Dynamin-dependent maintenance of epithelial integrity is essential for zebrafish epiboly

Abstract

Epiboly, the thinning and spreading of one tissue over another, is a widely employed morphogenetic movement that is essential for the development of many organisms. In the zebrafish embryo, epiboly describes the coordinated vegetal movement of the deep cells, enveloping layer (EVL) and yolk syncytial layer (YSL) to engulf the yolk cell. Recently, we showed that the large GTPase Dynamin plays a fundamental role in epiboly in the early zebrafish embryo. Because Dynamin plays a well-described role in vesicle scission during endocytosis, we predicted that Dynamin might regulate epiboly through participating in bulk removal of the yolk cell membrane ahead of the advancing margin, a proposed part of the epiboly motor. Unexpectedly, we found that Dynamin function was dispensable in the yolk cell and instead, it was required to maintain the epithelial integrity of the EVL during epiboly. Here, we present a model describing the maintenance of EVL integrity, which is required for the proper generation and transmission of tension during epiboly. Furthermore, we discuss the role of Dynamin-mediated regulation of ezrin-radixin-moesin (ERM) family proteins in the maintenance of epithelial integrity.

Introduction

A commonly employed morphogenetic movement in animal development is epiboly, the thinning and spreading of cells to engulf a yolk mass or envelop other cells.¹ Despite the widespread use of this fundamental cell movement during morphogenesis, our understanding of the cellular and molecular mechanisms that drive this process is limited. Epiboly is an essential movement during zebrafish gastrulation. The embryonic cells, which begin their development on top of a large yolk cell, move vegetally to engulf the yolk cell and close the blastopore by the end of gastrulation. Three distinct regions of the embryo undergo epiboly in a coordinated manner. The blastoderm consists of two such domains, the enveloping layer (EVL) and the deep cells. The EVL is a single-cell-thick epithelium that covers the loosely packed deep cells. The EVL margin is tightly attached to the yolk syncytial layer (YSL), a layer of syncytial nuclei that reside near the animal surface of the yolk cell, just beneath the blastoderm. Together, the EVL and YSL completely encase the deep cells.²

A long-held view is that the yolk cell provides the motive force that drives epiboly movements in the zebrafish. The YSL moves vegetally through the concerted actions of the yolk microtubule and actin cytoskeletons. The epibolizing YSL pulls the tightly attached EVL and encased deep cells toward the vegetal pole.^3-6 A localized region of active, bulk endocytosis, positioned just ahead of advancing blastoderm margin, likely acts to remove the exposed yolk cell membrane as epiboly proceeds.^5,6 This process can be visualized by submerging embryos in fluorescent dextrans, which are taken up by fluid-phase endocytosis. The resultant fluorescently labeled vesicles form a discrete circumferential band within the external YSL (E-YSL) and marginal EVL.⁶

It has been suggested that the bulk endocytic removal of the yolk cell membrane is a component of the yolk cell epiboly motor, contributing to the timely progression of epiboly.⁶ In our recent work, we sought to test this long held view by determining the effect of inhibiting yolk cell endocytosis on epiboly.⁷ To accomplish this, we centered our attention on Dynamin, a large multi-domain GTPase that has a well-established role in endocytosis.^8,9

Dynamin-Dependent Yolk Cell Endocytosis Does Not Contribute to Epiboly Progression

In our recent study, we used both drug-based (dynasore) and dominant-negative (DN) approaches to interfere with Dynamin function. We found that global inhibition of Dynamin function resulted in reduced marginal endocytosis, coupled with a highly penetrant epiboly delay. Both treatments resulted in delayed epiboly of the blastoderm, while epiboly of the YSL was only partially disrupted. Unexpectedly, targeted knock-down of Dynamin in the yolk cell had no effect on epiboly progression, despite inhibiting marginal endocytosis to the same extent as global Dynamin inhibition.⁷

In contrast to the prevailing model of zebrafish epiboly, these data suggest that Dynamin-mediated endocytosis in the yolk is dispensable for epiboly. However, as neither treatment completely abolished marginal endocytosis, it remains possible that other Dynamin-independent endocytic mechanisms are sufficient to drive epiboly movements. Alternatively, small volume endocytic vesicles, not detectable using the fluid-phase uptake assay, may compensate for the loss of the Dynamin-dependent fraction. To investigate this requires the identification of other components of the endocytic machinery that are involved in yolk cell endocytosis, and then systematically impairing them in a yolk-biased manner. Rab5 is a small GTPase that plays an important role in trafficking endocytic vesicles to the early endosome.¹⁰ Although we found Rab5-positive endocytic compartments within the yolk using immunohistochemistry, they were sparse in the YSL, the zone of marginal yolk endocytosis (SEL, unpublished data). Furthermore, yolk-biased expression of a dominant-negative Rab5 construct had no effect on epiboly progression (SEL, unpublished data). Although additional work is required, we propose that marginal endocytosis may not be a major contributor to the epiboly motor.

