Morphogenetic consequences of partial removal of blastomere cytoplasm during early embryonic development of the loach, Misgurnus fossilis L

The development of loach embryos is successfully regulated (normalized) after partial removal of the cytoplasm from one blastomere at the two- or four-cell stage or complete removal of one or two blastomeres at the stage of 8-16 cells. Using time-lapse video imaging and morphometric analysis, it has been shown that this regulation is a two-stage process. At the first stage, the ratio between the volumes of the blastodisk and yolk sac is rapidly (within one or two cell cycles) restored almost to the initial level; at the second stage, morphogenesis of the embryo is modified according to its new structural features acquired after the operation. After several rounds of cytokinesis, the cytoplasm remaining in the operated blastomere fuses with the marginal yolk syncytium (periblast),which at the blastula stage forms a distinct extension at the operation site. This extension marks the site of embryonic shield formation. The results of morphometric analysis show that restoration of the initial blastoderm volume in operated embryos leads to a reduction of active tension at the blastoderm--yolk boundary and an increase in the ratio of blastoderm surface to its volume at the moment of epiboly initiation. As a result, the convergence of blastoderm cells to the operation site and the embryonic shield formation begin at a lesser degree of epiboly, compared to the control.     

Morphogenetic consequences of partial removal of blastomere cytoplasm during early embryonic development of the loach, Misgurnus fossilis L.

The development of loach embryos is successfully regulated (normalized) after partial removal of the cytoplasm from one blastomere at the two- or four-cell stage or complete removal of one or two blastomeres at the stage of 8?C16 cells. Using time-lapse video imaging and morphometric analysis, it has been shown that this regulation is a two-stage process. At the first stage, the ratio between the volumes of the blastodisk and yolk sac is rapidly (within one or two cell cycles) restored almost to the initial level; at the second stage, morphogenesis of the embryo is modified according to its new structural features acquired after the operation. After several rounds of cytokinesis, the cytoplasm remaining in the operated blastomere fuses with the marginal yolk syncytium (periblast), which at the blastula stage forms a distinct extension at the operation site. This extension marks the site of embryonic shield formation. The results of morphometric analysis show that restoration of the initial blastoderm volume in operated embryos leads to a reduction of active tension at the blastoderm-yolk boundary and an increase in the ratio of blastoderm surface to its volume at the moment of epiboly initiation. As a result, the convergence of blastoderm cells to the operation site and the embryonic shield formation begin at a lesser degree of epiboly, compared to the control.     

Dorso-ventral polarity of the zebrafish embryo is distinguishable prior to the onset of gastrulation

We describe a set of observations on developing zebrafish embryos and discuss the main conclusions they allow:(1) the embryonic dorso-ventral polarity axis is morphologically distinguishable prior to the onset of gastrulation; and (2) the involution of deep layer cells starts on the prospective dorsal side of the embryo. An asymmetry can be distinguished in the organization of the blastomeres in the zebrafish blastula at the 30% epiboly stage, in that one sector of the blastoderm is thicker than the other. Dye-labelling experiments with DiI and DiO and histological analysis allow us to conclude that the embryonic shield will form on the thinner side of the blastoderm. Therefore, this side corresponds to the prospective dorsal side of the embryo. Simultaneous injections of dyes on the thinner side of the blastoderm and on the opposite side show that involution of deep layer cells during gastrulation starts at the site at which the embryonic shield will form and extends from here to the prospective ventral regions of the germ ring.     

Spatial-temporal dynamics of morphogenetic blastoderm potencies in early embryogenesis of the loach

The degree of differentiation of axial structures (notochord, neuroectoderm, and somites) in 24-hour explants (a total of 380) of the loach embryonic blastoderm was determined on histological sections according to a developed scale of estimates. Before the beginning of epiboly, axial structures were formed only from fragments of the dorsal sector of the blastoderm marginal zone. Its other sectors acquired the capacity of forming axial structure only with the beginning of epiboly, as the germ ring was formed in the marginal zone, unlike the cells outside the germ ring. The degree of differentiation of axial structures in the dorsal sector of marginal zone increased reliably with the appearance of embryonic shield, i.e. area of the convergence of cell flows. Here, statistically significant regional differences in morphogenetic potencies of the marginal zone first appeared, which corresponded to the differences in prospective significance of its materials; notochord and neuroectoderm better differentiate from the dorsal sector material, while somites better differentiate from the ventral sector material. Thus, distribution of morphogenetic potencies reflects precisely the spatial-temporal dynamics of collective movement of the blastoderm cells during the normal course of morphogenesis.     

