Origin, form and function of extraembryonic structures in teleost fishes

Abstract

Teleost eggs have evolved a highly derived early developmental pattern within vertebrates as a result of the meroblastic cleavage pattern, giving rise to a polar stratified architecture containing a large acellular yolk and a small cellular blastoderm on top. Besides the acellular yolk, the teleost-specific yolk syncytial layer (YSL) and the superficial epithelial enveloping layer are recognized as extraembryonic structures that play critical roles throughout embryonic development. They provide enriched microenvironments in which molecular feedback loops, cellular interactions and mechanical signals emerge to sculpt, among other things, embryonic patterning along the dorsoventral and left–right axes, mesendodermal specification and the execution of morphogenetic movements in the early embryo and during organogenesis. An emerging concept points to a critical role of extraembryonic structures in reinforcing early genetic and morphogenetic programmes in reciprocal coordination with the embryonic blastoderm, providing the necessary boundary conditions for development to proceed. In addition, the role of the enveloping cell layer in providing mechanical, osmotic and immunological protection during early stages of development, and the autonomous nutritional support provided by the yolk and YSL, have probably been key aspects that have enabled the massive radiation of teleosts to colonize every ecological niche on the Earth.

This article is part of the theme issue ‘Extraembryonic tissues: exploring concepts, definitions and functions across the animal kingdom’.

1. Introduction

Extraembryonic structures play a fundamental role in the development of the embryo. Broadly speaking, the term extraembryonic or embryonic annexes has historically been coined to designate any biological structure that develops from the zygote but is not part of the embryo, larvae or mature fetus, often located outside it, and which is functionally associated with providing protection and means of transport of nutrients and waste. This early definition then evolved following conceptual and technological advances in the field of developmental biology, to now include cell lineage criteria and the potential to instruct early embryonic development. From a comparative perspective, the developmental trajectory an animal follows has a critical impact on the type, number and form of extraembryonic structures it can develop. In teleost fishes, the focus of this review, a conserved meroblastic cleavage determines the early structural pattern of the egg from which extraembryonic structures emerge. Here we describe the origin, conformation and function of these structures in teleosts, and discuss how embryo–extraembryonic interfaces represent enriched microenvironments of interaction upon which the co-construction and canalization of embryonic ontogenetic programmes unfold.

2. The extraembryonic concept

The extraembryonic concept has changed throughout history. The first recognition of an extraembryonic structure was probably based on topological criteria, i.e. any visible membrane, appendage or fluid sac related to, but separated from, the fetus. The placenta and umbilical cord were recognized as extraembryonic as early as 1400 BC in ancient Egypt [1]. Subsequently, this topological criterium incorporated conserved functions by recognizing them as a source of nutritional support and protection for the developing embryo. In ancient Greece, the physician Hippocrates (c460 BC–c370 BC) suggested that the embryo obtained its blood supply from the placenta and developed by taking in moisture and breath from the mother [2]. Later work by Albertus Magnus of Cologne (AD c1200–AD 1280) and William Harvey (1578–1657) reinforced this idea. Based on systematic descriptions of the chick egg using low-power lenses, Harvey deduced that the amniotic fluid plays a critical role in nutrition and protection [2]. With the advent of chemical embryology and the subsequent discovery of the fundamental role of genes in embryo development [2–5], the extraembryonic concept was expanded to include cell lineages derived from the zygote but separated from it on the basis of different patterns of gene expression. In this new context, the gene regulatory networks involved in the specification of extraembryonic lineages began to be elucidated and the instructive potential of some extraembryonic lineages in embryonic development became evident for the first time [6–8]. More recently, biophysical studies and embryonic stem cells-based synthetic embryology approaches have revealed that extraembryonic structures are not only a source of chemical and molecular signals but also provide geometric and mechanical boundary conditions needed for embryo development [9,10]. Therefore, over the course of history, the definition of extraembryonic structure has evolved following the conceptual and technological advances in the fields of embryology, molecular and developmental biology, from being based initially on conserved topological and functional (nutrition and protection) criteria to now include criteria of cell lineage and instructive potential to guide embryonic development. In the following section, we will use this combined criteria to assess the definition of extraembryonic structures in teleost fishes.

