5-Bromodeoxyuridine does not affect development of the sea urchin, Arbacia punctulata

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Experimental

5-BROMODEOXYURIDINE

Cell Research 114 (1978) 85-93

DOES NOT AFFECT

OF THE SEA URCHIN,

ARBACIA

DEVELOPMENT

PUNCTULATA

IRENE M. EVANS’ and PAUL R. GROSS Department

of Biology,

University

of Rochester,

Rochester,

NY 14627, USA

SUMMARY Development of Arbacia punctulata embryos in the presence of bromodeoxyuridine (BUdR) was investigated under conditions which allowed about 20% substitution of BUdR for thymidine residues in DNA. Development was essentially normal from fertilization to the feeding larva. There appeared to be no restricted stretches of DNA which failed to incorporate BUdR. Even very early stages were permeable to BUdR, which entered all the nuclei. The failure of BUdR substitution to block development in Arbacia is discussed in the light of evidence that this analogue blocks the onset of differentiative product synthesis in many cellular systems.

In many systems incorporation of 5’-bromodeoxyuridine (BUdR) into DNA inhibits cellular differentiation without substantially lowering cell viability, proliferation, or macromolecular synthesis. The wide range of cellular systems responsive to BUdR has been reviewed by Rutter et al. [ 11.Included are erythropoiesis [24], myogenesis by chick muscle cells [5, 61, melanin synthesis by melanocytes [7], chondroitin sulfate production by chondrocytes [8], and zymogen synthesis by rat pancreatic cells [9]. These effects all require ongoing DNA synthesis. In the absence of DNA synthesis, BUdR causes neuroblastoma cells to form dendritic processes [IO]. The analogue also induces latent cellular virus particles (review in [ 111)and alters the surface properties of many cultured cells. BUdR is toxic for some mammalian systems, in which it inhibits and then blocks DNA synthesis and cell division [12-151. The effects of BUdR on intact embryos have been studied less thoroughly. Developmental arrest has been observed after

BUdR incorporation in sand dollar embryos [16, 171, in Xenopus embryos [18, 19, 211, in embryos of the sea urchins Strongylocentrotus purpuratus and Paracentrotus lividus [20, 211, and in mouse embryos [22]. Early inhibition of development in the first two of these systems seems to be associated with abnormalities of mitosis and subsequent inhibition of cell division. In contrast to many proliferating cellular systems studied in culture, embryos often exhibit difficulties of cleavage in the presence of the drug. However, in other embryo systems, such as the sea urchin and mouse, BUdR treatment, while causing some cleavage delay, does allow divisions to proceed, but causes eventual blockade at later stages. The effects of BUdR on differentiating systems have generally been interpreted in terms of specific suppression of the syn’ To whom reprint requests should be addressed: Department of Pharmacology and Toxicology, University of Rochester Medical Center, Rochester, NY 14642,USA. Exp Cell Res 114 (1978)

