Late Tailbud Stage Research Articles

Chromosome 1 in crested and marbled newts (Triturus)

The heteromorphic chromosomes 1 of Triturus cristatus carnifex and T. marmoratus were studied in mitotic metaphase after staining with the Giemsa C-banding technique and with the fluorochromes, DAPI (AT-specific) and mithramycin (GC-specific). They were also examined in the lampbrush form under phase-contrast before fixation and after fixation and staining with Giemsa. Chromosomes 1 of T.c. carnifex are asynaptic and achiasmatic throughout most of their long arms. They are also heteromorphic in most of their long arms for the patterns of Giemsa and fluorochrome staining and the distribution of distinctive lampbrush loops. The heteromorphic regions correspond to the regions that are asynaptic and achiasmatic. They stain more strongly with mithramycin and more weakly with DAPI than the remainder of the chromosomes, signifying that their DNA is relatively rich in GC. The patterns of staining with Giemsa and fluorochromes and the distributions of distinctive lateral loops vary from one animal to another in the same species and even in the same population. The asynaptic and achiasmatic regions of chromosomes 1 in T. marmoratus extend throughout the whole of the long arms and well beyond the heterochromatic region. Chiasmata form only in the short arm and occasionally in the short euchromatic segment at the tip of the long arms. The staining patterns of chromosomes 1 in T. marmoratus differ from those in T.c. carnifex although, like carnifex, their DNA is relatively GC-rich. The chromosomes 1 of T. marmoratus are more submetacentric than those of T.c. carnifex. In T. marmoratus chromosome 1B is about 12% shorter than 1A. There is a short paracentric inversion heterozygosity in the long arm of chromosome 1B in T. marmoratus which probably accounts for the lack of chiasmata in the euchromatin that separates the centromere from the start of the heterochromatin. In both carnifex and marmoratus, embryos that are homomorphic for chromosome 1 arrest and die at the late tailbud stage of development. The same applies to F1 hybrid embryos T.c. carnifex x T. marmoratus, and this has permitted identification of chromosomes 1A and 1B in both species. There is no correspondence between patterns of Giemsa or fluorochrome staining of the heteromorphic regions of chromosome 1 and any feature of the lampbrush chromosomes. However, the short euchromatic ends of the long arms of chromosomes 1 in both species are distinguished in the lampbrush form by a series of uniformly small loops of fine texture associated with very small chromomeres. The Giemsa C-staining patterns of both chromosomes 1A and 1B are different in each of the four subspecies of T. cristatus. T.c. karelinii stands out by having unusually large masses of Giemsa C-staining centromeric heterochromatin on all but 1 of its 12 chromosomes. A scheme is proposed for the evolution of chromosome 1 in T. cristatus and T. marmoratus, based on all available cytological and molecular data.

Mechanisms of Melanophore Induction in Amphibian Development: (pigment cells/ap /ap mutant/induction/mechanism).

Induction of melanophores was examined by the sandwich method of explantation with embryonic tissues of Xenopus laevis+/+ and the white mutant, aP /aP . Interspecific combinations of tissues of Triturus taeniatus and Xenopus borealis were also used. The ectoderm used as the reacting system was taken from embriyos at various stages and combined with various tissues known to be melanogenic inductors. The following results were obtained: 1) The sources of melanophore induction in both +/+ and ap /ap studied by sandwich explantation were the same in both retinal pigmented epithelium and dermal melanophores: 2) Melanophores were induced in epidermal material from embryos at stages from the early gastrula to the late tail bud stage: 3) The presence of melanoblasts together with other ectomesenchymal cells in the neural crest is not sine qua non for their determination and differentiation: 4) On isolation of reacting material from the late gastrula, melanophores appeared in all cases. This shows that two hours contact between inductor tissues and the ectoderm is necessary and sufficient for melanophore induction: 5) Melanophore induction is not species-specific, but occurred in Xenopus ectoderm under the action of endomesoderm of Tr. taeniatus or X. borealis, and vice versa. The shapes and structures of melanophores induced were typical for the species from which the ectoderm was taken: 6) Melanogenic activity in the late gastrula stage has a gradient of distribution with a maximum in the prechordal plate: 7) In the mutant only the primary source of melanogenic inductors, the prechordal plate (PrP1), was active in stages both before and after its invagination: 8) Despite the fact that skin melanophores and retinal melanocytes have different genesis in development, all the present data suggest the identity of the mechanisms of melanin synthesizing machinery in the two.

