Marie A. Di Berardino

The karyotype of Rana pipiens and investigation of its stability during embryonic differentiation

Abstract The Rana pipiens karyotype has been determined from embryonic cells in order to have a basis for detecting abnormal chromosomes in future studies and to investigate whether the karyotype undergoes any changes during the cellular differentiation that accompanies normal embryogenesis. Metaphases from the undifferentiated cells of the early gastrula and from the differentiated cells of the postneurula tail were analyzed from permanent squash preparations. The material was first exposed to prefixation treatment, then fixed, stained, and squashed. The 26 chromosomes have been classified into 6 pairs with median centromeres (group I nos. 1–4; II nos. 5–6) and 7 pairs with submedian centromeres (group III nos. 7, 8; IV nos. 9, 10; V, nos. 11–13). All the chromosomes can be identified individually with the exception of group II nos. 5, 6 and Group V nos. 11, 12 which are usually difficult to distinguish from each other. No heteromorphic pair of (sex) chromosomes is present. The karyotype of the undifferentiated early gastrula cells and the differentiated cells of the diploid and haploid androgenetic postneurula tails is basically identical. Two variations are present. Early gastrula chromosomes are longer than chromosomes of tail cells, and the secondary constriction present in the no. 10 chromosome is longer in gastrula chromosomes. A statistical analysis of relative lengths of chromosomes 1 and 4 showed that each of the chromosomes of the three cells types contributes the same percentage to the genome. The difference in absolute length at different stages is attributed to different degrees of chromosome despiralization. Centromere positions in Chromosomes 1 and 4 also do not differ significantly among the three cell types. However, the secondary constriction of no. 10 is significantly longer in early gastrula chromosomes. Chromosome length is discussed briefly in relation to ploidy and to nuclear and cell size. Chromosome studies on other Anura are related to those on Rana pipiens . The controversy concerning the existence of a pair of heteromorphic (sex chromosomes in Anura is reviewed.

Origin of chromosomal abnormalities in nuclear transplants—A reevaluation of nuclear differentiation and nuclear equivalence in amphibians

Abstract Previous transplantation tests of amphibian nuclei derived from advanced somatic and germinal cell types have demonstrated that most of these nuclei have acquired developmental restrictions during embryogenesis. The nature of these restrictions has been revealed in part through chromosomal studies of abnormal nuclear transplants. In these studies, it has been found that the developmental restrictions in the nuclear transplants are accompanied in most cases by karyotypic alterations. In order to determine the origin of these chromosomal aberrations and thereby gain some understanding of the chromosomal changes accompanying normal embryogenesis, we have studied the nuclear and chromosomal morphology of nuclear transplants within the first 8 hours after nuclear transplantation. These studies carried out on nuclei of determined endodermal cells demonstrate that in most cases the origin of these chromosomal aberrations is traceable to the initial responses of the transplanted nucleus to the cytoplasm of the recipient egg. Many transplanted endodermal nuclei fail to undergo sufficient nuclear enlargement and chromatin decondensation. As a result of these events, chromatid bridges arise during the first mitotic cleavage and subsequent to chromatid breakage at late anaphase, variable deletions in chromosomes occur. These different amounts of genetic losses then lead to variable abnormal development in nuclear transplants and ultimately are the cause of developmental arrest. The results of these studies indicate that during embryogenesis the chromosomes of many endodermal nuclei have acquired restrictions in the form of condensed or compact chromatin (heterochromatin). Furthermore, these restrictions are not reversed completely by transplantation into young egg cytoplasm; and consequently, the complete expression of the transplanted nucleus is prevented. The significance of these findings is discussed in relation to our current understanding of nuclear differentiation in amphibians.

Development and cellular differentiation of neural nuclear-transplants of known karyotype☆

Abstract Neural nuclei from the presumptive neural area of late gastrula and the definitive neural plate regions of early neurula and mid-neurula embryos of Rana pipiens were transplanted into enucleated eggs of the same species. The results obtained confirm the previous findings, that in the course of neural differentiation there occurs a gradual progressive change which restricts the capacity of most neural nuclei to promote the normal development of test eggs. In the current report, serial transplantation of neural nuclei from the open neural-fold stage demonstrated that the nuclear condition responsible for these developmental restrictions is stable and cannot be reversed to a more normal expression by repeated exposure to test egg cytoplasm. Chromosome analyses of abnormal neural nuclear-transplants revealed two karyotypic groups of abnormal individuals. One group, comprising the majority of neural nuclear-transplants, possessed recognizable karyotypic aberrations. In some cases the chromosome abnormalities were severe and development ceased prior to overt cellular differentiation. In other cases, the karyotypic abnormalities were less severe and the transplant embryos developed into abnormal post-neurulae and larvae, which displayed variable patterns of cellular deficiencies not consistent with the neural origin of their nuclei. A second group which represents 3 of the 17 individuals whose chromosome constitution and cellular deficiencies were analyzed possessed the standard karyotype, and exhibited cellular deficiencies primarily in mesodermal and endodermal derivatives. This pattern of deficiencies is distinct from that found in embryos resulting from the transplantation of nuclei from endoderm cells and is consistent with the neural origin of the donor nuclei. The probability that the characteristic deficiencies seen in neural nuclear-transplant embryos are a phenotypic expression of the differentiated state of the donor nucleus is discussed.