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thesis of differentiative gene products, per- [‘“C]TdR (57.2 mCi/mmol) and r3H]BUdR (28.6 Ci/ mmol) were obtained from New England Nuclear Co. haps by affecting transcriptional “switchUnless otherwise noted, all incubations were carried es”, which initiate transcription of the re- out at isotope concentrations of 2 &i/ml ([3H]BUdR quired genes [23]. If such proposals are li- and [3H]TdR) or 0.5 /.&i/ml ([14C]TdR). terally correct, i.e., a BUdR-sensitive step Autoradiography common to all differentiation programs, it Embryos were labeled for I h with 20 &i [3H]BUdR/ should not be possible to find a system ml HSW. After washing twice with sea water, embryos capable of completing a major develop- were fixed in freshly prepared methanol-acetic acid (3 : 1. v/v). dehvdrated in alcohols, treated with mental program, including cell specializa- xylene, and impregnated with paraffin. After sectiontion and synthesis of terminal differentia- ing, deparaffinization, and hydration, slides were dried and coated with Eastman Kodak emulsion type tion proteins, in the presence of BUdR. NTB-2. Slides were developed IO-14 days after coatWe have examined development of Ar- ing and scored for the presence or absence of labeled nuclei. bacia punctulata in the presence of BUdR. Our results are that incorporation of BUdR DNA isolation into the DNA of this species does not block Cultures showing 95% fertilization and proper first were divided and labeled with either [3H]or alter significantly progress to the swim- cleavage TdR, r3H]BUdR, or [Y]TdR plus 100 pg/ml of the ming larva (pluteus) stages. Other species appropriate unlabeled nucleoside. After development plutei the embryos were collected by centrifugaof sea urchins are more or less affected, and to tion and washed twice with HSW, and their DNA specifically so, depending upon the condi- was extracted. Embryos were lysed by the addition of lysis buffer (0.02 M EDTA, 0.15 M NaCI, 0.015 M tions of culture. Na citrate, pH 8) plus 0.5% sodium dodecyl sulfate. MATERIALS

AND METHODS

Embryo culture puncfulara were obtained from Florida Marine Biological Specimen Co. (Panama City, Fla). Strongylocentrotus purpuratus and Lytechinus pictus were obtained from Pacific Biomarine Co. (Los Angeles, Calif.). Shedding of eggs was induced by intercoelomic iniection of 0.55 M KCI. After collection. the eggs were washed three times with 20-40 vol of sea water (Harvey [24]; referred to subsequently as HSW), fertilized, and incubated at 16°C (L. pictus and S. purpuratus) or 18°C(A. punctulata) in the presence of 50 U/ml each of penicillin and streptomycin (Microbiological Associates). Embryos were cultured either aerated in Spinner Flasks or in a shallow, 25 ml layer of HSW in glass culture dishes at 1-5~ lo3 embryos/ml. Cultures showing less than 95% fertilization were discarded, as were all experiments in which controls did not develop to the pluteus stage.

Arbacia

Chemicals

After incubation with pronase (100 pg/ml) and RNAase (100 pg/ml), the lysate was extracted three times with either buffer-saturated phenol or chloroform/ isoamyl alcohol (24 : 1, v/v), extracted once with ether, and precipitated with ethanol kept at -20°C. After isolation, the DNA was dissolved in O.lXSSC (0.15 M NaCI, 0.015 M sodium citrate). Specific activity of the DNA was determined by trichloroacetic acid (TCA) precipitation, collection of the DNA filtrate on GF/A filters (Whatman Co.), and counting in a Nuclear Chicago Mark I scintillation counter.

Cesium chloride density gradient analysis of DNA Gradients were prepared by dissolving 4.36 g of CsCl in 3.36 ml DNA samples. Centrifugation of gradients was for 43-45 h in a Beckman type 50 rotor at 39000 rpm. Fractions were collected either by inserting a tube and pumping the gradient out from the bottom of the tube or by suction from the top, using a Buchler fractionator. Fraction densities were measured with a Bausch & Lomb refractometer, and radioactivity was assayed after 5% TCA precipitation by liquid scintillation counting.

BUdR and thymidine (TdR; both obtained from Sigma) were dissolved in HSW at concentrations of 1-5 mgl ml HSW and diluted appropriately for each experiRESULTS ment. The concentration of drug was checked before and after each experiment by reading the 280/260 Development of BUdR-treated absorbance in a Gilford model L spectrophotometer. Such readings showed that BUdR at 100 pg/ml was Arbacia embryos present in large excess, with over 99% of the original Incubation of developing A. punctulata concentration of BUdR remaining after embryos were bryos in 100 ,ug of BUdR/ml of HSW cultured to plutei. Tritiated thymidine (45 Ci/mmol),

emfrom

Bromodeoxyuridine

Fig. 1. Appearance of BUdR-treated and control em-

bryos in mid-development: prism stage whole mounts. Fertilized eggs were diluted to a concentration of IX lo3 embryos/ml and cultured in the presence of

in Arbacia embryos

87

(A) BUdR (100 &ml) or (B) TdR (100 pg/ml). The prism stages shown had similarly developed germ layers and skeletal systems, and all were actively motile. Bar, 50 I*.