Ascidian larval tunic: Extraembryonic structures influence morphogenesis.

The larval tunic of Corella inflata is composed of two cuticular layers, extracellular filaments and ground substance. It lies outside the epidermis and most of it is known to be produced by the epidermis. The dorsal, ventral and caudal fins are specialized parts of the tunic that are essential for larval locomotion. The following hypothesis was tested: Morphogenesis of the larval fins is dependent upon the presence of extraembryonic structures (test cells, chorion or follicle cells) before completion of the late tail bud stage of development. We tested this by dechorionating embryos of Corella inflata and Ascidia paratropa. The operation removes all extraembryonic structures. It was performed mainly on neurula, early tail-bud and late tail-bud stages. Fin formation is inhibited when neurulae are dechorionated but not when late tail-bud or older embryonic stages are dechorionated. Dechorionated neurulae produce all of the major components of the tunic (cuticular layers, filaments and ground substance) but they are unable to form functional fins. At the time of dechorionation, in all experiments, the embryos had no fins. Removal of the follicle cells does not inhibit fin formation. The test cells are known to secrete granular "ornaments" that attach to the surface of the tunic. The fibrous, acellular chorion may serve to contain the test cells and their products or products of the embryo that are not firmly attached. The test cells may induce or control the morphogenesis of the larval fins in ascidians before the late tail-bud stage of development. We suggest ways of testing this hypothesis and an alternative hypothesis.

The effects of Tunicamycin and 2-deoxy-D-glucose on the development ofXenopus laevis embryos.

Tunicamycin and 2-deoxy-D-glucose were applied toXenopus laevis embryos in the first cleavage furrow, blastula and early gastrula stages. No effect was observed with 2-deoxy-D-glucose up to the concentration 0.1 M. The effect of Tunicamycin is dose- and stage-dependent. At the concentration of 5 μg/ml cleaving embryos are arrested at the onset of gastrulation and their cells exhibit decreased intercellular adhesivity, while embryos treated in the blastula and early gastrula stages may develop up to the late neurula and tail-bud stage, respectively. Higher concentrations (up to 20 μg/ml) drastically affect cleavage. Concentrations of 4 to 1 μg/ml allow embryos to develop up to more advanced stages; however, developmental defects are the rule. Concentrations of less than 1 μg/ml do not affect development.

Heteromorphism for chromosome 1, a requirement for normal development in crested newts

In all adult males and females of T. cristatus most of the long arm of chromosome 1 is achiasmate and heteromorphic with respect to Giemsa C-staining pattern. This heteromorphism correlates well with the heteromorphism seen by previous investigators on the lampbrush chromosomes of adult females. The heteromorphism has nothing to do with sex determination. All those fertile eggs of T. cristatus that develop normally beyond blastula are capable of development through to late tail-bud stage, but 50% of them arrest at late tail-bud and ultimately die. Homomorphism for the long arm of chromosome 1 can only be found in those embryos that arrest at late tailbud. The same findings apply to T. marmoratus, which also shows heteromorphism for chromosome 1, but not to T. alpestris, which has homomorphic chromosomes 1 and a potential 100% developmental success rate.

Changes of lectin-binding sites on the embryonic muscle cell surface in the developing ascidian, Halocynthia aurantium

Lectin-binding sites on the muscle cell surface of an ascidian embryo were studied using the ferritin labeling technique. The embryos at 4-cell, gastrula, late tail-bud, and larval stages were dissociated in the Ca 2+- and Mg 2+-free solution with or without collagenase. Dissociated cells and fragments were prefixed, reacted with ferritin-lectin conjugates and processed for electron microscopy. Lectins used were concanavalin A (ConA) and Ricinus communis agglutinin. Ferritin particles showing lectin-binding sites were found singly or in the form of clusters on the cell surface exposed directly to the conjugates. Most of the particles of both conjugates were distributed singly and sparsely on entire surface areas of the 4-cell stage cells, whereas they were rich in population and tended to form clusters when embryos reached the gastrula stage. At the succeeding stages tested, tagged ferritin, which was single or clustering particles, was less in number as compared with those at the former stage; on the surface facing neighboring muscle cells, in particular, the ferritin particles were much fewer than those in areas of notochordal and epithelial sides. It is suggested that the embryonic muscle cells of the ascidian show stage-specific changes of cell surface carbohydrates. They have high reactivity to both lectins around the gastrula stage and bring out the regional difference of both lectin-binding sites in the tail-bud stage, namely during the period of histogenesis.

