changes in the nuclei of differentiating endoderm cells as revealed by nuclear transplantation

CHANGES IN THE NUCLEI O F DIFFERENTIATING ENDODERM CELLS AS REVEALED BY

NUCLEAR TRANSPLANTATION

ROBERT BRIGGS' AND THOMAS J. KING The Institute for Cancer Research and The Lankenau Hospital

Research Institute, Philadelph.ia, Pennsylvania

TWENTY-EIGHT FIQURES

In an effort to determine whether cell differentiation involves genetic changes in nuclei, we have carried out a series of experiments in which nuclei from various parts of frog embryos have been transferred to enucleated eggs. The earlier work along this line showed that living nuclei of undifferentiated blastula and early gastrula cells could be successfully transplanted (Briggs and King, '52, '53). The recipient eggs developed into normal embryos, showing that there had been no change in the properties of the nuclei during pre-gastrula development.

The next stage in the analysis involved the transplantation of nuclei from chorda-mesoderm and presumptive medullary plate of the late gastrula (King and Briggs, '54). Here the operation was more difficult t o do because of the fact that the donor cells were smaller and adhered more firmly to each other. However, about 10% of the attempted transfers were successful, giving rise to normal cleavage and blastula formation on the part of the recipient eggs. Of the blastulae produced, about half were arrested in blastula and gastrula

This investigation was supported in part by a research grant from the National Cancer Institute of the National Institutes of Health, United States Public Health Service, and in part by an institutional grant from the American Cancer Society.

Department of Zoology, Indiana University, Bloomington, Indiana. ' Present address :

269

270 ROBERT BRIQGS AND THOMAS J. KINQ

stages. The remainder developed into embryos displaying formation of all three germ layers. A few of the embryos developed into larvae, but the majority were arrested in various neurula and post-neurula stages.

The interpretation of these results posed a problem. I n previous experiments with blastula nuclei most of the recipient eggs which cleaved normally developed into larvae. I n the case of the late gastrula nuclear transfers, equally well cleaved eggs were frequently arrested in development as noted above. This could have been due to an intrinsic change in the late gastrula nuclei limiting their capacity to participate in morphogenesis. However, at the time we could not eliminate the possibility that the developmental deficiencies might be the result of nuclear damage during the operation. Therefore, emphasis was placed on the positive result, namely, that Rome of the recipient eggs developed normally, demonstrating that at least some of the late gastrula nuclei were unchanged and equivalent to nuclei at the beginning of development.

Before proceeding with an analysis of nuclei of post- gastrula embryos it was necessary to improve the transplantation procedure. This was done in the manner to be mentioned below. The improved method was first tested on nuclei of undifferentiated early gastrula cells with encouraging results. Previously, transfers of this type had resulted in normal cleavage in about 20% of the recipient eggs. With the new procedure the proportion of eggs displaying normal cleavage and development increased to about 40% (King and Briggs, '55). Next, the experiments with chorda-mesoderm nuclei of late gastrulae were repeated and it was found that here too the proportion of recipient eggs displaying normal cleavage was doubled-from 10% to 20%. This indicated that with the new procedure more of the nuclei were being transplanted in undamaged condition. However, despite this improvement there was no change in the later development of the blastulae, most of which were arrested in blastula gastrula, or early post-neurula stages, just as had been the case in the earlier experiments. This evidence indicated that the deficiencies in

TRANSPLANTATION O F ENDODERM NUCLEI 271

the development of the normally cleaved " chorda-mesoderm eggs" might be due to an intrinsic nuclear change, rather than an accidentally imposed one.

I n order to get conclusive evidence of nuclear change during differentiation we turned to a series of experiments with nuclei of endoderm cells. The endoderm was chosen for this test because it is determined early in development (Holtfreter, '38 and Kemp, '46)' and consists of large cells which are relatively easily managed in the transplantation operation. Even in the tail bud stage the endodcrni cells which we have used are still as large as the undifferentiated animal hemisphere cells of the early gastrula. The technical factors in the transplantation are therefore about the same, and a critical comparison can be made between nuclei of undifferentiated and differentiated cells.

METHODS

Nuclear transplantation involves two separate operations (Briggs and King, '53). First, the recipient eggs are activated and enucleated with a clean glass needle. This part of the method presents no serious problems. With practice one can be sure that all of the eggs operated on will actually be enucleated. The second phase of the procedure, the actual nuclear transfer, is carried out by picking up the donor cell in a micropipette with an inner diameter slightly smaller than that of the cell. When this is done properly the cell surface can be broken without dispersing the cytoplasm, which thus acts to protect the nucleus until it is injected into the enucleated egg. This part of the operation is quite difficult to do without diluting the cytoplasm with the medium and thereby damaging or killing the nucleus. Theoretically, the method could be improved by (a) the development of a medium less damaging to nuclei than the ones now available, or (b) by refinements in the methods of handling the doner cell so that the chance of diluting the cytoplasm and damaging the nucleus is minimized. Both attacks on the problem have been

272 ROBERT BRIGGS AND THOMAS J. KINQ

tried, and some improvements in technique have resulted which will require a separate paper to describe adequately. Here we may mention briefly the following points:

The medium. When the method was first developed we arbi- trarily selected the Niu-Twitty modscation of Holtfreter 's standard solution as a medium in which to carry out the transfer operation. This is a simple solution of the salts of Na, K, Ca, and Mg, plus bicarbonate and phosphate buffer (for formula see Niu and Twitty, '53). Numerous experiments have since been done with a variety of media, including those commonly used in biochemical work on isolated nuclei. The tests were not exhaustive but were adequate to show that none of the media tested was distinctly superior to the Niu-Twitty salt solution. I n all cases dilution of the donor cell cytoplasm appeared to be followed by nuclear damage and loss of mitotic activity. In view of this result we decided to continue for the time being with the Niu-Twitty solution as an operating medium, and to improve the technique of handling the donor cells.

Use of trypsim and versene for dissecting embryos and dissociating cells. Our early attempts to transfer nuclei from cells of post-gastrula embryos gave poor results partly because of the difficulty of dissecting out single cells of known type without damaging them. This difficulty has now been overcome by the use of the enzme, trypsin, and the chelating agent, versene (Ethylenediamine Tetraacetic acid), in the following way: the embryo (for example, a mid-neurula) is first dis- sected in sterile 0.5% trypsin-made up in a modified Niu- Twitty solution lacking Ca and Mg and buffered at pH 7.1 with phosphate. In this solution the layers or groups of cells making up the various parts of the embryo can be easily separated from each other, apparently as a result of a prefer- ential effect of the enzyme on the material cementing the layers together (Grobstein, '53). The enzyme, in this concen- tration, does not readily dissociate the cells in a given layer. In order to accomplish this dissociation a portion of the tissue being investigated is washed in modified Niu-Twitty

TRANSPLANTATION OF ENDODERM NUCLEI 273

solution, then placed in 5 x molar versene made up in the modified Xu-Twitty solution. Within 5 to 10 minutes the individual cells begin to round up. In this semi-dissociated condition they are transferred back into the regular Niu- Twitty solution. Individual cells can now be picked up in the micropipette and the nuclear transfers can be carried out in the usual way.

There is no indication that the trypsin-versene treatment damages the cells. When returned to the regular medium they reassociate within a few hours and continue their differentiation. In the case of versene, control transfers of blastula nuclei can be done directly in to molar versene without apparent damage to the nuclei, provided the usual care is taken to preserve the cytoplasmic protection until the nucleus is injected into the egg cytoplasm. The great advantage of the procedure is that it makes it possible to do accurate dissections of frog embryos, and to obtain isolated cells of known type with little or no cell or nuclear damage.

Construction of micropipettes. As we have already indicated, the most important single step in the transplantation operation is that in which the donor cell is pulled into the tip of the micropipette. If this is done carefully, and the pipette is of the right size and construction, the cell surface will be broken leaving the contents otherwise undisturbed. Recently this operation has been facilitated by improvements in the pipettes relating principally to the fitting of the pipettes to the donor cells, and the shaping of the tip. I n order to break the donor cell properly the inner diameter should be roughly Q to 8 that of the cell, depending on cell type. The tip of the pipette should be thin-walled with a sharp point on one side tapering approximately as do the tips of hypodermic needles. The orifice should be smooth to minimize the danger of accidentally ripping the cell surface.

