Fig 1 - uploaded by Ashley E Bruce
Content may be subject to copyright.
Schematic depiction of epiboly initiation and progression in the zebrafish embryo. (A) At sphere stage, the EVL and YSL have been created and the deep cells form a flat interface with the underlying yolk cell. (B) Upon epiboly initiation, the yolk cell domes and deep cells move radially outwards, forming a cap of cells over the yolk. (C-E) During the progression phase, the blastoderm continues to thin, expanding its surface area to envelop the yolk cell. (D) Once the blastoderm has covered approximately 50% of the yolk, deep cell epiboly temporarily pauses as cells begin to converge dorsally and gastrulation begins. (E) Concurrent with the other gastrulation movements, the deep cells, EVL and YSL move towards the vegetal pole in a coordinated manner, eventually closing the blastopore. Deep cells are shown in white, the EVL and YSL in blue and the yolk cell is shown in yellow. Black arrows indicate general direction(s) of organized cell movement. d, dorsal; dc, deep cells; dcm, deep cell margin; e-ysn, external yolk syncytial nuclei; ep, epiblast; hyp, hypoblast; i-ysn, internal yolk syncitial nuclei; vp, vegetal pole; yc, yolk cell.
Source publication
Gastrulation involves of a series of coordinated cell movements to organize the germ layers and establish the major body axes of the embryo. One gastrulation movement is epiboly, which involves the thinning and spreading of a multilayered cell sheet. Epiboly plays a prominent role in zebrafish gastrulation and studies of zebrafish epiboly have prov...
Contexts in source publication
Context 1
... to the onset of epiboly, the embryo is organized into three layers (Fig. 1A). A single-cell thick epithelium, the enveloping layer (EVL), envelops the deep cells that eventually give rise to embryonic tissues. The EVL gives rise to periderm and to dorsal forerunner cells which form Kupffer's vesicle (Oteíza et al., 2008), an organ analogous to the mammalian node (Essner et al., 2005;Kramer-Zucker et al., 2005). ...
Context 2
... layer, the yolk syncytial layer (YSL), an extra- embryonic syncytium populating the interface between the yolk and deep cells. The YSL forms around cell division 10 (3 hpf, midblastula transition), when marginal blastomeres fuse with and deposit their nuclei into the cortex of the underlying yolk cell ( Kimmel and Law, 1985). YSL nuclei (YSN, Fig. 1) undergo several more rounds of mitosis, and typically exit the cell cycle at division 14 (Kane et al., 1992). Initially, the YSN form a wide belt at the periphery of the blastoderm margin, the external-YSL (E- YSL), but by sphere stage (4 hpf) the YSN distribute beneath the blastoderm, forming the internal-YSL (I-YSL) (Kimmel et al., ...
Context 3
... Fundulus heteroclitus, which shares many developmental features with the zebrafish. In both organ- isms, epiboly can be considered a two-phase process ( Betchaku and Trinkaus, 1978;Strähle and Jesuthasan, 1993). The first phase, initiation, encompasses the transition from sphere stage (4 hours post-fertilization, hpf) to dome stage (4.3 hpf, Fig. 1 A,B) and the major cell movement during this transition is radial intercalation ( Warga and Kimmel, ...
Context 4
... flattens and the embryo assumes a spherical shape (Fig. 1A). At this time, the EVL becomes lineage restricted . In Fundulus, the EVL is held taut by tight junction attachments to the YSL, such that severing the connection between the EVL margin and YSL causes the EVL to retract towards the animal pole (Trinkaus, 1951). The situation appears to be similar in zebrafish and increased tension ...
Context 5
... EVL may act to maintain the lineage by keeping mitotic spindles oriented in the plane of the cell sheet ( Kane and Adams, 2002;Kimmel et al., 1990). During doming, the E-YSL contracts along the animal-vegetal axis, causing the YSN to crowd (SolnicaKrezel and Driever, 1994). Concurrently, the yolk cell rounds upwards into the overlying blastoderm (Fig. 1B) such that the blastoderm thins and forms an inverted cup on top of the yolk cell ( Warga and Kimmel, 1990). The mechanism by which the flat yolk-blastoderm interface domes is not understood. Deep cells become motile around the midblastula transition, exhibiting blebs and moving in random directions until the onset of epiboly when cells ...