3. Teleost egg organization and extraembryonic domains

Teleost fishes are the largest and most diverse group of vertebrates, with over 23 000 known species displaying a plethora of different phenotypes, ecologies and developmental strategies [11–13]. Although the study of embryonic development in teleosts has only covered a limited number of species within this large and diverse group of animals, teleost egg morphology appears to be largely conserved between species and their early developmental pattern shows common but highly derived features that are not present in other vertebrate lineages [14–17]. These unique characteristics are thought to be the result of an evolutionary shift in the pattern of cell cleavage from holoblastic (i.e. completely penetrating the egg) to meroblastic (i.e. involving only part of the egg). Such a change in cleavage polarity correlates with changes in the amount and organization of the yolk, resulting in the transformation of an ancestral plasmolecithal egg (i.e. with a relatively low content of yolk mixed with the cytoplasm) into a telolecithal egg (i.e. with a relatively high proportion of yolk arranged as a continuous mass separated from the cytoplasm) [15,17,18]. Although it remains unclear whether and how the emergence of the meroblastic cleavage pattern in the teleost lineage is causally related to the telolecithal condition [15,17], both ultimately defined the characteristic architecture of the teleost egg and the particularities of its early development and extraembryonic domains. The teleost egg is highly stratified, with most of its volume occupied by a continuous and uncleaved acellular yolk, above which is a small lens-shaped cytoplasm defining the animal pole. As a result of a series of meroblastic cleavages occurring exclusively at the animal pole, a mass of cells is formed, often referred to as the blastoderm or blastodisc [19–22]. In the blastula stage, three cellular domains emerge from the blastoderm: (i) a superficial layer of tightly adherent squamous epithelial cells, the enveloping layer (EVL) (also called cellular envelope, couche enveloppante or Deckschicht); (ii) an intermediate layer of cells giving rise to the embryo proper, the deep cell layer (DCL); and (iii) a multinucleated layer between the yolk and DCL, initially termed the periblast and now referred to as the yolk syncytial layer (YSL) [14,19,21–23] (figure 1). Among all acellular and cellular domains of the teleost egg, the yolk, YSL and EVL have historically been considered extraembryonic [15,17,24]. Based on the combined criteria defined above in §2, these structures are considered extraembryonic for different reasons. The yolk is an acellular lineage that separates early from the zygote, serves primarily a nutritional and immune function and is not incorporated (although its contents gradually are) into the embryo proper (see below, §§4a, 7a and 7b). The YSL, on the other hand, forms a separate multinucleate lineage at the beginning of the blastula stage, does not contribute cells to the embryo, influences the embryonic patterning and morphogenesis and assists in the uptake and transfer of nutrients and immune factors to the developing embryo and larva (see below, §§4b, 5a, 5b, 6, 7a and 7b). Finally, the EVL separates from the embryonic cell lineage in the blastula, mainly serves a protective function, influences embryo patterning and morphogenesis and, although it does contribute a restricted number of derived cell lineages to the embryo, these are only transient and are then reabsorbed during ontogeny (see below, §§4c, 5c, 5d, 6, 7b and 7c). It should be noted that two structural elements surrounding the teleost egg, the chorion (also known as egg envelope, vitelline envelope, zona radiata and zona pellucida, among many other names) [25] and the perivitelline fluid (PVF), play important roles in embryo protection against mechanical and osmotic stress and the infection by microorganisms (see below, §7c). These two elements are non-embryonic in nature, however, according to our definition they cannot be considered as extraembryonic as they are not derived from the zygote.

Figure 1.

Archetypical organization of the extraembryonic and embryonic domains in the early teleost egg. (left) Schematic of the late blastula stage of an archetypical teleost egg showing the three extraembryonic domains: the acellular yolk, the syncytial YSL and the epithelial EVL. (right) Sagittal section through the same egg showing in addition to the extraembryonic domains, the embryonic DCL domain located between the EVL and YSL. It can also be seen that YSL contains outer (eYSL) and inner (iYSL) portions. For further details and references, see text.