88

Evans and Gross

Table 1. Percent replacement of thymidine by bromodeoxyuridine in Arbacia punctulata DNA Percent substitution (mole %)

0 1,-,-,+<.~I._ 0 lo

I

\ ‘..P: \\ I-’ i \ \- *..> ,.,,.,.!..“..“....“‘...“i” B *--i;:,,,, 40 30 20

50

Figs 2,3. Abscissa: fraction no.; ordinate: (Lefr) cpmx lo+; (right) density CsCl (g/cm3). Fig. 2. Percent replacement of thymidine by BUdR

as determined by CsCl density gradient centrifugation. Arbacia eggs were fertilized and diluted into either 100 pg of BUdR/ml plus 2 &i of [3H]BUdR/ml (---); or 100 pg of TdR/ml plus 0.5 $i of [‘*C]TdR/ml (. . .). After development to plutei, the DNA was extracted and centrifuged to equilibrium (isopycnic CsCl density gradient sedimentation). Gradients were fractionated and the density and radioactivity of fractions determined by refractometry and scintillation counting. Percent substitution determined by examining the specific activity of DNA obtained in this experiment was 16.0.

Expt

BUdR cont. in HSW @g/ml)

Radioactive uptake

Density shift

: 3 4

500 250 100 loo 50

17.1 18.0 18.6 16.4 16.0

20.0 22.5 21.7 20.8 21.5

eggs were fertilized, suspended in 50-500 pg of BUdR/ml plus 2 &i of [3Hj13UdR/ml; or 50-500 pg of TdR/ml plus 0.5 &i of [Y]TdR/ml, and allowed to develop to plutei. The DNA was then extracted, and the percent BUdR substitution was determined as described in the text.

Arbacia

ment. Higher doses of BUdR (250 and 500 pg/ml) were also tried, but they, too, did not alter or block successful development. Percent thymidine

the fertilized egg to pluteus stage caused no significant developmental abnormality (fig. I). Cleavages were normal and showed approximately the same timing in experimental and control animals. Gastrulation of the embryos was unperturbed, as was development of mesenchymal cells, skeleton formation, and further development to the prism and pluteus stages. No gross effect of BUdR on the size of the embryo or on the number of cells per embryo was observed. The drug did cause a tendency for cells to be slightly less adhesive than normal. This is, at least, the impression gained from observation of the epithelioid surfaces at all post-blastula stages. Otherwise there were no cytological alterations evident at the light microscope level. Embryos fertilized in BUdR (100 pg/ml) and incubated in its presence similarly showed normal developExp Cell Res I14 (1978)

replacement

in genome

The extent of BUdR substitution for thymidine was calculated by two methods. The first involved dividing the specific activity

Fig. 3. CsCl density gradient centrifugation of embryo

DNA incubated in BUdR using [3H]TdR as tracer. After fertilization, sea urchin embryos were cultured in 100 pg of BUdR/ml plus tracer [3H]TdR (2 &i/ ml) (- --). After development to plutei, the embryos were collected and washed, and their DNA was extracted. The DNA was then centrifuged to equilibrium in a CsCl density gradient and treated as described in fig. 2. (. . .) [*“C]TdR-DNA marker.

Bromodeoxyuridine in Arbacia embryos

89

Fig. 4. Autoradiography of l&cell embryos incubated in [3HDUdR. After fertilization. embrvos were allowed to develou to the I&cell stage and incubated for 30 min with L3H]BUdR (20 #J/ml). The embryos were then washed, fixed, and prepared for autoradiography, as described. Microscopy at various focal levels revealed that all intact nuclei were radioactive, i.e., had silver grain clusters in the overlying emulsion, which is here in focus. Sections lightly stained with toluidine blue.

