Early Tailbud Embryos Research Articles

Stress-generating tissue deformations in Xenopus embryos: Long-range gradients and local cell displacements

BackgroundAlthough the role of endogenous mechanical stresses in regulating morphogenetic movements and cell differentiation is now well established, many aspects of mechanical stress generation and transmission in developing embryos remain unclear and require quantitative studies. ResultsBy measuring stress-bearing linear deformations (caused by differences in cell movement rates) in the outer cell layer of blastula – early tail-bud Xenopus embryos, we revealed a set of long-term tension-generating gradients of cell movement rates, modulated by short-term cell-cell displacements much increasing the rates of local deformations. Experimental relaxation of tensions distorted the gradients but preserved and even enhanced local cell-cell displacements. During development, an incoherent mode of cell behavior, characterized by extensive cell-cell displacements and poorly correlated cell trajectories, was exchanged for a more coherent regime with the opposite characteristics. In particular, cell shifts became more synchronous and acquired a periodicity of several dozen minutes. ConclusionsMorphogenetic movements in Xenopus embryos are mediated by mechanically stressed dynamic structures of two different levels: extended gradients and short-term cell-cell displacements. As development proceeds, the latter component decreases and cell trajectories become more correlated. In particular, they acquire common periodicities, making morphogenesis more coherent.

T-type Calcium Channel Regulation of Neural Tube Closure and EphrinA/EPHA Expression.

A major class of human birth defects arise from aberrations during neural tube closure (NTC). We report on a NTC signaling pathway requiring T-type calcium channels (TTCCs) that is conserved between primitive chordates (Ciona) and Xenopus. With loss of TTCCs, there is a failure to seal the anterior neural folds. Accompanying loss of TTCCs is an upregulation of EphrinA effectors. Ephrin signaling is known to be important in NTC, and ephrins can affect both cell adhesion and repulsion. In Ciona, ephrinA-d expression is downregulated at the end of neurulation, whereas, with loss of TTCC, ephrinA-d remains elevated. Accordingly, overexpression of ephrinA-d phenocopied TTCC loss of function, while overexpression of a dominant-negative Ephrin receptor was able to rescue NTC in a Ciona TTCC mutant. We hypothesize that signaling through TTCCs is necessary for proper anterior NTC through downregulation of ephrins, and possibly elimination of a repulsive signal.

P99. Paraxial T-box genes, Tbx6 and Tbx1, are required for cranial chondrogenesis and myogenesis

We previously reported that Tbx6, a T-box transcription factor expressed in nascent nonaxial mesoderm, is required for the differentiation of ventral body wall muscle and for segment formation and somitic muscle differentiation in Xenopus laevis . Here, we show that Tbx6 is also involved, at later stages, in cartilage differentiation from the cranial neural crest and head muscle development. In Tbx6 knockdown embryos, the cranial neural crest was shown to be correctly induced at the border of the neural plate and migrated in a slightly delayed manner, but finally reached positions in the pharyngeal arches nearly identical to those in the normal embryos as revealed by in situ hybridization and the neural crest-transplantation experiments. However, the neural crest cells failed to maintain Sox9 expression. In order to clarify the reasons why Tbx6 knockdown affects the development of head and pharyngeal muscles and cartilages, we noticed on Tbx1, another T-box gene expressed in more anterior paraxial structures, and a gene responsible for human DiGeorge syndrome, as a candidate to mediate Tbx6 function. Tbx6 knockdown reduced the expression of Tbx1. Next, Tbx1 knockdown caused phenotypes milder but similar to those of Tbx6 morphants, including reduced formation of head muscles and cartilages, and attenuated Sox9 expression. Furthermore, the phenotypes caused by Tbx6 knockdown were partially rescued by Tbx1 plasmid injection. Tbx6 expression in Tbx1 knockdown embryos was not reduced at all, but rather expanded in anterior direction, suggesting that Tbx6 is epistatic to Tbx1. This expansion of Tbx6 expression in Tbx1 morphants, when considered together with the fact that there was a gap between the anterior Tbx1-expressing and posterior Tbx6-expressing areas in normal neurula to early tailbud embryos, implys the presence of interaction between the anterior and posterior paraxial mesodermal tissues. These results suggest that Tbx6 is involved in the cranial cartilage and head muscle development by regulating anterior paraxial genes such as Tbx1 and Sox9.

