Changes in DNA and RNA during embryonic urodele development

Experimental

Cell Research

CHANGES

21,

523-534

523

(1.960)

IN DNA AND RNA DURING URODELE DEVELOPMENT’

EMBRYONIC

P. S. CHEN Institute

of

Zoology

and Comparative Received

Anatomy, December

University

of

Ziirich,

Switzerland

10, 1959

STUDIESon

the nucleic acids during embryonic development demonstrated that there is a cytoplasmic reserve of desoxyribonucleic acid (DXa) in the egg of sea urchin [22, 33, 41, 58, 651, nematode [45], insect [al, 461, frog [lo, 34, 571 and chicken [33, 53, 541. This reserve is used for the embryonic nuclei in the early developmental stage. However, the amount of DNA in the egg cytoplasm differs in various animals. In sea urchin the DNA store within the cytoplasm is very small, perhaps enough only for the first few cell divisions [60]. The frog egg, on the other hand, contains about 5000-9500 times as much DNA as the sperm or the liver nucleus [34, 571. It seems that the time of starting DNA synthesis depends on the quantity of the cytoplasmic reserve. For instance, in sea urchin DNA increases already in the early cleavage stage :l, 601, whereas in amphibia active DNA synthesis begins only in the late blastula or early gastrula stage [25, 341. Rut Agrell and Persson [l] pointed out, based upon their study on different sea urchin species, that it is not the amount of DNA reserve which determines the beginning of synthesis, but the developmental stage. It seems that the deciding factor for initiating the ne\v DNA synthesis has not yet been established. dccording to data given by Kutsky [36] and Sze [57] for Rantr pipiens, there is a continuous increase of DNA from the beginning of development in spite of the large cytoplasmic reserve in the egg. In contrast to this result, Gregg and Lovtrup [29], who worked on the same species, reported that total DS.4 increases only at the end of segmentation. In Rnna platyrrhenn and Rancr firsca this increase occurs still later, at about the beginning of gastrulaLion [34, 551. Thus, the stage at which DNA synthesis appears is still controversa1 and varies perhaps in different species. Information concerning the behaviour of ribonucleic acid (RNA) is like\\-ise contradictory. Rrachet :7] observed at first a steady increase of penlose from fertilization to early larvae in Rnna fuscn and the axolotl. How1 This investigation was aided by a grant to Prof. E. Hadorn and Prof. H. K. Mitchell

from the “Karl Hescheler Stiftung”. Thanks for a critical reading of the manuscript. Experimental

Cell

are due

Rrseareh

21

I’. S. Chen ever, according to Steinert [55], in R. fusca and R. esculenta R?JA does not increase until the neurula stage or even still slightly later. Rodenstein and Kondritzer [5] observed also a gradual increase of RNA in iimblystomcr punctntum, but their analysis started only from the Harrison stage 28. Working on the same species, Krugelis et rtl. [35] found that the RI\‘A synthesis initiates at the gastrula stage. Results from different studies on amphibians do not only vary, but are also in sharp contrast to those found for the sea urchin development. According to all data available, the RXA content of the sea urchin embryo remains constant until as late as the pluteus stage [17, 23, 49, 591. In view of the conflicting results and the large differences found for various animals, it appears that a comparative RNA analysis in the early development of different species under comparable conditions is desirable. Most biochemical data given in the literature are based upon results from bulk analyses of homogenates of a large number of eggs. It is known that the amphibian eggs from different females within the same species differ greatly in their nucleic acid contents. Even eggs laid by the same female sho\v different developmental rates and thus vary in their RNA and DS.2 contents. Such a variation makes the analysis of the assay data very difficult. ‘I’herefore, a microchemical method was devised for determining both nucleic acids in microgram quantities in the same sample. By using this method only a single egg is needed for each determination, and its morphogenetic state can be carefully controlled before being subjected to the estimation procedure. The present paper deals with a description of the analysis steps together with the assay data obtained for three European urodeles during their early embryonic development.