Fig. 5. Late gastrula of L.vtechinus &us developing in BUdR. Whole mount. A, Animal pole; V, vegetal pole, with (bl) blastopore. The embryo shows a normal archenteron, blastocoele, primary and secondary mesenchyme, and (.S) skeletal spicules being elaborated by the mesenchyme, concomitant with emerging bilateral symmetry. The ectoderm is ciliated and these embryos were actively motile. Bar, 15p.

of the isolated DNA (dpmlpg) by the molar specific activity of BUdR in HSW (dpm/ Fmol). This quotient was then divided by the molar thymine content of unsubstituted DNA (pm01 TdR/pg DNA) and multiplied by 100 to give mole percent BUdR substitution for TdR. For these calculations, thymidine in sea urchin DNA was taken to be 33 % [25]. The percent substitution of isolated DNA in five experiments is shown in table 1. Using the above method, it was estimated from five such determinations that 16-19% of the thymine residues were regularly replaced by BUdR. The second method for calculating the percent BUdR substitution employed the equation of Rownd [26] for the density of

DNA which has 100% of its thymidine residues replaced by BUdR. The density of fully substituted DNA containing BUdR in both strands of the duplex is: gBU :BU= 1.758 g/cm3+1.134 T, where T represents the mole fraction of thymidine in DNA. The density shift, representing 100% BUdR substitution, was then divided into the density shift calculated using DNA isolated from Arbacia embryos which develop in sea water plus BUdR, to obtain the percent BUdR substitution. The density of such DNA was obtained by centrifuging the DNA in a calibrated CsCl gradient. The increase in DNA density due to BUdR substitution is shown in fig. 2 and table 1. The reported density of unsubstituted duplex Exp Cell Res I14 (1978)

90

Evans and Gross

DNA from Arbacia embryos is 1.695 [27]. Our values are slightly higher (1.698-1.702). Using the equation of Rownd [26], fully substituted DNA would be expected to have a density of 1.802. Development of embryos to plutei in 100 pg of BUdR/ml resulted in a shift of the DNA density from 1.700 to 1.723, corresponding to 21.5 % thymidine DNA replacement (fig. 2). Incubation of embryos in increased doses of BUdR of 250 pg/ml or 500 pg/ml did not alter the degree of thymidine replacement significantly (table 1). Sea urchin embryos are believed to take up nucleosides by a carrier-mediated transport mechanism [28]; the failure of increased DNA substitution with increased dose might, perhaps, be due to saturation of the transport system at the lowest concentration of BUdR tested (50 dml) . The discrepancy of substitution values estimated from radioactivity and density shift of the DNA, respectively, is difficult to explain. Any one of several systematic errors, undetected despite a search for them, might be responsible (e.g., reduced specific activity of the tracer BUdR due to H-T exchange during storage, refractometry errors, etc.). In the circumstances, we can do no better than to report an overall mean value of substitution-19%which is nevertheless well within the range of those levels demonstrated to be effective in producing the characteristic effects of BUdR on differentiating cells in culture. Uniformity

of DNA substitution

It is possible that the resistance ofArbacia development to BUdR substitution in their DNA is due to differential incorporation of BUdR into specific regions of the genome not necessary for larval development. Fig. 2 shows that the DNA isolated from BUdRtreated embryos had a unimodal distribuE.rp Cell RES 114 (1978)