Context 6
... largest increase in blastoderm surface area occurs during epiboly progression which begins with the completion of doming and continues until the blastopore closes at the vegetal pole (4.3 -10 hpf, Fig. 1C-E). During this phase, stages are defined by the percentage of yolk surface that is covered by the spreading blastoderm. The relative positions of the deep cell, EVL and YSL margins dynamically change throughout the course of epiboly progression; however, the tightly attached YSL and EVL are always located vegetal to the blastoderm margin ...
Context 7
... and YSL margins dynamically change throughout the course of epiboly progression; however, the tightly attached YSL and EVL are always located vegetal to the blastoderm margin (Solnica-Krezel and Driever, 1994). Between 30% and 40% epiboly, the E-YSN progressively become covered by the blastoderm (Solnica-Krezel and Driever, 1994). At 50% epiboly (Fig. 1D), the blastoderm expands to engulf the widest part of yolk and shortly after, deep cell epiboly temporarily ceases as the other movements of gastrulation are initiated (Solnica-Krezel and Driever, 1994;Warga and Kimmel, 1990). This lag in deep cell epiboly allows the EVL and YSL to extend past the blastoderm margin such that the EVL and ...
Context 8
... to extend past the blastoderm margin such that the EVL and YSL cover 65% and 70% of the yolk surface, respectively, when the deep cells have reached 60% epiboly (Solnica-Krezel and Driever, 1994). As a result of internalization, the deep cells become orga- nized into two layers -an outer ectodermal epiblast and an inner mesendodermal hypoblast (Fig. 1E). Unlike initiation, which occurs in isolation from other gastrulation movements, the majority of the progression phase is concurrent with ingression and convergence. Once the blastoderm has spread beyond the equator of the yolk, the margin begins to constrict circumferentially, closing the blasto- ...
Similar publications
A new era in developmental biology has been ushered in by recent advances in the quantitative imaging of all-cell morphogenesis in living organisms. Here we have developed a light-sheet fluorescence microscopy-based framework with single-cell resolution for identification and characterization of subtle phenotypical changes of millimeter-sized organ...
In mammalian ES cells, the transcription factors Klf4 and Klf2 contribute to maintenance of pluripotency and self-renewal and are regulated by Pou5f1/Oct4. In the early zebrafish embryo Pou5f1/Oct4 is necessary for expression of three Klf2/4 family members, klf2a, klf2b and klf17 (previously klf4b), similar to the regulation reported for mammalian...
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 br...
Citations
... Many factors implicated in epiboly are maternally derived (Lepage and Bruce, 2010). The phenotype observed in MZvgll4a embryos was not observed in Zvgll4a mutant embryos obtained from heterozygous in-crosses (data not shown), suggesting that maternal but not zygotic vgll4a contributes to epiboly progression. ...
Gastrulation in zebrafish embryos commences with the morphogenetic rearrangement of blastodermal cells, which undergo a coordinated spreading from the animal pole to wrap around the egg at the vegetal pole. This rearrangement, known as epiboly, relies on the orchestrated activity of maternal transcripts present in the egg, compensating for the gradual activation of the zygotic genome. Epiboly involves the mechano-transducer activity of yap1 but what are the regulators of yap1 activity and whether these are maternally or zygotically derived remain elusive. Our study reveals the crucial role of maternal vgll4a, a proposed Yap1 competitor, during zebrafish epiboly. In embryos lacking maternal/zygotic vgll4a (MZvgll4a), the progression of epiboly and blastopore closure is delayed. This delay is associated with the ruffled appearance of the sliding epithelial cells, decreased expression of yap1-downstream targets and transient impairment of the actomyosin ring at the syncytial layer. Our study also shows that, rather than competing with yap1, vgll4a modulates the levels of the E-cadherin/β-catenin adhesion complex at the blastomeres’ plasma membrane and hence their actin cortex distribution. Taking these results together, we propose that maternal vgll4a acts at epiboly initiation upstream of yap1 and the E-cadherin/β-catenin adhesion complex, contributing to a proper balance between tissue tension/cohesion and contractility, thereby promoting a timely epiboly progression.