4. Ontogenic origin of extraembryonic domains in teleosts

(a) . Vegetal segregation of the acellular yolk

Unfertilized teleost eggs, mostly translucent, contain cytoskeletal networks and maternally derived mRNAs and proteins intermixed with a set of organelles collectively referred to as yolk granules, which are involved in the regulation of embryonic metabolism after fertilization [19,21,24,26–29]. Normal embryonic development requires the separation of the yolk material from the rest of the cytoplasmic components, including the germinal vesicle, i.e. the oocyte nucleus [30,31]. This process of ooplasmic segregation is initiated during oogenesis, but is completed after oocyte activation, triggered by sperm entry or by a chemical or mechanical stimulating agent [30–33]. Studies in zebrafish (Danio rerio) show that at one pole of the egg, just about 15–25 min after being activated by sperm entry, a prominent and translucent lens-shaped cytoplasm emerges defining the animal pole or embryonic disc. Biomechanical events favour embryonic disc expansion in this species (figure 2a) [26,27,31,34]. Bulk actin polymerization waves have been shown to travel from the animal to the vegetal pole, coupled and synchronized with the blastoderm cell cycle [34]. As these waves pull the ooplasm towards the animal pole, increased actin polymerization on the surface of yolk granules promotes their movement towards the vegetal pole [34]. Unlike zebrafish, the eggs of members of Atheriniformes and Cyprinodontiformes, including medaka (Oryzias latipes) and annual killifishes, respectively, contain numerous oil droplets [35]. In medaka, the oil droplets fuse together and form large globules that move towards the vegetal pole at the time of fertilization. Much remains to be elucidated about the mechanisms responsible for oopmasmic segregation in medaka, although it has been suggested that, as in zebrafish, there is a cell cycle coordinated mechanism but in this case it appears to depend on the activity of microtubules [33,36,37]. In summary, ooplasmic segregation ultimately reorganizes the teleost zygote, not only morphologically but also molecularly, reinforcing the establishment of an embryonic domain located at the animal pole, from which all embryonic and extraembryonic cell lineages will form, leaving the extraembryonic acellular yolk domain towards the vegetal pole (figure 2a; reviewed in [38]).

Figure 2.

(Caption overleaf.) Specification and lineage separation of the extraembryonic acellular yolk, syncytial YSL and epithelial EVL. (a) Ooplasmic segregation defines a vegetally located acellular yolk and an embryonic blastodisc on top. Unfertilized teleost eggs contain various organelles, mRNAs and proteins of maternal origin (coloured circles) intermixed with a significant amount of yolk granules (left panel). Shortly after fertilization a prominent, translucent lens-shaped blastodisc (Bd) emerges that defines the animal pole (middle panel). In zebrafish, a mechanical mechanism based on cytoskeletal actin polymerization waves promotes blastodisc expansion by creating high-velocity, animal-directed flux streamers located within both the yolk (dark brown arrows) and the ectoplasm (light brown arrows in the middle and right panel). Although most of the maternally derived material is transported toward the blastodisc, a smaller amount of material remains in vegetal positions maintained by retrograde, low-velocity fluxes (blue arrows in the middle and right panel). (b) Peripheral and partially cleaved cells of the blastoderm contribute to the formation of the YSL. During early meroblastic cell divisions the cleavage furrows progress rapidly from the animal pole (blue line in peripheral cells), but become slower and more horizontally oriented as they approach the yolk (red line in peripheral cells). In zebrafish, peripheral cells remain cytoplasmically connected to the yolk (green nuclei) from the 4-cell stage (left panel) and up to the early blastula stage (middle panel). The YSL forms between the 512-cell and 1 k-cell stages by the contribution of these partially connected peripheral cells that appear to coalesce or collapse, depositing their nuclei and most of their cytoplasmic contents in the underlying yolk space (green nuclei in the right panel). After the first row of YSL nuclei is formed, the second row of cells of the blastoderm becomes the new YSL contact zone, but remains cytoplasmically separated from the YSL (dark brown line in the right panel). (c) EVL cell specification is the result of a combination of genetic networks and cell/tissue properties. The top panels show that in zebrafish the EVL originates in the early blastula stage (left panel) and becomes lineage restricted in the late blastula (middle panel), whereas commitment to EVL fate occurs at later stages (right panel). Lower panels show that the position on the surface, the presence of lateral cell contacts and the elongated shape along the surface, which generate in-plane divisions, promote the separation of the EVL lineage from the DCL. The proposed gene regulatory networks involved in EVL specification begin during early blastula stages in zebrafish. Through the synergistic action of maternal region-non-specific genes (Pou5f1) and region-specific EVL inducers, Krüppel-like transcription factors (Klf17) are activated, which, in turn, lead to the expression of EVL differentiation genes, such as keratins. For further details and references, see text.