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.

Alkaline phosphatase isozymes of Xenopus laevis embryos and tissues

Alkaline phosphatase was obtained by treating embryos of Xenopus laevis with n-butanol at different developmental stages from gastrula to tadpole; the enzyme was also obtained from adult kidney, liver, and intestinal mucosa. Purification was carried out by gel filtration and polyacrylamide gel electrophoresis. The enzyme activity is chromatographically spearated into two peaks, with molecular weights of approximately 200,000 and 400,000. Alternatively, two groups may be characterized on the basis of their electrophoretic mobilities, which correspond to the different molecular weight classes. Effects of pH, temperature, inhibitors, and substrate concentration were studied. The kinetic and physical properties of the two alkaline phosphatase isozymes are similar, and are comparable to the properties reported for this enzyme from other vertebrates. Alkaline phosphatase activity increased sharply at the gastrula stage and reached a plateau at the late tailbud stage. During this period there was an 18-fold increase in activity.

Changes in protein during development of Triturus embryos: I. Contents and syntheses of soluble or basic protein of cell components

ABSTRACT The present paper reports basic data on DNA content, protein content, and protein synthesis in Triturus pyrrhogaster embryos during development from cleavage to the hatching stage. Except for measurements of DNA and total protein contents, embryos were labeled with sodium carbonate-14C for 10 h and fractionated into embryonic cell components, i.e. cytoplasmic mass, yolk and pigment granules, and nuclei, in a discontinuous density gradient of sucrose. The protein content and the radioactivity incorporated into protein were measured in each fraction. Those fractions combining protein soluble in buffer at pH 8·3 and in 0·25 N-HCl were further studied with polyacrylamide gel electrophoresis. In the newt embryo, four stages of active DNA increase were observed when cultured at constant temperature; they were gastrula, neurula, late tail-bud, and before-hatching stages. Total protein per embryo decreased from 3 to 2 mg during the development studied. The content of cytoplasmic soluble protein per embryo was low and constant throughout development. Synthesis of the fraction was observed at the earliest stage of development studied though the rate was not high and specific activity of the soluble protein increased during development. Qualitative changes in the newly synthesized protein were observed. With the yolk fraction, synthesis of protein, other than from probable contamination with the cytoplasmic fraction, was not detected and a detailed description was omitted. Changes were observed at two stages of development in the synthesis of nuclear protein soluble in buffer at pH 8·3, the first at gastrulation and the second at late tail-bud stage. The change at gastrulation seemed to be the start of syntheses of the nuclear soluble proteins, while quantitative enhancement rather than qualitative change was noticed at late tail-bud stage. Most of the nuclear protein soluble in 0·25 N-HCl was histone. The histone content increased in accordance with increase in the DNA content and the rate of DNA accumulation was accompanied by proportionate incorporation of radioactivity into histone. Among histone fractions, unique behaviour of the very lysine-rich histone was observed. The availability of [14C]sodium carbonate in rough estimations of protein synthesis in embryos and significance of the data obtained have been discussed.

Developmental cytology of chromatophores in the red-spotted newt.

AbstractThe cytology of pigment‐bearing cells is followed in the integument of the red‐spotted newt, Notophthalmus viridescens viridescens Rafinesque, with observations beginning in the late embryo and continuing through to the adult. Dermal melanophores appear in late tailbud stages, and dermal xanthophores differentiate prior to hatching. In young larvae, both become widely and evenly distributed in the dorsal skin, but in older larvae a pattern develops so that melanophores are absent from certain circumscribed lateral regions designated as melanophore voids. Iridophores differentiate and accumulate among the xanthophores which have remained in the voids. Those xanthophores then are transformed into proerythrophores; that is, their form is altered from stellate to punctate, their nuclei change from fusiform to ovoid, and their pterinosomes are largely replaced by lipid droplets. In some of the voids, the proerythrophores migrate into the epidermis to become the erythrophores of the red spots. The remaining voids cease to exist when their proerythrophores disappear and are replaced by dermal melanophores. At the same time, some dermal melanophores accumulate to form dorsal black spots, and a morphologically different type of melanophore differentiates within the epidermis. In the interspot regions of the eft, xanthophores hypertrophy and pack the dermis, giving an orange color to the skin. In the interspot regions of the adult newt, xanthophores with fewer organelles produce a yellow color on the ventral surface, and, in combination with melanophores and iridophores, produce a greenish color in the dorsum.

