Gastrulation of Gastrotheca riobambae in comparison with other frogs

Blastopore formation, the embryonic disk, archenteron and notochord elongation, and Brachyury expression in the marsupial frog Gastrotheca riobambae was compared with embryos of Xenopus laevis and of the dendrobatids Colostethus machalilla and Epipedobates anthonyi. In contrast with X. laevis embryos, the blastopore closes before elongation of the archenteron and notochord in the embryos of G. riobambae and of the dendrobatid frogs. Moreover, the circumblastoporal collar (CBC) thickens due to the accumulation of involuted cells. An embryonic disk, however, is formed only in the G. riobambae gastrula. We differentiate three gastrulation patterns according to the speed of development: In X. laevis, elongation of the archenteron and notochord begin in the early to mid gastrula, whereas in the dendrobatids C. machalilla and E. anthonyi the archenteron elongates at mid gastrula and the notochord elongates after gastrulation. In G. riobambae, only involution takes place during gastrulation. Archenteron and notochord elongation occur in the post gastrula. In the non-aquatic reproducing frogs, the margin of the archenteron expands anisotropically, resulting in an apparent displacement of the CBC from a medial to a posterior location, resembling the displacement of Hensen's node in the chick and mouse. The differences detected indicate that amphibian gastrulation is modular.     

The gastrocoel roof plate in embryos of different frogs

The morphology of the gastrocoel roof plate and the presence of cilia in this structure were examined in embryos of four species of frogs. Embryos of Ceratophrys stolzmanni (Ceratophryidae) and Engystomops randi (Leiuperidae) develop rapidly, provide comparison for the analysis of gastrocoel roof plate development in the slow-developing embryos of Epipedobates machalilla (Dendrobatidae) and Gastrotheca riobambae (Hemiphractidae). Embryos of the analyzed frogs develop from eggs of different sizes, and display different reproductive and developmental strategies. In particular, dorsal convergence and extension and archenteron elongation begin during gastrulation in embryos of rapidly developing frogs, as in Xenopus laevis. In contrast, cells that involute during gastrulation are stored in the large circumblastoporal collar that develops around the closed blastopore in embryos of slow-developing frogs. Dorsal convergence and extension only start after blastopore closure in slow-developing frog embryos. However, in the neurulae, a gastrocoel roof plate develops, despite the accumulation of superficial mesodermal cells in the circumblastoporal collar. Embryos of all four species develop a ciliated gastrocoel roof plate at the beginning of neurulation. Accordingly, fluid-flow across the gastrocoel roof plate is likely the mechanism of left-right asymmetry patterning in these frogs, as in X. laevis and other vertebrates. A ciliated gastrocoel roof plate, with a likely origin as superficial mesoderm, is conserved in frogs belonging to four different families and with different modes of gastrulation.     

Self-organization in the determination of the size of the axial structures in the embryogenesis of the clawed toad

Experiments were performed using X. laevis embryos during gastrulation and neurulation (stages 10, 11 1/2, 12 1/2, 13 1/2, 15 and 18). Part of presumptive epidermis and lateral plate mesoderm was removed, and embryos raised until stage 25. The size of axial structures (notochord, somite mesoderm, central nervous system) was determined using serial histological sections and compared with that of control embryos. In experimental embryos, the size of axial structures was decreased. Until a specific stage of development, close correlation was found between the volume of embryonic compartment corresponding to a particular, structure and the volume of presumptive epidermis and lateral plate mesoderm. This stage is individual for each axial organ: middle gastrula (stage 11 1/2) for notochord, late gastrula (stage 12 1/2) for somite mesoderm, and late neurula (stage 18) for central nervous system. This data suggest that differentiation pattern of ecto-mesodermal rudiment is subject to regulation during gastrulation-neurulation, and subdivision of ectoderm and mesoderm into axial and non-axial tissues is a self-organizing process.     

A comparative analysis of notochord formation in amphibian embryos]

We studied the origin, structure, and development of the notochord in Pleurodeles waltlii (Urodela) and Xenopus laevis (Anura) embryos. The notochord rudiment is formed in both species at the early gastrula stage as a cluster of polarized chorda-mesoderm cells located along the sagittal plane of the embryo. In Pl. waltlii the notochord rudiment is separated from the gastrocoele roof as a result of contraction of apical cell surfaces. The contraction wave spreads forward and backward along craniocaudal axis, i.e., segregation of the notochord rudiment progresses in two directions simultaneously. Similar process takes place in X. laevis embryos; however, propagation of the contraction wave in the anterior part of the body somewhat differs from that in the posterior part. While the "anterior" contraction wave resembles that in Pl. waltlii embryos, progression of the wave in the posterior part of the body is distinguished by a closer association of the notochord rudiment with ectoderm and the presence of its delamination boundaries with the somite mesoderm.     

