Comparison of variability of <Emphasis Type="Italic">Rana temporaria</Emphasis> (Amphibia,Anura) gastrula from different populations developing under the conditions of antropogenic pollution

The gastrulation of Rana temporaria embryos was studied at the natural spawning sites of Moscow city and Moscow district by means of morphometric analysis. We demonstrated that anthropogenic pollution plays an important role in morphogenesis of Anura. The measurements of embryos from the Moscow ponds made it possible to show an increased variability of gastrulation. Moreover, the level of gastrulation variability depends on the degree of pollution of a spawning pond and is coupled with increased correlations between the morphological features. This increase of correlations could be due to: (1) an increased number of correlating features (appearance of new correlations) and (2) strengthening of the existing correlations. The first way makes the gastrulation process more coordinated, and the second one causes the appearance of morphological structures, which are normally formed only at the next developmental stages.__________Translated from Ontogenez, Vol. 36, No. 2, 2005, pp. 110–122.Original Russian Text Copyright © 2005 by Severtsova, Severtsov.     

Interpopulation comparison of variations in gastrulation of Rana arvalis and Rana ridibunda occurring under the conditions of anthropogenic contamination of water bodies

The results of morphometric analysis of gastrulation in city populations of Rana arvalis and Rana ridibunda suggest that under the conditions of anthropogenic contamination of water bodies, the early development is more variable, if we compare less variable sizes of their eggs in the city and suburb populations. The variability of gastrulation appears to depend on the degree of contamination of spawning water bodies and is accompanied by increased correlation between the forming structures. This latter may be determined by two causes: (1) increased number of interdependent features, which leads to coordinated differentiation of structures and regulation of variability, and (2) increased coefficients of correlation within a small group of features, which leads to the formation of structures characteristic for the subsequent developmental stages, including the appearance of heterochronies.     

Interpopulation comparison of variations in gastrulation of <Emphasis Type="Italic">Rana arvalis</Emphasis> and <Emphasis Type="Italic">Rana ridibunda</Emphasis> occurring under the conditions of anthropogenic contamination of water bodies

The results of morphometric analysis of gastrulation in city populations of Rana arvalis and Rana ridibunda suggest that under the conditions of anthropogenic contamination of water bodies, the early development is more variable, if we compare less variable sizes of their eggs in the city and suburb populations. The variability of gastrulation appears to depend on the degree of contamination of spawning water bodies and is accompanied by increased correlation between the forming structures. This latter may be determined by two causes: (1) increased number of interdependent features, which leads to coordinated differentiation of structures and regulation of variability, and (2) increased coefficients of correlation within a small group of features, which leads to the formation of structures characteristic for the subsequent developmental stages, including the appearance of heterochronies.     

The natural variability of morphogenesis: a tool for exploring the mechanics of gastrulation movements in amphibian embryos

The investigation of natural variability of metric morphological characters in frog gastrulation revealed that in genetically and environmentally homogeneous samples of embryos their variability is of a higher order of magnitude than that known for quantitative metric characters in adult organisms. Matching the coefficients of variation of characters under consideration to the specific rates of their changes in normal development revealed a strong positive correlation between the rates of morphological change and the amount of morphological variance. The increase in the variance is mainly in characters concerned with shaping of moving embryonic areas and arises as a result of a positive feedback between the movement of a given area and recruitment of cells from surrounding areas into the movement. The account of natural variation suggests a new model of amphibian gastrulation whose essential feature is the intimate connection between the movement and shaping of the dorsal blastopore lip of the gastrula.     

Accumulation and Distribution of Extracellular Matrix as Revealed by Scanning Electron Microscopy in Freeze-Dried Newt Embryos Before and During Gastrulation

A scanning electron-microscopic study was carried out on the extracellular matrices (ECMs) in freeze-dried newt embryos from the cleavage to the gastrula stage. The results revealed the appearance, accumulation and distribution of two types of ECMs, a fibrillar ECM in the blastocoel and an amorphous ECM on the inner surface of the blastocoelic wall (BW). The fibrillar ECM first appeared in the blastocoel at the cleavage stage and increased notably in quantity at the blastula and gastrula stages. On the other hand, the amorphous ECM was initially detected on the inner surface of the BW at the beginning of gastrulation and it increased in quantity during gastrulation. With the progress of archenteron invagination, the amorphous ECM was found to be deposited in the space between the BW and migrating cells.     