Enhancer of split-related-2 mRNA shows cyclic expression during somitogenesis in Xenopus laevis

Somitogenesis is responsible for production of the segmented body plan typical of vertebrate embryos. The somites are blocks of mesoderm, produced by this process, that give rise to the vertebrae and ribs, the dermis of the skin and all the skeletal muscle of the body. Many genes that regulate somitogenesis have been identified in chick and mouse, whereas considerably fewer are known in Xenopus laevis. The expression of Hairy/Enhancer of split-related genes is known to cycle during somitogenesis and provides a mechanism for the regular formation of somites. In this project, in situ hybridizations were carried out on bowline, Thylacine1, Enhancer of split-related-1 (ESR1), ESR2 and ESR-5 in order to study their expression in relation to somitogenesis. All genes were found to be expressed during somitogenesis, even as early on as late gastrula stages in some cases. In addition, the expression of ESR2 is shown to be oscillating in the presegmented mesoderm of neurula and early-tailbud embryos. This study has identified ESR2 as the second known gene (after esr9) to show periodic oscillations of gene expression which can be considered as cycling during somitogenesis in X. laevis.

XHAPLN3 plays a key role in cardiogenesis by maintaining the hyaluronan matrix around heart anlage

Hyaluronan matrix plays an important role during vertebrate cardiogenesis. Transcripts for the hyaluronan synthase Has2 gene are expressed in heart anlage, and disruption of either Has2 or versican, a hyaluronan matrix component, abrogates normal cardiac morphogenesis. However, the mechanisms by which hyaluronan matrix contributes to early heart development are largely unknown. Here we show that Xenopus hy aluronan and proteoglycan-binding li nk protein 3 (XHAPLN3) helps to maintain hyaluronan matrix around the cardiac anlage, and thereby contribute to cardiogenesis. XHAPLN3 mRNA transcript localization overlapped with the mRNA expression of both Xhas2 and Xversican at the heart anlage of early tailbud (stage 23) embryos. Furthermore, knockdown of XHAPLN3 or Xhas2 with morpholino antisense oligos caused a heart deficiency in developing tadpoles. Our results show when and how components of the hyaluronan matrix function in cardiogenesis, improving our understanding of how extracellular matrix participates in embryogenesis.

Contactin 1 knockdown in the hindbrain induces abnormal development of the trigeminal sensory nerve in Xenopus embryos

In Xenopus tailbud embryos, the mandibular branch of trigeminal sensory nerve has a transient pathway innervating the cement gland. This pathway is settled by pioneer neurons in the trigeminal ganglion and along which extend later-growing axons from the trigeminal ganglion and the hindbrain. Axons in this branch express a neuronal recognition molecule, Contactin 1, from the initial stage of its outgrowth in early tailbud embryos and form a tightly joined, strongly Contactin 1-positive fascicle in the later stages. When the expression vector encoding the enhanced green fluorescent protein was electrotransfected into the brain neurons of early tailbud embryos, the fluorescence was detected in the hindbrain and the trigeminal nerve at late tailbud stages. Cotransfection of antisense vector caused knockdown of Contactin 1 concurrent with defasciculation and misguidance of the sensory axons in the trigeminal mandibular branch. The results suggest that Contactin 1 is required for the growing axon of hindbrain sensory neurons to recognize and follow the pathway settled by the pioneer neurons.

Large-scale cDNA analysis of the maternal genetic information in the egg of Halocynthia roretzi for a gene expression catalog of ascidian development.

