Transplantation studies of a putative lymphosarcoma of Xenopus.

The fate of the putative transplantable Xenopus lymphosarcoma (M. Balls, Cancer Res., 22: 1142-1154, 1962) was studied under three experimental conditions: (a) xenotransplantation, i.e., transplantation of live "tumor" tissue between adults of Xenopus borealis and X. laevis; (b) inoculation of live "tumor" cells from X. borealis into the blastocoele of X. laevis embryos; and (c) transplantation of "tumor" tissue into recipient adults immunologically unresponsive to the donor tissue antigens. This condition is fulfilled by using X. laevis-X. gilli (LG) hybrids [H.R. Kobel and L. Du Pasquier. In: J.B. Solomon and J.D. Horton (eds.), Developmental Immunology, pp. 229-306. The Netherlands: Elsevier/North-Holland Biomedical Press, 1977] as donors, and triploid X. laevis-X. gilli and X. borealis (LGB) hybrids [C.H. Thiébaud, Dev. Biol., 98: 245-249, 1983] as recipients. In all transplantation experiments, donor and recipient cells could be unambiguously distinguished upon quinacrine staining that yields typical nuclear patterns, for instance bright patchiness in X. borealis, visible also in LGB cells. The results of xenotransplantation between X. borealis and X. laevis indicated that all developing "tumors" were composed of the recipient cell phenotype. The inoculation of live "tumor" cells from X. borealis "tumor" into the blastocoele of X. laevis embryos resulted in "tumor" formation in the recipient tadpoles and in metamorphosed animals. The cell constituting these "tumors" all were of recipient, X. laevis cell phenotype. Finally, "tumor" tissues from LG clones transplanted into LGB hosts were replaced by "tumors" formed of cells with recipient, LGB phenotype. These experiments indicate that this Xenopus tumor-like growth is a transmissible and not a transplantable disorder.

Neural crest development in the Xenopus laevis embryo, studied by interspecific transplantation and scanning electron microscopy.

The Xenopus borealis quinacrine marker and scanning electron microscopy have been used to study the appearance, migration, and homing of neural crest cells in the embryo of Xenopus. The analysis shows that the primordium of the neural crest develops from the nervous layer of the ectoderm and consists of three segments at early neurula stages. This primordium is located in the lateral halves of the neural folds behind the prospective eye vesicles. The histological and experimental evidence shows that the neural crest cells also originate from the medial portion of the neural folds. The neural crest segments in the cephalic region start to migrate just before the closure of the neural tube. Isotopic and isochronic unilateral grafts of X. borealis neural crest into X. laevis embryos were performed in order to map the fate of the cranial crest segments and the vagal-truncal neural crest. The analysis of the X. laevis host embryos shows that the mandibular crest segment contributes to the lower jaw (Meckel's cartilage), quadrate, and ethmoid-trabecular cartilages, as well as to the ganglionic and Schwann cells of the trigeminus nerve, the connective tissues, the mesenchymal and choroid layers of the eye, and the cornea. The hyoid crest segment is located in the ceratohyal cartilage and in ganglia VII and VIII. The branchial crest segment migrates from the caudal part of the otic vesicle and divides into two portions which contribute to the cartilages of the gills. The vagal-truncal neural crest starts to migrate later at stage 25. It migrates by means of the vagus complex in a ventral direction and penetrates into the splanchnic layer of the digestive tract. The trunk neural crest cells disperse into three different pathways which differ from those of the avian embryo at this level.

Diploid gynogenesis in Xenopus laevis and the localization with respect to the centromere of the gene for periodic albinism ap.

Diploid gynogenetic Xenopus laevis were obtained by inseminating the eggs with u.v. irradiated spermatozoa, and treating them with hydrostatic pressure to inhibit the expulsion of the second polar body. A u.v. dose of 3000 ergs/mm2 genetically inactivates the spermatozoa without loss of their ability to activate egg development. The use of a genetic marker on very large samples of eggs made it possible to verify the efficiency of the methods employed. The comparison of the development of diploid gynogenetic embryos with that of haploid gynogenetic, triploid and diploid controls makes it very probable that the relatively high mortality or abnormal development obtained with the pressurized eggs is not due to partial homozygosity but rather to physical damage to the egg structure by this treatment. Altogether about 2500 developing eggs were used. In addition diploid gynogenetic reproduction from females heterozygous for a known mutation allows the mapping of the gene concerned relative to the centromere on the basis of the recombination rate. For this experiment we used the recessive mutation causing periodic albinism (ap) and found the position of this gene to be between 44 map units from the centromere and the end of the chromosome.