Intercellular bridges in vertebrate gastrulation

The developing zebrafish embryo has been the subject of many studies of regional patterning, stereotypical cell movements and changes in cell shape. To better study the morphological features of cells during gastrulation, we generated mosaic embryos expressing membrane attached Dendra2 to highlight cellular boundaries. We find that intercellular bridges join a significant fraction of epiblast cells in the zebrafish embryo, reaching several cell diameters in length and spanning across different regions of the developing embryos. These intercellular bridges are distinct from the cellular protrusions previously reported as extending from hypoblast cells (1-2 cellular diameters in length) or epiblast cells (which were shorter). Most of the intercellular bridges were formed at pre-gastrula stages by the daughters of a dividing cell maintaining a membrane tether as they move apart after mitosis. These intercellular bridges persist during gastrulation and can mediate the transfer of proteins between distant cells. These findings reveal a surprising feature of the cellular landscape in zebrafish embryos and open new possibilities for cell-cell communication during gastrulation, with implications for modeling, cellular mechanics, and morphogenetic signaling.     

Gastrulation in the sea urchin embryo requires the deposition of crosslinked collagen within the extracellular matrix

This study demonstrates that a collagenous extracellular matrix (ECM) is necessary for gastrulation in the sea urchin embryo. The approach taken was to disrupt collagen processing with two types of agents (a lathyritic agent, beta-aminopropionitrile (BAPN), and three types of proline analogs: dehydroproline, cis-OH-proline, and azetidine carboxylic acid) and to assess the effect on embryogenesis by morphological, immunological, and biochemical criteria. Embryos chronically exposed to either of the agents following fertilization displayed no detectable developmental abnormalities before the mesenchyme blastula stage. These embryos, however, did not gastrulate nor differentiate any further and remained at the mesenchyme blastula stage for at least 36 hr. Upon removal of the agents, the embryos resumed a normal developmental schedule and formed pluteus larvae that were indistinguishable from control embryos. By immunofluorescence studies with monospecific antibodies to type I and type IV collagens it is seen that the lathyritic agent BAPN reduces the accumulation of collagens within the ECM. This effect is confirmed and quantitated by use of an ELISA and by a biochemical determination of OH-proline. When the agents are removed from the inhibited embryos, collagen deposition returns to normal, coincident with gastrulation. Western-blot analysis, using monospecific antibodies to collagen, demonstrates that the effect of the lathyritic agent is to reduce the stability of the extracellular collagen by inhibiting the intra- and intermolecular crosslinking of collagen molecules. BAPN exhibits a dose-dependent effect on morphogenesis, but has no effect on respiration nor on protein synthesis of the embryos throughout development. Although the lathyritic agent affects collagen deposition, it is shown to not affect the expression of other molecules of the ECM, nor that of several cell surface molecules. However, a cell surface molecule that is expressed specifically in the endoderm, termed Endo 1, is not expressed in the inhibited embryos. Endo 1 is expressed after removal of the lathyritic agent and its appearance is coincident with gastrulation in the recovered embryos. These results suggest that a collagenous ECM is important for gastrulation and subsequent differentiation in the sea urchin, but not for earlier developmental processes. In addition, the dependence of Endo 1 expression on the collagenous ECM raises the possibility that this cell surface molecule is in some way regulated by interactions of the presumptive endodermal cells with the ECM.     

Developmental variability during early embryonic development of zebra fish, Danio rerio

Early vertebrate embryos pass through a period of remarkable morphological similarity. Possible causes for such similarity of early embryos include modularity, developmental constraints, stabilizing selection, canalization, and exhausted genetic variability. Supposedly, each process creates different patterns of variation and covariation of embryonic traits. We study the patterns of variation of the embryonic phenotype to test ideas about possible evolutionary mechanisms shaping the early embryonic development. We use the zebra fish, Danio rerio, as a model organism and apply repeated measures of individual embryos to study temporal changes of phenotypic variability during development. In particular, we are looking at the embryonic development from 12 hours post fertilization until 27 hours post fertilization. During this time period, the development of individual embryos is documented at hourly intervals. We measured maximum diameter of the eye, length of embryo, number of somites, inclination of somites, and the yolk size (as a maternal effect). The coefficient of variation (CV) was used as a measure of variability that was independent of size. We used a principal component analysis for analysis of morphological integration. The experimental setup kept environment x genotype interactions constant. Nongenetic parental contributions had no significant effects on interindividual variability. Thus all observed phenotypic variation was based on additive genetic variance and error variance. The average CV declined from 14% to 7.7%. The decline of the CV was in particular expressed during 15-19 h post fertilization and occurred in association with multiple correlations among embryonic traits and a relatively high degree of morphological integration. We suggest that internal constraints determine the patterns of variability during early embryonic development of zebra fish.     

