examples may be of continually deciding systems,

116

DEVELOPMENTALBIOLOGY

and, two, that BUdR is never added early enough in ouo to affect the program. Here BUdR seems to act only on the withdrawal mechanism to which this program appears to be tied in the eye. Yet, in acting so, it must affect a whole group of regulatory (rate control) and structural (premelanosomal proteins) genes without in any way disturbing them. Regulatory genes are affected, as there obviously is a delay in the change of the rate of synthesis, but they are not disturbed, i.e., altered, as when the change finally occurs the rate seems to appear to approach that of the control. Similarly, the structural genes involved do not seem to be impaired as judged by their products’ ability to incorporate [l%]TU. (Further evidence indicating the continued production of premelanosomal proteins in the retinal pigment epithelium in the presence of BUdR may be seen in the electron micrographs presented in Zimmerman et al., 1974.) This non-altering action could account for the recovery of cells that are released from BUdR suppression (Stockdale et al., 1964; Coleman et al., 1970; Koyama and Ono, 1971; Mayne et al., 1971; Zimmerman et al., 1974). As discussed in the introduction, cellular activity may be viewed in terms of luxury molecule synthesis. Prior to this we have seen evidence of a single decision class (red blood cells) and a continuing decision class (chondrocytes in vitro). Here we have evidence for a new class, a somewhat affected class, in which BUdR acts but does not stop the decision from being carried out. The above data suggest that the actual decision to tie increased melanin synthesis to cellular withdrawal occurs earlier than 3.5 days in ouo. Further work using FUdR to mimic cell withdrawal resulted in increased levels of melanogenic activity as early as 2.5 days. The resolution of the assay system is not sufficient when FUdR is added at 2 days to determine whether or not such an increase occurs when FUdR is added then. What seems clear is that

VOLUME44, 1975

withdrawal from the cell cycle acts as an intermediary, or mediates, the production of large amounts of melanin and that the decision-making division linking these two actions occurs more than 24 hr before withdrawal, while the cells are still rapidly dividing. (This conclusion is, of course, influenced by the assumption that cell death is not a factor involved.) This is not to say melanin is not synthesized when cells divide, but rather that the increased levels of melanin synthesis that occur are not brought about unless the cells withdraw (Ebeling, 1924; Doljanski, 1930). If BUdR keeps the cells in the cell cycle, it will not disturb their “melanogenic program” but it will prevent an increase until the cells withdraw; while FUdR may be used to cause the cells to withdraw early and so increase their rate of synthesis. Further work in determining more precisely when the two decision-making divisions occur, and if they occur together, is being carried out. Also, work is being done to determine the cause of the varying effect of BUdR, i.e., no effect (2-3 days) and a delaying effect (3.5-4.5 days). It is possible that different amounts of BUdR are incorporated at the different times, or that the protein-DNA bindings are altered, or unaltered when they should change at withdrawal, by the presence of BUdR in the DNA. APPENDIX

That part of the initial population which is unarrested by colcimid (1 ~ metaphase arrest index) is equivalent in number to that part of the final population which would be unlabeled in the presence of thymidine (1 - labeling index). Therefore, let 100 = original population, and let x = new population at the end of the generating period. Then the correction factor F = xl 100 or (100 + y/100), where y = the number of cells generated; and (1 - metaphase arrest index) 100 = (1 - labeling index) (x) = the total number of cells unchanged

JOEL ZIMMERMAN

Initiation

during the generation time. Then (1 - metaphase arrest index)/1 labeling index) = x/100 = F. This work was done in partial fulfillment of the degree of Doctor of Philosophy from the University of Pennsylvania. The work was supported by a grant from the USPHS (No. HD-00030) to Dr. H. Holtzer. REFERENCES ABBOT, J., and HOLTZER, H. (1968). The loss of phenotypic traits by differentiated cells. V. The effect of 5-bromodeoxyuridine on cloned chondrocytes. Proc. Not. Acad. Sci. USA 59,1144-11X. AFJBO~, J., MAYNE, R., and HOLTZER, H. (1972). Inhibition of cartilage development in organ cultures of chick somites by the thymidine analog, 5-bromodeoxyuridine. Deuelop. Biol. 28, 430-442. BISCHOFF,R., and HOLTZER, H. (1969). Mitosis and the process of differentiation of myogenic cells in uitro. J. Cell Biol. 41, 188-200. CAMPBELL, G. LEM., WEINTRALTB,H., MAYALL, B. H., and HOLTZER, H. (1971). Primitive erythropoiesis in early chick embryogenesis. II. Correlation between hemoglobin synthesis and the mitotic history. J. Cell Biol. 50, 669-681. CLARK, S. L. (1971). The effects of cortisol and BUdR on cellular differentiation in the small intestine in suckling rats. Amer J. Anat. 132, 319-338. COLEMAN, A. W., COLEMAN, J. R., KENKEL, D., and WERNER, I. (1970). The reversible control of animal cell differentiation by the thymidine analog, 5-bromodeoxyuridine. hp. Cell Res. 59, 319-328. COLJLOMBRE,A. J. (1955). Correlations of structural and biochemical changes in the developing retina of the chick. Amer. J. Anat. 96, 153-190. DOLJANSKI, L. (1930). Sur le rapport entre la prolifkration et l’activitk pigmentoptine dans les cultures d’Bpith8lium de I’iris. C. R. Sot. Biol. 105,343-345. EBELING, A. H. (1924). Cultures pures d’bpithblium proliferant in vitro depuis dix-huit mois. C. R. Sot. Biol. 90, 562-564. GEHRING, W. (1968). The stability of the determined state in cultures of imaginal disks in drosophilia. In “The Stability of the Differentiated State,” Vol. 1, (H. Urspring, ed.), Springer-Verlag, New York. HADORN, E. (1965). Problems of determination and transdetermination. Brookhaven Symp. Biol. 18, 148-161. HAMILTON, H. (1952). “Lillie’s Development of the Chick.” Halt, Rinehart and Winston, New York. HARRISON, J. R. (1951). In uitro analysis of differentiation of retinal pigment in the developing chick embryo. J. Exp. Zool. 118, 209-241. HOLTZER, H. (1968). Induction of chondrogenesis, a concept in quest of mechanisms. In “Epithelial

