Adherens junction function and regulation during zebrafish gastrulation.

The adherens junction (AJ) comprises multi-protein complexes required for cell-cell adhesion in embryonic development and adult tissue homeostasis. Mutations in key proteins and mis-regulation of AJ adhesive properties can lead to pathologies such as cancer. In recent years, the zebrafish has become an excellent model organism to integrate cell biology in the context of a multicellular organization. The combination of classical genetic approaches with new tools for live imaging and biophysical approaches has revealed new aspects of AJ biology, particularly during zebrafish gastrulation. These studies have resulted in progress in understanding the relationship between cell-cell adhesion, cell migration and plasma membrane blebbing.

Introduction

Cell-cell adhesion plays a critical role in many processes in embryonic development, adult homeostasis and diseases. Defining the molecular mechanisms involved in cell-cell adhesion is, therefore, critical to understanding many fundamental problems in tissue organization, dynamics and function. While there are many different cell-cell adhesion proteins, the cadherin family of cell-cell adhesion proteins is thought to play a central role in initiating cell-cell adhesion and controlling cellular dynamics and fate. The cadherin family comprises trans-membrane proteins that share multiple copies of an external domain called the EC domain. Classical cadherins have five EC domains that mediate trans-interactions with the extracellular domain of cadherins on opposing cells to form the adherens junction (AJ). The cytoplasmic domain binds proteins that belong to the catenin family that includes p120-catenin, β-catenin and α-catenin. p120-catenin is necessary for the stabilization of the cadherin complex at the plasma membrane, and together with β-catenin associates with cadherins during their intracellular transport to the plasma membrane. β-catenin binds α-catenin, and α-catenin mediates interactions with the actin cytoskeleton. Recent studies have revaluated the role of mammalian α-catenin from a simple static molecular bridge between AJs and the actin cytoskeleton, to an active role in regulating actin cytoskeleton and membrane dynamics;- it is unknown whether zebrafish α-catenin has properties similar to the mammalian isoform. Additional functions for α-catenin may include mechanical transduction and cell polarization., The catenin proteins are also involved in signal transduction pathways where they work as a sensor between the adhesive property of the plasma membrane and gene expression.-

In order to gain insights about the role and regulation of the AJs it is important to explore in vivo models. In this respect, Danio rerio (zebrafish) gastrulation has become an excellent model system. Gastrulation is a critical stage of animal development at the end of which the embryo acquires the future body plan including the anterior-posterior and dorsal-ventral axes, and the separation of the three germ layers (ectoderm, mesoderm and endoderm)., Gastrulation involves cell migration, cell sorting and tissue remodeling all of which require very fine regulation of cell-cell adhesion.,

Zebrafish gastrulation consists of four morphogenetic processes: epiboly, internalization, convergence and extension. Epiboly is defined as the spreading of a one tissue over another that causes the thinning of the initial tissue. Epiboly in zebrafish involves two processes: spreading of the most external cell layer (the enveloping layer, EVL) and, independently, spreading of deep cells over the yolk cell until the yolk is completely covered by the two cell layers. Spreading of the EVL cell layer may be driven by the changing morphology of yolk syncytial layer (YSL)., The YSL is a cytosolic, yolk-free region of the yolk cell found immediately under the blastoderm and connected to the EVL through tight junctions., The YSL may provide a pulling force on the EVL margin through an actin ring located just below the EVL margin. The main process that drives deep cell epiboly is radial intercalation, which involves the movement of deep cells from deeper layers to most superficial layers (Fig. 1). This causes thinning of the presumptive ectoderm and, as a consequence, the presumptive ectoderm spreads over the yolk.