Histone synthesis during development of Triturus embryos

ABSTRACT Synthesis of histone fractions and one basic protein fraction, which moved fast on gel electrophoresis and had been reported to increase in nuclei accompanying a decrease in cytoplasm during development, were studied with radioactive carbon dioxide as a tracer. Acid-extractable proteins of nuclei or cytoplasm, isolated from labelled embryos, were fractionated by polyacrylamide disc electrophoresis and the histone fractions and the fastmoving basic protein were identified. Radioactivities in these fractions and DNA were determined. Synthesis of the fast-moving basic protein was not detected throughout the period of development studied and this fraction was thought to move in from the cytoplasm to the nucleus during development. Syntheses of histone fractions were observed as early as the blastula stage. Rates of syntheses of four histone fractions (f3, f2b, f2a2 and f2al) per embryo increased thereafter, keeping pace with the increase in the rate of DNA synthesis with advancing development. The rate of the very lysine-rich f1 histone synthesis per embryo did not increase after the gastrula stage and the rate remained almost constant until the late tail-bud stage. Compositions of newly synthesized histones, calculated from the radioactivities incorporated into histone fractions, were almost the same during development and among different regions of neurula or tail-bud-stage embryos, with the exception of the f1 fraction, which varied depending on the stage and region of the embryos. The results are discussed in relation to the possible roles of the histone fractions in developing embryos.

The role of water-regulating mechanisms in the development of the haploid syndrome in Xenopus laevis

ABSTRACT The uptake of water by haploid and diploid sibling embryos of Xenopus laevis has been investigated by measuring the density changes which occur during the development of intact embryos from the blastula to the late tail-bud stage, and of explants from which most of the presumptive endoderm has been removed. The results show that up to the mid-gastrula stage there is no difference between the haploid and diploid embryos; but from then on, whereas the diploid volume increases steadily, the haploid gastrulae undergo a series of cyclical volume changes due to loss of fluid through the blastopore. It is concluded that this is the result of an excessive inflow of water through the haploid ectoderm, because it was found that the volume of haploid ectodermal explants increased much more rapidly than the volume of similar diploid explants. Excess flow through the haploid ectoderm also accounts for other characteristics of the haploid syndrome-microcephaly and lordosis. It is suggested that it is the doubling of the cell number in haploid embryos with the consequent 25% increase in aggregate cell membrane area which accounts for the difference between the uptake of water by the two types of embryos. It is also suggested that changes in the rate of water flow through the ectoderm and endoderm which are thought to account for the accumulation of water in the blastocoel and archenteron in the normal diploid embryo arise in a similar way.

Determination of the mesonephric kidney

AbstractThe determination of the mesonephric kidney in Taricha torosa involves both inductive and suppressive interactions between the kidney mesoderm and other embryonic tissues. The analysis of kidney determination was made by explanting the mesonephrogenic mesenchyme in epidermal vesicles either alone or in combination with the endoderm, notochord, neural tube, neural crest, lateral plate mesoderm, somite or nephric duct. It was found that the endoderm, notochord, early somite, and early lateral plate mesoderm are inductors of the mesonephros. Also, both premigratory and migratory neural crest cells, neural tube, older somite, and older lateral plate mesoderm were identified as suppressors of the development of the mesonephros.Most of the above interactions occur before the nephric duct makes contact with the mesonephrogenic mesenchyme. Too much emphasis has been placed on the nephric duct as being the inductor of the mesonephros. The mesonephric kidney is determined by late tailbud stages; a result of the inductive and suppressive interactions it has experienced.

ACTINOMYCIN D INHIBITION OF CELL DIFFERENTIATION IN THE AMPHIBIAN SUCKER.