EXPERIMENTAL

MATERIAL

AND

CHEMICAL

METHOD

The three urodeles studied were Trifon alpestris, Triton palmatus and Triton cristatus. Two days before the collection of eggs the female was injected with gonadotropic hormone (200 I.U., Ciba, Basel). For each series only eggs from the same female and laid at about the same time were used. After collection, the eggs were kept in tap water at 18°C until the desired stage was reached. The first two cleavages were controlled in order to get normally fertilized eggs and to estimate the time of fertilization. Unfertilized eggs were obtained by opening the injected female shortly after the beginning of the egg-laying process. During sampling the developmental stage of the embryos was examined according to the table of development originally prepared by Harrison for Amblystoma punctaturn (cited in Lehmann [37]). The embryos were dissected out of the jelly immediExperimental

Cell Research 21

DNA

and RNA

in urodele development

ately before being subjected to the analysis procedure. For stages younger than the late blastula, the vitelline membrane was not removed in order to avoid the loss of material by operation. The investigation included stages ranging from the early cleavage to the late neurula. Unfertilized eggs of T. alpesfris were also analyzed. Chemical method.-Recently Scott, Fraccastoro and Taft [52] described a method for the analysis of nucleic acids in microgram amounts of tissue sections. This method has been successfully applied for determining both RNA and DNA in the sea urchin hybrids [2 a, 171. However, when the same method was used for the urodele embryos, no satisfactory RNA extraction could be obtained. Perhaps degradation products of the yolk proteins interfere with the measurement of the ultraviolet (UV) absorption. The method of differential extraction of RNA and DNA with perchloric acid (PCA), first suggested by Ogur and Rosen 1471, has been shown to be adequate for assaying nucleic acids in the amphibian egg [15, 55, 641. The RNA separation was therefore effected by using PCA instead of the alkaline hydrolysis as described by Scott, Fraccastoro and Taft [52]. The analysis steps are as follows. All micropipettes and glass reaction tubes used were coated with silicone (General Electrical Dri-Film SC-87). The egg or embryo taken out from the jelly membrane was put into a small reaction glass tube (ca 300 ~1 in volume), and fixed in 90 ~1 of ice-cold X0 per cent ethanol for at least two hours. The acid-soluble compounds were removed by treating the egg with 55 ~1 of cold 0.3 N PCA for one hour (at 2’C). The fat-soluble substances were extracted with three changes of 90 ,LL~ice-cold ethanol-ether mixture (3 volumes 96 per cent ethanol + 1 volume ethyl-ether). During extraction the reaction tube was kept at 2°C for about 12 hours. After removing the ethanol-ether mixture, the egg was carefully crushed under a binocular with a glass rod, and 50 ,LL~of 0.8 N PCA added for the extraction of RNA. The content was thoroughly mixed by stirring, and the reaction tube allowed to stand exactly 3; hours at 25% Following centrifugation at 4000 rpm for one minute, the supernatant was removed into a 100 ~1 pipette. The residue was washed once more with 24 /II of 0.8 N PCA, again centrifuged, and the supernatant collected into the same 100 ~1 pipette. After filling the pipette to volume with 0.8 N PCA, the RNA concentration in the collected sample was estimated by reading the UV absorption in a microcuvette. For the extraction of DNA, 50 ~1 of 1.6 N PCA was added to the sediment in the reaction tube. It was then mixed thoroughly by stirring, and the tube heated at 60” for exactly 15 minutes, cooled immediately in ice bath, and again centrifuged. The supernatant was drawn into a 100 ,LL~pipette. Following washing the sediment with 26 ,LL~of 1.6 N PCA and centrifugation, the supernatant was also collected in the same 100 ,LJ pipette, the pipette filled to volume with 1.6 N PCA, and the content transferred to a microcuvette for estimation of U\’ absorption. For alpestris and crislatu.s eggs the final sample had to be diluted 1:l with the extracting reagents. Zeller [64] performed the RNA extraction by treating the amphibian egg in foto with 5 per cent PCA for 20 hours at room temperature. Since in the present study the embryos had been first thoroughly crushed in the reaction tube, the time of extraction could be shortened. Preliminary test showed that RNA extraction was complete at the end of 3 & hours (Fig. 1 a). For the same reason the time of 15 minutes was chosen for the extraction of DNA (Fig. lb). Experimental

