Zebrafish Epiboly Research Articles

NADPH oxidases regulation of vesicular trafficking during zebrafish epiboly.

NADPH oxidases regulation of vesicular trafficking during zebrafish epiboly.

Maternal vgll4a regulates zebrafish epiboly through Yap1 activity.

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.

Actin-mediated endocytosis in the E-YSL helps drive epiboly in zebrafish.

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.

In preprints: buckling under pressure during gastrulation.

In preprints: buckling under pressure during gastrulation.

Multiple asters organize the yolk microtubule network during dclk2-GFP zebrafish epiboly

It is known that the organization of microtubule (MT) networks in cells is orchestrated by subcellular structures named MT organizing centers (MTOCs). In this work, we use Light Sheet Fluorescence and Confocal Microscopy to investigate how the MT network surrounding the spherical yolk is arranged in the dclk2-GFP zebrafish transgenic line. We found that during epiboly the MT network is organized by multiple aster-like MTOCS. These structures form rings around the yolk sphere. Importantly, in wt embryos, aster-like MTOCs are only found upon pharmacological or genetic induction. Using our microscopy approach, we underscore the variability in the number of such asters in the transgenic line and report on the variety of global configurations of the yolk MT network. The asters’ morphology, dynamics, and their distribution in the yolk sphere are also analyzed. We propose that these features are tightly linked to epiboly timing and geometry. Key molecules are identified which support this asters role as MTOCs, where MT nucleation and growth take place. We conclude that the yolk MT network of dclk2-GFP transgenic embryos can be used as a model to organize microtubules in a spherical geometry by means of multiple MTOCs.

Rab5ab-Mediated Yolk Cell Membrane Endocytosis Is Essential for Zebrafish Epiboly and Mechanical Equilibrium During Gastrulation.

Morphogenesis in early embryos demands the coordinated distribution of cells and tissues to their final destination in a spatio-temporal controlled way. Spatial and scalar differences in adhesion and contractility are essential for these morphogenetic movements, while the role that membrane remodeling may play remains less clear. To evaluate how membrane turnover modulates tissue arrangements we studied the role of endocytosis in zebrafish epiboly. Experimental analyses and modeling have shown that the expansion of the blastoderm relies on an asymmetry of mechanical tension in the yolk cell generated as a result of actomyosin-dependent contraction and membrane removal. Here we show that the GTPase Rab5ab is essential for the endocytosis and the removal of the external yolk cell syncytial layer (E-YSL) membrane. Interfering in its expression exclusively in the yolk resulted in the reduction of yolk cell actomyosin contractility, the disruption of cortical and internal flows, a disequilibrium in force balance and epiboly impairment. We conclude that regulated membrane remodeling is crucial for directing cell and tissue mechanics, preserving embryo geometry and coordinating morphogenetic movements during epiboly.

MondoA regulates gene expression in cholesterol biosynthesis-associated pathways required for zebrafish epiboly.

The glucose-sensing Mondo pathway regulates expression of metabolic genes in mammals. Here, we characterized its function in the zebrafish and revealed an unexpected role of this pathway in vertebrate embryonic development. We showed that knockdown of mondoa impaired the early morphogenetic movement of epiboly in zebrafish embryos and caused microtubule defects. Expression of genes in the terpenoid backbone and sterol biosynthesis pathways upstream of pregnenolone synthesis was coordinately downregulated in these embryos, including the most downregulated gene nsdhl. Loss of Nsdhl function likewise impaired epiboly, similar to MondoA loss of function. Both epiboly and microtubule defects were partially restored by pregnenolone treatment. Maternal-zygotic mutants of mondoa showed perturbed epiboly with low penetrance and compensatory changes in the expression of terpenoid/sterol/steroid metabolism genes. Collectively, our results show a novel role for MondoA in the regulation of early vertebrate development, connecting glucose, cholesterol and steroid hormone metabolism with early embryonic cell movements.

Mechanisms of zebrafish epiboly: A current view.

