Development of the primary mouth in Xenopus laevis

The initial opening between the gut and the outside of the deuterostome embryo breaks through at the extreme anterior. This region is unique in that ectoderm and endoderm are directly juxtaposed, without intervening mesoderm. This opening has been called the stomodeum, buccopharyngeal membrane or oral cavity at various stages of its formation, however, in order to clarify its function, we have termed this the "primary mouth". In vertebrates, the neural crest grows around the primary mouth to form the face and a "secondary mouth" forms. The primary mouth then becomes the pharyngeal opening. In order to establish a molecular understanding of primary mouth formation, we have begun to examine this process during Xenopus laevis development. An early step during this process occurs at tailbud and involves dissolution of the basement membrane between the ectoderm and endoderm. This is followed by ectodermal invagination to create the stomodeum. A subsequent step involves localized cell death in the ectoderm, which may lead to ectodermal thinning. Subsequently, ectoderm and endoderm apparently intercalate to generate one to two cell layers. The final step is perforation, where (after hatching) the primary mouth opens. Fate mapping has defined the ectodermal and endodermal regions that will form the primary mouth. Extirpations and transplants of these and adjacent regions indicate that, at tailbud, the oral ectoderm is not specifically required for primary mouth formation. In contrast, underlying endoderm and surrounding regions are crucial, presumably sources of necessary signals. This study indicates the complexity of primary mouth formation, and lays the groundwork for future molecular analyses of this important structure.     

Development of behaviour in the hindlimb of Xenopus laevis

The paper is one of a series of studies of the ontogeny of the innervation of the vertebrate limb in which the histogenesis of the nerves is correlated with the development of the pattern of behaviour in the limb. Here, the motility of the developing limb in tadpoles of Xenopus laevis is described, both in the normal larva and those in which the spinal cord is isolated from the brain. In spinal tadpoles the responses of the limb to electrical stimulation are correlated with its normal behaviour.     

Protamine polymorphism in Xenopus laevis laevis

Protamines from individual frogs of the subspecies Xenopus laevis laevis were compared by electrophoresis on polyacrylamide gels containing acetic acid, urea, and Triton X-100 to determine if the expression of protamine genes differs among individuals. Two electrophoretic bands, SP2a and SP2b, appeared to be expressed as allelic variants. Of 33 frogs, 19 expressed only SP2a, 11 expressed both SP2a and SP2b, and three expressed only SP2b. Electrophoretic analysis of partial V8 protease digests could not distinguish the peptides released from SP2a and SP2b. Differences in sperm development between individuals were not detected by light or electron microscopy. The results suggest that protamine polymorphism can exist among individuals of a species without an apparent effect on sperm development or sperm function.     

Development of functional sex differences in the larynx of Xenopus laevis

Three laryngeal properties associated with the production of masculine song--laryngeal muscle tension, fiber twitch type, and fiber recruitment--are markedly sexually dimorphic in adult Xenopus laevis frogs. To elucidate the pattern of sexual differentiation, tension and fiber recruitment in male and female larynges and fiber twitch type in male larynges were examined throughout postmetamorphic development. Masculinization of male laryngeal properties begins early in postmetamorphic development and continues until adulthood. In contrast, tension and fiber recruitment in females do not change after the end of metamorphosis. Laryngeal muscle tension and fiber type are gradually and progressively masculinized; the temporal pattern of masculinization is very similar for these properties. Fiber recruitment, on the other hand, appears to masculinize in a stepwise manner. Masculinization of all three properties is highly correlated with larynx weight in males. We have used this relation to divide postmetamorphic development into seven stages associated with key events in sexual differentiation. This staging scheme provides an important experimental tool for studying the hormonal regulation of sexual differentiation, the subject of the accompanying paper.     