Figure 1. Radial intercalation. (A) Deep cells from the lower layer (red) move upwards and intercalate between the deep cells in the upper layer. (B) Radial intercalation drives expansion of the upper layer and triggers the overall epiboly of the deep cell tissue. (C) In αE-catenin morphants and E-cadherin mutant/morphant embryos, the deep cells are able to undergo radial intercalation but some cells migrate back in the lower layer (revRI). The revRI affects the proper expansion of the upper layer, resulting in a delay or block in epiboly of the deep cell tissue.,

Internalization occurs when the meso-endoderm moves through the blastoderm beneath the prospective ectoderm., Around mid-gastrulation, convergence and extension movements take place. Convergence narrows the tissue medio-laterally while extension elongates the embryo anterio-posteriorly. The different subtypes of mesodermal precursor undergo different cell movements that contribute to the final convergence and extension.

Adherence Junction Function during Zebrafish Gastrulation

The role of E-cadherin in gastrulation has been examined in mutant embryos, and in morphant embryos depleted of the protein by morpholino (an antisense oligo used to block translation or splicing of the targeted mRNA). Strong mutant alleles and a high concentration of morpholino cause epiboly arrest or delay, and strong defects in gastrulation.- Epiboly arrest is due to defects in radial intercalation. E-cadherin mutant and morphant cells undergo radial intercalation but they are not able to establish stable cell-cell contacts with the upper layer cells, and move back in the lower layer (reverse radial intercalation or de-intercalation; Fig. 1)., The deep cells in the most external layer establish cell-cell contacts with the opposing basal membrane of the EVL cells. In E-cadherin mutant/morphant embryos, the EVL/deep cells interactions are impaired, which might contribute to the overall defect in radial intercalation of the deep cells.,

A low concentration of morpholino is useful to dissect the role of E-cadherin in mesoderm migration. E-cadherin is required for the collective migration of the pre-chordal plate progenitor. During their migration, the pre-chordal plate progenitors need to establish contact with the prospective ectoderm located over them and E-cadherin is required to mediate this interaction. Moreover, morphant cells fail to properly elongate and decrease their rate of migration. The progenitors of the pre-chordal plate are surrounded by another subpopulation of mesodermal cells that have a lower level of E-cadherin. Increasing the level of E-cadherin in the latter cells affects the correct cell migration of the pre-chordal plate progenitors indicating that differentially expression of E-cadherin may finely modulate the correct migration of mesodermal precursors as a whole tissue. Finally, E-cadherin plays a role in coordinating the convergence and extension movement of the YSL nuclei with the movement of the mesodermal precursors., N-cadherin is required for lateral mesoderm migration and a gradient of bone morphogenic protein (BMP) that is present in the gastrula might regulate N-cadherin mediated cell migration.,

Depletion of αE-catenin by morpholino causes a delay in epiboly. The delay is caused by defects in both radial intercalation and EVL epiboly. αE-Catenin morphant cells, like E-cadherin morphant cells, undergo radial intercalation but move back into the lower layer (Fig. 1). In spite of similar defects in cell-cell adhesion, the structure of the deep cells layers appears different in the E-cadherin morphant and αE-catenin morphant (Fig. 2). These differences could reflect different cell behaviors and the presence of E-cadherin in the αE-catenin morphant cells. Additionally, αE-catenin depleted cells exhibit protracted plasma membrane blebbing (see below for further discussion). The delay in EVL epiboly correlates with the failure of the EVL cells at the margin to properly elongate (changing in cell morphology that correlates with epiboly progression). The fact that αE-catenin causes an epiboly defect in the EVL could be due to: (1) αE-catenin plays some role in regulating the actin cytoskeleton similar to what described in tissue culture cells or (2) αE-catenin depletion affects the functionality of other adhesion complex such as tight junction. This additional role of α-catenin might occur in the EVL and YSL.