(b) . Origin and organization of the yolk syncytial layer

The YSL is a prominent and transient syncytial belt-like structure characteristic of all teleost eggs [15,17,22,39], which forms at the interface of the yolk and blastoderm after cleavages during the early blastula period [14,39–41]. Based on their relative position, the YSL has been divided into external (eYSL) and internal (iYSL) to refer to the group of nuclei occupying marginal or central positions, respectively (figure 1). Importantly, it is not only the positional signals that differ between these two populations of YSL nuclei, but also their transcriptional and morphogenetic behaviours (see below, §§5a and 6).

(i) . External yolk syncytial layer

Detailed studies of early meroblastic cleavages of the teleost egg revealed that cleavage furrow progresses rapidly and vertically oriented near the animal pole, but becomes slower and horizontally oriented as it approaches the yolk [22,40,42]. This topological difference in the cleavage pattern generates two types of blastodermal cells with variable connection to the yolk and distinct potential to generate the YSL. At peripheral regions, partially cleaved blastodermal cells remain cytoplasmically connected to the underlying yolk, significantly contributing to the later eYSL (figure 2b). By contrast, inner blastodermal cells are completely detached from the yolk and most of them will form the DCL [22,40,42]. In fact, if a blastoderm cell leaves the margin or remains centrally located, its progeny never contribute to the formation of the eYSL, although its possible contribution to the iYSL remains to be determined [41].

In zebrafish, the eYSL forms between the 512-cell and 1 k-cell stages, i.e. between the 10th and 11th cleavage, by a mechanism that is not fully understood. A variable proportion of marginal blastomeres appear to coalesce or collapse, depositing their nuclei and most of their cytoplasmic contents in the underlying yolk space (figure 2b) [22,39–41]. This phenomenon is associated with cellular behaviours that affect only marginal cells and include a bias of cell division orientation along the animal–vegetal (A-V) axis, the prevalence of cytokinesis failures and a tendency for lateral membranes fusion events [39–41,43,44]. Once a single row of eYSL nuclei has appeared, no further collapse or A-V oriented mitotic divisions are observed. eYSL nuclei then divide metachronously, moving as mitotic waves, but only for a restrictive number of three to five times to finally become postmitotic just before the morphogenetic movement of epiboly takes place (see below, §6a) [39,41]. This change in mitotic behaviour has been associated with a sharp increase in the nucleus-to-cytoplasm ratio within the YSL. Indeed, in a wide range of living organisms and cell types [39,45–52] the nucleus-to-cytoplasm ratio is responsible for regulating cell cycle length and the timing of cell divisions, as well as the activation of transcription (e.g. during the teleost midblastula transition, MBT, a developmental period in which the cell cycle lengthens and loses synchrony as zygotic transcripts become activated [53]).

(ii) . Internal yolk syncytial layer

In contrast to the knowledge of the cellular and molecular processes involved in the formation of eYSL, the origin of the iYSL remains elusive in most known teleost species. The prevailing view in Fundulus is that the iYSL is formed, at least partially, by the contribution of nuclei initially located in the marginal regions [39]. Two lines of evidence support this view. First, the initial iYSL nuclei are observed preferentially occupying marginal regions and their appearance temporally coincides with mitotic events within the eYSL, suggesting that some eYSL nuclei slip into the yolk cell cytoplasm below the blastoderm margin. Indeed, the number of eYSL nuclei after each cell division is almost invariably less than twice the number of the mother nuclei [39]. Second, nuclei of the eYSL show an equidistant spacing and this seems to depend on the mitotic activity and cytoskeletal arrangements of microtubules [39,41,43,44]. Just when mitotic activity ceases, prior to the onset of epiboly, a strong A-V contraction of the eYSL occurs favouring not only the crowding of nuclei, but the displacement of several nuclei from marginal to more central positions below the blastoderm, thus contributing to the pool of iYSL nuclei [39,41]. It remains to be elucidated whether a similar mechanism is responsible for iYSL formation in other teleosts such as zebrafish, medaka and annual killifishes, and whether internal blastomere-derived fusion events also contribute to this process.