With the technical improvements mentioned above, the method now gives very satisfactory results. For example, in the experiments reported in this paper 60% of the control

274 ROBERT BRIGGS AND THOMAS J. KING

nuclear transfers resulted in genuine cleavage and development of nucleated blastulae, two-thirds of which were complete blastulae of normal appearance. I n more recent experiments the percentage of successful transfers is higher still. However, it should be emphasized that results on this order cannot be obtained by a beginner. The technique requires a lot of practice and constant attention to detail to be successful.

Rearing of experimental embryos. Each egg, after receiving its transplanted nucleus, was transferred to a small Stender dish containing charcoal treated tap water (Kemp, '51). Development occurred throughout at 18"-19"C. Observations were made at frequent intervals during cleavage, two or more

TABLE 1

Development of controls

A. Diploid controls Total no. eggs = 1168 Normal embryos = 1116 (95.5%)

B. Androgenetic haploid controls Total no. eggs = 272 No. of embryos = 263 No. of haploids = 263 (100% of embryos)

times a day during the next two days, and one or more times a day thereafter. The embryos were allowed to develop to the young tadpole stage (St. 25 of Shumway, '40) or until they were definitely arrested. They were then fixed in Smith's or Zenker's fluid, sectioned at 10 p and stained with the Feulgen reagent and fast green, or with the azure stain of Flax and Himes ( '52).

Controls were of two types: (a) Normally fertilized eggs which served as controls for the donor embryos and for the recipient enucleated eggs : Ninety-six per cent of these controls developed in perfectly normal fashion (table 1), indicating that only very rarely would the experimental embryos display aberrant development because of the condition of the eggs, (b) Normally fertilized eggs enucleated in the same manner


as were the experimental eggs: These controls are particularly important since they provide us with a check on the success of the enucleation operation. If the operation is successful the eggs develop as androgenetic haploids, while failures are readily apparent because the eggs then develop as diploids. I n each experiment some of the eggs were fertilized and enucleated in this fashion, and were observed later (ca. 4 days) when haploids and diploids were easily distinguished from each other. The results, summarized in table 1, show that all the eggs developed as haploids, and indicate that the enucleation operation is practically 100% successful. It may be mentioned that controls of this type have been run for numerous other experiments in addition to the ones reported here, and that only very rarely indeed does one of them develop as a diploid. I n such cases it appears that some of the eggs contain two nuclei, one of which may not be seen at the time of the operation. However, with careful observation it is possible to avoid such eggs, and to be quite certain of the success of the operation.

RESULTS

According to Holtfreter ('38) the endoderm in Amphibian embryos is determined in the early gastrula stage. By the mid-neurula stage the gut displays regional determination (Holtfreter, '38 and Kemp, '4-6) and should be highly stabilized, if not irreversibly set, in its differentiation. The main purpose of our experiments was to find out whether or not this determination of the endoderm involves irreversible changes in the nuclei of the constituent cells. If such nuclear changes do mot occur, then the eggs into which the nuclei are transferred should cleave normally and display normal differentiation of all cell types. On the other hand, if the nuclei are irreversibly changed we may expect any of the following results : (1) Absence of cleavage or abnormal cleavage of the recipient eggs - indicating a specialization of the nuclei such that they will no longer go through normal mitoses


in egg cytoplasm. (2) Normal cleavage and blastula formation followed by arrest of development at blastula or gastrula stages. This result would be obtained if the nuclei were capable of participating in cleavage, but not in the formation of chorda-mesoderm or medullary plate. (3) Normal development through gastrulation with the formation of all germ layers, followed by a failure of differentiation in all except the endodermal tissue. This result would be obtained if the nuclear specialization were limiting only with respect to cytodifferentiation during neurula and post-neurula stages. The experimental results indicate that all of these types of nuclear changes occur during the differentiation of endoderm. It will be simplest to consider first the information on the cleavage of the test eggs, and then to give a description of their later development.

Cleavage of eggs injected with e d o d e r m nuclei

Figure 1 gives the details on the manner in which the experiments were performed. Table 2 summarizes some of the information on the cleavage of test and control eggs. These results show that as differentiation proceeds there is a definite decrease in the capacity of the nuclei to participate in cleavage and blastula formation (column d, table 2). Eggs injected with late gastrula endoderm nuclei display genuine cleavage in 58% to 68% of the cases -values comparable to the 63% obtained in control experiments with nuclei of undifferentiated cells. When mid-neurula endoderm nuclei are transplanted only 33% of the test eggs develop into blastulae. This proportion drops still further, to 1770, in the case of endoderm nuclei of tail bud embryos. This decrease in transplantability cannot be attributed to technical factors, As we have already mentioned, the endoderm cells are as large as, or larger than, the undifferentiated control cells of the early gastrula animal hemisphere (see table 3). This means that the cytoplasmic protection afforded the nuclei during the transfer operation should be adequate in all cases, and indi-

TRANSPLANTATION O F ENDODERSI NUCLEI 277

Scheme of Endoderm Nuclear Transplantation

ACTlVATlO N

T R A N S P L A N T A T I O N

ENUC LE A T I ON

LATE GASTRULA MID- NEURULA POST - NEURULA

Fig. 1 Nuclei of endoderm cells, taken from the floor of the anterior midgut of late gastrula, mid-neurula, and post-neurula embryos, were transplanted into activated and enucleated eggs of thc frog, Rana pipiens. See text for details.

TABLE 2

Cleavage of mucleated eggs injected with endoderm nuclei

a. C. b. “AOBBOMO- B~sTU- a. TOTAL

RtT:GyNT U N C ~ E * ~ D ABORTIVE SOMAL” TYPE O F FUCLEUS

cLmAvAGm RLT:ucE partial Complete

Control-St 10 Early gast. Animal hemi.

Endoderm-St 1 2 Late gast.

Endoderm-St 14 Mid-neurula

Endoderm-St 17 Tail-bud

92 (100%)

67 (100%)

(100%)

(100%)

(100%)

88

98

130

19 1 14 19 39 63%

4 15 9 13 26 58%

68% 10 8 10 7 53

30 28 8 16 16 3370

31 39 38 13 9 17%

Note: The data for St, endoderm nuclear transfers were obtained from two separate groups of experiments as indicated.


cates that the decrease in the transplantability of the endoderm nuclei is the result of an intrinsic change which somehow limits the capacity of the nuclei to go through normal mitosis when transferred to egg cytoplasm.

TABLE 8

Diameters of donor cells ~ ~~

(EANQE) MEAN CELL DIAMETER 01DLL TYPE STAGE OB

DmVELOPMENT

10 animal (Early gast.) hemisphere

/r 42 (39.8-44.3)

12 Floor of archenteron 64 (60.4-69.3) (Late gast.) (presumptive mid-gut)

14 Floor of mid-gut 48 (43.8-50.9) (Mid-neur.)

17 Floor of mid-gut 42 (39.8-43.5) (Tail bud)

Note: The measurements were made with a Filar micrometer ocular on living cells which had been disaggregated in 5 X 10-’M Versene and were spherical in shape. A total of 100 cells from 5 different embryos were measured to give each of the values listed above.

I n addition to the decrease in transplantability there are some other differences in the cleavage behavior of test and control eggs. I n both series the eggs fall into 4 categories:

Eggs which fail completely to cleave. Eggs displaying a few abortive cleavages which fade away within 12 to 20 hours (fig. 7). Eggs in which cleavage occurs at a retarded rate, leading to the formation of partial blastulae made up largely of achromosomal’ ’ cells. These blastulae are arrested toward the end of the first day of development, and resemble closely the achromosomal blastulae previously described (Briggs, Green and King, ’51). Finally, there are the genuinely cleaved eggs which give rise to nucleated blastulae (figs. 2, 4, 6).