... Effect of M. bealei (KT09) extract on epiboly process. Epiboly is characterized by spreading of the cell mass over the yolk sac-an important process that plays a prominent role in embryo gastrulation and specification of the dorsoventral axis [20,52]. It is the first morphogenetic movement in zebrafish and amphibian embryos, leading to the expansion of blastoderm/ectoderm cells along the animal-vegetal axis, thereby covering the yolk cells and closing the blastopore. ...
... One of the earliest defects observed to some extent with all extracts was the delayed epiboly, giving rise to dumb-bell shaped yolks at 8 and 10hpf. As explained above, epiboly is the first morphogenetic movement of cells in zebrafish that depends on the dynamic regulation of cortical F-actin (filamentous actin) in the EVL [52]. Focusing on the treatment with KT09, we confirmed a significant reduction of the F-actin signal and a clear delay in the epiboly progress at 9hpf (Fig 6). ...
... Focusing on the treatment with KT09, we confirmed a significant reduction of the F-actin signal and a clear delay in the epiboly progress at 9hpf (Fig 6). These F-actin networks are essential for the circumferential constriction of the margin during late epiboly stages, and for the structural integrity of the yolk cell [52,54,63]. Treating embryos with the actin-depolymerizing agent cytochalasin at 50% epiboly delayed epiboly, blocked blastopore closure, and led to yolk cell lysis [54], while inhibiting signaling through the small G protein Rac1 disrupted the F-actin organization and/or delayed epiboly [55]. ...
Evaluating the risks and benefits of using traditional medicinal plants is of utmost importance for a huge fraction of the human population, in particular in Northern Vietnam. Zebrafish are increasingly used as a simple vertebrate model for testing toxic and physiological effects of compounds, especially on development. Here, we tested 12 ethanolic extracts from popular medicinal plants collected in northern Vietnam for their effects on zebrafish survival and development during the first 4 days after fertilization. We characterized more in detail their effects on epiboly, hatching, growth, necrosis, body curvature, angiogenesis, skeletal development and mostly increased movement behavior. Finally, we confirm the effect on epiboly caused by the Mahonia bealei extract by staining the actin filaments and performing whole genome gene expression analysis. Further, we show that this extract also inhibits cell migration of mouse embryo fibroblasts. Finally, we analyzed the chemical composition of the Mahonia bealei extract and test the effects of its major components. In conclusion, we show that traditional medicinal plant extracts are able to affect zebrafish early life stage development to various degrees. In addition, we show that an extract causing delay in epiboly also inhibits mammalian cell migration, suggesting that this effect may serve as a preliminary test for identifying extracts that inhibit cancer metastasis.
... Starting at the end of the blastula period (i.e. at~4.3-4.7 h post-fertilization; hpf), epiboly is the first of the large-scale coordinated morphogenetic cell movements that occur during the development of zebrafish (Danio rerio) embryos (Kimmel et al., 1995;Solnica-Krezel, 2006;Lepage and Bruce, 2010;Bruce, 2016;Bruce and Heisenberg, 2020). At this time, the embryo comprises a large yolk cell on the top of which (at the animal pole; AP), sits a mass of cells called the blastoderm. ...
In zebrafish, a punctate band of F-actin is reported to develop in the external yolk syncytial layer (E-YSL) during the latter part of epiboly in zebrafish embryos. Here, electron microscopy (EM) and fluorescence confocal microscopy were conducted to investigate dynamic changes in the E-YSL membrane during epiboly. Using scanning EM, we report that the surface of the E-YSL is highly convoluted, consisting of a complex interwoven network of branching membrane surface microvilli-like protrusions. The region of membrane surface protrusions was relatively wide at 30% epiboly but narrowed as epiboly progressed. This narrowing was coincident with the formation of the punctate actin band. We also demonstrated using immunogold transmission EM that actin clusters were localized at the membrane surface mainly within the protrusions as well as in deeper locations of the E-YSL. Furthermore, during the latter part of epiboly, the punctate actin band was coincident with a region of highly dynamic endocytosis. Treatment with cytochalasin B led to the disruption of the punctate actin band and the membrane surface protrusions, as well as the attenuation of endocytosis. Together, our data suggest that, in the E-YSL, the region encompassing the membrane surface protrusions and its associated punctate actin band are likely to be an integral part of the localized endocytosis, which is important for the progression of epiboly in zebrafish embryos.