tion on a CsCl gradient, which suggests, but does not prove, that the BUdR was uniformly distributed in the DNA. Since, however, endogenous production of TdR probably continued, and since selection in favor of TdR over BUdR has been shown to occur under certain conditions in other biological systems [3, 291, a second experiment was performed to investigate this point further. Cultures were incubated with 100 pg BUdR/ml plus trace amounts of [3H]TdR. After isolation, the purified DNA was again run on CsCl gradients. The results (fig. 3) show the same shift of DNA density and the same unimodal distribution found when [3H]BUdR was used as tracer. This result implies that for DNA of the molecular weight employed here, the internal distributions of TdR (which is of course uniform) and BUdR do not differ significantly. A more incisive investigation (employing systematic shearing of the DNA) might, of course, reveal inhomogeneities for BUdR and none for TdR, but, again, the substituted DNA studied as described behaves as does brominated DNA from other systems in which the analogue has exerted its full and characteristic effect [3]. Correlation of BUdR incorporation with developmental stage

The failure of BUdR to block development might be understood if for some reason BUdR was not taken up during early development. Gontcharoff & Mazia [20] have shown that S. purpuratus embryos become resistant to BUdR if the analogue is added after the embryos have become blastulae. To test whether early embryos were impermeable to BUdR, 8-16 cell stages were incubated with [3H]BUdR, and autoradiograms of sectioned embryos prepared as de-

Bromodeoxyuridine

scribed in Materials and Methods. The results show that all nuclei are radioactively labeled. There is substantial incorporation of BUdR at this early stage, and, as is evident from the autoradiograms (fig. 4), the incorporation occurs uniformly. This result agrees with earlier findings that the uptake of both thymidine [30] and BUdR [31] increases sharply after fertilization and remains constant during early development of S. purpuratus and P. lividus sea urchins. The pattern of labeling also indicates that BUdR labels nuclear DNA very specifically and that the tracer is not recycled significantly for use in other pathways. Development of S. purpuratus and L. pictus embryos in BUdR

Mazia & Gontcharoff [ 171reported that cultures of S. purpuratus sea urchins do not develop to plutei when BUdR is added to the incubating sea water. Their report suggests that the BUdR effect involved chromosomal bridging and subsequent abnormalities of cleavage. We repeated these experiments, and our results for S. purpuratus are in agreement with theirs. BUdR induced delays of cleavage as early as the 16-cell stage. Incubation of L. pictus embryos in BUdR did not cause delay of cleavage, but developmental abnormalities appeared in postgastrula stages, and few embryos became regular prism larvae. The timing of arrest in this species seemed to depend on culture conditions: if embryos were crowded (e.g., cultured at >104 embryos/ml), arrest at the late blastula stage was routine; but at low embryo densities (5x lo* to 1x 103/ml), development generally proceeded to the late gastrula or early prism stages. Fig. 5 is a photograph of an intact L. pictus gastrula developing in BUdR. In the case of L. pictus, therefore, BUdR incor-

in Arbacia embryos

91

poration seems to institute a form of stress, rather than a discrete developmental block. DISCUSSION The failure of Arbacia embryos to be affected by -20% substitution of BUdR for TdR in their DNA is puzzling, since BUdR does appear to block with some specificity the onset of “differentiative” product synthesis in a variety of cellular systems. Furthermore, it has been demonstrated by others, and we have verified, that BUdR alters development in two other species of sea urchin, S. purpuratus and L. pictus [17]. Also, Tencer & Brachet [21] found that development of Paracentrotus lividus is inhibited by BUdR at a substitution ratio of 13.7-37 % BUdR/TdR (percent substitution calculated from ref. [21] p. 60 graphs 1 and 2) which is within the broad range of our own estimates. BUdR blocks a variety of differentiation programs, such as erythropoiesis, melanogenesis, and myogenesis. Postgastrula sea urchin embryos produce differentiated cells and functional tissues-e.g., esophagus, stomach, stomo- and proctodeum, epidermis, skeleton, and pigment cells-and BUdR incorporation might have been expected to affect these processes. In two species of sea urchins developmental abnormalities are observed; in S. purpuratus these anomalies seem to result from cleavage difficulties associated with persistent chromosomal bridging, while in L. pictus they may be due in part to metabolic stress in overcrowded conditions, to which that species seems particularly sensitive. Since BUdR blocks differentiation in many systems via a mechanism that absolutely requires its incorporation into the DNA, it is widely believed that a specialized and particularly sensitive DNA target