Test and control eggs of groups (a), (b), and (c) do not differ significantly from each other in appearance. However, in group (d) , the genuinely cleaved eggs, there are the following differences. The control eggs, injected with undifferentiated nuclei, cleave in normal fashion. In some cases the cleavages may not include the entire egg but the form of the furrows is quite regular and indistinguishable from that of the furrows in naturally fertilized eggs. Some of the endoderm eggs also cleave in this fashion (fig. 2), but in many the first two or three furrows are distinctly abnormal. Most frequently the first furrow will show at intervals puckerings of the surface coat (fig. 4). The furrow itself is faint and appears to be interrupted or absent over part of the animal hemisphere. The second cleavage furrow usually displays the same abnormal form, at which time the first furrow becomes exceedingly faint. I n other eggs the first and second cleavages make excessively wide depressions in the egg surface, with extensive puckering of the surface coat along the length of the furrows (fig. 6). Occasionally the first cleavage extends over one-half of the egg, and the second half only begins to cleave a t the time of the third cleavage or later. Also, in a few instances the first cleavage is equatorial, or may even occur diagonally in the vegetal hemisphere. I n all of these eggs (of group d ) the cleavages later occur in normal fashion (fig. 3) and give rise to perfectly regular nucleated blastulae

The frequency of occurrence of abnormal cleavages, among eggs which later develop into normal blastulae, is given in table 4. The great majority of the control eggs cleave normally and develop into normal embryos, as we shall point out in the next section of this paper. Eggs injected with late gastrula endoderm nuclei display abnormal furrows in about one-half of the cases, while eggs containing endoderm nuclei from more advanced stages show the aberrant cleavages in higher frequency. However, repetitions of the experiments on endoderm nuclei of a given stage (late gastrula) show considerable variation in the proportion of test eggs display-

(fig. 5 ) .

280 ROBERT BBIGQS AND THOMAS J. EINQ

ing abnormal cleavage (see table 4). The significance of the differences between stages is therefore uncertain.

TABLE 4

SOURCE OF NUCLEI

OLEAVAQE O F RECIPIENT EGQS

Normal furrows -4bnormal furrows

Undifferentiated animal 27 hem. cells. Early gastrula

Lato gastrula Endoderm 17

(St 12) 17 Endoderm 7

Mid-neurula (St 14)

Endoderm 1 Tail-bud

(St 17-18)

2

9

33 a

7

Note: This table includes only the data on type of cleavage in eggs which later developed into complete blastulae. See test for description of abnormal cleavages. The data for S k endoderm nuclear transfers were obtained from two separate groups of experiments, as indicated.

We were interested to know if the irregularities in the early cleavages of endoderm eggs were correlated with developmental abnormalities in later stages, and have listed the types of embryos derived from the normally and abnormally cleaved eggs. This list (table 5) shows that there are no striking differences between the two groups of eggs. Both express a range of developmental potentialities, which will be described in detail below.

TABLE 5

Development of normally and abnormally cleaved endoderm eggs

TYPE OF CLEAVAGE

FURROW

D 1D V E L O P M E N T

Arrested Arrested Arrested Normal blastulae gastrulae post-neurulae embryos

Normal A b n o r m d

3 11 21 7 5 20 26 6

Note: This table includes only the data on the development of complete blastulae, with normal or abnormal cleavage histories as indicated.

TRANSPLANTATION OF ENWDERM NUCLEI 281

In addition to the observations on the form of the cleavage furrows we also recorded the times at which the eggs began the first two or three cleavages. It will be recalled that these eggs were first activated and enucleated. At some later time, roughly 40 to 90 minutes after activation, the nuclear transfers were carried out. When the cleavages were subsequently timed we found that the eggs fell into two groups. In one group the eggs began the first cleavages three hours after activatiort; i.e. at the normal time for eggs kept a t 18"-20°C. In the second group the first cleavage was delayed one hour, but once started the cleavages occurred at the normal intervals thereafter.

Table 6, summarizing our information on the cleavage times, shows that the endoderm eggs were more frequently delayed in initiating cleavage than were the controls. Again, we were interested to know if the delay in cleavage was correlated with the character of the later development, but found that both groups of eggs displayed the same general types of development, ranging from arrest at late blastula stage

TABLE 6

S O W C E OB NUCLEI

NO. OF BECIPIENT

EGGS

ISTCLEAVAGE 1STCLEAVAGE

TIME 1 XOUR AT NORMAL DELAYED

Early gastrula 42 35 7 Animal hemisphere (100%) (83%) (17%)

Late gastrula Endoderm

Mid-neurula Endoderm

38 19 19 (100%) (50%) (50%)

44 31 13 (100%) (70%) (30%)

32 12 20 (100%) (38%) (62%)

Tail-bud 18 6 12 Endoderm (100%) (33%) (67%)

Note: The above figures include all eggs that went through genuine cleavage. Data on late gastrula endoderm transfers were obtained from two separate groups of experiments as indicated.

282 ROBERT BRIQGS AND THOMAS J. KIN0

to normal development through all embryonic stages (table 7). However, there was a very definite correlation of cleavage delay with an increase in chromosome number (table 8). Practically all of the eggs which cleaved at the normal time developed as diploids, while those that exhibited a delayed first cleavage were tetraploids. Thus during the one hour delay the chromosome number was doubled in the absence of nuclear or cell division. This happened both in eggs injected with endoderm nuclei and in those injected with undifferentiated nuclei, but was a more frequent occurrence in the endoderm eggs.

TABLE 7

Development of endoderm eggs which ( a ) begin cleavage at normal time, and ( b ) are delayed one hour in beginning cleavage

TIME OF FIBST

CLEAVAGE

D E V E L O P M E N T

Arrested Arrested Arrwted abnormal Normal blastulae gastrulae post-neurulae embryos

3 hours 4 hours

6 15 24 8 1 14 15 4

Note: This table includes only the data on the development of complete blastulas with cleavage histories as indicated.

TABLE 8

RPla.tionship between cleavage time and ploidy

1 S T OLEAVAGE DELAYED ISTCLEAVAGEAT NOBMAL TIME SOURCE O F

N U C L I I Diploid Tetraploid Diploid Tetraploid embryos embryos embryos embrvos

Early gastrula Animal hemisphere 28 17 1 6 Late gastrula Endoderm Mid-neurula Endoderm Tail bud Endoderm

13 0 0 12 23 0 2(1 6

3 0 0 5

2 0 0 0 Totals 69 l? 1 (+3V 29

Note: This table includes all eggs that showed genuine cleavage and developed differentiated epidermis. Ploidy was determined on the basis of cell size (Briggs, '47). The data for St,, endoderm nuclear transfers were obtained from two separate groups of experiments as indicated.


Later developmed of “eiadoderm embryos”

From what has been presented above it is clear that endodermal differentiation involves a change in the mitotic behavior of the nuclei, eventually rendering them incapable of dividing normally in egg cytoplasm. However, this nuclear change does not occur in all cells at once, at the time when the endoderm as a tissue is determined. Rather it appears to occur in a small number of cells at first, and then in more and more as differentiation progresses. Thus, in the late gastrula the endoderm is probably already determined (Holt- freter, ’38). Yet the majority of the nuclei retain the ability to elicit genuine cleavage and blastula formation in the test eggs. Even in the neurula stage, when the endoderm exhibits regional determination, one-third of the nuclei are still capable of going through mitosis and of catalyzing normal cleavage in egg cytoplasm (table 2). This suggests that the change in mitotic behavior is a relatively late manifestation of nuclear specialization during differentiation.

