INITIATION OF RIBOSOMAL RNA SYNTHESIS AND CELL DIVISION IN XENOPUS LAEVIS EMBRYOS

Isolated cells from animal or vegetal pole regions of Xenopus blastulae were cultured, and the timings of rRNA synthesis and cell division were determined. rRNA synthesis was measured by the incorporation of (³H)methionine into rRNA, and cell division was studied by the decrease in cell size and rRNA content. Nucleoli in these cells were also examined. It was found that these animal and vegetal cells continue to divide under the present conditions in the same temporal pattern as that of intraembryonic cells, and that rRNA synthesis begins soon after the cells have undergone the division which probably corresponds to the 15th division following fertilization. At this time, the rRNA content and concentration of the animal and vegetal cells are significantly different. These results seem to support the view that cell division is involved in some way in the mechanism determining the timing of rRNA synthesis in early embryonic development.     

THE TIMING OF MEIOSIS AND DNA SYNTHESIS DURING EARLY OOGENESIS IN THE TOAD, XENOPUS LAEVIS

Recently metamorphosed female Xenopus laevis toads were injected with tritiated thymidine. Animals were kept at 20°C and were sacrificed 1–23 days after isotope injection. Radio-autographs of squash preparations of the ovaries were made. The progress of labeled germ cell nuclei was followed to obtain information on the time course of early meiosis and extra-chromosomal DNA synthesis. Premeiotic S was estimated to take not more than 7 days. Leptotene takes 4 days, zygotene takes 5 days, and pachytene was estimated to be completed in about 18 days. The major period of amplification of the extrachromosomal DNA occurs in pachytene and takes about 13 days. A low level of synthesis was observed before and after this period, in zygotene and late pachytene-early diplotene, extending the total time for extrachromosomal DNA synthesis during meiosis to about 18 days. These data allowed the calculation to be made that one round of replication of the amplified DNA takes between 1.2 and 3.0 days. It was also found that in both oogonial and premeiotic interphases, the nucleolus-associated DNA shows asynchronous (probably late) labeling with respect to the chromosomes.     

CONTROL OF 5S RNA SYNTHESIS DURING EARLY DEVELOPMENT OF ANUCLEOLATE AND PARTIAL NUCLEOLATE MUTANTS OF XENOPUS LAEVIS

Ribosomes of all eukaryotes contain a single molecule of 5S, 18S, and 28S RNA. In the frog Xenopus laevis the genes which code for 18S and 28S RNA are located in the nucleolar organizer, but these genes are not linked to the 5S RNA genes. Therefore the synthesis of the three ribosomal RNAs provides a model system for studying interchromosomal aspects of gene regulation. In order to determine if the synthesis of the three ribosomal RNAs are interdependent, the relative rate of 5S RNA synthesis was measured in anucleolate mutants (o/o), which do not synthesize any 18S or 28S RNA, and in partial nucleolate mutants (p^l-1/o), which synthesize 18S and 28S RNA at 25% of the normal rate. Since the o/o and p^l-1/o mutants have a complete and partial deletion of 18S and 28S RNA genes respectively, but the normal number of 5S RNA genes, they provide a unique system in which to study the dependence of 5S RNA synthesis on the synthesis of 18S and 28S RNA. Total RNA was extracted from embryos labeled during different stages of development and analyzed by polyacrylamide gel electrophoresis. Quite unexpectedly it was found that 5S RNA synthesis in o/o and p^l-1/o mutants proceeds at the same rate as it does in normal embryos. Furthermore, 5S RNA synthesis is initiated normally at gastrulation in o/o mutants in the complete absence of 18S and 28S RNA synthesis.     

STUDIES ON THE GASTRULATION OF AMPHIBIAN EMBRYOS: NUMERICAL CRITERIA FOR STAGE DETERMINATION DURING BLASTULATION AND GASTRULATION OF XENOPUS LAEVIS

The changing external features of Xenopus laevis embryos were examined from the first cleavage through the late gastrula as a means to develop quantitative criteria for the determination of stages for this period. During blastulation, the cell number along arcs centered on the animal or vegetal pole and representing one-sixth of a meridian - called the MCNo. - can be used as such a criterion. During gastrulation, the width of the blastopore divided by the diameter of the whole embryo can be used as such a criterion.     