Dynamin is Required For Normal EVL Cell Morphology and Epithelial Integrity

Our results suggested that the epiboly delay observed in response to global Dynamin inhibition was the consequence of defects in the blastoderm rather than the yolk cell. Moreover, we suspected that there were defects in the EVL, as fluid-phase markers often passed into the deep cell layer in dynasore-treated embryos, indicating that the barrier function of the EVL was compromised. In addition, we occasionally observed cells surpassing the EVL margin. We suspected that these were deep cells, which escaped because of impaired connections between the EVL and YSL.¹¹

The importance of epithelial integrity for normal morphogenesis has been demonstrated in many systems,¹² and an increasing body of work, from our lab and others, point to the importance of an intact and properly differentiated EVL for normal epiboly.^13-17 The EVL is under considerable tension during epiboly and must withstand substantial stretching. Strong adhesion not only between EVL cells themselves, but also between EVL cells and the YSL is essential for the efficient transmission and generation of tension within the EVL sheet and is required for the maintenance of its integrity. Concomitantly, this adhesion must be dynamic, to permit the cell rearrangements that occur to reduce the number of cells at the EVL margin to facilitate blastopore closure.¹¹ Thus, the EVL must be strong, yet flexible in the face of considerable mechanical challenges.

EVL integrity was disrupted in dynasore-treated embryos. Phalloidin staining revealed that scattered EVL cells possessed rounded morphologies and F-actin-rich basal protrusions. In live time-lapse movies of treated embryos, EVL cells were observed abnormally losing and reforming cell-cell contacts and, on occasion, cells basally extruding from the epithelium. The localization of the tight junction marker ZO-1 was disrupted in dynasore-treated embryos.⁷ This is likely to be significant, as our previous work on another tight junction component, Claudin E, demonstrated the importance of EVL tight junctions for normal epiboly progression.¹⁷ In addition, disrupted osmoregulation, caused by a systemic failure to differentiate the EVL, is believed to underlie the epiboly defect in poky mutants.¹⁴

Because EVL cells transiently lost contact with each other upon dynasore treatment, we expected the adherens junctions to be severely disrupted. Furthermore, inhibiting the endocytic recycling of adherens junction components is known to disrupt epithelial integrity.^18,19 Unexpectedly, adherens junction components, Cadherin and β-Catenin, were enriched in the EVL; however, both proteins were abnormally shifted toward the basal side of EVL cells. Epithelial cells are normally coupled through a transcellular actin network, linked together through the adherens junction.²⁰ We hypothesize that the mislocalization of adherens junction components prevents the effective mechanical coupling between cells in the EVL. This, in conjunction with the disruption to tight junctions, leads to defects in the generation and transmission of tension in the EVL, leading to disrupted epiboly.

Ezrin-Radixin-Moesin (ERM) Proteins and EVL Integrity

The most obvious defect detected by antibody staining dynasore-treated embryos was the reduction in cortically localized activated ERM in the EVL cells. ERM proteins play important roles in cortical actin organization and in actin filament crosslinking to the plasma membrane.^21,22 Notably, in several other systems, impaired ERM function results in defects in actin organization and disrupted apical-basal polarity. In particular, in the wing imaginal disc of Drosophila moesin mutants, cells are extruded basally from the epithelium.²³ Given the striking similarities between the EVL defects in dynasore-treated embryos and the epithelial defects in other systems upon disruption of ERM function, we investigated whether ERMs were involved in the epiboly defects seen in embryos with compromised Dynamin function. To accomplish this, we used a previously characterized morpholino against erzin b (formerly ezrin 2) to knock down its function.^24,25 We found that ezrin b morphant embryos closely resembled dynasore-treated embryos. In some contexts, ERM proteins negatively regulate Rho A function,^23,26 and consistent with these studies, we were able to rescue epithelial integrity and epiboly in dynasore-treated embryos by injecting dominant-negative Rho RNA.

Thus, our data are in keeping with work in other systems demonstrating that ERM protein function is critical for the maintenance of epithelial cell morphology and integrity.^23,26-28 Furthermore, ERM might maintain epithelial integrity through antagonizing Rho activity. Although the link between Dynamin and Ezrin B remains unclear, and may in fact be very indirect, these experiments highlight an important role for Ezrin B in epithelial integrity and polarity in the EVL, which in our view warrants further investigation. Future work to extend our findings would enable us to learn more about this potentially conserved role for ERM proteins in epithelial morphogenesis.