A reliable new cell marker in Xenopus.

A new reliable and durable method for marking cells in Xenopus is described. It is based on the differential staining of the nuclei of different Xenopus species, e.g., X. laevis and X. borealis, with the fluorescent dye quinacrine. This method permits us to recognize with certainty each cell in mitosis and interphase of X. borealis origin in any tissue combination with most of the other Xenopus species tested so far. This holds for all stages of development following grafting experiments, including adult tissues. The method is applicable in smears and squash preparations as well as in microtome sections. The method is particularly useful for marking migrating cells which are difficult to track, for instance, in embryos and in the circulatory system.

The intra-nucleolar localization of amplified rDNA in Xenopus laevis oocytes.

A fluorescent Feulgen-stain was adapted in order to demonstrate DNA-containing structures inside the amplified nucleoli of Xenopus laevis. At all stages of oogenesis this method reveals granules or complex structures of DNA in each nucleolus. The micronucleoli which do not stain with this method and which do not reveal an internal structure in low molarity saline, unlike real nucleoli are considered as nucleolus-like bodies. The DNA-containing structures in the nucleoli can be composed of one or several granules, or they can be arranged in a linear, reticulated or circular form, independant of any correlation with the stage of oogenesis.

Quantitative determination of amplified rDNA and its distribution during oogenesis in Xenopus laevis.

The number of extra-chromosomal nucleoli and their rDNA content were determined during oogenesis in Xenopus laevis. The highly variable number of nucleoli (500 to 2,500) in oocytes of the same stage and from the same female or of different stages or from different females is not a measure of the extent of amplification. In all oocytes examined, a inversely proportional relation was found between the number of nucleoli in an oocyte and their mean rDNA content. These results indicate that there is no variation of the rDNA content of oocytes during oogenesis nor between oocytes of different females. The varying nucleolar numbers found in oocytes result thus from fusion and fission of pre-existing nucleoli. The determination of the rDNA content, in absolute units (35 pg), after amplification which occurs at the beginning of oogenesis, makes it possible to calculate the rDNA content of one nucleolus. This ranged from 0.7.10(-2) pg to 15.10(-2) pg, corresponding to about 500--11,000 cistrons of rDNA. No distinct size classes between these two extremes were observed.

Cytophotometric study of nuclear differentiation during pollen development in Tradescantia paludosa.

During pollen development the dry weight, total protein, histone, DNA, arginine, and lysine content were analysed by cytophotometric methods in partially isolated nuclei. The amount of analysed substances increased from the end of the meiosis to the mitosis of the microspores to the double of the initial values. After mitosis the ratio histone/DNA remained almost unchanged in both vegetative and generative nuclei. On the other hand a large difference in the ratio non-histone protein/DNA could be observed, the vegetative nucleus containing more non-histone protein than the generative nucleus. The rate of RNA synthesis being higher in the vegetative nuclei, these non-histone proteins may have some function in nuclear activation. The DNA of the generative nucleus is duplicated before anthesis, whilst in the vegetative nucleus the DNA content remains constant.

DNA content in the genus Xenopus.

Nuclear DNA amounts were determined by cytofluorometry for twelve species and subspecies of the genus Xenopus. Absolute values, in pg per nucleus, were obtained by direct comparison with human lymphocyte nuclei. The lowest DNA amount (3.55 pg) was found in X. tropicalis, which possess only 20 chromosomes, and the highest (16.25 pg), in the hexaploid X. ruwenzoriensis, with 108 chromosomes. The two recently discovered tetraploid species, X. sp.n. and X. vestitus have, respectively 12,57 and 12.83 pg of DNA. Among the species and subspecies with 36 chromosomes, the DNA content ranges from 6.35 to 8.45 pg.