Epiboly is a conserved gastrulation movement describing the thinning and spreading of a sheet or multi-layer of cells. The zebrafish embryo has emerged as a vital model system to address the cellular and molecular mechanisms that drive epiboly. In the zebrafish embryo, the blastoderm, consisting of a simple squamous epithelium (the enveloping layer) and an underlying mass of deep cells, as well as a yolk nuclear syncytium (the yolk syncytial layer) undergo epiboly to internalize the yolk cell during gastrulation. The major events during zebrafish epiboly are: expansion of the enveloping layer and the internal yolk syncytial layer, reduction and removal of the yolk membrane ahead of the advancing blastoderm margin and deep cell rearrangements between the enveloping layer and yolk syncytial layer to thin the blastoderm. Here, work addressing the cellular and molecular mechanisms as well as the sources of the mechanical forces that underlie these events is reviewed. The contribution of recent findings to the current model of epiboly as well as open questions and future prospects are also discussed.

Spatiotemporal characterization of dynamic epithelial filopodia during zebrafish epiboly

During zebrafish epiboly, the embryonic cell mass, or blastoderm, spreads to enclose the yolk cell. The blastoderm consists of an outer epithelial sheet, the enveloping layer (EVL), and the underlying deep cell layer (DEL). Studies have provided insights into the mechanisms of EVL and deep cell epiboly, but little is known about the interactions between the two cell layers and what role they may play during epiboly. We used live imaging to examine EVL basal protrusions. We identified them as filopodia based on f-actin content and localization of fluorescently tagged filopodial markers. A spatiotemporal analysis revealed that the largest number of EVL filopodia were present during early epiboly at the animal pole. In functional studies, expression of a constitutively active actin-bundling protein resulted in increased filopodial length and delayed gastrulation. We identified protrusions on the basal surface of EVL cells as filopodia and showed that they are present throughout the EVL during epiboly. The largest number of filopodia was at the animal pole during early epiboly, which is when and where deep cell radial intercalations occur to the greatest extent. These findings suggest that EVL filopodia may function during epiboly to promote deep cell rearrangements during epiboly initiation.

CDK5RAP3, a UFL1 substrate adaptor, is crucial for liver development.

Protein modification by ubiquitin and ubiquitin-like proteins (UBLs) regulates numerous biological functions. The UFM1 system, a novel UBL conjugation system, is implicated in mouse development and hematopoiesis. However, its broad biological functions and working mechanisms remain largely elusive. CDK5RAP3, a possible ufmylation substrate, is essential for epiboly and gastrulation in zebrafish. Herein, we report a crucial role of CDK5RAP3 in liver development and hepatic functions. Cdk5rap3 knockout mice displayed prenatal lethality with severe liver hypoplasia, as characterized by delayed proliferation and compromised differentiation. Hepatocyte-specific Cdk5rap3 knockout mice suffered post-weaning lethality, owing to serious hypoglycemia and impaired lipid metabolism. Depletion of CDK5RAP3 triggered endoplasmic reticulum stress and activated unfolded protein responses in hepatocytes. We detected the in vivo interaction of CDK5RAP3 with UFL1, the defined E3 ligase in ufmylation. Notably, loss of CDK5RAP3 altered the ufmylation profile in liver cells, suggesting that CDK5RAP3 serves as a novel substrate adaptor for this UBL modification. Collectively, our study identifies CDK5RAP3 as an important regulator of ufmylation and suggests the involvement of ufmylation in mammalian development.

Prmt7 regulates epiboly and gastrulation cell movements by facilitating syntenin.

Epiboly spreads and thins the blastoderm over the yolk cell during zebrafish gastrulation. Despite of its fundamental function, little is known about the molecular mechanisms that control this coordinated cell movement. In this study, we investigated protein arginine methyltransferase 7 (Prmt7) morphants with an epibolic delay defect in zebrafish. The ratio of morphants with epiboly delay phenotypes increased as the dose of the injected morpholino (MO) increased. Here, syntenin transcripts are maternally deposited and ubiquitously expressed from the oocyte period to the early larva stage. Furthermore, we demonstrated that Prmt7 modulates epibolic movements of the enveloping layer by regulating F-actin organization. These defects can be partially rescued by re-expression of Prmt7 or syntenin protein. Analysis of the earliest cellular defects suggested a role of Prmt7 in the autonomous vegetal expansion of the yolk syncytial layer and the rearrangement of the actin cytoskeleton in extra-embryonic tissues. By a combination of knockdown studies and rescue experiments in zebrafish, we showed that epiboly relies on the molecular networking of Prmt7 by facilitating syntenin, which acts as a regulator for cytoskeleton. This study identifies the important function of the Prmt7 for the progression of zebrafish epiboly and establishes its key role in directional cell movements during early development.