... ; https://doi.org/10.1101/2023.06.30.547135 doi: bioRxiv preprint over the massive yolk cell syncytium [2]. Different cell adhesion molecules are required for epiboly [33] among which E-cadherin has been widely studied due to its relevance. ...
Epiboly is an evolutionary conserved morphogenetic massive cell movement in zebrafish that initiates at the onset of gastrulation. The regulation of cell adhesion/cohesion of blastoderm cells during this developmental stage is essential for the coordinated cell movements that characterize gastrulation. Here we show that inhibition of NADPH oxidase (Nox) activity leads to a significant delay in epiboly progression, displays altered E-cadherin and F-actin localization at the enveloping layer (EVL) cell margins and a significant decrease in embryo survival; suggesting that EVL cells have increased endosomal endocytosis. We confirmed this effect since the VAS2870 effects are fully rescued by down-regulating dynamin 2 activity. In addition, we show that proteasome pharmacological inhibition partially rescues the effects on epiboly due to Nox activity inhibition. Overall, our results show that Nox derived reactive oxygen species participate in the regulation of E-cadherin trafficking required for epiboly progression during early zebrafish development.
HIGHLIGHTS
– NADPH oxidases (Nox) are required for epiboly and localization of E-cadherin and F-actin
– Dynamin 2 inhibition rescues early developmental defects due to Nox down regulation
– Proteasome inhibition partially rescues epiboly defects due to Nox down regulation
– Nox derived reactive oxygen species participate in E-cadherin trafficking required for epiboly
... Treatment with 2,5-DCBQ might exert effects on the germ layer development, and disturb the subsequent fundamental body formation. Epiboly is a pivotal morphogenetic movement, which entails the spreading of mesendoderm cells over the yolk and radial cell intercalation (Ulrich et al. 2003;Lepage and Bruce 2010). These biological processes were regulated by the CAMs pathway (Arboleda-Estudillo et al. 2010). ...
2,5-Dichloro-1,4-benzenediol (2,5-DCBQ) is a putative disinfection by-product that belongs to the halogenated benzoquinone class. However, its developmental toxicity and related mechanism remained unclarified. In our study, we used zebrafish embryos as the model and exposed them to graded concentrations of 2,5-DCBQ (100, 200, 300, 400 μg/L). We found that the rate of epiboly abnormalities increased significantly in a concentration-dependent manner. The results of whole-mount in situ hybridization (WISH) indicated that the expression patterns and levels of chordin (dorsoventral marker), foxa2 (endodermal marker), eve1 (ventral mesodermal marker), and foxb1a (ectodermal marker) were altered, suggesting that 2,5-DCBQ might affect the germ layer development of zebrafish embryos. Integrated transcriptomic and metabolomic analyses were adopted to explore the molecular mechanisms of embryonic developmental delays. The results showed that 2,5-DCBQ exposure induced 1163 differentially expressed genes (DEGs) and 37 differential metabolites (DEMs). Bioinformatic analysis enriched the most affected molecular pathways (Wnt signaling pathway, cell adhesion molecules, actin cytoskeleton regulation) and metabolic pathways (purine metabolism, aminoacyl-tRNA biosynthesis, arginine and proline metabolism) in zebrafish embryos. To summarize, our findings broadened the molecular mechanisms of 2,5-DCBQ embryotoxicity through multi-omics and bioinformatic analyses.
Graphical Abstract
... During this reorganization, cells experience several collective displacements, and with that, are exposed to a number of forces that in turn guide tissue morphogenesis [96,97]. One of the most studied gastrulating systems in terms of mechanics is the zebrafish epiboly, a process characterized by blastoderm cells engulfing and expanding over the yolk [98,99] (Fig. 3B). Epiboly starts when the blastoderm transitions from a sphere to a dome stage through a process driven by active surface cell expansion and cell intercalation [100]. ...