Spatial-temporal dynamics of morphogenetic blastoderm potencies in early embryogenesis of the loach

The degree of differentiation of axial structures (notochord, neuroectoderm, and somites) in 24-hour explants (a total of 380) of the loach embryonic blastoderm was determined on histological sections according to a developed scale of estimates. Before the beginning of epiboly, axial structures were formed only from fragments of the dorsal sector of the blastoderm marginal zone. Its other sectors acquired the capacity of forming axial structure only with the beginning of epiboly, as the germ ring was formed in the marginal zone, unlike the cells outside the germ ring. The degree of differentiation of axial structures in the dorsal sector of marginal zone increased reliably with the appearance of embryonic shield, i.e. area of the convergence of cell flows. Here, statistically significant regional differences in morphogenetic potencies of the marginal zone first appeared, which corresponded to the differences in prospective significance of its materials; notochord and neuroectoderm better differentiate from the dorsal sector material, while somites better differentiate from the ventral sector material. Thus, distribution of morphogenetic potencies reflects precisely the spatial-temporal dynamics of collective movement of the blastoderm cells during the normal course of morphogenesis.     

SEM observation of the External Yolk Syncytial Layer in Blastopore Closure of the Medaka, Oryzias latipes

The embryonic surface of the teleost, Oryzias latipes , was observed by scanning electron microscopy (SEM) to examine the last phase of epiboly or blastopore closure. The surface of the external yolk syncytial layer (E–YSL), a surface cytoplasmic layer encompassing the yolk sphere situated beyond the blastoderm, was highly undulated with surface folds of random orientation throughout most of epiboly (st. 14–20). Scattered microvilli were observed on the surface of the margin of the yolk plug in st. 18–20. The microvilli, 1 to 6 μm in length, were projected in a bunch at the end of blastopore closure (st. 20–21). The appearance of these microvilli in the last phase of epiboly is discussed with respect to the mechanism of epiboly.     

Dynamics and variability of early morphogenesis in the loach according to observations of individual developmental trajectories

The dynamics and variability of quantitative morphological characters (morphological variables), which undergo changes upon epiboly, were studied by means of vital observations and measurements of developing loach (Misgurnus fossilis L.) embryos within equal time intervals. None of morphological variables, which characterize the dynamics of blastoderm shape, had monotonous dependence on time. In each individual embryo, the intervals of changes in morphological variables in the "normal" direction corresponding to the change of their mean values during the normal course of epiboly alternated with arrests, as well as with the changes of morphological variables in the reverse direction. The dynamics of morphological variables in time, which reflect the sequence of morphological states of the same embryo, and those of individual variations (variations of morphological states of different embryos on the same temporal section) had identical structure. This suggests instability of individual trajectories of morphogenesis or, strictly speaking, their actual absence. It was shown for the first time on the basis of analysis of individual trajectories of morphogenesis that its dynamics corresponded to so-called "determined chaos", which was previously discussed only as a theoretical possibility. The data obtained suggest that upon approach to the equatorial area of the embryo, the blastoderm marginal zone was elongated in the longitudinal direction and contracted across the axis of its movement. As a result, a positive feedback arises between the cooperated cell movement and the change of shape of the surface, over which the cells move. This leads, due to unstable radial symmetry of this movement, to the formation of embryonic shield.     