Cell I
21

P. S. Chen For measuring the UV absorption a microattachment (type P. Siitterlin, Zurich) designed for use with the Beckman Model DU spectrophotometer was employed. The quartz microcuvettes had a width of 3 mm and a path length of 10 mm. PCA (0.8 N) served as blank for estimating the RNA fraction, and 1.6 N PCA for estimating the DNA fraction. For each measurement the complete absorption spectrum from 220 to 300 mp was read. The difference between maximum absorbance at 260 rnp

1 a.

1 b.

Fig. l.-The rate of extraction of the nucleic acids from a single Triton pulmafus gastrula (H.ll). (a) Extraction of RNA by 0.8 N PCA at 25°C; (b) Extraction of DNA by 1.6 N PCA at 60°C. Ordinate: Difference between absorbance at 260 mp and that at 240 m,u. Abscissa: Time after the beginning of extraction.

and minimum absorbance at 230 or 240 m/l was used to calculate the nucleic acid concentration (see [64]). In all determinations a distinct minimum absorbance was observed, indicating that there was no serious contamination of interfering substances at 23Oj240 rnp (see absorption curves in Fig. 2). Furthermore, estimations of standards showed a linear relation between the nucleic acid concentration and the difference of maximum and minimum absorbance values at the wavelengths mentioned. As standards, ribonucleic acid from yeast and desoxyribonucleic acid from herring sperm (Nutritional Chemicals) were used. The substance was dissolved in water at 60°C until the solution became water clear. For RNA 0.2 ml 1 N NaOH had to be added. The RNA solution was diluted to a final concentration of 0.03 per cent, and the DNA solution 0.02 per cent. Adequate aliquots of standards (corresponding to 1-6 pg of nucleic acids) were pipetted into the reaction tubes and subjected to the same procedure as described above for the unknowns, beginning with the acid hydrolysis at 25°C and 60°C for RNA and DNA respectively. Standards have been also prepared by dissolving the substance in 10 per cent PCA at 90°C for 30 minutes. Adequate aliquots of these standards were transferred to microcuvettes and their UV absorption was read directly. Experimental

Cell

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DNA

and RNA

527

in urodele development

As the extraction of nucleic acids with PCA has been shown to be satisfactory for amphibian eggs by previous investigations [15, 55, 641 and the analysis procedure employed in this study was largely the same as that described by Scott, Fraccastoro and Taft (521, no control experiments by using other methods were carried out. Sze [57] reported that the PCA extraction of DNA gave too high values for R. pipiens. However, there are several facts which indicate that the present assay data are valid.

Fig. 2.

Fig. 3.

Fig. 2.-The IJV- absorption spectrum of RN.4 and DiX\iX extracts from a single Y’rifon palmalus embryo at the 4-cell stage (two samples). Fig. 3:-Changes in DiXL4 content during the early urodele development. fertilization at 18°C. 0, T. dpestris; 0, T. pdmatus; x , T. cristatus.

Abscissa:

hours after

The samples were always controlled by reading the whole absorption spectrum and found to be typical for nucleic acids. The overall 260/280 ratio of density is 1.54 and thus indicates an adequate separation of the nucleic acids from the proteins. The RNA and DNA values estimated in this study are in reasonable agreement with those found for other amphibian eggs (see 531). Furthermore, a test made by measuring the RNA contents of two embryos at the 4-cell stage from the same female gave very close absorption values (Fig. 2). The small difference between separate determinations is apparently within the range of biological variation. As a basis for comparison, both fresh and dry weights as well as the total nitrogen (N) of embryos at various developmental stages were determined. Total N was estimated according to the ultramicro-Kjeldahl method described by Roe11 and Shen [4]. Experimental

Cell Research

21

528

P. S. Chen RESULTS

From the curves presented in Figs. 3 and increase in DNA and RNA is the same for of both nucleic acids remains constant from stage. Shortly before gastrulation begins,