The process by which biological systems such as cells, tissues and organisms acquire shape has been named as morphogenesis and it is central to a plethora of biological contexts including embryo development, wound healing, or even cancer. Morphogenesis relies in both self-organising properties of the system and in environmental inputs (biochemical and biophysical). The classical view of morphogenesis is based on the study of external biochemical molecules, such as morphogens. However, recent studies are establishing that the mechanical environment is also used by cells to communicate within tissues, suggesting that this mechanical crosstalk is essential to synchronise morphogenetic transitions and self-organisation. In this article we discuss how tissue interaction drive robust morphogenesis, starting from a classical biochemical view, to finalise with more recent advances on how the biophysical properties of a tissue feedback with their surroundings to allow form acquisition. We also comment on how in silico models aid to integrate and predict changes in cell and tissue behaviour. Finally, considering recent advances from the developmental biomechanics field showing that mechanical inputs work as cues that promote morphogenesis, we invite to revisit the concept of morphogen.
... The initial major morphogenetic movement during the gastrulation stage of embryonic development in some organisms is termed epiboly where the blastoderm grows and covers the yolk. In zebrafish, epiboly involves an organized movement of embryonic cells between 4.3 and 10 h post fertilization (hpf) during which a layer of epithelial cells, are also referred to as the Enveloping Layer (EVL), spreads and covers the yolk cell [36][37][38]. At the start of the epiboly process, the blastoderm, a single multilayer of cells is located at the animal pole of the embryo on top of the yolk cell. ...
... As proliferation takes place, the EVL thins and increases in area [38]. The yolk syncytial layer (YSL) stays in contact with the yolk cell, causing spherical spreading observed in microscopy images such as in [36,37]. In addition to cell proliferation, other mechanisms that contribute to the epiboly process are the polymerization of actin filaments at the leading edge of the EVL, and myosin-driven contraction on the actin belt that forms at junction between the EVL and the YSL [38,39]. ...
We propose a PDE-constrained shape registration algorithm that captures the deformation and growth of biological tissue from imaging data. Shape registration is the process of evaluating optimum alignment between pairs of geometries through a spatial transformation function. We start from our previously reported work, which uses 3D tensor product B-spline basis functions to interpolate 3D space. Here, the movement of the B-spline control points, composed of an implicit function describing the shape of the tissue, yields the total deformation gradient field. The deformation gradient is then split into growth and elastic contributions. The growth tensor captures the addition of mass, i.e. growth, and evolves according to a constitutive equation which is usually a function of the elastic deformation. Stress is generated in the material due to the elastic component of the deformation alone. The result of the registration is obtained by minimizing a total energy functional which includes: a distance measure reflecting similarity between the shapes, and the total elastic energy accounting for the growth of the tissue. We apply the proposed shape registration framework to study zebrafish embryo epiboly process and tissue expansion during skin reconstruction surgery. We anticipate that our PDE-constrained shape registration method will improve our understanding of biological and medical problems in which tissues undergo extreme deformations over time.
... The initial major morphogenetic movement during the gastrulation stage of embryonic development in some organisms is termed epiboly where the blastoderm grows and covers the yolk. In zebrafish, epiboly involves an organized movement of embryonic cells between 4.3 and 10 hours post fertilization (hpf) during which a layer of epithelial cells, are also referred to as the Enveloping Layer (EVL), spreads and covers the yolk cell [33][34][35]. At the start of the epiboly process, the blastoderm, a single multilayer of cells is located at the animal pole of the embryo on top of the yolk cell. ...
... As proliferation takes place, the EVL thins and increases in area [35]. The yolk syncytial layer (YSL) stays in contact with the yolk cell, causing spherical spreading observed in microscopy images such as in [33,34]. In addition to cell proliferation, other mechanisms that contribute to the epiboly process are the polymerization of actin filaments at the leading edge of the EVL, and myosindriven contraction on the actin belt that forms at junction between the EVL and the YSL [35,36]. ...