Geometry and mechanics of teleost gastrulation and the formation of primary embryonic axes

Examination of normal shaping dynamics and immediate and long-term responses to blastoderm cutting in zebrafish and loach embryos prior to the onset of gastrulation and during the course of epiboly revealed that anteroposterior (AP) and dorsoventral (DV) polarity formation is connected with shaping of the blastoderm circumferential region, which stretches along and shrinks across its movement axes and originates the non-isotropic fields of tensile stresses. Based on data from cutting experiments and quantitative morphology, we reconstructed the movement-shaping patterns of epiboly and embryonic shield formation. We revealed that AP and DV axes originate as a mass cell movement subject to the movement-shaping equivalence principle, which means the spatial series of differently shaped areas corresponding to the time succession of the same area shaping. Maintenance of the main body axes in orthogonal orientation depends on the mechanical equilibrium principle allowing for converting shape asymmetry into that of tensile stresses and vice versa. The causal relationship between the main movement-shaping axes and that of embryonic polarity was proved in cutting experiments in which the DV axis direction was subject to rearrangement so as to adjust to the new direction of mass cell movement axes induced by healing the wound in the blastoderm circumferential region.     

Cell movements during epiboly and gastrulation in zebrafish

Beginning during the late blastula stage in zebrafish, cells located beneath a surface epithelial layer of the blastoderm undergo rearrangements that accompany major changes in shape of the embryo. We describe three distinctive kinds of cell rearrangements. (1) Radial cell intercalations during epiboly mix cells located deeply in the blastoderm among more superficial ones. These rearrangements thoroughly stir the positions of deep cells, as the blastoderm thins and spreads across the yolk cell. (2) Involution at or near the blastoderm margin occurs during gastrulation. This movement folds the blastoderm into two cellular layers, the epiblast and hypoblast, within a ring (the germ ring) around its entire circumference. Involuting cells move anteriorwards in the hypoblast relative to cells that remain in the epiblast; the movement shears the positions of cells that were neighbors before gastrulation. Involuting cells eventually form endoderm and mesoderm, in an anterior-posterior sequence according to the time of involution. The epiblast is equivalent to embryonic ectoderm. (3) Mediolateral cell intercalations in both the epiblast and hypoblast mediate convergence and extension movements towards the dorsal side of the gastrula. By this rearrangement, cells that were initially neighboring one another become dispersed along the anterior-posterior axis of the embryo. Epiboly, involution and convergent extension in zebrafish involve the same kinds of cellular rearrangements as in amphibians, and they occur during comparable stages of embryogenesis.     

A Force Balance Can Explain Local and Global Cell Movements during Early Zebrafish Development

Embryonic morphogenesis takes place via a series of dramatic collective cell movements. The mechanisms that coordinate these intricate structural transformations across an entire organism are not well understood. In this study, we used gentle mechanical deformation of developing zebrafish embryos to probe the role of physical forces in generating long-range intercellular coordination during epiboly, the process in which the blastoderm spreads over the yolk cell. Geometric distortion of the embryo resulted in nonuniform blastoderm migration and realignment of the anterior-posterior (AP) axis, as defined by the locations at which the head and tail form, toward the new long axis of the embryo and away from the initial animal-vegetal axis defined by the starting location of the blastoderm. We found that local alterations in the rate of blastoderm migration correlated with the local geometry of the embryo. Chemical disruption of the contractile ring of actin and myosin immediately vegetal to the blastoderm margin via Ca²⁺ reduction or treatment with blebbistatin restored uniform migration and eliminated AP axis reorientation in mechanically deformed embryos; it also resulted in cellular disorganization at the blastoderm margin. Our results support a model in which tension generated by the contractile actomyosin ring coordinates epiboly on both the organismal and cellular scales. Our observations likewise suggest that the AP axis is distinct from the initial animal-vegetal axis in zebrafish.     