The ascidian egg is a well-known mosaic egg. In order to investigate the molecular nature of the maternal genetic information stored in the egg, we have prepared cDNAs from the mRNAs in the fertilized eggs of the ascidian, Halocynthia roretzi. The cDNAs of the ascidian embryo were sequenced, and the localization of individual mRNA was examined in staged embryos by whole-mount in situ hybridization. The data obtained were stored in the database MAGEST (http://www.genome.ad.jp/magest) and further analyzed. A total of 4240 cDNA clones were found to represent 2221 gene transcripts, including at least 934 different protein-coding sequences. The mRNA population of the egg consisted of a low prevalence, high complexity sequence set. The majority of the clones were of the rare sequence class, and of these, 42% of the clones showed significant matches with known peptides, mainly consisting of proteins with housekeeping functions such as metabolism and cell division. In addition, we found cDNAs encoding components involved in different signal transduction pathways and cDNAs encoding nucleotide-binding proteins. Large-scale analyses of the distribution of the RNA corresponding to each cDNA in the eight-cell, 110-cell and early tailbud embryos were simultaneously carried out. These analyses revealed that a small fraction of the maternal RNAs were localized in the eight-cell embryo, and that 7.9% of the clones were exclusively maternal, while 40.6% of the maternal clones showed expression in the later stages. This study provides global insights about the genes expressed during early development.

Mechanical Design of Fiber-Wound Hydraulic Skeletons: The Stiffening and Straightening of Embryonic Notochords

The notochord can play an important mechanical role in shape changes during early morphogenesis of vertebrates. For example, osmotic inflation of notochords elongates and straightens the axis of frog early tail-bud embryos. In Xenopus laevis, the sheath of cross-helically arranged fibers around the notochord limits the shape changes it undergoes when inflating, causing the notochord to stiffen and straighten (Adams et al., 1990; Koehl et al., 1990). We used physical models of stage 24 X. laevis notochords to explore the mechanical consequences of different arrangements of the sheath fibers on the behavior of such curved hydraulic cylinders. All the models straightened upon inflation regardless of initial fiber angle (𝛉 = angle of the fibers to long axis of the cylinder). Notochord models with 𝛉 > 54° lengthened and narrowed as they straightened; although they could push, the forces they exerted were limited by their tendency to buckle, which increased the greater the 𝛉. In contrast, models with 𝛉 < 54° shortened and widened as they straightened and showed pronounced increases in flexural stiffness. The mean 𝛉 of X. laevis early tail-bud notochords is 54°, a fiber angle that permits an increase in the end-to-end distance of the model (along the anterior-posterior axis of the embryo) as it straightens and pushes when pressurized, but that is less prone to Euler and local buckling than are models with higher 𝛉's. Nonetheless, a 𝛉 of 54° in notochords may simply be the result of osmotic swelling.

Mechanical Design of Fiber-Wound Hydraulic Skeletons: The Stiffening and Straightening of Embryonic Notochords1

The notochord can play an important mechanical role in shape changes during early morphogenesis of vertebrates. For example, osmotic inflation of notochords elongates and straightens the axis of frog early tail-bud embryos. In Xenopus laevis, the sheath of cross-helically arranged fibers around the notochord limits the shape changes it undergoes when inflating, causing the notochord to stiffen and straighten (Adams et al., 1990; Koehl et al., 1990). We used physical models of stage 24 X. laevis notochords to explore the mechanical consequences of different arrangements of the sheath fibers on the behavior of such curved hydraulic cylinders. All the models straightened upon inflation regardless of initial fiber angle (θ = angle of the fibers to long axis of the cylinder). Notochord models with θ > 54° lengthened and narrowed as they straightened; although they could push, the forces they exerted were limited by their tendency to buckle, which increased the greater the θ. In contrast, models with θ < 54° shortened and widened as they straightened and showed pronounced increases in flexural stiffness. The mean θ of X. laevis early tail-bud notochords is 54°, a fiber angle that permits an increase in the end-to-end distance of the model (along the anterior-posterior axis of the embryo) as it straightens and pushes when pressurized, but that is less prone to Euler and local buckling than are models with higher θ's. Nonetheless, a θ of 54° in notochords may simply be the result of osmotic swelling.

Xenopus Hand2 expression marks anterior vascular progenitors but not the developing heart.

We have isolated cDNAs encoding the bHLH protein Hand2 in the amphibian Xenopus laevis and analysed Hand2 expression in early development from the onset of gastrulation to feeding tadpole stages. XHand2 is expressed in the branchial arch mesenchyme and also in small bilateral populations of cells in the anterior, ventrolateral region of early tailbud embryos. At later stages, these punctate Hand2-expressing cells are located at the sites of the forming common cardinal veins, suggesting that they may constitute progenitors of vascular smooth muscle cells. Other Hand2-expressing cells are also associated with further components of the forming anterior vasculature but are not detected in mature blood vessels. Interestingly, no myocardial expression of XHand2 can be detected in the developing tadpole heart, in marked contrast to results obtained with chick and mouse embryos.