We propose a PDE-constrained shape registration algorithm that captures the deformation and growth of biological tissue from imaging data. Shape registration is the process of evaluating optimum alignment between pairs of geometries through a spatial transformation function. We start from our previously reported work, which uses 3D tensor product B-spline basis functions to interpolate 3D space. Here, the movement of the B-spline control points, composed with an implicit function describing the shape of the tissue, yields the total deformation gradient field. The deformation gradient is then split into growth and elastic contributions. The growth tensor captures addition of mass, i.e. growth, and evolves according to a constitutive equation which is usually a function of the elastic deformation. Stress is generated in the material due to the elastic component of the deformation alone. The result of the registration is obtained by minimizing a total energy functional which includes: a distance measure reflecting similarity between the shapes, and the total elastic energy accounting for the growth of the tissue. We apply the proposed shape registration framework to study zebrafish embryo epiboly process and tissue expansion during skin reconstruction surgery. We anticipate that our PDE-constrained shape registration method will improve our understanding of biological and medical problems in which tissues undergo extreme deformations over time.
... Zebrafish has been widely used as a model organism for various developmental processes, such as epiboly, bone generation and pigment patterningjust to name a few (Bruce and Heisenberg, 2020;Lepage and Bruce, 2010;Marques et al., 2019;Patterson and Parichy, 2019). In zebrafish development, ERK is also activated downstream of RTKs, especially FGF receptors. ...
The extracellular signal-regulated kinase (ERK) pathway governs cell proliferation, differentiation and migration, and therefore plays key roles in various developmental and regenerative processes. Recent advances in genetically encoded fluorescent biosensors have unveiled hitherto unrecognized ERK activation dynamics in space and time and their functional importance mainly in cultured cells. However, ERK dynamics during embryonic development have still only been visualized in limited numbers of model organisms, and we are far from a sufficient understanding of the roles played by developmental ERK dynamics. In this Review, we first provide an overview of the biosensors used for visualization of ERK activity in live cells. Second, we highlight the applications of the biosensors to developmental studies of model organisms and discuss the current understanding of how ERK dynamics are encoded and decoded for cell fate decision-making.
... In the yolk cell, an initially a-nuclear lipid sphere of roughly 700 µm in diameter [1], MTs are also present and account for transport of molecules essential for early development [2][3][4][5]. The current view for the MTs organization in the yolk cell describes a network of parallel MTs that emerge from the marginal blastomeres, extend towards the vegetal pole and are located at the superficial region of the yolk cell, in the Yolk Cytoplasmic Layer (YCL) [3,[6][7][8][9]. Reaching the 512-cell stage, a syncytium forms in the yolk, called Yolk Syncytial Layer (YSL) [1], containing the Yolk Syncytial Nuclei (YSN) [8], which derive from the collapse of the marginal cells and the release of their content into the yolk mass. ...
... The current view for the MTs organization in the yolk cell describes a network of parallel MTs that emerge from the marginal blastomeres, extend towards the vegetal pole and are located at the superficial region of the yolk cell, in the Yolk Cytoplasmic Layer (YCL) [3,[6][7][8][9]. Reaching the 512-cell stage, a syncytium forms in the yolk, called Yolk Syncytial Layer (YSL) [1], containing the Yolk Syncytial Nuclei (YSN) [8], which derive from the collapse of the marginal cells and the release of their content into the yolk mass. YCL MTs are believed to be associated with the most vegetally located external YSN (eYSN) once the YSL has been formed. ...
... These parallel MT arrays have been studied mainly for their role in the mechanism of epiboly, and have been proved to be necessary for the correct migration of the eYSN toward the vegetal pole [7], through associated motor proteins [9]. Besides that, the presence of a MTs mesh in the internal YSL (iYSL) between the internal YSN (iYSN) and eYSN, has also been described [6][7][8]. However, little is known about the organization and changes that the yolk MT network presents at cleavage and blastula stages. ...
During its first hours of development, the zebrafish embryo presents a large microtubule array in the yolk region, essential for its development. Despite of its size and dynamic behavior, this network has been studied only in limited field of views or in fixed samples. We designed and implemented different strategies in Light Sheet Fluorescence microscopy for imaging the entire yolk microtubule (MT) network in vivo. These have allowed us to develop a novel image analysis from which we clearly observe a cyclical re-arrangement of the entire MT network in synchrony with blastoderm mitotic waves. These dynamics also affect a previously unreported microtubule array deep within the yolk, here described. These findings provide a new vision of the zebrafish yolk microtubules arrangement, and offers novel insights in the interaction between mitotic events and microtubules reorganization.