4 it can be seen that the pattern of all three urodeles. The contents fertilization until the late blastula new synthesis appears. During

Fig. 4.-Changes in RNA contenl during the early urodele development. Abscissa: hours after fertilization at 18°C. x , T. cristatus; 0, T. alpestris; l , T. palmatus (-- 1. batch; - - - 2. batch).

gastrulation and neurulation there is a rapid increase in total amounts of DNA, and a slower increase in that of RNA. However, the relative increase varies in the three species. From 20 to 80 hours the increase in DNA is 3.1 times for T. alpestris, 3.5 times for T. palmatus, and 6.70 times for T. cristatus. The RNA values are more similar. For the developmental period mentioned, the relative increase in RNA is 1.5 fold for T. alpestris, 1.9 fold for T. palmatus and 1.3 fold for T. crisfafus. The nucleic acid determinations on T. cristafus are less extensive, especially for stages prior to the nucleic acid increase. As described above, the assay data show that there is a more rapid increase in DNA than in RNA from the early gastrula to the late neural stage. Consequently, the ratio DNA/RNA varies as development proceeds. For instance, for 7’. nlpestris it is 0.87 before the synthesis sets in, and changes to 1.91 at the end of neurulation. The corresponding values are 0.79 and 1.53 for T. palmafus, and 0.32 and 1.65 for T. crisfatus. Only in the earlier stage of T. crisfatus the DNA/RNA value is lower. Otherwise the quantitative relation between both nucleic acids appears to be similar for all three urodeles at corresponding developmental periods. According to evidence available, the large bulk of RNA and DNA in the early developing egg of amphibian are located in the cytoplasm. In this connection it would be of interest to know how the nucleic acid contents are Experimental

Cell Research

21

DNA

and RNA

529

in urodele development

related to the egg size. Data of both fresh and dry Jveights as dell as total N determined for T. alpestris and T. palmatus are presented in Tables I and II. Taking the biological variation into consideration, the vveight values are in reasonable agreement with those reported for the same species in previous studies [14, 161. Apparently, there are no significant changes in both lveights

TABLE

I. Average fresh and dry weights of Triton pahnatus during embryonic development. Triton

Age in hours at 18°C

Developmental stage

palmatus

n

and Triton

Triton Fresh weight (w per embryo)

Dry

weight (mg per embryo)

Age in hours at 18°C

alpestris

alpesfris

Developmental stage

6

3.51

1.10

Dry weight (mg per embryo)

34

early tH.

gastrula 9)

5

2.15

0.71

33

early (H.

53

late gastrula (H. 12C-13)

4

2.38

0.73

4s

mid-gastrula (H. 12b)

5

3.34

1.04

62

early (H.

5

2.17

0.72

65

early (H.

5

3.68

0.98

n, number

neurula 14)

of determinations;

H, Harrison

stage for Amblystoma

gastrula 9-10)

n

Fresh weight (w per embryo)

neurula 14) pundatum.

and total N at the stages examined. The same conclusion was reached in an earlier investigation [16], Gregg and Ballentine [28] reported also no changes of total N during the early embryonic development of R. pipiens. The same is true for Amblystoma mexicanum [39]. According to the present data, the fresh vveight of palmatus embryo is 64 per cent of alpestris, the dry weight 69 per cent, and the total N 67 per cent. Previous to the late blastula stage, 7’. palmatm contains 62 per cent as much DKA and 69 per cent as much RNA as T. alpestris. By 80 hours, i.e. at end of neurulation, the per cent value of DNA is 65 per cent and that of RNA 83 per cent. Based upon these data, it can be concluded that the concentration of both nucleic acids at the beginning of development is nearly the same for these two species. The higher per cent value of RNA for T. palmatus at 80 hours is obviously due to its relatively more rapid synthesis at the later period. Experimental

Cell Research

21

P. S. Chen DISCUSSION

A comparison between the present data and those obtained by earlier investigators for other amphibians is not possible because of the different materials and the different methods and units used. However, a review of the data available in the existing literature can serve to check the validity of the ‘~AULE