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is responsible for controlling transcription (or post-transcriptional stabilization) of certain kinds of mRNAs that encode terminal differentiation proteins. Such specialized DNA sequences might control differentiation via specific coding sequences and associated regulatory regions, unequally active in the elaboration of differentiative gene products. Several mechanisms have been proposed by which BUdR might affect these regions. It might interfere directly with the transcription of differentiation sequences that are already present [32-341, or it might alter the synthesis of such DNA regions so that they are underrepresented in precursors to the terminally differentiated cell [35, 361. Both mechanisms have been assumed to act via a universal “differentiation switch’. The problem is thus to explain why sea urchin embryos-Arbacia in particular-do not respond in the characteristic manner to substitution by BUdR. None of the above proposals for the mechanism of BUdR action explains how it can fail to block development in Arbacia embryos. Terminal differentiative programs (e.g., gut, epidermis, skeleton, pigment, and digestive organs) must surely be activated for development to the feeding larva. One is left with the arguments either that Arbacia development differs in some fundamental mechanism from other systems, or that the effects of BUdR are less specific, and more complex, than present hypotheses suggest. Several alternatives may be proposed to deal with this problem. Since, for example, no protein associated with terminal differentiation of larval cells is known with certainty to be obligately synthesized in the post-gastrula embryo, it might be argued that terminal differentiation, as it is usually defined, does not occur in sea urchin development at all. If that were correct, the Exp Cell Res 114 11978)

definition in common use would have to be modified to a much more restrictive one: it is difficult to believe, but is of course not impossible, that an array of morphologically specialized tissues that function in motility, support, and alimentation, and most of which are literally terminal (in the sense that they will die at metamorphosis), can have attained that state without significant synthesis of specialized products. Alternatively, there might well be a distinct and limited target for BUdR in DNA, functioning solely in the regulation of “luxury” protein synthesis, but the physiology of that target might depend upon the affinity of particular chromosomal proteins for it. The Arbacia case would then be one of a species-specific peculiarity of regulatory proteins such that, in this case (alone to date), the presence of the brominated thymidine analogue in DNA has no effect upon their function. This is a real possibility, and perhaps the most likely one. We are bound, nevertheless, to consider it with caution, since development of other sea urchins in BUdR does not mimic particularly well the behavior of prototype cell culture systems undergoing terminal differentiation, and since there is a notable paucity of comparable studies on other embryos of the same general kind, i.e., those which (like most animal embryos) complete their development without an increase in mass at the expense of a nutrient medium or a very large supply of yolk. Current thinking about the remarkable BUdR effect depends, in any case, upon the implicit assumption of selective transcription of the DNA as the elementary fact of differential gene expression, and certainly the indirect evidence favoring such an assumption is overwhelming [37]. Nevertheless, a rigorous proof that differential transcription of DNA, rather than differ-

Bromodeoxyuridine

ential activity at some proximal post-transcriptional level, occurs in cellular differentiation, has not been obtained. In the case of sea urchins, at least, recent investigations [38] of sequence content in immediate transcription products appear to be inconsistent with selective transcription, at least of sequences encoding “complex class” RNA. Whether shifts from the complex class to “prevalence” are really transcriptional (e.g., [39]) remains in doubt. Our conclusion must be that while the explanation for the relatively normal and complete development of Arbacia embryos with heavily brominated DNA is likely to be peculiar to the species, and not inconsistent with the presence in DNA of BUdR targets concerned with terminal differentiation, that may not after all be so. Further and more detailed studies on macromolecule synthesis in whole embryos exposed to the analogue should certainly be done. This investigation was supported in part by USNCI Grant Number CA-1 1198and by NICHD Grant Number HD-08652-02,03.

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