NADPH-Oxidase-derived reactive oxygen species are required for cytoskeletal organization, proper localization of E-cadherin and cell motility during zebrafish epiboly

Cell movements are essential for morphogenesis during animal development. Epiboly is the first morphogenetic process in zebrafish in which cells move en masse to thin and spread the deep and enveloping cell layers of the blastoderm over the yolk cell. While epiboly has been shown to be controlled by complex molecular networks, the contribution of reactive oxygen species (ROS) to this process has not previously been studied. Here, we show that ROS are required for epiboly in zebrafish. Visualization of ROS in whole embryos revealed dynamic patterns during epiboly progression. Significantly, inhibition of NADPH oxidase activity leads to a decrease in ROS formation, delays epiboly, alters E-cadherin and cytoskeleton patterns and, by 24 h post-fertilization, decreases embryo survival, effects that are rescued by hydrogen peroxide treatment. Our findings suggest that a delicate ROS balance is required during early development and that disruption of that balance interferes with cell adhesion, leading to defective cell motility and epiboly progression.

A cargo model of yolk syncytial nuclear migration during zebrafish epiboly.

In teleost fish, the multinucleate yolk syncytial layer functions as an extra-embryonic signaling center to pattern mesendoderm, coordinate morphogenesis and supply nutrients to the embryo. External yolk syncytial nuclei (e-YSN) undergo microtubule-dependent movements that distribute the nuclei over the large yolk mass. How e-YSN migration proceeds, and the role of the yolk microtubules, is not understood, but it is proposed that e-YSN are pulled vegetally as the microtubule network shortens from the vegetal pole. Live imaging revealed that nuclei migrate along microtubules, consistent with a cargo model in which e-YSN are moved down the microtubules by direct association with motor proteins. We found that blocking the plus-end directed microtubule motor kinesin significantly attenuated yolk nuclear movement. Blocking the outer nuclear membrane LINC complex protein Syne2a also slowed e-YSN movement. We propose that e-YSN movement is mediated by the LINC complex, which functions as the adaptor between yolk nuclei and motor proteins. Our work provides new insights into the role of microtubules in morphogenesis of an extra-embryonic tissue and further contributes to the understanding of nuclear migration mechanisms during development.

Dynamic Alterations in DNA Methylation Precede Tris(1,3-dichloro-2-propyl)phosphate-Induced Delays in Zebrafish Epiboly.

Tris(1,3-dichloro-2-propyl)phosphate (TDCIPP) is an organophosphate flame retardant that impacts zebrafish epiboly - an effect that may be associated with genome-wide hypomethylation. Using zebrafish as a model, the objectives of this study were to (1) quantify concentration-dependent impacts of TDCIPP on epiboly; (2) determine whether co-exposure with folic acid (FA) - a methyl donor - mitigates TDCIPP-induced impacts; and (3) using ten previously identified TDCIPP-susceptible loci, rely on bisulfite amplicon sequencing (BSAS) to monitor CpG methylation dynamics across multiple TDCIPP concentrations in the presence or absence of FA. Embryos were exposed to TDCIPP from 0.75 h post-fertilization (hpf) to 2, 4, 6, or 24 hpf in the presence or absence of 1 mM FA. Although TDCIPP delayed epiboly up to 3 h by 6 hpf and induced malformations by 24 hpf, FA was unable to mitigate TDCIPP-induced effects at all stages evaluated. Moreover, while no differences in global methylation were detected using a 5-methylcytosine (5-mC) DNA ELISA, BSAS revealed that TDCIPP-induced effects on CpG methylation were dependent on concentration and developmental stage, and that early effects on methylation do not persist despite continuous exposure. Our findings demonstrate that TDCIPP delays zebrafish epiboly, a phenotype that is preceded by complex, dynamic alterations in DNA methylation.

