A novel POZ/zinc finger protein, champignon, interferes with gastrulation movements in Xenopus.

We have cloned a novel krüppel-like transcription factor of Xenopus that encodes POZ/zinc finger protein by expression cloning. Overexpression of mRNA resulted in interference with gastrulation. Because the injected embryo looks like a mushroom in appearance at the neurula stage, we have named this gene champignon (cpg). In cpg-injected embryos, the blastopore appeared normally, but regressed thereafter. The injected embryos then elongated along the primary dorsoventral axis during the tailbud stage. Histologic sections and reverse transcription-polymerase chain reaction analysis showed that cpg had no effect on the cell differentiation. The animal pole region of cpg-injected embryos was thick during the gastrula stage, and mesodermal cells remained in the marginal zone. Furthermore, neither Keller-sandwich explants nor activin-treated animal cap explants excised from cpg-injected embryos elongated. These results suggest that cpg acts as a potent inhibitor of cell migration and cell intercalation during gastrulation.

Injection of a sperm extract triggers egg activation in the newt Cynops pyrrhogaster.

Unfertilized eggs of the newt Cynops pyrrhogaster are arrested at the second meiotic metaphase. The primary signal for egg activation is a transient increase in [Ca2+](i), which is triggered by the fertilizing sperm and propagates over the egg cortex as a Ca2+ wave. We injected an extract of Cynops sperm (SE) into unfertilized eggs and induced a wave-like [Ca2+](i) increase which resulted in activation and resumption of meiosis. The SE-injected eggs showed degradation of cyclin B1 and DNA replication. When SE was boiled or treated with proteinase K before injection, it was unable to cause egg activation. Preinjection of Ca2+ -chelator BAPTA before SE injection inhibited egg activation. These results indicate that a heat-labile and proteinaceous factor in the sperm cytoplasm induces a transient increase in [Ca2+](i) which is required for egg activation. Injection of IP3 into unfertilized eggs caused an increase in [Ca2+](i) and egg activation, but injection of cADP-ribose did not. These results support the hypothesis that Ca2+ release at fertilization occurs via IP3 receptors.

Expression and characterization of Xenopus type I collagen alpha 1 (COL1A1) during embryonic development.

A cDNA encoding Xenopus type I collagen alpha 1 (Xenopus COL1A1) has been isolated from an ovary cDNA library. The COL1A1 cDNA is approximately 5.7 kb pairs and encodes 1447 amino acids. The putative COL1A1 polypeptide shares high identities of amino acid sequence with other vertebrate COL1A1 proteins. The level of Xenopus COL1A1 transcripts was increased markedly in the posterior region of the embryo at the tail-bud stage, then gradually spread to the anterior region. Histological observations of the tail-bud embryos showed that COL1A1 was mainly expressed in the inner layer of the posterior dorsal epidermis exposed to the somite mesoderm, except for in the dorsal fin. Less intense signals were also detected in the outer layer of the dorsal epidermis and dermatome. The expression of COL1A1 was increased in posteriorized embryos resulting from treatment with retinoic acid but decreased in hyper-dorsalized embryos resulting from lithium chloride treatment. These results suggest that COL1A1 is a major component of the dorsal dermis exposed to the somite in Xenopus embryos, but its expression is not related to the temporal sequence of somite segregation.

Oscillation of intracellular free calcium in cleaving and cleavage-arrested embryos of Xenopus laevis.

Changes in the intracellular free calcium levels in the cortical region of cleaving Xenopus eggs were examined using semi-synthetic aequorin. We detected periodic oscillations in the free calcium levels accompanying the cleavage cycle as early as the first cleavage onward. The calcium levels began to increase at early interphase and were maximal during metaphase of mitosis. Eggs injected with colchicine to inhibit cleavage showed periodic oscillations in the calcium levels similar to those of normal eggs. This fact indicates that the furrowing process is not necessary for the periodic changes in the intracellular free calcium levels, but the changes are associated with the autonomous periodic cytoplasmic activity which controls cell cycle events.

A female sterile mutant, unfertilizable (uf), in Xenopus laevis.