Expression pattern of the Brachyury gene in the arrow worm paraspadella gotoi (chaetognatha)

Arrow worms (the phylum Chaetognatha), which are among the major marine planktonic animals, are direct developers and exhibit features characteristic of both deuterostomes and protostomes. In particular, the embryonic development of arrow worms appears to be of the deuterostome type. Brachyury functions critically in the formation of the notochord in chordates, whereas the gene is expressed in both the blastopore and stomodeum invagination regions in embryos of hemichordates and echinoderms. Here we analyzed the expression of Brachyury (Pg-Bra) in the arrow worm Paraspadella gotoi and showed that Pg-Bra is expressed in the blastopore region and the stomodeum region in the embryo and then around the mouth opening region at the time of hatching. The expression of Pg-Bra in the embryo resembles that of Brachyury in embryos of hemichordates and echinoderms, whereas that in the mouth opening region in the hatchling appears to be novel.     

The Dorsalization of Spermann's Organizer Takes Place during Gastrulation in Xenopus laevis Embryos

Suramin, a polyanionic compound, which is thought to inhibit the binding of growth factors to their receptors, prevents the differentiation of the dorsal blastopore lip of early gastrulae into dorsal mesodermal structures as notochord and somites. Suramin treated blastopore lips form ventral mesodermal structures, mainly heart structures. Several cases showed rythmic contractions ("beating hearts"). Of special interest is the fact that blastopore lips isolated from middle gastrulae followed by suramin treatment differentiate in about 50% of the cases brain structures without the presence of notochord. These data suggest that suramin prevents the differentiation of the dorsal blastopore lip into notochord up to the early middle gastrula stage but no longer the formation of head mesoderm, which is the prequisite for the induction of archencephalic brain structures. Treated chordamesoderm with overlaying ectoderm from late gastrulae will differentiate as untreated controls, namely into dorsal axial structures like notochord, somites and brain structures. The results indicate that primarily a more general or ventral mesodermal signal is transferred from the dorsal vegetal blastomeres (Nieuwkoop center) to the dorsal marginal zone. The dorsalization, which enables the blastopore lip to differentiate into head mesoderm and notochord and in turn to acquire neuralizing activity, takes place during the early steps of gastrulation.     

Characterization of embryonic cardiac pacemaker and atrioventricular conduction physiology in Xenopus laevis using noninvasive imaging

Congenital heart defects often include altered conduction as well as morphological changes. Model organisms, like the frog Xenopus laevis, offer practical advantages for the study of congenital heart disease. X. laevis embryos are easily obtained free living, and the developing heart is readily visualized. Functional and morphological evidence for a conduction system is available for adult frog hearts, but information on the normal properties of embryonic heart contraction is lacking, especially in intact animals. With the use of fine glass microelectrodes, we were able to obtain cardiac recordings and make standard electrophysiological measurements in 1-wk-old embryos (stage 46). In addition, a system using digital analysis of video images was adapted for measurement of the standard cardiac intervals and compared with invasive measurements. Video images were obtained of the heart in live, pharmacologically paralyzed, stage 46 X. laevis embryos. Normal values for the timing of the cardiac cycle were established. Intervals determined by video analysis (n = 53), including the atrial and ventricular cycle lengths (473 +/- 10 ms and 464 +/- 19 ms, respectively) and the atrioventricular interval (169 +/- 5 ms) were not statistically different from those determined by intrathoracic cardiac recordings. We also present the data obtained from embryos treated with standard medications that affect the human conduction system. We conclude that the physiology of embryonic X. laevis cardiac conduction can be noninvasively studied by using digital video imaging. Additionally, we show the response of X. laevis embryonic hearts to chronotropic agents is similar but not identical to the response of the human heart.     