Primordial germ cell development: is the urodele pattern closer to mammals than to anurans?

All animals can be classified into three types depending on their modes of germ cell formation; epigenetic, intermediate and preformistic. In urodeles, which show the intermediate mode, primordial germ cells (PGCs) are morphologically recognized at first in early tailbud embryos. The PGCs, which are located within the lateral plate mesoderm, are induced as part of the regional induction of the mesoderm by the vegetal yolk endoderm. No cytologically distinctive, germ cell-specific germ plasma can be detected during early development of urodeles. 'Nuage' materials, which are specific to germ line cells in almost all animals, do, however, appear in the cytoplasm of the urodele PGCs during later embryogenesis. In contrast, PGCs in anurans are preformistically established under the influence of germ plasma. Because all germ cells, once established, show virtually identical behavior, regardless of whether different modes of germ cell formation are employed, the basic mechanism of germ cell formation and differentiation in all animals could be similar at the molecular levels. Although the molecules involved in germ cell formation in amphibians have not been identified, many aspects of germ plasm formation in anurans are similar to Drosophila, in which three classes of genes involved in germ cell formation have been identified: Class I genes are necessary for pattern specification during germ cell formation, Class II for the assembly of germ plasm components, and Class III for germ cell segregation. Assuming that germ cell formation in all animals requires the expression of all such genes, the three modes of germ cell formation mentioned above could be explained in terms of spatio-temporal expression of genes which are similar to those that have been identified in Drosophila. A tentative model of gene regulation for the three different modes of germ cell formation has been proposed in terms of temporal expression of these three classes of genes.

An ascidian homologue of vertebrate BMPs-5–8 is expressed in the midline of the anterior neuroectoderm and in the midline of the ventral epidermis of the embryo

The ascidian tadpole larva is thought to be the prototype for the ancestral chordate. Although ascidians show a highly determinate mode of development, recent studies suggest significant roles of cell-cell interaction during embryogenesis. To elucidate the signaling molecules responsible for the cellular interaction, we investigated an ascidian homologue of the transforming growth factor β (TGF-β) superfamily. HrBMPa is an ascidian member of the 60A subclass of the BMP subfamily. Molecular phylogenetic analysis suggested that HrBMPa branched prior to further divergence of vertebrate BMPs-5–8. The zygotic expression of HrBMPa was initiated around gastrulation. HrBMPa transcripts were first evident in precursor cells of the spinal cord, notochord, epidermis and nervous system, although signals in the first two regions quickly disappeared. In neurulae and early tailbud embryos, transcripts were evident in the adhesive organ, midline of the anterior dorsal neuroectoderm and midline of both ventral and dorsal ectoderm, suggesting that HrBMPa plays a major role in neuroectodermal cell differentiation during embryogenesis. This HrBMPa expression profile resembled that of Xenopus BMP-7, implying a primordial function of BMP-7 among vertebrate BMPs-5–8.

Identification of members of the HSP30 small heat shock protein family and characterization of their developmental regulation in heat‐shocked xenopus laevis embryos

In the present study we have characterized the synthesis of members of the HSP30 family during Xenopus laevis development using a polyclonal antipeptide antibody derived from the carboxyl end of HSP30C. Two-dimensional PAGE/immunoblot analysis was unable to detect any heat-inducible small HSPs in cleavage, blastula, gastrula, or neurula stage embryos. However, heat-inducible accumulation of a single protein was first detectable in early tailbud embryos with an additional 5 HSPs at the late tailbud stage and a total of 13 small HSPs at the early tadpole stage. In the Xenopus A6 kidney epithelial cell line, a total of eight heat-inducible small HSPs were detected by this antibody. Comparison of the pattern of protein synthesis in embryos and somatic cells revealed a number of common and unique heat inducible proteins in Xenopus embryos and cultured kidney epithelial cells. To specifically identify the protein product of the HSP30C gene, we made a chimeric gene construct with the Xenopus HSP30C coding sequence under the control of a constitutive promoter. This construct was microinjected into fertilized eggs and resulted in the premature and constitutive synthesis of the HSP30C protein in gastrula stage embryos. Through a series of mixing experiments, we were able to specifically identify the protein encoded by the HSP30C gene in embryos and somatic cells and to conclude that HSP30C synthesis was first head-inducible at the early tailbud stage of development. The differential pattern of heat-inducible accumulation of members of the HSP30 family during Xenopus development suggests that these proteins may have distinct functions at specific embryonic stages during a stress response.