In our recent work, we were hampered in our ability to investigate the function of Ezrin B in the EVL due to our inability to rescue the morphant phenotype. Recently, ezrin b mutant lines were generated as part of the Sanger Institute Mutation Project.²⁹ This line will be a useful tool for future studies. Previously, we were unable to validate that activated Rho A levels were increased in dynasore-treated embryos. We attempted to detect activated Rho A in live embryos by global expression of RNA encoding GFP-tagged Rhotekin-GBD³⁰ and by performing activated Rho pull-down assays. However, the basal level of EVL Rho A activity is below the threshold of what the GFP-tagged Rhotekin-GBD can detect, and neither approach was sensitive enough to detect changes in Rho A activity. For technical reasons, global RNA injection often does not result in extensive overexpression in EVL cells. Further, global delivery methods do not allow us to differentiate between EVL- or deep cell-specific protein functions. Thus, the krt18: KalTA4-ERT2 transgenic line, first reported in our study,⁷ will serve as an invaluable tool by allowing various constructs to be specifically expressed in the EVL. Specifically, dominant-negative ERM protein constructs³¹ should be targeted to the EVL to assess their effect on cell morphology and epithelial integrity. In addition, it would be interesting to determine whether EVL-targeted overexpression of ERM proteins can restore the dynasore-induced epithelial defects. Expression of constitutively active (CA)-Rho caused similar EVL defects as those observed in dynasore-treated embryos and ezrin morphants, but also caused severe deep cell defects (SEL, unpublished data). CA-Rho and DN-Rho constructs could also be expressed in the EVL alone or in combination with DN-Ezrin B. If Rho is negatively regulated by Ezrin B, then expression of CA-Rho would be expected to cause similar defects as ezrin b loss-of-function, whereas DN-Rho would be anticipated to rescue the effects of ezrin b loss-of-function.

In the Drosophila blastoderm, it has been shown that the synaptotagmin Bitesize is responsible for recruiting Moesin to the apical membrane during cell polarization.²⁷ Future work should address whether synaptotagmin plays a similar role in recruiting Ezrin B to the EVL membrane. Several synaptotagmin genes are present in the zebrafish genome (zfin.org); however, none of these genes have been examined at early developmental stages. Thus, the first step would be to determine whether any synaptotagmins are expressed in the EVL during epiboly. If so, then morpholinos or other genetic approaches can be used to block synaptotagmin function, and determine if recruitment of Erzin B to the membrane is impaired and EVL integrity is affected.

Concluding Remarks

Epiboly is a widely employed cell movement in animal development, yet there is still much to learn regarding its underlying cellular and molecular mechanisms. The zebrafish is an excellent model in which to study the process as it occurs in three distinct contexts: the EVL, a monolayered epithelium; the deep cells, a multilayered cell mass; and the YSL, a multi-nucleate syncytium. Our recent work has called into question the long held, but never experimentally tested, view that a discrete region of marginal endocytosis in the yolk cell is part of the epiboly motor. At the same time, our study further highlights the importance of EVL polarity and integrity for normal epiboly progression. Our current working model (Fig. 1) proposes that the recruitment of Ezrin B to the EVL apical cortex, potentially by synaptotagmin, is required to organize and stabilize the cortical actin cytoskeleton in the sub-apical domain. Proper actin organization is required for the maintenance of adherens junctions, which is in turn essential for the maintenance of the transcellular actin network and tissue integrity.

Figure 1.A model for Dynamin-dependent maintenance of epithelial integrity. Possibly through the regulation of synaptotagmin (blue), Dynamin localizes activated ERM proteins (red) to the cortex. ERM proteins anchor the cortical F-actin belts (green) in the subapical region. The cadherin/catenin complex (purple) links the cortical actin belts of adjacent cells, forming a confluent transcellular network. Upon Dynamin inhibition, tight junctions (yellow) are disrupted, compromising the barrier function of the epithelium. ERM protein activation and/localization is eliminated from the subapical region, leading to a disorganized cortical actin network. The cadherin/catenin complex becomes mislocalized, causing inefficient mechanical coupling of cells and the breakdown of epithelial integrity.

Glossary

Abbreviations:

DN

dominant negative

ERM

ezrin-radixin-moesin

EVL

enveloping layer

YSL

yolk syncytial layer

Lepage SE1, Tada M, Bruce AE. Zebrafish Dynamin is required for maintenance of enveloping layer integrity and the progression of epiboly. Dev Biol. 2014;385:52–66.