Figure 2. Tissue morphogenesis during radial intercalation. (A–D) Structure of the deep cell layers at the onset of epiboly (A) and at 60% epiboly in wild-type (B), and E-cadherin (C) and αE-catenin morphants (D). (A) Deep cells are organized in multiple layers with weak cell-cell contacts and have a rounded morphology; the deep cells of the deeper layer migrate toward the upper layer (the arrow indicates the direction of migration); the horizontal line defines the focal plane for the images shown in (A’–D’) (see below). (B) At 60% epiboly in a wild-type background, deep cells stabilize cell-cell contacts and form one or two compact layer., (C) At 60% epiboly in E-cadherin (cdh1) morphant/mutant embryos, the deep cells fail to stabilize cell-cell contacts and some cells undergo revRI, (direction of arrow). (D) At 60% epiboly in αE-catenin (ctnna1) morphant embryos, the deep cells undergo revRI (direction of arrow) but also exhibit extensive membrane blebbing. (A’–D’) Confocal images of the most external deep layers below the EVL. The horizontal bar in (A) indicates the focal plane. Cells express a membrane bound GFP to highlight the plasma membrane, and images are shown in reverse contrast. (A’) Wild-type external layer at the onset of epiboly; some deep cells are rounded and have not yet engaged in stable cell-cell contacts. (B’) Wild-type external layer at 60% epiboly; the deep cell form a compacted layer of cells. (C’) The external cell layer in an E-cadherin morphant at 60% epiboly; the deep cells are rounded and fail to form an external compacted layer of cells. (D’) The external cell layer in an αE-catenin morphant at 60% epiboly; the deep cells are more engaged in cell-cell contacts than the E-cadherin morphants, but they also fail to form a compacted layer of cells and have extensive plasma membrane blebbing (cells marked by an asterisk).

Adherens Junction Regulation during Zebrafish Gastrulation

An example of E-cadherin regulation is the repression of gene expression by Snail during epithelial-to-mesenchyme transition (EMT).- Two studies highlight this type of regulation of cell-cell adhesion in zebrafish., The first study focused on the role of the two zebrafish Snail isoforms, each of which regulates the repression of E-cadherin in different regions of the mesoderm. The second study highlighted E-cadherin repression by a pathway activated by prostaglandin. Interfering with prostaglandin signaling by depleting its receptor results in broad overexpression of E-cadherin, which caused defects in gastrulation. The increased E-cadherin expression is due to snail degradation by the proteasome.

An increasing number of reports highlight the importance of post-translational regulation of proteins during morphogenesis to enable the adhesive properties of cells to be changed rapidly. Regulation of cadherin by intracellular trafficking is an efficient pathway to quickly change cell-cell adhesion. For example, pre-chordal plate progenitor cells respond to a migratory cue from Wnt11, that in turn regulates the E-cadherin intracellular localization through Rab5. Depletion of zebrafish prion protein also causes defects in radial intercalation similar to E-cadherin mutants due to the relocation of E-cadherin to intracellular Rab11-positive vesicles.

Another way to influence cell-cell function is to functionally block the interaction between E-cadherin and the cytoplasmic catenin complex. In zebrafish, overexpression of Gα12/13 negatively regulates E-cadherin-dependent cell-cell adhesion, and in vitro inhibits binding of β-catenin to E-cadherin., Moreover, Wnt signaling in necessary to stabilize β-catenin and in turn β-catenin stabilized E-cadherin at the plasma membrane during the onset of epiboly. Finally influencing the plasma membrane domain to which the AJ is localized can influence cell polarity. For example, the adhesion molecule epCAM is necessary for the enrichment of E-cadherin at the basal membrane of the EVL.

Does the Adherens Junction Play a Role in Plasma Membrane Blebbing?