ULTRASTRUCTURE OF THE 'GERMINAL PLASM' IN XENOPUS EMBRYOS AFTER CLEAVAGE

The endodermal location of 'germinal plasm'-bearing cells (GPBCs) and the ultrastructure of the 'germinal plasm' were studied in Xenopus laevis embryos at gastrula, neurula, tailbud and younger tadpole stages. Primordial germ cells (PGCs) of feeding tadpoles were also observed ultrastructurally.
GPBCs were found in the inner endoderm and in the yolk plug region at the late gastrula stage, in the middle and in the dorsal part of the endoderm cell mass at the late neurula and late tailbud stages, respectively. At the younger tadpole stage they were observed in the uppermost dorsal part of the endoderm. Germinal granules were always present in GPBCs at all stages examined but were not found in PGCs of feeding tadpoles. Irregularly shaped-stringlike bodies (ISBs) which seemed to have changed from germinal granules were first noticed in GPBCs at the late neurula stage, and were still present in PGCs of tadpoles, while 'granular materials' were not seen in GPBCs until the feeding tadpole stages. These facts and ultrastructural similarities shared by these organelles lead us to conclude that the change of the germinal granule through ISB, to the 'granular material' takes place during the differentiation of GPBCs into PGCs.     

SCANNING ELECTRON MICROSCOPICAL APPEARANCE OF CELLS FROM XENOPUS LAEVIS EMBRYOS OF DIFFERENT STAGES

The scanning electron microscopical appearances of cells isolated from different regions of Xenopus laevis embryos of different stages, and cultured in vitro have been compared. Blastula inner ectoderm cells initially show filopodia, then become flattened onto the substrate and then form pseudopodia. Blastula outer ectoderm cells are initially similar, but do not form pseudopodia. Most of the ectoderm cells from gastrulae and neurulae are featureless. Endoderm cells from blastulae do not initially form filopodia, but later form pseudopodia. Most of the endoderm cells from gastrulae and neurulae show neither filopodia nor pseudopodia, but in the gastrula some elongated, cylindrical cells are observed. Thus cells change their appearance after the three hour culture period; cells from different regions of embryos of the same stage show different appearances in vitro ; and cells from equivalent regions of embryos of different stages show different behaviours in vitro.     

CORTICAL GRANULES PERSISTING IN GERM CELLS OF XENOPUS LAEVIS AFTER THE CLEAVAGE STAGES

Cortical granules were demonstrated, in two successive Epon sections (0.7 μm thick) stained with PAS reagent and the triple staining method respectively, to persist beyond the cleavage stages of development to the tadpole stages in Xenopus laevis. They were also examined by electron microscope. The granules which are similar both cytochemically and ultrastructurally to the cortical granules of the unfertilized eggs were observed not only in germ cells, pPGCs and PGCs, but also in somatic cells at all the stages examined. An ultrastructural similarity between the granules found in the PGCs at the tadpole stages and chromatoid body was discussed.     

A METHOD FOR THE REMOVAL OF THE JELLY AND VITELLINE MEMBRANE FROM THE EMBRYOS OF XENOPUS LAEVIS

A simple procedure is described for removing the jelly and vitelline membrane of Xenopus laevis embryos. The method is based on the observation that incubation of the embryos in the mixed solution of trypsin and sodium thioglycolate at pH 8.0 causes effective dissolution of these structures. This solution is equally effective in this respect on the embryos at different developmental stages. Normal development is obtained from all of the denuded neurulae and from many of the denuded earlier embryos. Some chemical properties of the jelly and the vitelline membrane of Xenopus laevis are discussed based upon these observations.     