Introduction and Expression of Recombinant Genes in Ascidian Embryos.

In order to examine the expression of exogenous genes introduced into ascidian eggs, two recombinant plasmids pmiwZ and pHrMA4aCAT were microinjected into the cytoplasm of fertilized eggs of Ciona savignyi and Halocynthia roretzi, respectively. The plasmid pmiwZ contains the coding sequence of bacterial β-galactosidase gene (lac-Z) fused with animal gene promoters, while pHrMA4aCAT was constructed by fusing about 1.4-kb long 5' flanking region of H. roretzi muscle actin gene HrMA4a with bacterial chloramphenicol acetyltransferase gene (CAT). Injection of approximately 160 pl of 10 μg/ml pmiwZ DNA into Ciona eggs did not affect the embryogenesis, although introduction of the same volume of 30 μg/ml pmiwZ DNA resulted in abnormal development of injected eggs. When the expression of lac-Z was examined by histochemical detection of the enzyme activity, the expression was evident in the early tailbud embryos and later stage embryos, and larvae, irrespective of linear or circular form of the plasmid. The enzyme activity appeared in various cell-types including epidermis, nervous system, endoderm, mesenchyme, notochord, and muscle. In contrast, when pHrMA4aCAT was introduced into Halocynthia eggs and the appearance of CAT protein was examined later by the anti-CAT antibody, the CAT expression was restricted to muscle cells. These results indicate that the recombinant genes introduced into ascidian eggs could express during embryogenesis and that the 1.4-kb long 5' flanking region of HrMA4a contains regulatory sequences enough for the appropriate spatial and temporal expression of the gene.

The mechanics of notochord elongation, straightening and stiffening in the embryo of Xenopus laevis

We have examined the biomechanical development of the notochord of Xenopus early tail-bud embryos by: (1) quantifying morphological and mechanical changes in the embryo during stages 20-28, and (2) conducting manipulative experiments to elucidate mechanical roles of various components of the notochord. The notochord, which is composed of a stack of flat cells surrounded by a connective tissue sheath, elongates dramatically and begins straightening between stages 21 and 25. At this time the fiber density in the notochord sheath goes up, the osmotic activity of the notochord cells increases, vacuoles within these cells swell, the internal pressure of the notochord increases 2- to 3-fold, and the flexural stiffness of the notochord rises by an order of magnitude. We suggest that the tendency of the notochord cells to osmotically swell is resisted by the sheath, thereby permitting the internal pressure to rise. This pressure increase results in the greater stiffness that permits the notochord to elongate and straighten without being buckled by the surrounding tissues.

Developmental Fates of Blastomeres of Eight-Cell-Stage Xenopus laevis Embryos: (intracellular injection/horseradish peroxidase/developmental fate/Xenopus embryo).

The developmental fates of animal, vegetal, dorsal, and ventral egg regions of Xenopus laevis embryos were examined. For this purpose, a tracer enzyme (horseradish peroxidase) was injected bilaterally into pairs of eight-cell-stage blastomeres and the clonal organization of marked cells in the early tail-bud embryos was examined. The epidermis over most of the body originated from animal-ventral micromeres, but that in the head originated from animal-dorsal blastomeres and that in the area surrounding the anus originated from vegetal-ventral blastomeres. The neural tube originated from animal-dorsal, vegetal-dorsal, and animal-ventral blastomeres. These results were consistent with those of previous studies. But in contrast to previous findings, results showed that the entire notochord is derived from animal-dorsal micromeres and that the somites originate from all four bilateral pairs of blastomeres in the eight-cell stage. These results are discussed in relation to the current maps of prospective fates based on results of vital-dye staining. Morphogenetic movements are also discussed on the basis of the clonal organization demonstrated in the present study.