Blebbing is a type of plasma membrane protrusion driven by detachment of the plasma membrane from the cell cortex and cytoplasmic hydrostatic pressure, which cause the plasma membrane to transiently protrude outwards. Plasma membrane blebbing, unlike lamellipodial formation, does not involve actin polymerization in the initial protruding phase. Once the membrane bleb is formed, ERM proteins (ezrin or moesin) are recruited to the plasma membrane to reconnect the membrane to the actin cytoskeleton in the cell cortex (membrane-to-cortex attachment, MCA).- Finally, activation of Rho signaling, myosin II activity and F-actin assembly cause the retraction of the membrane bleb.,,

The zebrafish has been particularly useful in gaining new insight into the mechanisms underlying plasma membrane blebbing.,- The regulation of blebbing plays a critical role in tuning the timing and direction of the migration of cells during gastrulation;,- blebbing is also used by the primordium germs cells (PGC) to reach the future gonad., The role of cell-cell adhesion during plasma membrane blebbing is poorly understood. Depletion of E-cadherin leads to defects in the migration of PGC, and a high concentration of morpholino impairs blebbing. In contrast, increased E-cadherin levels, and hence cell-cell adhesion, correlates with increased plasma membrane blebbing.

Depletion of αE-catenin causes protracted membrane blebbing by deep cells undergoing radial intercalation. Depletion of E-cadherin does not cause blebbing, whereas co-depletion of E-cadherin and αE-catenin inhibits membrane blebbing. To understand the relationship between αE-catenin and E-cadherin in plasma membrane blebbing, transplantation experiments have been performed in which a group of cells are transferred between embryos of different genetic backgrounds to test if a phenotype is cell autonomous. Morphant cells depleted of αE-catenin have protracted membrane blebbing in a wild-type background. In contrast, when these cells are transfer into an E-cadherin morphant embryo they do not exhibit membrane blebbing. These results indicate that membrane blebbing requires E-cadherin-mediated regulation of cortical tension and, perhaps, cell-cell contact. Significantly, E-cadherin depletion also inhibited increased blebbing caused by ezrin depletion, which is required for MCA.,

Based on this result, αE-catenin may have a role in regulating the dynamic connection between the plasma membrane and the cell cortex. During radial intercalation, deep cells migrate in the upper deep cell layer and establish cell-cell contact/adhesion with neighboring cells. The plasma membrane of deep cells is very dynamic and possible under tension. Under those conditions, membrane blebbing can still be triggered but with low frequency. Depletion of αE-catenin, or ezrin, destabilizes the connection between the plasma membrane and the cell cortex and, therefore, relieves the suppression of membrane dynamics that is not matched by opposing forces at the cell cortex. Reducing the level of E-cadherin decreases plasma membrane activity/tension, therefore reducing the requirement for factors that normally suppress blebbing.

Two recent reports appear to support this model. The first report showed that deep cells mutant for eomesodermin, a transcription factor needed to trigger the onset of epiboly, displayed more blebbing than control cells. Interestingly the increased blebbing in the eomesodermin mutant did not correlate with increased E-cadherin levels (tested by western blotting) although the morphology of the most external deep cell layer resembled cells with increased cell-cell adhesion. The second report showed that mesodermal cells after internalization undergo a phase in which they bleb followed by a transition to a mesenchymal morphology and behavior. Mesodermal cells mutant for spadetail (a transcription factor) remain much longer in the blebbing phase than wild-type cells. Again, these cells do not show any increase of cadherins at the membrane, however they are more adhesive than control cells. These observations suggest that inactivation of MCA factors it is not matched by decreased adhesive property of involuting mesodermal cells. Interestingly, the adhesive properties are not dictated by the amount of cadherins present in the cell but most likely by post-translation modifications (see above). Taken together these reports indicate that blebbing might be regulated by tuning the interaction between the cell cortex and cell-cell adhesion complexes.

Perspective

In past 10 years, many studies have highlighted the function and regulation of AJs during zebrafish gastrulation. These studies started with the characterization of mutants and morphants to dissect AJ functions in complex morphogenetic process. The advance of new methods allowed the study of cellular behaviors in parallel with studies of the biophysical properties of cells. These advances have led to a new appreciation of the role of cell-cell adhesion complexes and their link to the actin cytoskeleton in plasma membrane dynamics and cortical tension during complex developmental processes such as collective cell migration and cell sorting. In this respect, the study of zebrafish gastrulation has much to offer the cell biologist in the future.