Localization of two IQGAPs in cultured cells and early embryos of Xenopus laevis

Mammalian IQGAP1 is considered to modulate organization of the actin cytoskeleton under regulation of signaling proteins Cdc42 or Rac and calmodulin [Bashour et al., 1997: J Cell Biol 137:1555-1566; Hart et al., 1996: EMBO J 15:2997-3005] and also to be involved in cadherin-based cell adhesion [Kuroda et al., 1998: Science 281:832-835]. However, its function in the cell has not been clear. In order to clarify the function of IQGAP, we investigated IQGAP in Xenopus laevis cells. We isolated two Xenopus cDNAs encoding homologues of mammalian IQGAP, XIQGAP1, and XIQGAP2, which show high homology with human IQGAP1 and IQGAP2, respectively. Immunofluorescent localization of XIQGAPs in Xenopus tissue cultured cells (XTC cells) and in developing embryos was examined. In XTC cells, XIQGAP1 was colocalized with F-actin at cell-to-cell contact sites, membrane ruffles in lamellipodia, and filopodia. During development of embryos, XIQGAP1 was concentrated in the borders of all embryonic cells. An intense staining for XIQGAP1 was found in regions undergoing active morphogenetic movements, such as the blastopore lip of gastrulae, and the neural plate, the notochord, and the somite of neurulae. These results suggest that XIQGAP1 is involved in both cell-to-cell adhesion and cell migration during Xenopus embryogenesis and in cultured cells. On the other hand, the localization of XIQGAP2 in XTC cells was distinct from that of XIQGAP1 although it was also seen in lamellipodia, filopodia, and borders between cells. In addition to these regions, strong nuclear staining was observed in both XTC cells and embryonic cells.     

The floor plate is sufficient for development of the sclerotome and spine without the notochord

Danforth'sshort-tail (Sd) mouse is a semi-dominant mutation affecting the development of the vertebral column. Although the notochord degenerates completely by embryonic day 9.5, the vertebral column exists up to the lumber region, suggesting that the floor plate can substitute for notochord function. We previously established the mutant mouse line, Skt(Gt), through gene trap mutagenesis and identified the novel gene, Skt, which was mapped 0.95cM distal to the Sd locus. Taking advantage of the fact that monitoring notochordal development and genotyping of the Sd locus can be performed using the Skt(Gt) allele, we assessed the development of the vertebra, notochord, somite, floor plate and sclerotome in +-+/+-Skt(Gt), Sd-+/+-+, Sd-Skt(Gt)/+-+, Sd-Skt(Gt)/+-Skt(Gt), Sd-+/Sd-+ and Sd-Skt(Gt)/Sd-Skt(Gt) embryos. In Sd homozygous mutants with a C57BL/6 genetic background, the vertebral column was truncated in the 6th thoracic vertebra, which was more severe than previously reported. The floor plate and sclerotome developed to the level of somite before notochord degeneration and the number of remaining vertebrae corresponded well with the level of development of the floor plate and sclerotome. Defects to the sclerotome and subsequent vertebral development were not due to failure of somitogenesis. Taken together, these results suggest that the notochord induced floor plate development before degeneration, and that the remaining floor plate is sufficient for maintenance of differentiation of the somite into the sclerotome and vertebra in the absence of the notochord.     

Prospective Neural Areas and Their Morphogenetic Movements during Neural Plate Formation of Xenopus Embryos. I. Development of Vegetal Half Embryos and Chimera Embryos

Development of animal cap-less Xenopus gastrulae was examined. In vegetal halves from which the animal cap was removed 0.6 mm above the blastopore, an apparently normal array of craniocaudal structures developed. Histological examination showed differentiation of central nervous system (CNS) structures in the cap-less embryos, but differentiation of sensory organs, such as a lens and ear vesicle in only a few embryos. Only the dorsal midline of the embryos was covered with epidermis, and its lateral-ventral areas consisted of bare endoderm and mesoderm. The development of animal cap was also investigated by exchanging the animal cap of X. laevis embryos with that of X. borealis embryos, which can be distinguished by quinacrine fluorescence staining. The central nervous system of chimera embryos consisted mainly of X. laevis cells stained homogeneously with quinacrine but a small number of punctately-stained X. borealis cells was in the anterior tip of the forebrain. Cells of the lens and ear vesicle were punctately stained. More than two-thirds of the epidermal area consisted of punctately-stained cells and only the dorsal midline of the posterior head- and trunk-epidermis consisted of homogeneously-stained cells.
Areas of the prospective central nervous system and their movement during embryogenesis of Xenopus are discussed.     