Retarded gastrulation and altered subsequent development of neural tissues in heparin-injected Xenopus embryos

Gastrulation was examined in Xenopus embryos injected with various polysaccharides into the blastocoel cavity. The progression of gastrulation was assessed by observing pigmentation and yolk plug size in vegetative view embryos. In heparin- or dextran-sulphate-injected embryos, gastrulation was significantly retarded. This was further confirmed in tissue sections of embryos. In contrast, no such retardation was found in embryos injected with hyaluronic acid or chondroitin sulphate. A quantitative analysis showed that the extent of retardation in heparin- or dextran-sulphate-injected embryos was dose-dependent and that, after the initial retardation of up to 2-3 h, gastrulation progressed at a similar rate to controls. At the time when untreated sibling embryos hatched, embryos injected with heparin or dextran sulphate showed abnormalities in their external appearance and swimming behavior in a dose-dependent manner. When these embryos were examined histologically or immunohistochemically using tissue-specific monoclonal antibodies, it was found that central nervous system (CNS), especially the brain and eye structures, were most severely damaged. The extent of damage was again dose-dependent. In contrast, neural-crest-derived melanophores were abundant even in aneural larvae. No such change was found in embryos injected with hyaluronic acid or chondroitin sulphate.     

Analysis of extraembryonic mesodermal structure formation in the absence of morphological primitive streak

During mouse gastrulation, the primitive streak is formed on the posterior side of the embryo. Cells migrate out of the primitive streak to form the future mesoderm and endoderm. Fate mapping studies revealed a group of cell migrate through the proximal end of the primitive streak and give rise to the extraembryonic mesoderm tissues such as the yolk sac blood islands and allantois. However, it is not clear whether the formation of a morphological primitive streak is required for the development of these extraembryonic mesodermal tissues. Loss of the Cripto gene in mice dramatically reduces, but does not completely abolish, Nodal activity leading to the absence of a morphological primitive streak. However, embryonic erythrocytes are still formed and assembled into the blood islands. In addition, Cripto mutant embryos form allantoic buds. However, Drap1 mutant embryos have excessive Nodal activity in the epiblast cells before gastrulation and form an expanded primitive streak, but no yolk sac blood islands or allantoic bud formation. Lefty2 embryos also have elevated levels of Nodal activity in the primitive streak during gastrulation, and undergo normal blood island and allantois formation. We therefore speculate that low level of Nodal activity disrupts the formation of morphological primitive streak on the posterior side, but still allows the formation of primitive streak cells on the proximal side, which give rise to the extraembryonic mesodermal tissues formation. Excessive Nodal activity in the epiblast at pre‐gastrulation stage, but not in the primitive streak cells during gastrulation, disrupts extraembryonic mesoderm development.     

Dynamic morphogenetic events characterize the mouse visceral endoderm

Several lines of evidence suggest that the extraembryonic endoderm of vertebrate embryos plays an important role in the development of rostral neural structures. In mice, neural inductive signals are thought to reside in an area of visceral endoderm that expresses the Hex gene. Here, we have conducted a morphological and lineage analysis of visceral endoderm cells spanning pre- and postprimitive streak stages. Our results show that Hex-expressing cells have a tall, columnar epithelial morphology, which distinguishes them from other visceral endoderm cells. This region of visceral endoderm thickening (VET) is found overlying first the distal and then one side of the epiblast at stages between 5.5 and 5.75 days post coitum (d.p.c.). In addition, we show that the epiblast has an anteroposterior-compressed appearance that is aligned with the position of the VET. Intracellular labeling of VET/Hex-expressing cells reveals an anterior and anterolateral shift from their distal epiblast position. VET/Hex-expressing cells are first localized to the anterior side of the epiblast by 5.75 d.p.c. and form a crescent on the anterior half of the embryo at the onset of gastrulation. Subsequently, VET descendants are distributed along the embryonic/extraembryonic boundary by headfold stages at 7.5 d.p.c. The morphological characteristics and position of VET/Hex-expressing cells distinguishes the future anteroposterior axis of the embryo and provide landmarks to stage mouse embryos at preprimitive streak stages. Moreover, the morphological characteristics of pregastrulation mouse embryos together with the stereotyped shift in the position of visceral endoderm cells reveal similarities among amniote embryos that suggest an evolutionary conservation of the mechanisms that pattern the rostral neurectoderm at pregastrula stages.     