In order to see whether the change in mitotic activity is preceded by a change in the capacity of the nuclei to promote differentiation we have followed the later development of the “endoderm blastulae” (table 9). In this study only completely cleaved blastulae of normal appearance were considered. For controls we had the complete blastulae derived from eggs injected with undifferentiated nuclei (i.e. nuclei of early gastrula animal hemisphere cells). Although the two groups of blastulae were identical in appearance, their later development was strikingly different. The large majority (85%) of the controls developed into normal embryos and larvae (figs. 13,16). By contrast, the large majority of the endoderm blastulae were arrested in their development, as indicated in table 9. Blastulae containing late gastrula endoderm nuclei mere arrested in blastula and gastrula stages (26%), or as abnormal neurula and post-neurula embryos (53%) (figs. 10, 12, 14, 15). Only a small proportion (20%) succeeded in differentiating normally (fig. 11) and developing to the larval

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stage. When the endoderm nuclei were from mid-neurula and tail bud donors, the development was still more abnormal. About 70% of the blastulae were arrested in blastula and gastrula stages (figs. 8, 9), 30% were arrested later on, and only one embryo succeeded in transforming into a larva. There appeared to be no qualitative difference between comparable classes of “endoderm embryos” initiated by late gastrula, neurula or post-neurula endoderm nuclei.

Embryos arrested in blastula and early gastrula stages

The abnormal “endoderm embryos ” have been studied in some detail. Let us consider first those that were arrested in late blastula or early gastrula stages. These embryos remained intact for two or more days and showed no signs of differentiation. Eventually they underwent a degeneration characterized by a withdrawal of pigment from the surface of the animal hemisphere, followed by a generalized rounding up and loss of cells.

Twelve of the arrested blastulae and early gastrulae were fixed after they had been in the arrested state for one day (total age = two days). They were sectioned and stained with the Feulgen reagent and fast green, or with azure (Flax and Himes, ’52). A study of the sections showed that the embryos were completely cleaved and consisted of cells of regular size. Some of the cells of the blastocoel roof were partially rounded up, and some free cells were present in the blastocoel proper (figs. 17, 18). Otherwise the blastulae appeared to be of normal form.

Particular attention was paid to the condition of the nuclei of the arrested blastulae. So fa r as could be determined all cells were nucleated (fig. 17) and in the majority of blastulae the nuclei were of uniform size (fig. 19) indicating that the cleavage mitoses had been of the usual equational type. How- ever, at the time when the blastulae were fixed mitotic activity had largely ceased, and the condition of the nuclei was ap-

286 ROBERT BRIOGS AND THOMAS J. KINQ

praised by comparing them with the corresponding interphase nuclei of control embryos of the same age (two days, late gastrula stage). In the controls the vegetal nuclei differ from those of the animal hemisphere. They are more irregular in outline, stain less intensely but with a deeper hue of red or purple (Feulgen and fast green), and sometimes have the chromosomes concentrated at the periphery and sparse in the central portion of the nucleus. Nuclei of this type were found in the vegetal cells of both the experimental and control embryos, and there was no clear evidence of a difference between them.

In the animal hemisphere, on the other hand, the nuclei of the “endoderm blastulae” were definitely abnormal. These nuclei in the controls have the chromatin distributed throughout the nucleus (figs. 20, 22) and, in azure stained prepara- tions, display clearly one or two nucleoli. The corresponding nuclei of the experimental embryos usually have the chromatin concentrated at the periphery (figs. 19, 21), resembling in this respect some of the endoderm nuclei. In azure stained nuclei it is usually impossible to see nucleoli. An even more abnormal structure is exhibited by the nuclei of the free cells in the blastocoel, and occasionally by nuclei in the wall of the blastula as well. In these nuclei the chromosomal structure appears to have been completely lost, and all of the chromatin is concentrated in a few discrete, intensely Feulgen-positive bodies arranged around the inner surface of the nuclear membrane (fig. 18).

The description given above holds for the nuclei of arrested late blastulae. Embryos arrested in the early gastrula stage (St. 10 of Shumway, ’40) display the same nuclear conditions except in the area of the dorsal lip of the blastopore. Here the interphase nuclei have the normal appearance, and some mitotic figures can be seen. Elsewhere in the animal hemisphere there are no mitotic figures, and the interphase nuclei show the abnormalities noted above, while in the yolk cells of the vegetal hemisphere the nuclei are again apparently normal.


Embryos arrested i m later stages of developmerd

I n addition to the embryos which were arrested in late blastula and early gastrula stages, there were a few that proceeded part way through gastrulation, were then arrested, and survived in the arrested state for several days (fig. 9). These embryos developed ciliated epidermis, but otherwise showed no signs of differentiation.

Most of the embryos that developed beyond the early gastrula stage went on to complete gastrulation in normal fashion. A small number continued to develop normally (fig. 11) and transformed into larvae as indicated in table 9. Sections of these larvae showed that they possessed all of the organ systems in normally differentiated form. The majority of completed gastrulae, however, soon began to show deficiencies. In the more severely affected embryos these were first apparent early in neurulation, when a symmetrical but abnormally small medullary plate developed. Later, the neural folds formed at a retarded rate. These too, were smaller than normal, particularly in the head region. The subsequent post- neurula development of these embryos was strongly retarded (fig. 10) and eventually stopped completely after the embryos had attained a length of 4 mm to 6 mm (controls = 7 mm to 9 mm). At this time they displayed the following pronounced abnormalities (fig. 12) : (1) A small head with little or no external evidence of the development of sense organs. (2) Gill plates, but no external gills. (3) Absence of the pro- nephric protuberance. (4) The dorsal fin of the trunk and tail, and the ventral tail fin, very poorly developed or absent. ( 5 ) Loss of the integrity of the epidermis. At first (i.e. in neurula stages) the epidermis is normal in appearance and displays ciliary activity, but later it becomes thickened in some places and very thin or absent in others. (6) Spontane- ous small amplitude twitchings of the trunk musculature. I n addition to embryos of this type there were others which lived longer and displayed many or all the deficiencies mentioned above, but in a less pronounced form (figs. 14, 15).


In order to determine more exactly the nature of these deficiencies 16 of the endoderm embryos were fixed and sectioned serially. Twelve of the embryos (referred to as group A) displayed in pronounced form all of the gross abnormalities listed above. An additional group of 4 embryos (group B) were less severely inhibited, but still not capable of developing into larvae.

Let us consider first the 12 more severely affected embryos (group A). The main result of the microscopic study was to show that the ectodermal derivatives in these embryos were very poorly differentiated and were undergoing degenerative changes at or prior to the time when development was arrested. On the other hand, the endodermal and endomeso- dermal structures were more nearly normal, and did not display degenerative changes. The detailed observations upon which this statement is based can best be presented in the form of a list summarizing the condition of the organ systems in the 12 embryos.

1. E ct od ervnal derivatives

(a) The epidermis in all 12 embryos was abnormal, being absent or very thin in some places, and considerably thickened in others (figs. 25,27). Where thinned the epidermis consisted of very thin, flat cells; while in the thickened areas the cells were cuboidal. The cells were uniformly nucleated. However, there appeared to be no mitotic activity, and some degenerating nuclei were always present. The degeneration, or pycnosis, involved a loss of chromosomal structure and the accumulation of the chromatin in the form of an amorphous condensed mass of intensely Feulgen-positive material.

One or two small suckers were presented in 10 out of 12 embryos.

(b) The central nervous system was deficient and abnormal in all embryos. The brain was absent (1 case) ; or represented only by disorganized neural tissue (5 cases) ; or recognizable but very poorly developed compared with the control brains (6


cases). The spinal cord consisted of a small irregular rod with no neurocoel (3 cases) ; or with the neurocoel present only along a part of its length (5 cases). I n the remaining 4 embryos the cord displayed the neurocoel along most or all of its length, but was still small and poorly differentiated (fig. 27). As in the case of the epidermis, the nervous system appeared to be uniformly nucleated, but many of the nuclei were pycnotic.

(c) The neural crest derivatives were either completely lacking or, if present, they were very poorly developed and displayed much nuclear degeneration. None of the embryos developed spinal nerves. Some dorsal mesenchyme was present, but it was very deficient in amount and contained many pycnotic nuclei (figs. 23, 27). I n the trunk this deficiency was associated with the absence of the dorsal fin and with the abnormal juxtaposition of somites, spinal cord, and notochord (see below and figs. 25 and 27). In the head region the dorsal mesenchyme was also absent or in a degenerating condition and was here associated with the absence of visceral skeletal rudiments and external gills.