We describe a female sterile mutant, unfertilizable (uf), in Xenopus laevis whose eggs cannot be fertilized by either natural mating or artificial insemination. The uf eggs were able to be activated by pricking with a fine glass needle. Ultraviolet-solubilized jelly from the uf eggs could permit the fertilization of the dejellied wild-type eggs by artificial insemination, while uv-solubilized jelly from the wild-type eggs did not permit the fertilization of the dejellied uf eggs. The uf eggs, whose jelly coats and vitelline envelopes were removed, could be fertilized by insemination in the uv-solubilized jelly from either wild-type or uf eggs. These results show that the eggs and the jelly coats are intact but the vitelline envelopes are abnormal in uf mutant eggs. One- and two-dimensional gel electrophoretic comparisons of vitelline envelopes from wild-type and uf eggs demonstrated that five protein components, having estimated molecular masses of about 26, 64, 69, 78, and 250 kDa, were present in the vitelline envelopes of uf eggs but were absent in those of wild-type eggs. All of these components were polymorphic in terms of isoelectric points. Thus the cause of unfertilizability of uf eggs resides in the vitelline envelopes whose protein components exceed those of the wild-type eggs.

Furrow-related contractions are inhibited but furrow-unrelated contractions are not affected in af mutant eggs of Xenopus laevis.

In embryos from af mutant females of Xenopus laevis, the cleavage furrows stayed on the surface and cytoplasmic divisions did not take place at all, while nuclear divisions continued (Kubota et al., 1991). To gain insights into the roles of the normal product of af on early development, contractile events which have been observed in the period from fertilization until first cleavage in wild-type eggs were examined in af mutant eggs. Activation waves, activation contraction, and surface contraction waves which were identical to those in wild-type eggs were observed in af eggs by time-lapse video recording. However, second polar body elimination was inhibited in af eggs, although a sign of the polar body formation was indicated by the cytoplasmic bulge of the egg surface as seen by light and electron microscopy. These results indicate that the normal product of af regulates furrow-related contractile events which involve formation of the contractile ring, but exerts no effects on furrow-unrelated contractions in early Xenopus eggs.

Cytological and biochemical analyses of the maternal-effect mutant embryos with abnormal cleavage furrow formation in Xenopus laevis.

We describe an embryonic lethal mutation in Xenopus laevis that provokes regression of cleavage furrow formation. The mutant females (designated as af) were obtained by the back-cross of a female with one of her sons. All the fertilized eggs laid by the mutant females, regardless of the wild-type male used in the mating, failed to cleave although each furrow ran at a proper position superficially. Light and electron microscopic observations of the embryos revealed that the cleavage furrows stayed on the surface and cytoplasmic divisions did not take place at all, while nuclear divisions did. Two-dimensional gel-electrophoretic comparisons of af and wild-type embryos demonstrated that two proteins, having estimated molecular masses of about 38 kDa (pI 6.6) and 78 kDa (pI 7.6), were missing in af embryos. Microinjection of clear cytoplasm from a wild-type egg into fertilized af eggs provoked partial surface contraction and cleavage furrow formation in recipient af eggs. The results showed that the af females carry a lethal maternal-effect mutation which causes cleavage furrow regression by being deficient in a few proteins, and that cytoplasm of wild-type eggs can partially rescue the cleavage furrow formation of af eggs by furnishing the corrective material, presumably a product of the normal allele of af.

The expression of epidermal antigens in Xenopus laevis.

Five kinds of monoclonal antibodies that are specific for the epidermis of Xenopus embryos were produced. Epidermis-specific antibodies were used to investigate the spatial and temporal expressions of epidermal antigens during embryonic and larval development. The cells that were recognized by the antibodies at the larval stage are as follows: all of the outer epidermal cells and cement gland cells were recognized by the antibody termed XEPI-1, all of the outer and inner epidermal cells, except the cement gland cells, were recognized by XEPI-2 antibody, the large mucus granules and the apical side of the outer epidermal cells, except for the ciliated epidermal cells, were recognized by XEPI-3 antibody, the large mucus granules and basement membrane were recognized by XEPI-4 antibody, and the small mucus granules contained in the outer epidermal cells as well as extracellular matrices were recognized by the antibody termed XEPI-5. All of the epidermal antigens, except XEPI-4, were first detected in the epidermal region of the late gastrula or early neurula. The XEPI-4 antigen was first detected in stage-26 tail-bud embryos. None of these antigens were expressed by the neural tissues at any time during embryonic development. Only the XEPI-2 antigen continued to be expressed after metamorphosis, while the expression of the other antigens disappeared during or before metamorphosis. The specificity of the antibodies allowed us to classify the epidermal cells into four types in early epidermal development. The four types of epidermal cells are (1) the outer epidermal cells that contain small mucus granules, (2) the ciliated epidermal cells, (3) the outer epidermal cells that contain large mucus granules and (4) the inner sensorial cells.