The cholinergic system in the early development of chickens. 1. Choline acetyltransferase and acetylcholinesterase as the key regulators of acetylcholine metabolism

Acetylcholine esterase (AChE, EC 3.1.1.7) and choline acetyltransferase (CAT, EC 2.3.1.6) activities were studied in the early chick embryos. Gastrulation is accompanied by a sharp increase in the AChE activity which was most pronounced in anterior hypoblast. Three molecular of AChE (4.7, 6.8 and 10.9 S) were identified in the extract of chick embryos using a sucrose density gradient centrifugation. The CAT activity remained unchanged during gastrulation but increased twice at the end of gastrulation.     

Induction of increase in intracellular calcium concentration of embryonic cells and acceleration of morphogenetic cell movements during amphibian gastrulation by a 50-Hz magnetic field

The influence of an alternating electromagnetic field (EMF) on early development of amphibian embryos was examined. When the embryos developed under the influence of a low-frequency EMF (50 Hz, 5-30 mT), the rate of early development was accelerated. The effect of EMF was exerted preferentially at the gastrula stage, and the period of gastrulation was shortened. Histological observations showed that EMF promoted morphogenetic cell movements during the gastrulation. The concentration of intracellular free Ca2+ ([Ca2+]i) in the embryonic cells under the influence of EMF was analyzed using Fura-2, an indicator of the intracellular concentration of calcium ions. The influence of EMF on [Ca2+]i was analyzed in embryonic cells isolated from blastula, gastrula, and neurula, EMF increased a [Ca2+]i particularly in the cells isolated from gastrula. Our results suggest that EMF specifically increased the [Ca2+]i of gastrula cells, thereby, accelerating the rate of morphogenetic cell movements during gastrulation.     

Inhibition of the synthesis of glycosphingolipid by a ceramide analogue (PPMP) in the gastrulation of Bufo arenarum

In the present study the role of glycosphingolipids (GSL) in amphibian development was investigated. We analysed the de novo synthesis of neutral GSL and gangliosides through the initial stages of Bufo arenarum embryo development and their participation during gastrulation using 1-phenyl-2-palmitoyl-3-morpholino-1-propanol (PPMP), a potent inhibitor of glucosylceramide synthase. Ganglioside synthesis began at the blastula stage and reached a maximum during gastrulation (stages 10-12) while neutral GSL synthesis showed a slight gradual increase, the former being quantitatively more significant than the latter. Ganglioside synthesis was reduced by 90% while neutral GSL synthesis was inhibited by 65% when embryos at blastula stage were cultured for 24 h in 20 microM PPMP. The depletion of GSL from amphibian embryos induced an abnormal gastrulation in a dose-dependent manner. We found that PPMP had a pronounced effect on development since no embryos exhibited normal gastrulation; their developmental rate either slowed down or, more often, became totally arrested. Morphological analysis of arrested embryos revealed inhibition of the gastrulation morphogenetic movements. Analysis of mesodermal cell morphology in those embryos showed a severe decrease in the number and complexity of cellular extensions such as filopodia and lamellipodia. Mesodermal cells isolated from PPMP-treated embryos had very low adhesion percentages. Our results suggest that glycosphingolipids participate in Bufo arenarum gastrulation, probably through their involvement in cell adhesion events.     

Localized axis determinant in the early cleavage embryo of the goldfish, Carassius auratus

 The teleost dorsoventral axis cannot be distinguished morphologically before gastrulation. In order to examine whether the yolk cell affects axis determination, we bisect early cleavage embryos of the goldfish, Carassius auratus. When the vegetal yolk hemisphere is removed by bisection along the equatorial plane at the 2-cell stage, the embryos develop abnormally and exhibit a symmetrical morphology. No dorsal structures, such as notochord, somites and neural tube, differentiate and no embryonic shield is formed during gastrulation. In addition, no goosecoid mRNA is expressed before gastrulation. The frequency of abnormality decreases as the age at which the vegetal yolk hemisphere is removed increases. Most embryos removed at the 32-cell stage develop normally. Their morphological phenotype is similar to that of a Xenopus ventralized embryo generated by ultraviolet irradiation on the vegetal hemisphere soon after fertilization. We also observed that, when the embryos were bisected along the first cleavage plane at the 2-cell stage, the proportion of pairs of embryos of which one embryo developed normally was 44.8%. These results indicate that the vegetal yolk hemisphere of the early cleavage embryo of the goldfish contains axis determination factor(s), which are necessary for generation of dorsal structures. Furthermore, it is suggested that these determinant(s) are distributed asymmetrically within the vegetal yolk hemisphere. Received: 25 May 1996 / Accepted: 19 September 1996     