(d) Sense organs: Nasal pits were absent in all embryos. Eyes were absent in 9 embryos and very deficient in the other 3. Ears were also absent in 8 embryos and present in the form of small otic capsules in the other 4. As in the case of the other ectodermal derivatives, the sense organs when present, contained some pycnotic nuclei.

2. Mesodermal derivatives

The organs derived from the endomesoderm (i.e. the in- vaginated mesoderm) did mot contain significant numbers of degenerating nuclei, and were more nearly normal in their differentiation than were the ectodermal organs. This was particularly true of the structures which differentiated earliest (notochord and somites) (fig. 27), while those developing later on (pronephros, blood system) were more deficient (fig. 25).

290 ROBERT BRIOOS AND THOMAS J. KINQ

(a) The notochord was present in all 12 embryos. I n 8 of them it was of normal size and form (fig. 27) while in the remaining 4 embryos it was somewhat reduced in size and retarded in its differentiation.

(b) Somites were differentiated in 9 (possibly 10) of the embryos. In 6 cases the mass of somite material appeared in cross section to be quite large, perhaps larger than it was in the controls, However, the form of the organ was definitely abnormal (compare figs. 27 and 28). In the controls the somites extended dorsally alongside the spinal cord, but well separated from it by the intervening dorsal mesenchyme. In the endoderm embryos both the spinal cord and the dorsal mesenchyme were very deficient, as we have seen above, and the somites correspondingly did not show their normal dorsal extension. Furthermore, there was little or no mesenchpe separating the somites from the spinal cord, so that the two organs frequently were fused to each other (figs. 25 and 27). This direct contact of somite with central nervous system may account for the spontaneous twitchings noted in some of the endoderm embryos.

(c) Pronephroi were present in 8 of the 12 embryos but were quite deficient, being represented usually in the form of one or two tubules on one side only (compare figs. 25 and 26).

(d) The heart was absent in 9 cases and developed as a rudimentary straight tube in 3. There were no blood cells in any of the embryos.

3. Ercdoderm The endoderm tissue in all embryos appeared to be normal

and contained no pycnotic nuclei (fig. 27). I n the trunk region the form of the gut was normal. In the head region differentiation was retarded, no doubt as a result of the general retardation stemming from the degenerative changes in the ectodermal organs and the absence of heart and blood cells. The pharynx was present in 8 cases, and rudimentary branchial pouches in 4 or 5.


I n addition to the 12 embryos described above (Group A), there were the 4 embryos of Group B which developed somewhat longer and displayed less pronounced abnormalities. These embryos were arrested after 8 to 9 days of development, at which time they had assumed an appearance similar to that shown in figures 14 and 15. A study of the serial sections showed that while these embryos were more advanced in their differentiation than were those of Group A, they still exhibited the same pattern of abnormalities. The notochord and somites were well developed, and the endoderm tissue was normal in appearance, although morphogenesis was retarded. As for the ectodermal derivatives, the central nervous system and sense organs were better developed than in the Group A embryos, but still deficient in comparison with the corresponding organs of the controls. The ectomesenchyme was present around the dorsal and dorso-lateral portions of the brain and cord. It contained numerous pycnotic nuclei as indicated in figure 23. The central nervous system and sense organs also showed considerable nuclear degeneration, but not so much as was present in the ectomesenchyme. The epidermis, on the other hand, contained relatively few pycnotic nuclei and was more nearly normal in appearance (compare fig. 12 with 14 and 15, and fig. 23 with 25 and 27).

These observations on the structure of endoderm embryos have been confirmed in the course of a separate study involving serial nuclear transfers (see King and Briggs, '56, in press). In these experiments sections of 44 post-neurula endoderm embryos showed in varying degrees deficiencies and degenerative changes, especially of ectodermal derivatives. A series of 31 sectioned controls, derived from eggs injected with late blastula animal hemisphere nuclei, showed the following. I n 22 embryos all organs were normally differentiated. I n 5 embryos there was a general, non-speczc, retardation of differentiation, but no degenerative changes. I n 2 embryos, also retarded in development, the central nervous system was reduced in size and the neural crest derivatives definitely contained pycnotic nuclei, as in the endoderm embryos but


to a smaller degree. Finally, 2 retarded embryos displayed a very small amount of nuclear degeneration in the crest derivatives. Thus, some of the deficiencies characteristic of the endoderm embryos may occasionally appear in the development of control eggs injected with blastula nuclei, but this is a relatively rare event.

Developmefit of eggs injected w i t h endoderm cytoplasm. As was mentioned in the Methods section of this paper, successful nuclear transplantation can only be accomplished if the nucleus is protected by its own cytoplasm until the moment when it is liberated into the cytoplasm of the recipient egg. Thus, the donor cell cytoplasm is of necessity transferred along with the nucleus. The volume of cytoplasm so transferred is very small, being only about 1/40,000 or less of the volume of the recipient egg. None the less, it is necessary to determine if it contains substances or particles which might by themselves alter the development of the test eggs in the manner described in the preceding parts of this paper. For this reason experiments have been carried out in which endoderm cytoplasm was transplanted into both enucleated and normally nucleated eggs. The technique of transfer involved pulling the cytoplasmic portion of the cell carefully into the tip of the micropipette, cutting off the end containing the nucleus, and then transferring the cytoplasm alone into the test eggs. Twelve enucleated eggs were so injected. Al- though the number was small the result was unequivocal, for none of the eggs cleaved, showing that the cytoplasm by itself is unable to catalyze cleavage in non-nucleated eggs. This confirms an earlier experiment in which it was shown that cytoplasmic granules from tail bud stage embryos or blood cells also lacked the ability to elicit cleavage in enucleated eggs (Briggs, Green and King, '51; Briggs and King, '53).

A larger number of eggs, 68 in all, were first fertilized in the usual way, and then about one hour later were injected with endoderm cytoplasm taken from late gastrula, mid- neurula, and tail bud stage donors. The development of these eggs did not differ from that of the uninjected control eggs.


In both cases the majority (68% to 75%) of eggs developed into larvae while the remainder displayed the same types of common abnormalities. None of the embryos in either group showed the combination of abnormalities characteristic of the embryos developing with endoderm nuclei. There was thus no evidence that endoderm cytoplasm by itself can either elicit cleavage in enucleated eggs, or alter the cleavage or differentiation of normally nucleated eggs.

DISCUSSION

The main objective of this work was to obtain, by means of nuclear transplantation, a critical comparison of the properties of nuclei from undifferentiated and differentiated cells. To this end nuclei of undifferentiated early gastrula cells were compared with those of endoderm cells at later stages of development. The results of the experiments demonstrate that there is a real difference in the properties of the nuclei from the two sources. Those from early gastrula cells are the equivalent of nuclei at the beginning of development, as is shown by the fact that they participate normally in cleavage and differentiation following transfer to enucleated eggs. The endoderm nuclei, on the other hand, are definitely changed. The change involves first a limitation of their potentialities for differentiation. For example, late gastrula endoderm nuclei, transferred to enucleated eggs, participate normally in cleavage and blastula formation. A few of these endoderm blastulae continue to develop normally, showing that a small proportion of the nuclei are undifferentiated. A larger number of the test eggs complete gastrulation and develop the inductor system (notochord and somites), but display pronounced deficiencies especially in the central nervous system and other ectodermal derivatives, indicating that these nuclei have lost the capacity to participate normally in the differentiation of this class of structures. Another large group of test eggs also develop into “normal blastulae,” but are arrested at this stage or more frequently early in gastrulation. In such cases the endoderm nuclei are apparently unable to promote

294 ROBERT BRIGGS AND THOMAS J. HINQ

the differentiation of chorda-mesoderm. Theoretically these nuclei should retain only the ability to elicit endodermal differentiation, but whether they actually display such a rigid restriction of differentiative activity can only be told when the appropriate grafting experiments are done with portions of the blocked gastrulae.