(c) . Enveloping layer lineage specification

The EVL originates in the blastula from the outermost layer of the blastoderm in a developmental process that transforms a rounded/cuboid pluripotent cell arrangement into a differentiated flat epithelium over several metasynchronous cell cycles. Studies in zebrafish show that EVL specification begins shortly after the MBT, while the EVL becomes lineage restricted in the late blastula [24,54] and commitment to EVL fate occurs 1–2 h later [55]. EVL specification involves a combination of genetic mechanisms based on maternal factors and morphogenetic cues based on cell/tissue properties. With respect to the genetic mechanisms, several maternal genes have been shown to be crucial for EVL specification [56–62]. Among these, non-region-specific maternal genes, such as the POU Class 5 Homeobox 1 (Pou5f1; also known as Oct4) [57] have been proposed to act synergistically with region-specific EVL inducers (e.g. interferon regulatory factor 6) [58] to activate closely related Krüppel-like transcription factors [62]. These, in turn, trigger the expression of key EVL differentiation genes, such as epithelial keratins, while presumably also blocking the establishment of other embryonic cell fates [62] (figure 2c). Recently, using single-cell RNAseq combined with a computational method based on simulated diffusion, it was possible to reconstruct cell developmental trajectories in the early zebrafish embryo based on covariant gene modules demonstrating that the EVL is discernible as a separate cellular trajectory already at the high stage [63]. Although most EVL cells express keratins [62,64–68] and subsequently give rise to the periderm [24,69–71], there is some degree of regional heterogeneity along the dorsoventral (D-V) axis. Subsets of dorsal cells of the EVL in the early zebrafish embryo show a unique set of transcripts (e.g. crestin) [72] and Ca⁺² transients [73,74], with some of them having an increased endocytic activity [75] and being internalized to give rise to dorsal forerunner cells (DFCs), the progenitors of the laterality organ (see below, §5d) [75–78]. Such regionalization of the EVL appears to be regulated by TGFß Nodal signalling, which seems to be restricted dorsally by members of Sox, Max and Snail protein families [72,76].

Besides the role of genetic determinants, cell/tissue properties such as cell–cell contact, relative cell position and cell shape all play relevant roles in EVL specification (figure 2c). Cell–cell contact or close cell proximity is required for EVL specification and, consequently, surface blastoderm cells in zebrafish embryos with defective tight junctions [79], transplanted as single cells into host embryos [55] and dissociated from embryo explants [66] fail to commit and differentiate as EVL. Furthermore, only the most superficial cells of the blastoderm, which are contacted with other cells basolaterally but not apically, are able to specify as EVL cells, indicating that such positional asymmetry probably related to differences in surface tension is necessary for proper differentiation [24,55,66,68]. In fact, even in cell aggregates of the ectodermal germ layer where EVL cells differentiate de novo they do so only on the surface [68]. Furthermore, a change in the orientation of cell division from initially occurring out-of-plane (i.e. generating one daughter EVL cell on the surface and one DCL cell below) to being in-plane (i.e. generating two daughter EVL cells on the surface) is essential for EVL lineage separation. Importantly, in vivo imaging studies and mathematical modelling show that this change in division orientation is not determined by lineage, but by cell shape (figure 2c), being the result of a closed morphogenetic feedback loop between cell shape and cell number that couples geometric constraints through mechanical interactions [54]. Notably, prospective EVL cells in some species of annual killifishes show early fusion events generating multinucleated surface cells [21,80,81], that increase their nuclei number by later rounds of mitosis with failed cytokinesis [81]. These multinuclear cells only follow the EVL fate and do not contribute to the DCL lineage [81], suggesting that multinuclearity could be critical for the separation of the EVL cell lineage in this group of teleosts, as has been previously noted for the lineage separation of the YSL in zebrafish (see above, §4b). The syncytial nature of EVL cells appears to be a highly derived character that has so far only been observed in annual killifish species [21,80,81]. However, in zebrafish, multinuclear EVL cells have been reported as a result of affecting cell division either chemically [82] or by blocking the expression of membrane recycling small GTPases Rab25a/b [83]. During epiboly, the orientation of the mitotic spindle in EVL cells is strongly influenced by the main axis of tissue tension and therefore one of the main functions of cell division in expanding epithelia is related to the release of anisotropic tissue tension [82]. Consequently, blocking cell division or exposing embryos to ectopic tension is sufficient to trigger EVL cell–cell fusion events that seems to compensate, at least partially, for the lack of cell divisions [82]. Although further studies are needed, the mechanisms responsible for the generation of multinucleated EVL cells in zebrafish and annual killifish appear to be different, as in the latter most fusion events occur before epiboly, when anisotropic tension appears not to be as significant as during epiboly [21,80,81].