XENOPUS LAEVIS AND DEVELOPMENTAL BIOLOGY

1. Because of the present popularity of Xenopus laevis for research in developmental biology, a review of the literature on this animal has been undertaken which emphasizes the anatomical, physiological and developmental features in which it differs from other anuran Amphibia. The need for caution in generalizing from observations on Xenopus to other vertebrates is stressed. 2. Earlier literature and the use of Xenopus for pregnancy testing have been surveyed briefly. Some of the peculiarities of this genus are: the prevalence of pulmonary rather than cutaneous or branchial respiration in the larva, with concomitant modifications of the vascular system; the larval filter-feeding mechanism; the unusual development of the forelimbs, outside the gill chamber; and a number of features of musculature and skeleton in the adult which may be regarded either as primitive or as neotenous, or as specializations for aquatic life. Urodele-like features of the morphology of the pituitary and pineal glands are also mentioned. 3. Recent work on the germ cells and their origin in Xenopus is reviewed in Section III. The germ plasm has been traced from early cleavage stages into germ cells whose identity and genetic characteristics may be traced by reciprocal transplants between anucleolate and normal Xenopus. This plasm is thought to contain redundant copies of DNA from the maternal oocyte, which may thus get passed on to the next generation. During oogenesis, yolk proteins originate from maternal liver protein, and both yolk platelets and pigment granules appear to form in association with mitochondria. The yolk platelets evidently contain both DNA and RNA, and the mitochondria also contain both DNA, of a circular form, and ribosomal RNA. In the oocyte nucleus, special interest has been focused recently on the extrachromo-somal DNA which arises from the nucleolar organizer regions of chromosomes. This DNA later forms the cores of the nucleoli. A number of synthetic processes can take place in the oocyte cytoplasm in the absence of the nucleus, and in the presence of foreign messenger-RNA. Ribosomal RNA synthesis shows at first an excess of 5 s over 18 s and 28 s forms. 4. Spermatogenesis has been studied little in Xenopus. Two unusual features are the absence of seminal vesicles for sperm storage and the spiral shape of the sperm head. By techniques involving destruction of the female pronucleus with ultraviolet light, or suppression of polar-body formation, androgenetic haploids, as well as triploids and tetraploids, have been produced in this species. Paternal genes begin to act at the onset of gastrulation, when nucleoli appear and major rRNA synthesis begins. This situation is sometimes presumed to typify events in all Amphibia -perhaps all vertebrates - but the assumption is unjustified, since in mammals there is much variation in the time of onset of rRNA synthesis, from the evidence so far available. 5. During cleavage in Xenopus, which appears to follow the same pattern as in other Amphibia, septate junctions may serve as channels of communication between the cells. Cytoplasmic DNA is a source for the nuclear DNA synthesis, and the total DNA per cell decreases. As shown by nuclear transplantation experiments, cleavage nuclei, like those of later embryonic stages, remain capable of initiating development in an enucleated egg. Egg cytoplasm can also initiate DNA and RNA synthesis in adult nuclei. 6. Gastrulation in Xenopus is unusual in that the mesoderm migrates forward below the surface and the dorsal lip is lined superficially by endoderm. Neural inductors have been extracted from the dorsal lip of Xenopus, but have not been analysed biochemically. By the end of gastrulation the induced ectoderm is synthesizing high-molecular-weight RNA and also shows increased quantities of three antigenic proteins. 7. In the early processes of differentiation of tissue primordia, regional differences in rates of yolk breakdown, proteolysis, amino-acid activation, tRNA characteristics and rates of incorporation of individual amino acids into protein may be demonstrated. There are also differences in antigens and in isoenzyme patterns. One peculiar morphological feature of early tissue development is the rotatory mode of somite-formation, not so far seem in any other vertebrate. 8. Among several organs whose development has been studied in some detail in Xenopus are: the granular skin glands, which arise from clones of cells; the lateral-line organs, which persist in the adult and are controlled by sensory and motor nerves; and the epidermal cells, which transmit electrical discharges, probably through their zonulae occludentes. In connexion with the filter feeding, the gut is ciliated in the larva: so also are the pronephric ducts. The growth of the pronephros appears to be controlled by a tissue-specific ‘chalone’. 9. The development of the eye in Xenopus normally entails induction of the lens by the eye-cup, as in other vertebrates, but independent ‘free lenses’ may form, by aggregation of epidermal cells instead of invagination from a placode, when the eye-cup rudiment is removed. In the development of the retina there is little evidence of the large-scale cell death described in other vertebrates. Topographical relations between retina and tectum appear to be established long before the full complement of cells is present in either organ. This and other recent experimental evidence suggests that there are no specific point-to-point retinotectal connexions. 10. Studies of the development of motor and sensory elements in the spinal cord of Xenopus showed that there were some early sensory cells lying dorsomedially, and also that the proximal regions of the motor roots were orientated longitudinally: both features are unique to Xenopus. As in Urodela, ablation of the limb causes reduction in size of the lumbar motor horns: in Xenopus it has been shown that there is also increased cell death in the sensory ganglia. 11. Like other Amphibia, Xenopus can regenerate central nervous system, limbs and the lens of the eye. Limb regeneration is somewhat better than in other Anura but gradually declines with increasing age after metamorphosis and also with increasingly proximal levels of amputation. The lens may regenerate from the cornea, the neural retina or the iris, and the regenerates soon acquire lens antigens. 12. Events at metamorphosis in Xenopus are controlled by interactions between the anterior pituitary and the thyroid, as in other Amphibia: cells secreting thyroid-stimulating hormone and thyroid-releasing factor have been identified in the anterior lobe. In response to thyroxine, the isolated tail regresses in organ culture, and this regression is accompanied by increases in the activities of lytic enzymes. 13. Some physiological features of metamorphosis peculiar to Xenopus are: the continued increase in serum proteins for some time afterwards; the more gradual changes in haemoglobin than in other Anura; and the continued excretion of more ammonia than urea. Under conditions of dehydration, however, carbamyl phosphatase activity in the liver increases and a higher proportion of urea is produced. 14. It is concluded that the preferential use of Xenopus for research in developmental biology since the 1950s has led to some important advances in knowledge, but that there is now a need to use other species in order to find out to what extent the same mechanisms operate in them as in Xenopus.     