Transplantation of nuclei from the frog renal adenocarcinoma: II. Chromosomal and histologic analysis of tumor nuclear-transplant embryos

Abstract Chromosomal and histologic analyses were performed on abnormal frog embryos and larvae derived from the transplantation of a single nucleus from the frog renal adenocarcinoma into enucleated host eggs. These analyses were undertaken to determine the nature of the developmental restrictions exhibited by nuclei of tumors. All tumor nuclear-transplants were abnormal in organ differentiation and chromosome constitution; the extent of these deficiencies was directly related to the extent of development attained. Tumor nuclear-transplants which developed to larval stages were mainly diploids but on karyotype analysis were found to be abnormal and mosaic. Individuals which arrested at postneurula stages were variably aneuploid, and those which arrested at blastula and gastrula stages were also variably aneuploid, but in addition contained severe structural chromosome abnormalities. Histologic abnormalities were present in all of the three primary germ layer derivatives; and therefore, no specific pattern of cellular deficiencies can be associated with the tumor origin of their donor nuclei. The occurrence of chromosome abnormalities in the tumor nuclear-transplants can account for their developmental arrest and might also obscure, if present, specific deficiencies of the donor nucleus. The present analyses indicate that these chromosome aberrations occur after the tumor nucleus is transplanted into the egg host and at different times in embryonic development; however, their origin is unknown. The occurrence of abnormal chromosomes is interpreted as a reflection of the differentiated “state” of the donor tumor nucleus prior to transplantation. Supporting evidence for this interpretation comes from the fact that frequent chromosome abnormalities occur in transplant embryos derived from nuclei of differentiated cells. By contrast, chromosome abnormalities are only occasionally found in transplant embryos derived from nuclei of undifferentiated cells.

Nucleo-cytoplasmic exchange of non-histone proteins in amphibian embryos.

Abstract As a basis for understanding the role of non-histone proteins in nuclear differentiation, we have identified one period during embryogenesis when intense accumulation of non-histones occurs in nuclei of Rana pipiens. We then demonstrated, experimentally, the loss of non-histones from nuclei after transplantation into enucleated eggs. 3H-tryptophan or 3H-lysine was injected into blastocoeles of mid-blastulae and into archenterons of late gastrulae; embryos were subsequently studied autoradiographically. Nuclei of animal hemisphere cells from blastulae accumulated only small amounts of 3H-tryptophan within 3 h, whereas a large accumulation occurred in endodermal nuclei of gastrulae as early as 1 h, and after 3 h 95.9% ( x ) of the nuclei were densely labelled. Significant accumulation of 3H-lysine occurred in the majority of nuclei of both types within 3 h (blastulae x = 77.5% ; gastrulae x = 54.8% ). Controls, involving RNase or boiling TCA, demonstrated that the 3H-amino acids have been incorporated mainly into proteins. Endodermal nuclei labelled either with 3H-tryptophan or 3H-lysine after a 3 h incubation were transplanted singly into enucleated eggs. Autoradiograms demonstrated that most non-histones leave the nucleus during its reprogramming in the egg cytoplasm prior to first cleavage; whereas other types of proteins labelled with 3H-lysine remain for the most part in the nucleus. Cytochemical studies indicated that some of the non-histones which leave transplanted nuclei are acidic proteins; whereas some of those proteins which remain in the nucleus are histones. In addition to the above findings, the results of these studies demonstrate the feasibility in the future of studying the nucleocytoplasmic migration of different kinds of macromolecules in a developmental metazoan system and determining their roles in the establishment of nuclear differentiation.

Origin and Progress of Nuclear Transfer in Nonmammalian Animals

This chapter traces the origin and progress of nuclear transfer that later became the paradigm for cloning animals. Classic studies in cytology, embryology, or genetics spanning more than five centuries that led to nuclear transfers in unicellular animals and to those in oocytes of insects, fish and amphibians are reviewed. The impetus for the development of successful nuclear transfers in amphibian oocytes in 1952 was to determine whether or not differentiated somatic cell nuclei are developmentally equivalent to zygote nuclei. Experiments in amphibians demonstrated several important results: (1) specialized somatic cell nuclei are extensively multipotent; (2) fertile adult amphibians can be cloned from embryonic and larval nuclei; (3) serial cloning expands the number of clones; (4) transplanting nuclei into oocyte cytoplasm induces reprogramming of their gene function; and (5) amphibian cloning became the model for cloning mammals. Subsequent studies in mice, a more technically favorable species, revealed that specialized cell nuclei are equivalent to zygote nuclei.