At later stages of development these restrictions in capacity to promote differentiation become more pronounced. At mid- neurula and tail bud stages few or none of the endoderm nuclei are capable of promoting normal development, and the majority of the eggs that cleave are arrested in late blastula or early gastrula stages. Furthermore, an increasing proportion of the test eggs fail to cleave, indicating that many of the nuclei have now lost the capacity to go through normal mitosis when transferred to egg cytoplasm.

Taken together, this evidence suggests a progressive specialization of nuclear function during differentiation, which gradually limits the capacity of the nuclei to participate in types of differentiation other than that of the donor cell. Accompanying this change there may also be a specialization of the mitotic machinery such that eventually it cannot function except in its own cytoplasm. However, concerning this latter point we have only negative evidence - failure of test eggs to cleave following injection into them of nuclei from the older embryos. An alternative interpretation of this result can be made on the assumption that the mitotic rate decreases signscantly in the endoderm during development. I n this case there might be an accompanying decrease in the number of nuclei which, by chance, are in the appropriate phase to enter mitosis within a limited time following transfer to the test eggs.

An interesting feature of the nuclear changes described in this paper is that they do not occur in all cells at once, at the time when the endoderm is determined. According to grafting and explantation experiments, involving masses of fairly large numbers of cells, the endoderm appears to be determined as a tissue in the early gastrula stage, and is certainly stabilized


in its path of differentiation by the neurula stage (Holtfreter, Kemp and others). However, experiments of this type do not prove that all of the component cells are simultaneously differentiated. Grobstein ( '52) has pointed out that an organ area as a whole may be determined while fragments of it are still in a labile state of differentiation (see also the recent discussion of problems of cell vs. organ determination by Trinkaus, '56). Our evidence on the properties of the nuclei indicates further that a determined organ area may consist of cells in different stages of differentiation, or at least of cells containing nuclei of widely varying properties, as indicated above.

While it is quite certain that endoderm nuclei undergo the progressive specialization referred to above, there are many questions concerning its nature still to be answered. I n the first place there is the problem of the specificity of the change, i.e. of how it may differ in different types of cells. If nuclear differentiation is the basis of cell differentiation we should expect to find a gradual restriction of nuclear potencies so that eventually the nuclei would be capable of engaging only in those types of activities characteristic of given cell types. For example, endoderm nuclei would eventually be able to participate only in the synthetic activities of a particular type of enzyme-secreting cell, lens nuclei in activities specialized toward the formation of lens protein, and so on. Our knowl- edge of the nuclear changes in endoderm cells is consistent with this hypothetical statement, but does not yet specify the complete range of the changes. It should also be mentioned that we do not know how nuclei in other types of cells may change during differentiation. Transplantation experiments which have been done with mesodermal and neural nuclei indicate that in these tissues, as well as in endoderm, the nuclei undergo changes limiting their potentialities for differentiation. However, the nature of these changes has not yet been suflCiciently studied to allow us to say how they may differ from tissue to tissue.


In connection with this question of specificity we may mention two additional points that have been drawn to our attention. The first of these has to do with the possibility that the main mass of the midgut endoderm consists of “lethal” cells, destined to be sloughed off into the gut lumen and eventually digested by the intest.ina1 epithelium, which forms from the peripheral cells (Holtfreter, ’33). Through the kindness of Dr. G. Fankhauser we have been able to examine a series of sectioned embryos of Triturus viridescens, and have confirmed the fact that in this Urodele the main mass of midgut endoderm cells is eventually sloughed off and apparently destroyed in the lumen, leaving the peripheral cells which form the epithelium of the relatively short intestine, However, in the frog, Rana pipiens, the situation is different. Examination of sections of embryos at all stages, particularly toward the end of embryonic life (St. 23-25) when the gut is elongating rapidly and coiling, fails to reveal significant numbers of dead or dying cells. Rather, it appears that mitoses occur throughout the midgut mass, and that in effect all of the cells are utilized in the formation of the epithelium of the very long tadpole intestine.

A more direct check of the possibility of regional differences in the midgut endoderm mass has been made by transplanting nuclei, not only from the archenteron floor as in the experiments described above, but also from the ventral mass near the periphery. Transfers from the ventral mass of late gastrulae led to cleavage and formation of normal blastulae in 33 (59%) of 56 test eggs. Of these blastulae 42% were arrested in blastula or gastrula stages, 52% in abnormal post-neurula stages, while 6% developed into larvae. These results are consistent with the ones reported in the main body of this paper for nuclei from the archenteron floor.

A second point, relating to the possibility of diverse cell types being present in the presumptive midgut endoderm, has to do with the primordial germ cells. Since these cells arise from, or are associated with the endoderm in early stages (see reviews by Tyler, ’55 ; Burns, ’55) it has been sug-


gested that they may provide the minority of nuclei which, in our experiments, give complete and normal development in the test eggs. Certainly, the nuclei of these cells are acting as germ cell nuclei, in the sense that they are capable of being introduced into eggs and of participating normally in development. But this does not mean that they must come from germ cells as ordinarily defined, for occasionally we have also obtained normal development in test eggs injected with late gastrula or neurula nuclei from regions like the presumptive notochord and neural plate - i.e. regions which would not be expected to give rise to germ cells. In fact, on the basis of our experiments it appears that in the early gastrula all nuclei act as germ cell nuclei, and that in all regions of the embryo some nuclei remain unaltered for a time during the immediate subsequent stages, and presumably would be indistinguishable from germ cell nuclei. Eventually germ cells are segregated and must, of course, be genetically intact. How their nuclei might behave on transplantation to eggs has not yet been tested.

In the discussion so far we have spoken of the nuclear changes in differentiating endoderm cells without considering which part(s) of the nucleus arc involved, or what correlated changes there might be in the cytoplasm. The actual evidence shows that the change in potentiality for differentiation must occur in the nucleus, or in some nucleus-associated structure. The endoderm cytoplasm by itself apparently contains nothing that can alter the pattern of differentiation in normal eggs. This does not rule out the possibility that cytoplasmic entities play an important role in cell differentiation, but it does indicate that in such a role they would be dependent on nuclear factors. Of course, nucleus-dependent cytoplasmic entities are well known in certain microorganisms, and may well be involved in the establishment or maintenance of different cell types in Metazoa, although there is no evidence to this effect as yet.

The question still remains as to whether the changes we have described in this paper occur in the nucleus as such,

898 ROBERT BRIQGS AND THOMAS J. KING

or in some cytoplasmic entity present in the immediate vicinity of the nucleus. The entities which would be suspected are the Golgi apparatus and the centrosomes. So fa r as we are aware nothing is known about the Golgi apparatus that would indicate a controlling role for it in differentiation. Concerning the centrosome there is the common observation that it acts during mitosis as the center of organization for the spindle, and in some cells at least there is evidence that cytoplasmic elements are oriented toward the centrosome during the interphase, as if it were organizing the activities of the cytoplasm (Porter, '54). Whether it is actually determining the character of cytoplasmic structures is, however, quite con- jectural. In contrast to the lack of evidence of a role in differentiation for the Golgi apparatus and centrosome, there is a large body of information pointing to the importance of chromosomes and genes in the process. On this basis it seems very probable that the nucleus itself is the seat of the changes reported in this paper. We have therefore referred to the changes as nuclear changes, even though the peri-nuclear structures cannot be completely ruled out.

