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.     

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。     

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。     

KINETIC ANALYSIS OF ENTIRE OOGENESIS IN XENOPUS LAEVIS

In our breeding conditions (without artificial hormonal stimulation) two years after metamorphosis are necessary for a Xenopus laevis female to produce mature oocytes. Four periods of different growth rates can be distinguished into this first wave of oogenesis: 1) oocytes reach 120 μm in diameter (early stage I according to D umont (5)) in a few weeks after metamorphosis; 2) growth slows down and the size of 250–300 μm (late stage I) is obtained 6–7 months later; 3) a rapid growth resumes during vitellogenesis (stages II, III and IV) and a 1,000–1,100 μm diameter is reached in 2–3 months; 4) the last period spans for a year and the oocyte grows up to 1,200 μm. This phase covers many physiological changes and it should be critical to size carefully the oocytes for biochemical investigations and comparisons.
At last most of the oocytes in a young female do not proceed through this entire oogenesis, they are stopped at the end of the second growth phase (about 250 μm in diameter).     

THE ANATOMY OF PACHYDERMIA LAEVIS (ARCHAEOGASTROPODA: 'PELTOSPIRIDAE')

The anatomy of Pachydermia laevis Warén & Bouchet,1989 is investigated. It is a deep-sea gastropod and restrictedto hydrothermal vents of the East Pacific Rise. Its anatomyresembles that of Melanodrymia aurantiaca Hickman, 1984 (Melanodrymiidae)in most respects, except that its gonopericardial duct opensinto the renopericardial duct, not into the peri-cardial chamber,and that it lacks a copulatory organ. Examination of M. aurantiacamain confirms earlier work. The two species have a cerebralbuccal connective fused with the cerebral ganglion (not freefrom it), a synapomorphy that has not been described for anyother archaeogastropod. The investigation suggests that Melanodrymiaand Pachydermia are closely related and together with Cyathermiidaeand Neomphalidae form a monophyletic group within the Neomphalina.No evidence was found to support earlier speculations aboutclose relations of Neomphalina to Viviparoidea. (Received 10 January 1996; accepted 3 April 1997)     

CYCLIC SURFACE CHANGES IN THE NON-NUCLEATE EGG FRAGMENT OF XENOPUS LAEVIS

Fertilized uncleaved eggs of Xenopus laevis were divided into nucleate and non-nucleate egg fragments. Both fragments, together with the whole egg of the same batch, were observed by time-lapse cinematography.
Two kinds of cyclic surface changes, (1) rounding-up and relaxing movements and (2) surface contraction waves, accompanying each cleavage in the whole eggs and the nucleate fragments, were also observed even in the non-nucleate fragments although they do not cleave.
Cleavage intervals of the whole egg and the nucleate fragment were nearly equal, but the rounding-up intervals of the non-nucleate fragment were slightly but definitely longer than the cleavage intervals of the nucleate fragment and the whole egg.     

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.     

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.     

APOMIXIS IN AMELANCHIER LAEVIS,SHADBUSH (ROSACEAE,MALOIDEAE)

We studied ovule and megagametophyte development in tetraploid (n = 34) individuals of Amelanchier laevis in Maine. Nomarski differential interference contrast microscopy of cleared, whole ovules and conventional microscopy of sectioned, stained material show no clear evidence for the successful completion of meiosis. Instead, the megasporocyte or its derivatives degenerate and one to six nearby cells develop into aposporous initials. Usually more than one of these divide to form eight-nucleate, Polygonum-type megagametophytes. The egg apparently forms a proembryo parthenogenetically, but seed maturation requires pollination. This evidence for apospory and pseudogamy, the first to be reported in Amelanchier, conforms to the general pattern found in other apomictic genera of the Maloideae.     

PLASTID FATTY ACID BIOSYNTHESIS IN THE DIATOMS NITZSCHIA ALBA AND NITZSCHIA LAEVIS

Among the co-dominant molecular markers, microsatellite loci have a number of advantages in population genetic studies. However, the different methods to hunt these markers are expensive, time consuming, and they require sophisticated laboratory equipment. Using in one species the microsatellites primers originally described for another one saves time and reduces costs. Examples in the literature revealed that microsatellites described for Gracilaria gracilis from France have not worked for other members of the Gracilariales. The results were not very promising, at least for Gracilaria chilensis from Chile. In this study, a number of microsatellite loci described for Laminaria digitata (Laminariaceae) from France were amplified and sequenced in two Lessonia (Lessoniaceae) species from Chile. Preliminary results show a partial conservation of both, flanking and tandem repeat regions. Some polymorphism has also been detected in Lessonia spp. The higher molecular affinity (conservation of primer sites) observed in species belonging to different families of brown algae, respect to that observed among species of the same genus in the red algae, is surprising. Such a result is strikingly counterintuitive when observing the morphological disparity among the Laminariales, respect to the similarity observed in the Gracilariales. It also reminds one of an earlier discussion by Gary Saunders about "When a family is not a family".     

用DNA酶切小分子片段在非洲爪蟾卵提取物中实现非细胞体系核装配

用质粒ｐＢＲ３２２的ＤＮＡ酶切片段,长度分别为７５０、３７５和１８６ｂｐ作为外源ＤＮＡ在非洲爪蟾 卵提取物中实现了非细胞体系的核装配（核重建）。将分离纯化得到的 ＰＢＲ３２２ ＤＮＡ酶切后经低熔 点琼脂糖回收ＤＮＡ片段,长度为７５０、３７５和１８６ｂｐ,分别加入到爪蟾卵提取物再生系统中温育, 经ＤＡＰＩ染色、孚尔根染色及电子显微镜观察发现能装配大量的重建核。显微分光光度法研究表 明,ＤＮＡ片段在诱导形成重建核的过程中发生了明显的凝集－会凝集变化。α－３２Ｐ－ｄＣＴＰ掺入重建核 的液闪计数结果表明,ＤＮＡ酶切小分子片段诱导形成的重建核具有较高的ＤＮＡ复制活性,而且复 制活性在一定范围内与加入的ＤＮＡ片段长度呈正相关。实验结果表明,非细胞体系中诱导的核重 建不仅与外源ＤＮＡ的种类无关,与外源ＤＮＡ的长度也没有关系。     

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.     

移植非洲爪蟾胚胎细胞核引起花背蟾蜍成熟卵单性发育的研究

将非洲爪蟾胚胎细胞核移入花背蟾蜍成熟未受精卵后,得到了发育至各期的胚胎。对发育不同的时期的胚胎,进行了乳酸脱氢酶同工酶谱及染色体组型分析,结果一显示其均与受体一致。根据实验分析认为,非洲爪蟾的胚胎细胞进入花背蟾蜍成熟卵后,引起的是花背蟾蜍的单性发育。