BIOCHEMICAL AND CYTOLOGICAL EXAMINATION ON THE INITIATION OF RIBOSOMAL RNA SYNTHESIS DURING GASTRULATION OF XENOPUS LAEVIS

Embryos of Xenopus laevis at stage 6 were labeled with ¹⁴CO₂ for 4 hr and then allowed to develop under nonradioactive conditions until they reached stage 9, 10, 11 or 12. RNA was extracted and electrophoresed on a polyacrylamide-agarose gel. From the pattern of newly synthesized RNAs, the incorporation into 18S and 28S ribosomal RNAs was measured. At the same time, the specific radioactivity of nucleoside triphosphates in the acid-soluble fraction was determined. On the basis of the results obtained, the absolute amounts of the RNAs synthesized were calculated. The results show that the synthesis of the ribosomal RNAs begins, or is at least markedly activated, around stage 10. Moreover, cytological examination has shown that cells with nucleolated nuclei appeared between stages 9 and 10 and increased thereafter.
Thus, from the results of these studies along two different lines, it can safely be concluded that the initiation of 18S and 28S RNA synthesis takes place around stage 10.     

金黄仓鼠早卵裂期胚胎发育的研究

研究结果证明:(1)本研究制备和采用的金黄仓鼠(Mesocricetus aurilus)血清是体外培养金黄仓鼠胚胎的有效成分;(2)丙酮酸钠和乳酸钠加入TC-199液中可显著提高金黄仓鼠胚胎的发育率;(3)TC-199(Ⅱ)、台氏液和杜氏液加适量FCS或PHS都是培养金黄仓鼠胚胎的有效培养液;(4)本研究中已观察到4-细胞期胚胎在体外培养时的进一步发育;(5)金黄仓鼠2-细胞期胚胎移植至雌金黄仓鼠受体的输卵管中可继续发育,培养子兔输卵管中可存活44小时以上。     

爪蟾脊索细胞的决定

在两栖类,关于脊索对邻近组织的作用有大量的研究工作,但是,相反,关于邻近组织对脊索的影响过去几乎没有任何报道。因此,本工作企图通过外植的方法,研究脊索在早期发育是是如何决定的,是否也受邻近细胞的影响。结果发现,脊索的决定是一个渐进的过程,并提供了研究邻近组织影响的线索。     

爪蟾(Xenopus laevis)的四倍体及其子一代

在爪蟾受精卵第一次有丝分裂中期进行静液压处理,抑制细胞质分裂从而得到四倍体。实验中获得三只存活了三年以上的四倍体雌性爪蟾。其中一只雌性与二倍体雄性爪蟾作人工催青得到受精卵。现已产卵三批,发育的胚胎400余,经检查子代染色体为三倍体。     

OBSERVATIONS ON EARLY GERM CELL DEVELOPMENT AND PREMEIOTIC RIBOSOMAL DNA AMPLIFICATION IN XENOPUS LAEVIS

The induction of acute hepatocellular necrosis in rats resulted in the production of complement fixing, IgM autoantibodies directed toward inner and outer mitochondrial membranes, microsomal membrane, lysosomal membrane, nuclear membrane, cytosol, but not to plasma membrane. Utilizing selective absorption procedures it was demonstrated that each subcellular membrane fraction possessed unique autoantigenic activity with little or no cross-reactivity between the various membrane fractions. It is proposed that the development of membrane-specific autoantibodies may provide an immunological marker useful in the differential characterization of various subcellular membranes.     