Embryonic muscle development in rainbow trout (Oncorhynchus mykiss): a scanning electron microscopy and immunohistological study

Embryonic muscle development was studied in rainbow trout (Oncorhynchus mykiss) at low and high temperature using scanning electron microscopy (SEM) and immunohistology. Somite development was described starting at stage 16 (Vernier JM. 1969. Ann Embryol Morphogen 4:495-520) for both temperatures, with special interest in their shape and size. Muscle differentiation, associated with somite growth, is characterized by a larger increase in height compared to width and by acquisition of a chevron shape. Thin structures such as striation, sarcomeres, and myofibrils within muscle cells and myotubes were observed starting at the eyed stage (stage 24). Immunohistological analyses showed appearance of embryonic fast myosin at stage 20 in the deep part of the somite. The area where myosin was expressed extended in the somite throughout embryonic development and the presence of myosin was observed in the entire somite at hatching (stage 30). Slow myosin was expressed in a monolayer of superficial cells at the eyed stage and during the entire embryonic development. Then it was expressed in a few layers of cells located in the red muscle area. These results suggest that muscle differentiation, characterized by myosin expression, is engaged at stage 20. Myogenesis starts in the deep part of the somite, near the notochord and progresses laterally to cover the complete somite at hatching when the somite is composed of muscle fibres exhibiting a high degree of maturity. No significant difference was observed in terms of muscular development between low- and high-temperature conditions. J. Exp. Zool. 286:379-389, 2000.     

In vivo magnetic resonance microscopy of differentiation in Xenopus laevis embryos from the first cleavage onwards

Differentiation inside a developing embryo can be observed by a variety of optical methods but hardly so in opaque organisms. Embryos of the frog Xenopus laevis--a popular model system--belong to the latter category and, for this reason, are predominantly being investigated by means of physical sectioning. Magnetic resonance imaging (MRI) is a noninvasive method independent of the optical opaqueness of the object. Starting out from clinical diagnostics, the technique has now developed into a branch of microscopy--MR microscopy--that provides spatial resolutions of tens of microns for small biological objects. Nondestructive three-dimensional images of various embryos have been obtained using this technique. They were, however, usually acquired by long scans of fixed embryos. Previously reported in vivo studies did not cover the very early embryonic stages, mainly for sensitivity reasons. Here, by applying high field MR microscopy to the X. laevis system, we achieved the temporal and spatial resolution required for observing subcellular dynamics during early cell divisions in vivo. We present image series of dividing cells and nuclei and of the whole embryonic development from the zygote onto the hatching of the tadpole. Additionally, biomechanical analyses from successive MR images are introduced. These results demonstrate that MR microscopy can provide unique contributions to investigations of differentiating cells and tissues in vivo.     

Bowline, a novel protein localized to the presomitic mesoderm, interacts with Groucho/TLE in Xenopus

Cells in the prospective somite of Xenopus laevis embryos rotate in an orchestrated manner to form a segregated somite. The prospective somite boundaries are prepatterned by gene expressions in the unsegmented presomitic mesoderm (PSM). However, the roles of polarized gene expression in this boundary formation are not well elucidated. Here we identified a novel gene, bowline, which localizes to the anterior halves of S-II, III in the PSM of X. laevis. Bowline associated with corepressor XGrg-4, a Xenopus homolog of Groucho/TLE protein. A WRPW tetrapeptide motif in Bowline was prerequisite for coprecipitation with XGrg-4 and for downregulation of X-Delta-2 by bowline RNA injection. This study indicates that Bowline is a novel protein interacting with Groucho/TLE and may play a role in somitogenesis in X. laevis.     

Effects of hypoxia on embryonic development in two Ambystoma and two Rana species

Oxygen available to amphibian embryos fluctuates widely and is often very low. We investigated the effects of oxygen partial pressure (1. 3-16.9 kPa) on embryonic development and hatching of two salamander (Ambystoma) and two frog (Rana) species. In Ambystoma, chronic hypoxia resulted in slowed development, delayed hatching, and embryos that were less developed at the time of hatching. Although hypoxia was not lethal to embryos, temporary developmental abnormalities were observed in Ambystoma at oxygen partial pressures of 3.8 kPa and below. Posthatching survival decreased below 3.3 kPa. In Rana, hypoxia did not affect developmental rate, presumably because hatching occurs at a very early stage of development relative to Ambystoma. However, Rana embryos hatched sooner in hypoxia than in normoxia, resulting in less developed embryos at the time of hatching. The results suggest that embryonic hypoxia may negatively affect survival and fitness in these species.     