Dynamics and Variability of Early Morphogenesis in the Loach according to Observations of Individual Developmental Trajectories

The dynamics and variability of quantitative morphological characters (morphological variables), which undergo changes upon epiboly, were studied by means of vital observations and measurements of developing loach (Misgurnus fossilis L.) embryos within equal time intervals. None of morphological variables, which characterize the dynamics of blastoderm shape, had monotonous dependence on time. In each individual embryo, the intervals of changes in morphological variables in the “normal” direction corresponding to the change of their mean values during the normal course of epiboly alternated with arrests, as well as with the changes of morphological variables in the reverse direction. The dynamics of morphological variables in time, which reflect the sequence of morphological states of the same embryo, and those of individual variations (variations of morphological states of different embryos on the same temporal section) had identical structure. This suggests instability of individual trajectories of morphogenesis or, strictly speaking, their actual absence. It was shown for the first time on the basis of analysis of individual trajectories of morphogenesis that its dynamics corresponded to so-called “determined chaos,” which was previously discussed only as a theoretical possibility. The data obtained suggest that upon approach to the equatorial area of the embryo, the blastoderm marginal zone was elongated in the longitudinal direction and contracted across the axis of its movement. As a result, a positive feedback arises between the cooperated cell movement and the change of shape of the surface, over which the cells move. This leads, due to unstable radial symmetry of this movement, to the formation of embryonic shield.__________Translated from Ontogenez, Vol. 36, No. 3, 2005, pp. 211–221.Original Russian Text Copyright © 2005 by Cherdantsev, Tsvetkova.     

A Novel Role for MAPKAPK2 in Morphogenesis during Zebrafish Development

One of the earliest morphogenetic processes in the development of many animals is epiboly. In the zebrafish, epiboly ensues when the animally localized blastoderm cells spread, thin over, and enclose the vegetally localized yolk. Only a few factors are known to function in this fundamental process. We identified a maternal-effect mutant, betty boop (bbp), which displays a novel defect in epiboly, wherein the blastoderm margin constricts dramatically, precisely when half of the yolk cell is covered by the blastoderm, causing the yolk cell to burst. Whole-blastoderm transplants and mRNA microinjection rescue demonstrate that Bbp functions in the yolk cell to regulate epiboly. We positionally cloned the maternal-effect bbp mutant gene and identified it as the zebrafish homolog of the serine-threonine kinase Mitogen Activated Protein Kinase Activated Protein Kinase 2, or MAPKAPK2, which was not previously known to function in embryonic development. We show that the regulation of MAPKAPK2 is conserved and p38 MAP kinase functions upstream of MAPKAPK2 in regulating epiboly in the zebrafish embryo. Dramatic alterations in calcium dynamics, together with the massive marginal constrictive force observed in bbp mutants, indicate precocious constriction of an F-actin network within the yolk cell, which first forms at 50% epiboly and regulates epiboly progression. We show that MAPKAPK2 activity and its regulator p38 MAPK function in the yolk cell to regulate the process of epiboly, identifying a new pathway regulating this cell movement process. We postulate that a p38 MAPKAPK2 kinase cascade modulates the activity of F-actin at the yolk cell margin circumference allowing the gradual closure of the blastopore as epiboly progresses.     

Membrane microdomain formation is crucial in epiboly during gastrulation of medaka

Membrane microdomain (microdomain) was isolated from early gastrula embryos. The isolated microdomain was characterized by enrichment of cholesterol and sphingomyelin, and by the presence of huge glycoproteins containing Lewis X structure. Importance of the microdomain in the progress of epiboly was assessed using methyl beta-cyclodextrin (MBCD) and C2-ceramide that disrupt microdomains through different mechanisms. Both reagents efficiently disrupted the microdomain structure and concomitantly impaired epiboly. Interestingly, when embryos pretreated with MBCD, a cholesterol-binding molecule, were exogenously supplemented with cholesterol, the embryos underwent not only reconstitution of the microdomain, but also complete restoration to the normal epiboly. Thus, normal or impaired development is reversibly controlled by the cholesterol-dependent formation or disruption of microdomains. The most typical phenotype of the microdomain-disrupted embryos is detachment of cells from the blastoderm, suggesting that a major contribution of microdomains to epiboly is cell adhesion of blastodermal cells.     