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Mesenchymal Interactions” (R. Billingham, ed.), Williams and Wilkins, Baltimore. HOLTZER, H., and ABBOT, J. (1968). Oscillations of the chondrogenic phenotype in uitro. In “The Stability of the Differentiated State” (H. Urspring, ed.), Springer-Verlag, New York. HOLTZER, H., SANGER, J. W., ISHIKAWA, H., and STRAHS, K. (1973). Selected topics in skeletal myogenesis. Cold Spring Harbor Symp. Quant. Biol. 37, 549-566. HUMASON, G. L. (1967). “Animal Tissue Techniques.” W. H. Freeman, San Francisco. JIROUSEK, L. (1968). The reaction of thiouracil with P-lactoglobulin sulfenyl iodide. Biochim. Biophys. Acta 170, 152-159. KOYAMA, H., and ONO, T. (1971). Effect of 5-bromodeoxyuridine on hyaluronic acid synthesis of a clonal hybrid line of mouse and Chinese hamster in culture. J. Cell Physiol. 78, 265-272. LERNER, A. B., FITZPATRICK, T. B., CALKINS, E., and SUMMERSON,W. H. (1950). Mammalian tyrosinase: the relationship of copper to enzymatic activity. J. Biol. Chem. 187, 793-802. MAYNE, R., SANGER, J. W., and HOLTZER, H. (1971). Inhibition of mucopolysaccharide synthesis by 5-bromodeoxyuridine in cultured chick amnion cells. Deuelop. Biol. 25, 547-567. OKAZAKI, K., and HOLTZER, H. (1965). An analysis of myogenesis in vitro using fluorescein-labelled antimyosin. J. Histochem. Cytochem. 13, 726. SCHNEIDER, W. C. (1957). Determination of nucleic acids in tissues by pentose analysis. Methods Enzymol. 3, 680-684. SCHULTE-HOLTHAUSEN, H., CHACKO, S., DAVIDSON, E. A., and HOLTZER, H. (1969). The effect of 5-bromodeoxyuridine on expression of cultured chondrocytes grown in vitro. Proc. Nat. Acad. Sci. USA 63, 864-870. SILAGI, S., and BRUCE, S. (1970). Suppression of malignancy and differentiation in melanotic melanoma cells. Proc. Nat. Acad. Sci. U.S.A. 66, 72-78. STOCKDALE. F., OKAZAKI, K., NAMEROFF, M., and HOLTZER, H. (1964). 5-Bromodeoxyuridine; effect on myogenesis in uitro. Science 146, 533-535. TURKINGTON, R. W., MAJUMDER, G. C., and RIDDLE, M. (1971). Inhibition of mammary gland differentiation in vitro by 5-bromo-2’.deoxyuridine. J. Biol. Chem. 246, 1814-1819. WEINTRAUB, H., CAMPBELL, G. LEM., and HOLTZER, H. (1971). Primitive erythropoiesis in early chick embryogenesis. I. Cell cycle kinetics and the control of cell division. J. Cell Biol. 50, 652-668. WEINTRAUB, H., and HOLTZER, H. (1971). Fine control of DNA synthesis in developing chick red blood cells. J. Mol. Biol. 66, 13-35. WEINTRAUB, H., CAMPBELL, G. LEM., and HOLTZER, H. (1972). Identification of a developmental program using bromodeoxyuridine. J. Mol. Biol. 70,337-350.

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WESSELS,N. K. (1964). DNA synthesis, mitosis and differentiation in pancreatic acinar cells in uitra. J. Cell Biol. 20, 415-433. WHITTAKER, J. R. (1966). An analysis of melanogenesis in differentiating pigment cells of ascidian embryos. Deuelop. Biol. 14, l-39. WHIT~AKER, J. R. (1971). Biosynthesis of a thiouracil pheomelanin in embryonic pigment cells exposed to thiouracil. J. Biol. Chem. 246, 6217-6226. WILT, F., and ANDERSON, M. (1972). The action of 5-bromodeoxyuridine on differentiation. Deuelop.

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Biol. 38, 443-447. WRATHALL, J. P., OLIVER,C., SILAGI,S., and ESSNER, E. (1973). Suppression of pigmentation in mouse melanoma cells by 5.bromodeoxyuridine. Effects on tyrosinase activity and melanosome formation. J. Cell Biol. 57, 406-423. ZIMMERMAN, J., BRUMBALJGH, J., BIEHL, J., and HOLTZEX, H. (1974). The effect of 5-bromodeoxyuridine on the differentiation of chick embryo pigment cells. Exp. Cell Res. 83, 159-165.