LOCALIZATION OF DYNEIN IN SEA URCHIN EGGS DURING CLEAVAGE*

Detection and localization of dynein in cleaving sea urchin eggs were attempted using antidynein serum (prepared against a tryptic fragment of dynein, Fragment A, of sea urchin sperm flagella) and fluorescein conjugated goat antiserum to rabbit γ-globulin. In both unfertilized and newly fertilized eggs, fluorescence was distributed rather uniformly within the cells but was absent from the nuclei. At prophase, intense fluorescence was observed on both sides of nucleus, suggesting accumulation of dynein in developing asters. From metaphase to anaphase, the whole mitotic apparatus (MA) was stained with the exceptions of the chromosomes and pole areas. Fluorescence then again became dispersed within the eggs. Throughout the mitotic process and cytokinesis, the egg cortex including the cleavage furrow was stained intensely, presumably reflecting the presence of dynein in this region. Similar distributions of fluorescence were obtained with the isolated MAs. Neither non-immune serum nor the antiserum to which Fragment A was absorbed stained the eggs. Little staining was obtained with the antiserum against starfish egg myosin. The results, together with the finding that the chromosome motion in the isolated MAs was completely inhibited by anti-dynein serum, but not with the anti-myosin serum, suggest an active role played by a tubulin-dynein system in mitosis.     

爪蟾(Xenopus laevis)的雄性二倍体成体的产生

野生型爪蟾的色素基因是显性,a~p白化型的色素基因是隐性。用a~p型精子与野生型卵子受精,以UV照射卵子使卵核失活,再用静液压方法抑制受精卵的第一次核分裂,结果产生雄核发育的二倍体(androgenetic diploid)。它们是核质杂种。这些杂种发育时黑色素细胞出现时期同野生型一致。色素细胞的数目和颜色介于野生型和白化型之间。当白化型蝌蚪本来就极少的色素细胞消退时,杂种蝌蚪仍有许多色素细胞,直至变态完成两个月后色素才完全消退。因此核质杂种黑色素细胞的发育并维持到变态后,归因于野生型卵细胞质对a~p型核的作用。??实验还证明了仅具有父本基因组的爪蟾能发育至成体,性成熟,均为雄性。其精子与正常卵结合产生发育正常的下一代。现已变态成为幼蟾。     

爪蟾脊索在决定过程中和相邻细胞的关系

已经知道,对预定脊索的决定起重要作用的是位于它两侧的预定肌节。电子显微镜的观察指出,预定脊索和肌节细胞相互靠得很近,或者相隔一定距离,以突起相连形成腔隙。有被小窝和小泡在两类细胞的外缘常被观察到。最引人注意的是在肌节细胞近腔隙的部位或者附近,球状体的出现。它们大小不等,内含物主要是颗粒,有的松散分布,有的致密地充满整个球状体。这些颗粒的大小和电子染色与这时期胚胎细胞中的核糖体很相似。在预定脊索细胞中以及附近,未见上述结构,但是,观察到它们伸出突起包吞腔隙中物质的现象。讨论了这些球状体的出现与脊索决定之间可能存在的关系。     

CHANGES IN CELL FINE STRUCTURE DURING LENS REGENERATION IN XENOPUS LAEVIS

Changes at the level of cell fine structure have been studied during lens regeneration in the toad, Xenopus laevis, where cornea gives rise to the new lens. The transformation of these cells may be divided into three phases. (1) In the cornea, flattened cells become cuboidal and rough endoplasmic reticulum increases in amount. (2) In the new lens vesicle, cisternae of the rough ER break down into vesicles, smooth-walled vesicles and free ribosomes increase in number, and mitochondria can become enlarged and irregular, then centrally attenuated. Rudimentary cilia form. (3) As new lens fibers form, ribosomes become very numerous and low density fibrous elements and dense clumps appear in the cytoplasm. These phases are accompanied by marked nucleolar changes. The changes during the 3rd phase are similar to changes in the lens during normal development. The first two phases show an unexpected morphological complexity.