II. Average

vcrlues of total during

Trifon Age in hours at 18°C

II: for Triton palmatus cmd Triton alpestris

embryonic

development.

palmatus

Trifon

Developmental stage

n

Total N 0% Per embryo)

Age in hours at 18°C

alpesfris

Developmental stage

11

Total N (Pup Per embryo)

30

late blastula (H. 8+)

64.8

26

mid-blastula (H. X-S+)

‘2

105.1

54

late

63.4

48

mitl-gastrula (H. llbp12a)

3

!)3.3

67.8

82

late neurula (H. 16)

3

94.1

gastrula

(H. 65

n, number

12b-c)

mid-neurula (H. 15) of determinations;

H, Harrison

stage

for Amblysfoma

punctafum.

present method. Previous to this investigation, no comparative study of both DNA and RNA in the three European urodeles has been made, so far as I know. The RNA data estimated in this study are 1.9 ,ug per embryo for T. palmatus, 2.7 and 3.4 for T. alpestris and T. cristatus respectively. These values are lower than those given for other amphibians. However, the relative increase is in reasonable agreement with that determined by Brachet [7] for R. fusca and A. mexicanum. From fertilized egg to the early larva stage a 1.6-l .7 fold increase was observed by him for these two species, and in the present analysis a 1.3-l .9 fold increase for the three urodeles examined. It is known that the RNA content varies to a great extent in eggs from different species (j-20 pg per embryo [ho]), and even in different batches of eggs from the same species. DNA serves as a better basis for comparison. The more comparable data are the DNA values given by Sze [57] for the early stages of R. pipiens. Using the techniques of Schneider [-ill and Schmidt and Thannhauser [50], he reported that the total DN,4 of this anuran increaExperimental

Cell

Research

21

DNA

and RNA

531

in urodele development

ses from 0.96 to 6.1 ,~g per embryo from fertilization to the Shumway stage 19. For the same species, values from the study of Kutsky [36], using the method of Schmidt and Thannhauser [50!, are 1.2 ,ug per embryo at stage 2 to 6.5 ,ug at stage 18 (see [40]). The amount of DNA for the interval from fertilization to the end of neurulation estimated in this study is 1.1 to 7.4 ,~g per embryo for T. cristutus, 1.5 to 5.3 ,ug per embryo for T. pcllmatns, and 2.4 to 8.4 pugper embryo for T. trlpestris. During the periods included in the above studies, the relative increase is 5-6 fold for R. pipiens and 3.4-6.7 fold for the three urodeles. The values are therefore within the range expected for amphibian eggs by the use of chemical methods. In general, the pattern of increase in DNA observed for the three urodeles is in agreement \vith that found by Hoff-.Jergensen and Zeuthen [34] and Hoff-Jcrrgensen [33] for R. platyrrhena and Lclvtrup [40] for A. mexicrrnrzm. In spite of the increase in nuclei there is no change in total DNA until the late blastula stage. Obviously during this period the developing egg uses DNA reserve stored in its cytoplasm. Beginning from gastrulation, new synthesis of’ DNA sets in. This fact is in accordance with the finding of Steinert [56] that the egg of R. /&u starts to use free purines and mononucleotides at the early gastrula stage. The relative increase in total DNA seems to be higher in R. platyrrhenrr and A. me,ricrrnunt than in the urodeles examined in this study. i\s the above authors employed the microbiological method for assaying I)NA, it is difficult to decide if this dif’ference is species-specific or due to the different techniques used. Since no cell count in the present study was performed, it is also impossible to judge how the increase in DNA is correlated \\-ith the increase in the number of nuclei. Even if such data were available, it is still questionable if a strict correlation exists, because evidence has been shobvn that the cell-constancy of DNA, i.e. the same amount of DNA per haploid chromosome set, does not always hold true for embryonic tissues 119, 38, 42, 431. In a previous study, using a similar but less sensitive technique, a slight increase in DSA at the late cleavage stage of T. pulmrxtus was observed j15]. But this increase is very small compared to the synthesis at gastrulation and neurulation. Csing radioactive glycine and analogues of folic acid, Grant ‘26, 2i] found that in R. pipiens DNA svnthesis occurs to a slight extent prior to gastrulation and becomes intensified in later development. The same conclusion was reached by Moore [43]. With regard to RNA, the present data demonstrate clearly that its synthesis does not take place until the end of blastulation. This result is in agreement \\-ith that reported by Krugelis et ~1. [XI] for A. punctrrtum and Zeller 1641 Ezperimentnl