1. Early neurulae of the treefrog, Hyla regilla, were treated for 48 hours with actinomycin D (4–6 μg/ml) to inhibit the development of the oral sucker. 2. At the conclusion of the experiment (i.e. after 2 days) suckers in controls (late tailbud stage) were fully differentiated and functional paired disks composed of intensely pigmented, columnar, secretory cells; those in experimental embryos (early tailbud stage) were often single, median, weakly pigmented, poorly differentiated, non-adhesive elevations of cuboidal ectodermal cells. 3. The fine structure of suckers in controls was characterized by: large irregular nucleoli containing many incursions of nucleoplasm; endoplasmic reticulum of long cisternae studded externally with many ribosomes; abundant secretory droplets, some of which were being discharged to the exterior through the plasma membrane; and a reduction in number of glycogen (?) granules so abundant earlier in neurular cells. 4. The strongly inhibited cells of the sucker differed from those of the control in the following ultrastructural features: dense, spherical, often small nucleoli; endoplasmic reticulum of large smooth vesicles; few ribosomes; almost complete absence of secretory droplets; and glycogen (?) granules as numerous as in neurular cells. 5. The poor differentiation of fine structure in the actinomycin-inhibited adhesive disk was interpreted on the basis of an interference with DNA-dependent RNA synthesis.

Autonomy in the regionalisation of branchial endoderm in amphibia.

1. The branchial endoderm taken from late neurula or tail bud embryos developed into typical pharynx, when left to develop with head-mesenchyme, while in a purely ectodermal environment lacking mesodermal elements, it did not give rise to any identifiable structure. 2. The same endodermal piece was explanted together with lateral mesoderm. In this combination, the frequency of the occurrence of intestine decreased with increasing age of the donor, and the appearance of "pharyngoid" (the amorphous assemblage of pharyngeal cells) increased. 3. The piece of branchial endoderm was removed from an early neurula, and was isolated alone or together with cranial ectomesoderm for 24 or 48 hours, until the synchronous control had reached early or late tail bud stage; then pre-isolated piece of endoderm was combined with lateral mesoderm and cultivated further in an epidermal envelope. A decreased production of intestine and increased production of "pharyngoid" were obtained, according to the duration of the pre-isolation. 4. In the discussion, the establishment of regionalisation of endoderm in normal embryo was interpreted as anautonomous process, which can be accerelated by contact with head mesenchyme but which does not require it, while for the individuation of pharyngeal organisation, the presence of mesenchyme was considered to be indispensable.

On The Existence of Regionally Specific Evocators

ABSTRACT Spemann (1931) pointed out that different parts of the organization centre in the amphibian gastrula have different properties, in that the more anterior regions tend, other things being equal, to induce the formation of more anterior parts of the neural system, such as the brain and associated sense organs, while the posterior parts tend to induce the neural tube of the trunk or tail. Living organizer grafts frequently become more or less fused with the induced tissues, so that if a definite organ is finally produced, the individuation of it involves both inducing and induced materials, the former playing the leading part. When dead pieces of the organizer are used as grafts, they cannot be similarly incorporated, and any individuation which may occur must have arisen entirely within the reacting material. It might still be possible, however, that the dead organizer was able to specify which region of the embryonic axis was represented by the induction, and thus guide the individuating processes. It has been known, however, since the work of Holtfreter (1934a) that organizer material after being killed loses its power to induce mesodermal tissues. It evocates only the appearance of neural tissue. If this becomes individuated into any recognizable organ, it is usually found to represent some region of the brain or eye, while the organs resulting from secondary inductive effects of the brain (e.g. nose or ear) are also frequently found. It is certainly not impossible to find a dead material which can induce more posterior regions of the axis, since Holtfreter (1934b), Chuang (1938) and Toivonen (1949, 1950) have demonstrated such activity on the part of dead adult tissues or extracts from them. No phenomena of the kind have, however, been described as resulting from the action of dead parts of the gastrula organizer, and it has been usual to consider that this material loses its trunk-inducing powers, and becomes solely a specific head-organizer, when killed.

The First Appearance of Functional Activity in the Pars Intermedia in the Frog, Xenopus

SummaryObservations on the development of the melanophores in normal and hypophysectomized Xenopus show contraction of the melanophores in the latter at a late tail bud stage, about 48 hours after fertilization. The pituitary at this stage is undifferentiated. It is inferred that the secretion of melanophore hormone begins at or possibly before this time.