Developmental expression of Rab11, a small GTP‐binding protein in Drosophila epithelia

Intracellular membrane trafficking regulates a wide variety of developmental processes, including cell and tissue morphogenesis. Here we report developmental expression of Drosophila Rab11, a small GTP‐binding protein, required for both endocytic recycling and exocytosis. Rab11 is expressed in the epithelial cell types of diverse lineages at all developmental stages, beginning from the cellular blastoderm in early embryos to adult primordia and adult tissues, like the columnar epithelia lining male ejaculatory bulb. A robust expression of Rab11 is seen both in the amnioserosa and in the lateral epidermis during embryonic dorsal closure, a morphogenetic event that involves spreading and fusion of the contra‐lateral sides of epidermis. Rab11 mutant embryos fail to display the characteristic morphological changes in these two epithelial tissues during dorsal closure, providing a strong basis to dissect the role of Rab11 in coordinated epithelial sheet movements. genesis 47:32–39, 2009. © 2008 Wiley‐Liss, Inc.     

Rho mediates cytokinesis and epiboly via ROCK in zebrafish

To study the regulation of embryonic development by Rho, we microinjected Clostridium botulinum C3-exoenzyme (C3) into zebrafish embryos. We found that C3 inhibited cytokinesis during early cleavages. C3 inhibition appeared to be specific on RhoA, since the constitutively active RhoA could partially rescued the C3-induced defects. Distributions of actin and the cleavage furrow associated beta-catenin were disrupted by C3. Belbbistatin, a myosin II inhibitor, also caused blastomeres disintegration. It suggested that Rho mediates cytokinesis via cleavage furrow protein assembly and actomyosin ring constriction. Furthermore, C3 blocked cellular movements during epiboly and gastrulation as evident by the impairment on no tail and goosecoid expression in blastoderm front runner cells and the dorsal lip of blastopore, respectively. Y-27632, an antagonist of Rho-associated kinase (ROK/ROCK), had the similar inhibitory effects on zebrafish development as the C3 treatments. Taken together, these results suggest that Rho mediates cleavage furrow protein assembly during cytokinesis and cellular migration during epiboly and gastrulation via a ROK/ROCK-dependent pathway.     

CELLULAR BASIS OF EPIBOLY OF THE ENVELOPING LAYER IN THE EMBRYO OF MEDAKA, ORYZIAS LATIPES. I. CELL ARCHITECTURE REVEALED BY SILVER STAINING METHOD

A modification of silver nitrate staining, when applied to embryos of Oryzias latipes , was found to make clear not only the boundary of enveloping layer during epiboly, but also the outline of individual cells of the layer. The linear speed of advance of the enveloping layer was constant at fixed temperatures (20°, 25° or 30°C), except for the start and the end of the epibloy. Observation of the shape and arrangement of cells in the layer stained at successive stages of epiboly revealed that the enveloping layer expands uniformly over the yolk until late gastrula (3/4 epiboly). No cytokinetic figure was observed during epiboly until the blastopore was going to close. Total cell number of the layer remained constant during epiboly. Thus the expansion of the enveloping layer is accomplished without an accompanying increase in the number of constituent cells. In the last phase of epiboly, the surface area occupied by individual cells reduced locally at the region above the embryonic body, which suggests the occurrence in teleost of the convergence of cell sheets commonly observed in amphibian embryos.     

Characterization and expression of a presomitic mesoderm-specific<Emphasis Type="Italic"> mespo</Emphasis> gene in zebrafish

A complete zebrafish mespo cDNA encoding a protein of 131 amino acids with a bHLH domain in the C-terminal has been isolated. The bHLH domain of zebrafish Mespo is highly similar to those in the mouse, chick and Xenopus, sharing 82.4%, 80.4% and 74.5% amino acid identity, respectively. At 50% epiboly, the zebrafish mespo is first detected in the marginal zone of the blastoderm but excluding the prospective shield. Subsequently, mespo expression is intensified in the involuting mesoderm at 60% epiboly, and then restricted to the presomitic mesoderm (PSM) at 95% epiboly. At the 1-somite stage, mespo expression becomes reduced in the most rostral PSM. During segmentation, mespo expression is gradually downregulated at the most rostral segmental plate where cells are being coalesced to form somites. In spadetail mutant embryos, most of mespo-expressing cells were missing.