Free calcium wave upon activation in Xenopus eggs.

Eggs of Xenopus laevis were preloaded with aequorin and the spatial and temporal pattern of free calcium release in the egg cortex on artificial activation was determined by the aequorin luminescence emitted from the thin cortical layer of naturally opaque eggs. The aequorin luminescence was detected with a photonic microscope system consisting of a light microscope and a two-dimensional photon-counting system with an image processor. A free calcium increase was initiated around the point of prick activation. The state of increased Ca2+ propagated in the cortical cytoplasm of the egg as a wave with a velocity of about 8 micron/sec at 22 degrees C. This wave reached the antipode by 5 to 6 min of prick activation. The spatial pattern of the Ca2+ wave was similar to that of changes in brightness of the egg surface on activation, termed the "activation wave" by K. Hara and P. Tydeman (1979, Wilhelm Roux's Arch. Dev. Biol. 186, 91-94). To examine the temporal correlation between the Ca2+ wave and the activation wave, images of aequorin luminescence and those of the egg cortex taken by incident light illumination were recorded alternately in the same egg. The zone of free calcium increase corresponded to the light (relaxation) zone of the activation wave, where exocytosis of cortical granules and elongation of microvilli were taking place.

Structural basis of the activation wave in the egg of Xenopus laevis.

A series of changes in the surface of activated Xenopus eggs was observed. Within a few seconds of prick activation a light area appears near the pricking point and expands as a circular light zone (light wave). Some 60 s later this is followed by a dark area expanding as a circular dark zone (dark wave). Both waves travel at a rate of about 9 micron/s at 21 degrees C. In the light zone, cortical granules are breaking down, microvilli are elongating, and the egg surface is expanded. On the other hand, the elongated microvilli are reshortening to become globular and the egg surface is contracted in the dark zone.

Surface contraction waves in amphibian eggs.

Circular waves of change in brightness, known as 'surface contraction waves' (SCW-1 and SCW-2), propagate over the animal surface of amphibian eggs at each cycle of cleavage. Movement of carbon particles attached to the egg surface indicated that SCW-1 involves expansion of the egg surface, whereas SCW-2 accompanies surface contraction. Stiffness of the cortex as measured by applying negative pressure through a micropipette increased concomitantly with the passage of SCW-2. Measurement of stiffness at two loci on the egg surface with two sets of pipettes confirmed the spatio-temporal coincidence of the wave of stiffness and SCW-2. The stiffness showed either no change or even a slight decrease on passage of SCW-1. Thus SCW-2 is a genuine wave of 'contraction', but SCW-1 can more properly be called a 'surface relaxation wave'.

Temporal pattern of cleavage and the onset of gastrulation in amphibian embryos developed from eggs with the reduced cytoplasm.

Fertilized eggs of the Japanese newt, Cynops pyrrhogaster, were divided into two or four equal-sized parts with fine glass rods before the first cleavage. In such cases one of the egg fragments, at least, proceeds to cleavage and gastrulates. The temporal pattern of cell division and the onset of gastrulation in such half or quarter embryos were investigated and compared with normal development. The following results were obtained: (1) desynchronization starts two divisions earlier in quarter embryos and one division earlier in half embryos compared with whole embryos, (2) the time from the first cleavage to the onset of gastrulation does not widely vary among quarter, half and whole embryos and (3) the numbers of blastomeres which constitute embryos at the pigment stage decrease in proportion to the diminution of egg volume.

Cinematographical study of cell migration in the opened gastrula of Ambystoma mexicanum.

The migration of inner marginal cells was studied in the Ambystoma gastrula, using scanning electron micrography and time-lapse cinemicrography. Scanning electron micrographs of gastrulae which were fixed while intact revealed that the migrating cells have flattened lamellipodia at their anterior end and a rounded cell body, which can sometimes be seen to be attached to a neighbouring cell by a slender posterior process. Films of opened gastrulae showed actively moving cells, with the same features described above. Details of their movements are reported and discussed in relation to the mechanism of gastrulation.