Cell

Research

21

P. S. Chen

532

for T. palmatus and T. &status. The significance of the RNA increase at the beginning of gastrulation can be understood only when the morphogenetic events are considered. Since the first suggestion of Capersson [121 and Brachrt [B], many experiments have demonstrated that RNA plays an important role in the biosynthesis of proteins (for literature see [ 11,131). From various studies it is also known that the synthesis of specific proteins and enzymes in the developing egg starts at the early gastrula stage [18, 24, 30, 31, 481. In a previous investigation it was found that the total concentration of free amino acids in the three urodeles increases at the beginning of gastrulation [16]. That RN-4 synthesis starts at the time of intensified proteins metabolism al>pears therefore self-evident. As pointed out by Brachet [8], ribonucleoprotein plays an essential role in the embryonic induction. This concept is supported by the recent biochemical study of the inductive substance [32, 621, although it seems that it is the protein component which plays the more important role [(ill. It is thus understandable \vhy there is a rapid increase of RNA during gastrulation and neurulation. The fact that RX,4 synthesis begins at the time n-hen the content of total DKA increases suggeststhat there is possibly a causal relation in the synthesis of both nucleic acids. Very recently Zalokar [63] has brought evidence that cytoplasmic RNA is synthesized in the nucleus, the site where chromosomes and genes are located. Also the study of Hoff-Jarrgensen and Zeuthen [34j on K. platyrrhena indicated that DNA store in the egg cytoplasm is synthesized in the germinal vesicle during the growth of the oocyte. These results are inconsistent with the hypothesis that DNA serves as templates for the synthesis of RNA [‘LO]. A related problem concerns the interpretation of developmental failure in the lethal hybrids. Interspecific hybrids of amphibians usually cease to develop at the late blastula or the early gastrula stage [2, 1-1.. Biochemical analysis sho\ved that in these arrested hybrids the synthesis of nucleic acids either does not occur or is much reduced [3, 1.5, 43, ~51. It has been therefore postulated that an inhibition in nucleic acid synthesis is the main cause of the developmental block [Cl]. This concept is strengthened by the present study, showing that it is at this “critical period”, i.e. at the beginning of gastrulation, the cytoplasmic store of DNA and RNA becomes exhausted and new synthesis begins.

Experimentnl

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DNA

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533

in urodele development

SUMMARY

1. h microchemical method designed for the determination of nucleic acids in a single amphibian embryo has been described. It is based upon the principle of differential extraction of RNA and DNA used by Ogur and Rosen [Ji] and the analysis procedure suggested by Scott, Fraccastoro and Taft [S’L]. Values obtained by this method have been shown to be within the range expected for amphibian eggs. 2. The assay data indicate that RNA and DNA contents in the developing eggs of Triton alpestris, Triton pahatus and Triton cristcrtus remain constant from fertilization until the end of blastulation. .It the beginning of gastrulation synthesis of both nucleic acids sets in. The significance oi’ this ne\\ synthesis is discussed in relation to the morphogenetic processes.

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--~ ~~~~ ~-

Experimental

Cell Research

21

P. S. Chen 34.

HOFF-JGIRGENSEN, E. and E. .J., NICHOLAS,

35. KRUGELIS, 36. I
40. 41. 42. 43.

44. 45. 46. 47. 48.

49. 50. 51. 52. 53. .54. 5.5. 56.

57. 58. 59.

60. 61.

62. 63. 64. 65.

P. Is., J. &J>tl.

ZEUTHEN, E., i\‘ature J. S. and VOSGIAN,

169, 245 (1952). M.

E.,

J. Ezpt/.

Zoo!.

121,

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