Finally, we should like to consider briefly the question of how nuclear differentiation may be brought about during development. Early in development, during the first few cleavages, the nuclei in the various blastomeres are known to be equivalent. By constricting the egg or applying pressure to it the distribution of the cleavage nuclei can be changed without altering the pattern of the subsequent differentiation. The situation is different with the cytoplasmic areas. If these are deleted or shifted in position the pattern of differentiation is changed, and a great deal of evidence is available to show that the cytoplasmic localizations of the egg determine the positions of the organ areas in the embryo. However, it should be emphasized that these localizations are not capable by themselves of giving rise to differentiated structures. This is shown by the fact that in the absence of nuclei the egg can be stimulated to cleave and develop into a blastula, but fails to gastrulate. Therefore, the nuclei are essential if the pattern


of differentiation is to be realized, and a basic problem of embryology and genetics has been to determine the nature of the nuclear contribution. One of the more plausible hypothe- ses along this line is that the cytoplasmic localizations elicit changes in nuclear or gene activity, which in turn act recipro- cally upon the cytoplasm to promote cell differentiation (Driesch, 1894; Morgan, '34). That nuclear changes of this general type are elicited during development seems quite certain, for nuclei in different tissues are known to vary in their morphological and biochemical properties. It has frequently been assumed that these variations reflect reversible changes in gene activity. However, the possibility is not excluded that the nuclear changes might be in the form of relatively irreversible alterations in gene activity, and that stability of the cell in its final differentiated form might depend upon the stability of the gene changes. The experiments reported in this paper support the latter view. If the nuclear changes which occur in endoderm cells were readily reversible, the test eggs into which they were transplanted should have developed into normal embryos. The actual result was quite different. The test eggs revealed a reduction in the capacity of the endoderm nuclei to promote ectodermal and mesodermal differentiation, and eventually a reduction in the ability to participate in cleavage of egg cytoplasm.

More recent experiments, involving serial transplantation of endoderm nuclei, have shown further that the nuclei are highly stabilized in their altered states, that these alterations do not involve changes in chromosome number, and that the descendants of a single endoderm nucleus promote a uniform type of differentiation in contrast to the varied types observed in eggs injected with nuclei from different endoderm donor cells (King and Briggs, '56, in press). Thus, while much remains to be done to specify the nature of these nuclear changes, the fact that they occur and are stable enough to be expressed following nuclear transplantation seems well established.

300 ROBERT BRIGGS AND T H O M A S J. KING

SUMMARY

Nuclei of endoderm cells, taken from the floor of the anterior midgut at different stages of development, were transplanted into enucleated eggs of the frog, Rana pipiens.

When the nuclei were from the late gastrula the majority of the recipient eggs (63%) cleaved and developed into partial or complete blastulae. Although the complete blastulae (50% of total) were normal in appearance, they usually displayed pronounced abnormalities in later development. In some cases development stopped in blastula or gastrula stages ; in others gastrulation was completed and development was arrested later. The arrested embryos contained the inductor system (notochord and somites) and endoderm tissue of normal appearance, but displayed pronounced deficiencies of the ectodermal derivatives. A few of the embryos developed normally and transformed into larvae.

When the endoderm nuclei were derived from mid-neurulae the proportion of the recipient eggs displaying cleavage was reduced to 3376, and the development of the resulting blastulae was more restricted. The majority were arrested in blastula or early gastrula stages while the remainder (except for a single embryo) displayed the abnormalities mentioned above.

In the case of endoderm nuclei of tail bud embryos the capacity to elicit cleavage in the recipient eggs was reduced still further (to 17%). Only 9 complete blastulae were obtained in this series, 7 of which were arrested in blastula or early gastrula stages, and 2 in the abnormal embryonic stages.

Control experiments were done in which endoderm cytoplasm was transferred into enucleated eggs and normally nucleated eggs. The enucleated eggs failed to cleave, while the normally nucleated eggs developed in normal fashion; i.e. they did not differ from the uninjected controls.

These experiments show that endoderm nuclei undergo stabilized or “irreversible ” changes during differentiation. These changes appear to restrict first the ability of the nuclei


to promote the formation of ectodermal and ectomesenchymal structures. Later the capacity to promote gastrulation and chorda-mesoderm formation appears to be lost, and eventually the endoderm nuclei appear to lose the ability to elicit cleavage in egg cytoplasm.

We wish to thank Miss Marie DiBarardino for her valuable assistance in this work.

LITERATURE CITED

BRIQQS, R. 1947

BRIQQS, R., E. U. GREEN AND T. J. KINQ 1951

The experimental production and development of triploid frog embryos. J. Exp. Zool., 106: 237-266.

An investigation of the capacity for cleavage and differentiation in Rana pipiens eggs lacking “func- tional” chromosomes. J. Exp. Zool., 116: 455-500.

Transplantation of living nuclei from blastula cells into enucleated frogs’ eggs. Proc. Nat. Acad. Sci., 58: 455463. 1953 Factors affecting the transplantability of nuclei of frog

embryonic cells. J. Exp. Zool., 122’ 485-506. BURNS, R. K. 1955 Urinogenital System. In Analysis of Development. ed.

Willier, Weiss and Hamburger. Saunders Co. DRIESCH, H. 1894 Analytische Theorie der organischen Entwicklung. Leipzig. FLAX, M. H., AND M. H. HIMES 1952 Microspectrophotometric analysis of

metachromatic staining of nucleic acid. Physiol. Zool., 25: 297-311. GROBSTEIN, C. 1952 Effect of fragmentation of mouse embryonic shields on their

differentiative behavior after culturing. J. Exp. Zool., 12’U: 437-456. Inductive epithelio-mesenchymal interaction in cultured organ

rudiments of the mouse. Science, 118: 52-55. HOLTFRETER, J. 1933 Die totale Exogaatrulation, eine Selbstablosung des Ekto-

derms vom Entomesoderm. Entwicklung und funktionelles Verhalten nervenloser Organe. Arch. Entw-mech. Organ., I29 : 669-793.

Diff erenzierungspotenzen isolierter Teile der Anurengastrula. Arch. Entw-mech., 138: 657-738.

Regulation in the entoderm of the tree frog, Hyla regilla. Univ. Calif. Pub. Zool., 52: 159-183. 1951 Development of intestinal coiling in Anuran larvae. J. Exp.

Transplantation of living nuclei of late gastrulae into enucleated eggs of Rana pipiens. J. Embryol. Exp. Morph.,

Changes in the nuclei of differentiating gastrula cells, as demonstrated by nuclear transplantation. Proc. Nat. Acad. Sci., 41 :

BRIQGS, R., AND T. J. KINQ 1952

1953

1938

KEMP, N. E. 1946

ZOO^., 116: 259-287. KING, T. J., AND R. BRIQQS 1954

I: 73-80. 1955

321-325.


KINQ, T. J., AND R. BBIGGS Serial transplantation of embryonic nuclei. Cold Spr. Harb. Symp. Quant. Biol. (in press).

MORGAN, T. R. 1934 Embryology and genetics. Columbia Univ. Press, New Pork, N. Y.

NIU, M. C., AND V. C. TWITTY The differentiation of gastrula ectoderm in medium conditioned by axial mesoderm. Proc. Nat. Acad. Sci., 39:

1954 Cell and tissue differentiation in relation to growth (ani- mals). I n Dynamics of Growth Processes (11th Growth Symp.). ed. by E. J. Boell, pp. 95-110.

SHUXWAY, W. 1940 Stages in the normal development of Rana pipiens. I. External form. Anat. Rec., 78: 139-147.

TBINEAUS, J. P. The differentiation of tissue cells. Am. Nat., 90: 273-289. TYLER, A. 1955 Gametogenesis, fertilization and parthenogenesis. In Analysis

1956

1953

9 85-9 89, PORTER, K. R.

1956

of Development. ed. Willier, Weiss and Hamburger. Saunders 00.

PLATES

General note: Each of the “endoderm embryos” shown in the accompanying photographs was produced by first enucleating the recipient egg, and then transplanting into it a single endoderm nucleus.

Figures 2 through 16 are photographs of living embryos; figures 17 through 28 are photographs of Feulgen-fast green stained sections.

PLATE 1

EXPLANATION OF FIGUHEP

Cleavage of “endoderm eggs.’’ X 21.

2 Enucleated egg injected with late gastrula endoderm nucleus. Normal first cleavage furrow, delayed in appearance by one cleavage interval. This particular egg developed into a normal tetraploid embryo (fig. l l ) , but more frequently equally well clcaved eggs are arrested during gastrulation or in abnormal post-neurula stages. The same situation applies in the case of eggs displaying abnormal furrows (figs. 4, 6) which also sometimes develop into normal embryos, but more frequently arc arrested in gastrulation or later stages.