Cross-fertilization and structural comparison of egg extracellular matrix glycoproteins from Xenopus laevis and Xenopus tropicalis

While the anuran amphibian Xenopus laevis is a widely used vertebrate model system, it is not optimal for genetic manipulations due to its tetraploid genome and long generation time. A current alternative amphibian model system, Xenopus tropicalis, has the advantages of a diploid genome and a much shorter generation time. We undertook a comparative investigation of X. tropicalis egg extracellular matrix glycoproteins in relation to those already characterized in X. laevis. Fertilization methods and isolation of egg extracellular molecules were directly transferable from X. laevis to X. tropicalis. Cross-fertilizations were successful in both directions, indicating similar molecules involved in sperm-egg interactions. Egg envelopes analyzed by SDS-PAGE were found to have almost identical gel patterns, whereas jelly component profiles were similar only for the larger macromolecules (>90 kDa). The cDNA sequences for egg envelope glycoproteins ZPA, ZPB, ZPC, ZPD and ZPAX, and also egg cortical granule lectin involved in the block to polyspermy, were cloned for X. tropicalis and showed a consistent approximately 85% amino acid identity to the X. laevis sequences. Thus, homologous egg extracellular matrix molecules perform the same functions, and the molecular and cellular mechanisms of fertilization in these two species are probably equivalent.     

Endogenous lectin secretion into the extracellular matrix of early embryos of Xenopus laevis

An endogenous galactoside-binding lectin with subunit molecular weight of 43,000-45,000, previously detected in unfertilized eggs of Xenopus laevis, persists at high levels in embryos through gastrulation. During embryonic development the lectin is found in cytoplasmic vesicles, and then is secreted into extracellular matrix which is prominent around the blastopore and on the roof of the blastocoel. The lectin is also found in the extracellular material in the developing neural fold. The presence of lectin at sites of active morphogenetic movements raises the possibility that it participates in the formation of an extracellular matrix that influences these processes.     

Proteomic analysis of the porcine interphotoreceptor matrix

The interphotoreceptor matrix (IPM) is located between photoreceptors and pigment epithelium in the retina and is involved in fundamental functions of the visual cycle. These include visual pigment chromophore exchange, retinal adhesion, metabolite trafficking, and growth factor presentation. In general, IPM preparations are contaminated with intracellular proteins, as has also been described for other body fluids. This study aimed at identifying new components of the IPM by discriminating between truly secreted proteins and proteins that are part of the IPM for secondary reasons. "Soluble" porcine IPM was extracted from retina and pigment epithelium with PBS by two different procedures, followed by extraction with water alone that released "insoluble" IPM matrix sheets. Samples from all preparations were separated by 2-DE and a total of 140 protein spots were identified by MALDI-TOF and/or CapLC Q-TOF MS. Although identified proteins included several already known in the IPM, the majority had not been previously described in this structure. Gene ontology classifications allocated the identified proteins into nine different functional networks. The IPM preparations also included intracellular proteins from cells adjacent to the IPM, which may have resulted from cell disruption. This underlines the experimental difficulties of a biochemical analysis of the IPM as an intact compartment. We show here a strategy for predicting the probability of identified IPM proteins occurring in vivo by combined high-resolution protein separation methods with computational prediction methods. Thus, a set of potentially neuroprotective proteins could be extracted, including PEA-15, peroxiredoxin 5, alpha-B-crystallin, macrophage migration inhibitory factor, 78 kDa glucose-regulated protein (GRP78), protein disulfide-isomerase, and PEP-19, which have not been previously associated with the IPM. Furthermore, with immunohistochemical staining we could confirm the localization of GRP78 in the IPM on porcine eye sections, thus validating the proposed prediction method.     

Changes in Activity of Mitochondrial Enzymes during the Development of Xenopus laevis

Changes in activity of mitochondrial enzymes were studied during the embryonic development of Xenopus laevis.
The following enzymes were determined: malate dehydrogenase (MDH), isocitrate dehydrogenase (NAD⁺-dependent) (IDH), aspartate aminotransferase (GOT), cytochrome oxidase (COX), succinate dehydrogenase (SDH), rotenone-insensitive NADH cytochrome c reductase (NADH-red) and monoamine oxidase (MAO). IDH is constant throughout the period studied. COX and SDH, two enzymes of the inner membrane, are constant in pregastrula stages, and subsequently decrease significantly. MDH and NADH-red are highly active in the pregastrula stages and decline thereafter, while MAO is undetectable during early development and increases significantly only in the larvae. GOT increases during the cleavage stages, being most active in the gastrula stages, and decreases subsequently.
The results are discussed in the sense of mitochondrial differentiation during the early development of the amphibian embryo.     

