Transplantation of Living Nuclei of Late Gastrulae into Enucleated Eggs of Rana pipiens

The next step in this analysis was to test the nuclei from areas of the late gastrula which are regionally determined, namely, the chorda-mesoderm and the presumptive medullary plate. Embryological evidence indicates that the chorda-mesoderm is determined to differentiate into mesodermal structures, while the overlying presumptive medullary plate has at least a labile determination in the direction of neural differentiation. However, it is not known whether these regional determinations involve stabilized differentiation of the constituent cells. This question will be discussed after we have presented the results of experiments in which nuclei from chorda-mesoderm and presumptive medullary plate were transferred into enucleated eggs.

The areas from which nuclei were taken for transplantation are indicated in Text-fig. 1. Donor cells were obtained from the median plane, in a region approximately midway along the longitudinal axis of the embryo. In the case of the chorda we used only cells from the two uppermost layers, i.e. the layers nearest the overlying presumptive medullary plate. Presumptive neural nuclei were provided by the two lower cell-layers of the presumptive medullary plate, these being the layers adjacent to the chorda. In all cases the nuclei were transplanted by means of the technique previously described (Briggs & King, 1952, 1953 a, b). The procedure involves first enucleating the recipient egg, and then transferring into it a single nucleus from a donor cell. The nucleus must be protected by its own cytoplasm in the course of the transfer, which means that donor cell cytoplasm is introduced along with the nucleus into the recipient egg. Quantitatively the cytoplasm thus included is insignificant, being only about 1 / 600,000 the volume of the recipient egg. None the less, it must be considered in the interpretation of results, as we shall indicate below. Another important technical matter has to do with the fact that the operation is not perfect, and under the best circumstances results in damage to some of the transferred nuclei (see Briggs & King, 1953a). This must also be considered in appraising the significance of the results to be presented.

As we have reported previously (Briggs & King, 1952), enucleated eggs which are left untreated or are injected with cytoplasmic granules never display cleavage. When these eggs are injected with nuclei from late gastrulae the majority do show cleavage of one sort or another, as indicated in Table 1. In about one-fourth of the total number of cases the cleavage is abortive and fails to give rise to blastulae. The remainder of the cleaved eggs develop into partial or complete blastulae. Some of the partial blastulae are made up largely of cells lacking nuclei and resemble the ‘nucleusless’ blastulae produced by inseminating enu-cleated eggs with heavily irradiated sperm (Briggs, Green, & King, 1951). The remainder of the partial blastulae are normal in appearance except for the presence of an uncleaved area. This area is quite variable in size, ranging from less than one-tenth to roughly one-half of the blastula surface. Finally, the complete blastulae, comprising 8-9 per cent, of the total number of eggs, are of normal appearance. As will be apparent from the table, there is no significant difference in the behaviour of eggs containing transplanted chorda-mesoderm nuclei and those containing nuclei from the presumptive medullary plate.

From these results we can say that at least some of the nuclei from determined areas of the late gastrula are capable of participating normally in cleavage and blastula formation when transferred back into enucleated eggs. However, a larger number of nuclear transfers result in abnormal cleavage, partial cleavage, or in no cleavage at all. While we cannot exclude the possibility that some of these failures may be due to intrinsic properties of gastrula nuclei rendering them incapable of entering normally into the cleavage cycle of the egg, the evidence indicates that the majority of the failures actually result from damage inflicted on the nuclei in the course of the transfer. A detailed study, including cytological evidence of damage to the transplanted nuclei, has already been published (Briggs & King, 1953a). Here we should like only to point out that the main hazard in the transfer procedure is the inadvertent exposure of the nuclei to the medium in which the operation is carried out. With the relatively large cells of late blastulae the cytoplasmic protection afforded the nuclei during the transfer is such that about one-third of the attempted transfers are completely successful, leading to normal cleavage of the recipient eggs. With the smaller cells of early gastrulae, and the still smaller ones of late gastrulae, the amount of cytoplasm protecting the nucleus is diminished and the proportion of the attempted transfers which turn out to be successful is correspondingly reduced (see Table 2). This evidence, along with the cytological work referred to above, indicates that the majority if not all of the failures in cleavage are due to nuclear damage, and not to changes in the properties of the nuclei during development.