Elongation of axolotl tailbud embryos requires GPI-linked proteins and organizer-induced, active, ventral trunk endoderm cell rearrangements

Application of phosphatidylinositol-specific phospholipase C to early tailbud stage axolotl embryos reveals that a specific subset of morphogenetic movements requires glycosylphosphatidylinositol (GPI)-linked cell-surface proteins. These include pronephric duct extension, "gill bulge" formation, and embryonic elongation along the anteroposterior axis. The work of Kitchin (1949, J. Exp. Zool. 112, 393-416) led to the conclusion that extension of the notochord provided the motive force driving anteroposterior stretching in axolotl embryos, elongation of other tissues being a passive response. We therefore conjectured that axial mesoderm cells might display the GPI-linked proteins required for elongation of the embryo. However, we show here that removal of most of the neural plate and axial and paraxial mesoderm prior to neural tube closure does not prevent elongation of ventrolateral tissues. Tissue-extirpation and tissue-marking experiments indicate that elongation of the ventral trunk occurs via active, directed tissue rearrangements within the endoderm, directed by signals emanating from the blastopore region. Extension of both dorsal and ventral tissues requires GPI-linked proteins. We conclude that elongation of axolotl embryos requires active cell rearrangements within ventral as well as axial tissues. The fact that both types of elongation are prevented by removal of GPI-linked proteins implies that they share a common molecular mechanism.     

Role of TSC-22 during early embryogenesis in Xenopus laevis

Transforming growth factor-beta1-stimulated clone 22 (TSC-22) encodes a leucine zipper-containing protein that is highly conserved. During mouse embryogenesis, TSC-22 is expressed at the site of epithelial-mesenchymal interaction. Here, we isolated Xenopus laevis TSC-22 (XTSC-22) and analyzed its function in early development. XTSC-22 mRNA was first detected in the ectoderm of late blastulae. Translational knockdown using XTSC-22 antisense morpholino oligonucleotides (XTSC-22-MO) caused a severe delay in blastopore closure in gastrulating embryos. This was not due to mesoderm induction or convergent-extension, as confirmed by whole-mount in situ hybridization and animal cap assay. Cell lineage tracing revealed that migration of ectoderm cells toward blastopore was disrupted in XTSC-22-depleted embryos, and these embryos had a marked increase in the number of dividing cells. In contrast, cell division was suppressed in XTSC-22 mRNA-injected embryos. Co-injection of XTSC-22-MO and mRNA encoding p27Xic1, which inhibits cell cycle promotion by binding cyclin/Cdk complexes, reversed aberrant cell division. This was accompanied by rescue of the delay in blastopore closure and cell migration. These results indicate that XTSC-22 is required for cell movement during gastrulation though cell cycle regulation.     

Temperature and neuromuscular development in embryos of the trout (Salmo trutta L.)

Myogenesis and neural development were examined in the myotomes of trout (Salmo trutta L.) embryos reared at 2, 6 and 10 degrees C. The relative timings of myotube and muscle fibre formation were similar, with respect to somite stage, at all three temperatures. Myogenesis was seen to begin medially, adjacent to the notochord, and also in separate zones located near the outer surface of the myotomes, believed to be the sites of formation of future slow muscle fibres. Temperature did not affect the relative timings of most aspects of neural development, including HNK-1-immunoreactivity of myosepta, primary motor neuron axonogenesis, Rohon-Beard dendrite outgrowth, and expression of acetylcholinesterase in the spinal chord and at the myosepta. The posterior progression of the lateral line primordium was slightly but significantly delayed relative to somite stage in embryos reared at 10 degrees C compared to 6 and 2 degrees C, while formation of vacuoles in the notochord occurred relatively earlier at higher temperatures. No significant differences in neuromuscular development were observed between offspring of migratory and of non-migratory females.     

Novel regulation of yolk utilization by thyroid hormone in embryos of the direct developing frog Eleutherodactylus coqui

Thyroid hormone (TH) is required for metamorphosis of the long, coiled tadpole gut into the short frog gut. Eleutherodactylus coqui, a direct developing frog, lacks a tadpole. Its embryonic gut is a miniature adult form with a mass of yolky cells, called nutritional endoderm, attached to the small intestine. We tested the TH requirement for gut development in E. coqui. Inhibition of TH synthesis with methimazole arrested gut development in its embryonic form. Embryos treated with methimazole failed to utilize the yolk in their nutritional endoderm, and survived for weeks without further development. Conversely, methimazole and 3,3',5-tri-iodo-l-thyronine, the active form of TH, stimulated gut development and utilization and disappearance of the nutritional endoderm. In Xenopus laevis, the receptor for TH, TRβ, is upregulated in response to TH. Similarly, EcTRβ, the E. coqui ortholog, was upregulated by TH in the gut. EcTRβ expression was high in the nutritional endoderm, suggesting a direct role for TH in yolk utilization by these cells. An initial step in the breakdown of yolk in X. laevis is acidification of the yolk platelet. E. coqui embryos in methimazole failed to acidify their yolk platelets, but acidification was stimulated by TH indicating its role in an early step of yolk utilization. In addition to a conserved TH role in gut development, a novel regulatory role for TH in yolk utilization has evolved in these direct developers.