An Actomyosin-Arf-GEF Negative Feedback Loop for Tissue Elongation under Stress

In response to a pulling force, a material can elongate, hold fast, or fracture. During animal development, multi-cellular contraction of one region often stretches neighboring tissue. Such local contraction occurs by induced actomyosin activity, but molecular mechanisms are unknown for regulating the physical properties of connected tissue for elongation under stress. We show that cytohesins, and their Arf small G protein guanine nucleotide exchange activity, are required for tissues to elongate under stress during both Drosophila dorsal closure (DC) and zebrafish epiboly. In Drosophila, protein localization, laser ablation, and genetic interaction studies indicate that the cytohesin Steppke reduces tissue tension by inhibiting actomyosin activity at adherens junctions. Without Steppke, embryogenesis fails, with epidermal distortions and tears resulting frommyosin misregulation. Remarkably, actomyosin network assembly is necessary and sufficient for local Steppke accumulation, where live imaging shows Steppke recruitment within minutes. This rapid negative feedback loop provides a molecular mechanism for attenuating the main tension generator of animal tissues. Such attenuation relaxes tissues and allows orderly elongation under stress.

Spatiotemporal changes in viscoelasticity control tissue morphogenesis during zebrafish epiboly

Spatiotemporal changes in viscoelasticity control tissue morphogenesis during zebrafish epiboly

Flotillins control zebrafish epiboly through their role in cadherin-mediated cell-cell adhesion.

Zebrafish gastrulation and particularly epiboly that involves coordinated movements of several cell layers is a dynamic process for which regulators remain to be identified. We show here that Flotillin 1 and 2, ubiquitous and highly conserved proteins, are required for epiboly. Flotillins knockdown compromised embryo survival, strongly delayed epiboly and impaired deep cell radial intercalation and directed collective migration without affecting enveloping layer cell movement. At the molecular level, we identified that Flotillins are required for the formation of E-cadherin-mediated cell-cell junctions. These results provide the first in vivo evidence that Flotillins regulate E-cadherin-mediated cell-cell junctions to allow epiboly progression.

Alkbh4 and Atrn Act Maternally to Regulate Zebrafish Epiboly.

During embryonic gastrulation, coordinated cell movements occur to bring cells to their correct position. Among them, epiboly produces the first distinct morphological changes, which is essential for the early development of zebrafish. Despite its fundamental importance, little is known to understand the underlying molecular mechanisms. By generating maternal mutant lines with CRISPR/Cas9 technology and using morpholino knockdown strategy, we showed that maternal Alkbh4 depletion leads to severe epiboly defects in zebrafish. Immunofluorescence assays revealed that Alkbh4 promotes zebrafish embryonic epiboly through regulating actomyosin contractile ring formation, which is composed of Actin and non-muscular myosin II (NMII). To further investigate this process, yeast two hybridization assay was performed and Atrn was identified as a binding partner of Alkbh4. Combining with the functional results of Alkbh4, we found that maternal Atrn plays a similar role in zebrafish embryonic morphogenesis by regulating actomyosin formation. On the molecular level, our data revealed that Atrn prefers to interact with the active form of Alkbh4 and functions together with it to regulate the demethylation of Actin, the actomyosin formation, and subsequently the embryonic epiboly.

Polarized cortical tension drives zebrafish epiboly movements.

The principles underlying the biomechanics of morphogenesis are largely unknown. Epiboly is an essential embryonic event in which three tissues coordinate to direct the expansion of the blastoderm. How and where forces are generated during epiboly, and how these are globally coupled remains elusive. Here we developed a method, hydrodynamic regression (HR), to infer 3D pressure fields, mechanical power, and cortical surface tension profiles. HR is based on velocity measurements retrieved from 2D+T microscopy and their hydrodynamic modeling. We applied HR to identify biomechanically active structures and changes in cortex local tension during epiboly in zebrafish. Based on our results, we propose a novel physical description for epiboly, where tissue movements are directed by a polarized gradient of cortical tension. We found that this gradient relies on local contractile forces at the cortex, differences in elastic properties between cortex components and the passive transmission of forces within the yolk cell. All in all, our work identifies a novel way to physically regulate concerted cellular movements that might be instrumental for the mechanical control of many morphogenetic processes.

Issue Information

COVER PHOTOGRAPH: Confocal projection of a live embryo expressing a microtubule associated protein fused to GFP. From Zebrafish epiboly: Spreading thin over the yolk, Ashley E.E.Bruce, Developmental Dynamics 245:244–258.Read the full article at DOI: 10.1002/dvdy.24353