Specificity of cell-cell interactions in sea urchin embryos: Appearance of new cell-surface determinants at gastrulation

Studies on normal and hybrid sea urchin embryos show that, beginning at gastrulation, hybrid cells express cell-surface antigens specific to both species. The appearance of these antigens is shown to be correlated with a change in the adhesive specificity of hybrid cells: Beginning at gastrulation, hybrid cells recognize and adhere to embryonic cells of both normal genotypes. Prior to gastrulation, hybrid cells adhere to cells of the maternal genotype only. Two adhesion assays demonstrate these adhesive preferences. (i) When cell aggregates are placed together in a dish, Lytechnius aggregates fuse together, and Tripneustes aggregates fuse together, but aggregates of the two species do not fuse with each other. Hybrid cell aggregates, if they are past the beginning of gastrulation, fuse to both Tripneustes and Lytechinus aggregates. (ii) In a collection assay, midgastrula cells of the hybrid embryos are collected at a high rate to aggregates of either species. Pregastrula hybrid cells collect at a high rate to aggregates of the maternal species only. This change in adhesive preference is temporally correlated with the appearance of new cell surface antigens. Antiserum was prepared in rabbits against membranes from Lytechinus gastrulae. Indirect immunofluorescence tests show that hybrid cells of the cross (T♀ × L♂) express Lytechinus-specific antigens at the cell surface beginning at gastrulation. Furthermore, an apparent relationship between the new cell-surface antigens and adhesion exists in that Lytechinus cell adhesion is inhibited specifically after binding Fab fragments of the Lytechinus antiserum. The antiserum has no effect on Tripneustes adhesion. The Lytechinus adhesion-inhibiting activity can be removed by absorption of the antiserum with Lytechinus cells.     

Timing of spawning and early development of Palythoa tuberculosa (Anthozoa, Zoantharia, Sphenopidae) in Okinawa, Japan

The spawning behavior and early embryogenesis of Palythoa tuberculosa (Anthozoa, Zoantharia) were observed in August 2009 off Okinawa Island, Japan. P. tuberculosa released zygotes just after high tide around new moon nights. The mean diameter of zygotes was 365.6 ± s.d.14.8 μm, and zygotes did not contain any symbiotic algae (zooxanthellae). About 2 h after spawning, the first cleavage furrow appeared on one side of the zygotes, although it was uncertain when eggs were fertilized. After second cleavage, the arrangement of blastomeres was pseudospherical. At 9 h after spawning, the embryo became a concave-convex dish shape, then gastrulation occurred and the blastopore was formed. Seven-day old larvae were ellipsoid and about 700 μm long, with an open mouth at one end. Two weeks after spawning, the larvae developed a longitudinal band of long cilia (= ventral ciliate band) that is characteristic of zoanthella larvae. In P. tuberculosa, larvae show a non-radial body plan and then metamorphose to almost-radial (in outward appearance) polyps after settlement. These results may support a hypothesis that a common ancestor of Cnidaria had a bilateral body plan that has been secondarily lost in some extant cnidarians.     

Effect of incubation temperature on green sturgeon embryos, <Emphasis Type="Italic">Acipenser medirostris</Emphasis>

Regulation of river flow and the amount of winter rainfall are the major factors affecting the water temperature of the spawning grounds, for green sturgeon in the Klamath River. During the primary spawning period of green sturgeon, mid-April to June, the water temperature may vary from 8 to 21°C. To estimate the potential implications of this modified thermal regime, we examined the survival and development in three progeny groups of green sturgeon embryos from zygote to hatch, at constant incubation temperatures (11–26°C). Temperatures 23–26°C affected cleavage and gastrulation and all died before hatch. Temperatures 17.5–22°C were suboptimal as an increasing number of embryos developed abnormally and hatching success decreased at 20.5–22°C, although the tolerance to these temperatures varied between progenies. The lower temperature limit was not evident from this study, although hatching rate decreased at 11°C and hatched embryos were shorter, compared to 14°C. The mean total length of hatched embryos decreased with increasing temperature, although their wet and dry weight remained relatively constant. We concluded that temperatures 17–18°C may be the upper limit of the thermal optima for green sturgeon embryos, and that the river thermal regime during dry years may affect green sturgeon reproduction.