Elicitation of weak immune response in larval and adult Xenopus laevis by allografted pituitary.

To test the claim that tolerance to organ-specific self antigens is established during the ontogenic development of the immune system, pituitary anlagen were removed from early tailbud embryos of Xenopus laevis, and the resulting hypophysectomized tadpoles were grafted with pituitaries after the tadpoles had become immunocompetent. The result was that none of the histocompatible or allogeneic pituitary grafts was rejected throughout an observation period of 100 days. Subsequent experiments revealed that hypophysectomy does not affect the development of immune responsiveness. In addition, allogeneic pituitary grafts were usually not rejected by unoperated, normal tadpoles or froglets, although they suffered lymphoid invasion to various degrees. However, a significant number of allogeneic pituitaries was rejected when they were grafted, either after or at the same time as the grafting of skin from the pituitary donors. We conclude that the pituitary of Xenopus possesses weak transplantation antigens or expresses them in ways that make them less accessible to the immune surveillance system of the allogeneic host.

Types of Neural Tissues Induced through the Presumptive Notochord of Newt Embryo

Neural induction through the presumptive notochord was tested by means of the sandwich method. The result disclosed that the notochord was a potent inducer of neural tissues not only in the ectoderm of gastrula but also in the ventral ectoderm of neurula and early tail-bud embryos. Structures formed by the induced neural tissue varied greatly. They can be classified into three types. (1) Tubular: the neural tissues induced in explants containing abundant mesenchymes always formed long tubular structures. The shapes of these neural tubes showed considerable variation; moreover, they were atypical and none formed the regular structure of the spinal cord. This type was most frequent, being found in about 50% of the explants. (2) Inverted: this type was produced when the explant contained mesenchymal component. Consequently, the epithelium of explants was missing. Nevertheless, a considerable mass of neural tissue was always induced. It was noticed that the induced neural tissues were invariably inside out; this type was found in about 30% of the explants.(3) Archencephalic: this was the only type to form the regular structure, i.e., the archencephalon. Formation of the archencephalon was limited solely to those explants containing only a few mesenchymes; this type was found in about 20% of the cases. As described above, it was found that the neural tissues induced by the same inducer of the notochord were not uniform but varied in type. Further it was shown that the types of neural tissue differed according to different quantities of the surrounding mesenchyme. Based on these facts, it is to be concluded that it is not the inducer of notochord, but the surrounding mesenchyme that is of primary importance for the determination of the types of neural tissue.

Inter- and intramyotomal gap junctions in the axolotl embryo

In early tailbud embryos of the axolotl ( Ambystoma mexicanum), cells of the anterior myotomes begin to elongate and align along the longitudinal axis of the animal. Soon thereafter, gap junctions appear between the differentiating myotubes. These junctions occur between adjacent cells within a myotome (intramyotomal) and between the cells of adjacent myotomes which are separated from one another by narrow connective tissue septa (intermyotomal). The latter are found at the ends of the elongating cells where muscle-tendon insertion will occur and nerve-muscle synapses will form. The gap junctions are transient: They appear with the onset of myofibrillar formation at the time that nerve fibers enter the intermyotomal septa. The junctions last until the cells have differentiated into mature striated muscle cells and neuromuscular synapses are fully developed. These gap junctions may provide a means for the direct intercellular spread of electrical excitation between the differentiating muscle cells and so account for the observed myogenic contraction of myotomes. We also suggest that these junctions may form a means for cellular communication and interaction during the development of the axial musculature.

Z Bands in Developing Heart Muscle of the Mexican Salamander, Ambystoma mexicanum

Electron-dense plaques, often seen in association with the plasma membranes of pre-heartbeat salamander myocardial cells, appear to be directly involved in early Z band formation. In differentiating myocardial cells of higher vertebrates, Z band material has been observed in close proximity to the plasma membranes and as isolated electron-dense material in the deep cytoplasm. The present study explores the fine structural features of Z band formation in salamander myocardium from early tailbud embryos (Harrison stage 25) through the adult. Hearts were fixed In a 0.1 M phosphate buffered formaldehyde-glutaraldehyde-picric acid-styphnic acid mixture and were post-fixed and embedded using standard methods.