非洲爪蟾眼的形态发生研究

详细观察和描述了非洲爪蟾Xenopus laevis眼的发生和发育变化过程,并分别对各发育时期视网膜的厚度进行了定量分析.非洲爪蟾眼的发牛开始于眼原基的形成,进而形成视泡;晶状体的发生是在视杯外壁增厚的同时诱导覆盖其上的胚胎外胚层内层增厚,形成预定晶状体板;在视网膜和晶状体共同诱导下,预定角膜上皮变为透明的角膜.在视杯出现之前,预定RPE的厚度由厚变薄,NR层不断地增厚直至结构功能完善.     

Telomere Variation in Xenopus laevis

Eukaryotic telomeres are variable at several levels, from the length of the simple sequence telomeric repeat tract in different cell types to the presence or number of telomere-adjacent DNA sequence elements in different strains or individuals. We have investigated the sequence organization of Xenopus laevis telomeres by use of the vertebrate telomeric repeat (TTAGGG)_n and blot hybridization analysis. The (TTAGGG)_n-hybridizing fragments, which ranged from less than 10 to over 50 kb with frequently cutting enzymes, defined a pattern that was polymorphic between individuals. BAL 31 exonuclease treatment confirmed that these fragments were telomeric. The polymorphic fragments analyzed did not hybridize to 5S RNA sequences, which are telomeric according to in situ hybridization. When telomeric fragments from offspring (whole embryos) were compared to those from the spleens of the parents, the inheritance pattern of some bands was found to be unusual. Furthermore, in one cross, the telomeres of the embryo were shorter than the telomeres of the parents’ spleen, and in another, the male’s testis telomeres were shorter than those of the male’s spleen. Our data are consistent with a model for chromosome behavior that involves a significant amount of DNA rearrangement at telomeres and suggest that length regulation of Xenopus telomeres is different from that observed for Mus spretus and human telomeres.     

Gene-centromere mapping in Xenopus laevis

Gynogenesis was used to map eight loci to their centromeres in Xenopus laevis. Several loci remote from their centromeres were identified. This information may be useful in distinguishing gynogenetic diploid progeny produced by suppression of second polar bodies from gynogenetic diploid progeny homozygous at all loci produced by suppression of first cleavage of gynogenetic haploids.     

Subdivisions of the terminal nerve in Xenopus laevis

The majority of recent studies on the terminal nerve (nt) in various vertebrates either involved tracer injections into the nasal cavity or made use of the LHRH-/FMRFamide-like immunoreactivity (ir) of a portion of its fibers. The present investigation was designed to determine the extent of overlap between data rendered by the two methods in Xenopus. The findings reveal no overlap of nt projections visualized by the two experimental techniques. This result sheds doubt on the validity of current definitions of the nt system.     

NeuN immunoreactivity in the brain of Xenopus laevis

Neuronal nuclear antigen (NeuN), discovered in mice brain cell nuclei by Mullen et al. (1992), is used as an excellent marker of post-mitotic neurons in vertebrates. In this study, the expression pattern of NeuN was examined in the Xenopus brain to explore phylogenetic differences in NeuN expression. Anti-NeuN antibody showed selective staining in mouse and Xenopus brain extracts, but the number and molecular weight of the bands differed in Western blotting analysis. In immunostaining, anti-NeuN antibody showed selective staining of neurons, but not glial cells, in the Xenopus brain. Most neurons, including olfactory bulb mitral cells and cerebellar Purkinjie cells, which show no immunoreactivity in birds/mammals, showed NeuN immunoreactivity in Xenopus. This study revealed that anti-NeuN antibody is a useful marker of post-mitotic neurons in amphibians, but it also stains neurons that show no reactivity in more derived animals.