Having established the fact that chorda-mesoderm and pre-medullary plate nuclei can participate normally in cleavage, the next step in the investigation was to find out how they might function during later stages of development. For this purpose we followed the development of the eggs which cleaved normally following nuclear transfer. These gave rise to apparently normal complete blastulae, as has already been mentioned. The later development of these blastulae is summarized in Table 3, which lists the results for blastulae containing nuclei from the determined areas of the late gastrula. For comparison, the results for blastulae containing nuclei from an undetermined area of the early gastrula are also given. An inspection of the table and of the notes attached to it shows that there are no differences in the types of embryos derived from the three classes of blastulae. In all cases some of the embryos are arrested in blastula or gastrula stages. Sections were made of some of these and revealed mitotic irregularities and absence of nuclei in some parts of the embryos—abnormalities probably stemming from damage to the nuclei during transfer. The remaining embryos, representing one-half or more of the total number, continue their development to neurula and post-neurula stages and display differentiation of all three germlayers and derivatives thereof. Considering the embryos containing chordamesoderm or pre-medullary plate nuclei (lines 2 and 3 of Table 3), there are certainly no differences in extent or type of development that can be correlated with the different sources of the nuclei. In both cases the most successful embryos develop into larvae and display advanced differentiation of all cell types. Thus, nuclei from the presumptive medullary plate are not limited to participation in neural differentiation but may participate as well in mesodermal and endodermal differentiation. The same situation exists with respect to the chordamesoderm nuclei, which also participate in all types of differentiation. Embryos containing nuclei from early gastrulae (line 1, Table 3) are less apt to become arrested or abnormal during gastrulation and neurulation. This is to be expected since these nuclei are less likely to be damaged in transfer than are those of the late gastrulae. Otherwise, this group of embryos does not differ from the other groups listed in the table.

The conclusion to be drawn from these experiments is that some, and possibly all, of the nuclei of determined areas of the late gastrula are not themselves determined or differentiated. In other words, they have not undergone irreversible genetic changes limiting them to participation in only one type of differentiation. Since the experiments involved the introduction of cytoplasm along with the nuclei into the test eggs, it also appears that there are no cytoplasmic genetic units directing differentiation in the late gastrula, or that if there are such units they are no longer effective when transferred back into the egg cytoplasm.

Now, how is this result to be fitted in with the embryological evidence concerning the properties of the chorda-mesoderm and the presumptive medullary plate? The chorda-mesoderm of the late gastrula is quite certainly determined in a general way to form mesodermal structures, and will not give rise to neural or other structures normally derived from ectoderm (Holtfreter, 1933). On the other hand, the presumptive medullary plate, following exposure to the chordamesoderm, is determined to form neural tissue, although in the late gastrula this determination may still be labile (Marx, 1925: Mangold, 1929; Holtfreter, 1947). While the evidence indicates that the areas are thus determined, it does not tell us whether this determination depends primarily upon differentiation of the individual cells or is to be regarded instead as a property of the mass as a whole, the individual cells being still undifferentiated or in a labile state of differentiation. Recent evidence obtained by Grobstein (1952) and by Grobstein and Zwilling (1953) indicates that the second of these two possibilities may be the correct one. These investigators, working with cultured explants of chick blastoderm and mouse embryonic shield, find that the extent of differentiation depends upon the degree to which the explant cells are dispersed. For example, large explants will differentiate into neural tissue, but if these explants are divided into eighths or sixteenths the differentiation fails to occur or is poorly expressed. On the basis of this and other evidence it appears that in chick and mouse embryos organ determination occurs while the component cells are still undifferentiated or are in a labile state of differentiation. The same situation may very well exist in the amphibian gastrula. Our results support this view since they indicate that this phase of differentiation does not require specific irreversible changes in nuclear function, nor does it involve the elaboration of cytoplasmic elements which can direct differentiation when transferred back into eggs. Whether irreversible changes in genetic units are involved in cytodifferen-tiation during later stages of development remains to be worked out.

1. Living nuclei were transferred from determined areas of late gastrulae, the chorda-mesoderm, and presumptive medullary plate, back into enucleated eggs (Rana pipiens). In both cases about 8 to 9 per cent, of the attempted transfers resulted in normal cleavage of the recipient eggs. The rest of the eggs either failed to cleave or cleaved abnormally. The evidence indicates that most, if not all, of these failures were due to damage inflicted on the nucleus during the transfer procedure.

2. The normally cleaved eggs developed into complete blastulae. Some of these were arrested in blastula or gastrula stage, again as a result of nuclear damage in all probability. The remaining embryos, representing about one-half of the normally cleaved eggs, developed to neurula and post-neurula stages. These embryos displayed differentiation of all three germ-layers and their derivatives, regardless of whether their nuclei were derived from chorda-mesoderm or from presumptive medullary plate.

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

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