Morula stage of the same endoderm egg shown in figure 2.

Enucleated egg injected with mid-neurula endoderni nucleus. The first cleavage furrow is faint, interrupted, and shows puckering of the surface coat. After the first 2 or 3 cleavages eggs such as this begin to cleave in regular fashion.

Normal blastula developed from egg shown in figure 4.

Enucleated egg injected with late gastrula endoderm nucleus. The first cleavage furrow is broad and Rhows extensive puckering of the surface coat. Subsequent cleavages occur in normal fashion and give rise to perfectly regular nucleated blastulae.

Enucleated egg injected with mid-neurula endoderm nucleus, showing abortive cleavagcs which fade away within 12 to 20 hours.

3

4

5

6

7

304

TRAh’RFTANTATIOK OF ENDODERJI NUCLEI RORRRT RRIGDR AND THOMAS J. KING

PLATE 1

305

PLATE 2

EXPLANATION OF FIGURE6

Later development of endoderm blastulae

All of these embryos were apparently perfectly normal in the late blastula stage, hut then displayed the highly varied types of post-blastula development sliown in the photographs.

8

9

10

11

12

1 3

14

15

1G

Arrested early gastrula, age 54 hours. Nuelem from endoderm of midneurula donor. x 12.

Arrested gastrula, aye 44 days. One-half of the pigmented hemisphere shows epidermal differentiation corresponding with tha t of a normal embryo of about the same age (see fig. 11). The defect iii the vegetal hcniisphere was produced accidentally in removing the embryo from its membranes. Nucleus from rudoderm of mid-neurula donor. x 12.

Abnormal endoderm embryo, agc 4 days. Embryos of this type complete gastru1:ition normally ; then show deficiencies, notably in the central iiervous system, seiise organs, aiitl neural crest derivatives. Compare with normal endoderm embryo of the came age shown in figure 11. Nucleus from endoderni of late gastrula donor.

Normal cntlo&mn cmbryo, age 4 days. Niirleus from cndoderm of late gastrula donor X 12.

Same embryo as sliomn in figuro 10, photographed two days later a t age of 6 days, illustratitig rnaximuui clovclopment displayed by embryos of this type. Note epidermal defects, tlefieieiiey of tail :id trunk fins, absence of gills and scwse organs. X 12.

Control for embryo sliown i n figure 12. Age G days. X 12.

Abnormal endoderm embryo, age 8 days. Deficiencies of the same general type as displayed by embryo in figure 12, but lrvs severely expressed. Chin- pare with control shown in figuro 16. Kucleus from endoderm of late gastrnla donor. X 7.

Moderately abnorinal endoclerm embryo, age 8 diiys. Some rediiction of fins. Sciisc organs and gills present but tlefirirnt. Compare with control (fig. 16). x 7.

Control for embryos shown in figures 14 and 15. Age 8 clays. X 7.

X 12.

TRhZISI'L.\STATIOh'~~ OF ENDODERY PI'ITC'LEI ROBYRT RRIGGS AND THOMAS J. PINO

PLATE 2

307

PLATE 3

EXPLANATION OF FI(tliKE6

structure of arrested endoderm blastulae

Figures 17, 18, 19, and 21 show sections of a typical arrested late blastula, derived from an enuclentod egg injected with ail endoderm nucleus from :I mid- neurula donor. Developinent was apparently normal to the beginning of gastriila- tion (at about 26 hours), then was arrested with pigment accumulation at the dorsal lip bu t no invagination. Fixed n t 54 hours, at which time the control embryos (figs. 20, 22) were :it late gastrula stage.

17

18

19

20

21

22

Section of complete, regularly cleiived, arrested endoderm blastula. Cell8 appcar to be uniformly nueleated. By the time of fixation, a f te r about one day in the arrested state, some cells of the blastocoel roof have rounded up and lie free in the blastocoel.

Higher power photograph of rounded up cells in the blastocoel. Chromosome structure appears to be completely lost, all chromatin being concentrated in n few discrete, intensely Feulgen-positive bodies arranged around the inner surface of the nuelear membrane. X 270.

Portion of auimal hemisphere of a r r c s t d endoderm blastula. Note tha t nuclei are of a uniform size, comparable with the size of nuclei i n the control (fig. 20) bu t differing in appearance, with the chromatin concentrated a t the periphery and sparse or absent in the central region. X 270.

Portion of a control (late gastrula, two days old) including presumptive medullary plate above and ehorda-mesoderm below. X 270.

Nuclei of equatorial regiou of arrested endodrrm blastula, as seen under oil immersion. The nucleus on the right is in focus, and shows the “vacuo- lated ” condition, with chromatin concentrated around the periphery leaving the central portion relatively empty. Compare with fig. 22. X 610.

Same control as in figure 20, showing nuclei of chorda-mesoderm cells as seen under oil immersion. The nucleus on the left i s in focus. It contoins a nuclcolus, and chromatin distributed throughout the nucleus.

X 40.

X 610.

308

TH.\XSPLANTdTION 0)’ ESDODERX KUCLEI ROHRRT BRIOBS AND THOMAS J. RING

PLATE 3

309

PLATE 4

Structure of post-neurula endoderin embryos

Showii 011 ttir left (figs. 23, 25, 27) are cross sections of endoderm embryos, obtained in the usual way froin runcleated eggs iujected with endoderm nuclei. All of tho embryos shown gwtrulated normally, and then began t o show deficiencies in iieurula and post-neuruls stages. On the right (figs. 24, 26, 28) a re cross strtions through corresponding levels of control embryos.

23

24

25

26

27

“8

C‘ross section through the cye rrgion of an endoderm embryo; age 7 days; nucleus from endoderm of late g;istruk donor. In gross form this embryo rvscuiblrd tlie embryo shown in figure 14. Dorsiil me~e~ i rhyme shows extensivc degeneration and in the original prrparation can be s e n to contain numerous pycnotic nuclei. Brain and eyeu arc retarded and abnormal, and also contain pycnotic nuclei. The foregut epithelium, just outside the lower border of the photograph, and thc: adjacent ventral mesenchyme do not contain sig- nific:mt nunitwrn of ilegenrrnting nuclei. Compare with control (fig. 2 4 ) . x 43.

Cross section through eye region of control for embryo shown in figure 23. Age 7 days. X 44.

Cross section through proneptiric region of an endoderm embryo, age 7 (hys, nucleus froin cndoderm of post-neurula (tail bud) donor. Tn gross form this embryo resembled the one shown in figure 12, but IVM tiornewhat better developed. Epidermis is :ibiiornial, contdiis Some pycnotic nuclei, and shows differential thickenings. JIindbrain is smaller than normal and contains pycnotic nuclei. Dorsal nirsenchyme is deficient, and tha t wliich is present contailis numerous degenerating nuclei. Notochord is normal in size and well differentiatrd. Souiites apprnr more massive than in the control (fig. 2ti) but tihow :ibnorni:il form and :ire in sonie placrs fused t o the notochord and ncurnl tube. The aorta is a small, bloodless tubc, :ind the proncphros is represeiitcd by a single poorly differentiated tubule on c:ich sidr. x 43.

Control for embryo shown in figure 25, age 7 days. Cross srction through levrl of pronephros. x 43.

Cross section through the mid-trunk region of tlir tetraploid e.idoderiii rmljryo shown in figure 12, age 6 days. Gut, notochord, and somites are well developed, as diowii. The neural tube is drastically reduced in size, contains pycnotic nuclei, and is fused to the soniitrs. Dorsal meseiichpme is sparse and drgrnerat- ing. The epidermis is :ibnormal as shown, and contains sonip pycnotic nuclci. x 43.

Control for the rmhryo slionwl i n figure 27, age ti (1:1ys. Crow wetion through the mid-trunk region. X 43.

310

l'R.~SSPLASTATION O F EKDODERM NECLEI ROREHT 11IZIGI:S AND TH03IAS J. KING

PLATE 4

311

changes in the nuclei of differentiating endoderm cells as revealed by nuclear transplantation

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