In summary, EVL lineage specification is a consequence of the coordinated action of gene regulatory networks, cell shape changes, topological cues and morphogenetic behaviours that gradually separate the EVL and DCL cell lineages keeping the latter as an epithelial cell monolayer at the embryo surface (figure 2c).

5. Teleost extraembryonic domains and embryo patterning

(a) . Yolk, yolk syncytial layer and dorsoventral patterning

The interaction between the blastoderm and yolk begins as soon as fertilization is completed. As discussed above, a cell cycle-dependent mechanism of the blastoderm synchronizes the pulling of the yolk granules towards the vegetal pole and thus contributes to the reorganization of maternally derived molecular components during ooplasmic segregation (see above, §4a). Subsequently, the ordered pattern of cell cleavage events that follows in the blastoderm appear to require a minimal amount of yolk. A prominent set of experiments in which yolk supply was serially reduced using Fundulus, Carassius, Salmo and zebrafish eggs demonstrated that for embryo development to occur, the size of the yolk must at least exceed that of the blastoderm [19,84–88]. Manipulations of the yolk content at different stages also revealed that very early subtractions, i.e. before the 4-cell stage, severely affect the cell cleavage pattern resulting in a disorganized cell mass, originally known as hyperblastula. However, yolk subtractions at the 32-cell stage or later can generate exogastrulae (reviewed in [19]). Therefore, the presence of a minimal amount of yolk supply, which probably functions as a mechanical support structure, and the flow at critical stages of maternally derived material and information between the yolk and the blastoderm appear to be essential for initiating differentiation programmes and patterning in the blastoderm.

Dorsal expression of the mesodermal gene goosecoid (gsc) is a highly conserved molecular cue that marks the prospective location of the embryonic shield, i.e. the teleost structure homologous to the Spemann-Mangold organizer in amphibians and the node in mammals. In zebrafish, yolk-derived grafts that include the YSL are able to induce high levels of expression of gsc and of the pan-mesodermal gene no tail (ntl) in the animal pole region of host embryos, which normally do not express such markers [89]. Injection of RNase into the yolk affects the expression of mesodermal (gsc, ntl) and endodermal (gata5) genes but only in ventrolateral positions while the dorsal expression of gsc and ntl is unaffected [90], suggesting that dorsal specification is ensured by a mechanism that functions independently of the YSL. Indeed, a highly conserved maternal pre-patterning mechanism required for specification of the prospective dorsal side and for embryonic organizer establishment has been reported not only for teleosts but for other chordates [91–93]. A critical first step of this mechanism involves the nuclear accumulation of maternal b-catenin restricted to a circumscribed region of the blastoderm that includes a small cluster of DCL and prospective EVL cells, but which subsequently expands to include prospective dorsal nuclei within the YSL (figure 3a,b) [94,95]. Although it seems likely that b-catenin accumulation may be triggered by activation of canonical Wnt signalling, it has recently been reported that nuclear enrichment of b-catenin on the dorsal side of zebrafish and Xenopus laevis embryos can be driven by a Wnt signalling-independent mechanism involving the maternally derived transmembrane protein Huluwa [94,96]. One of the target genes resulting from the co-transcriptional activity of b-catenin is the homebox dharma/bozozok whose expression pattern follows the temporal dynamic of b-catenin, i.e. it first appears in the prospective dorsal blastoderm but later includes expression within the YSL (figure 3b) [97,98]. Based primarily on (i) a slightly complementary spatial expression of dharma and gsc at the shield stage in zebrafish, encompassing the dorsal YSL and blastoderm, respectively, and (ii) a higher gsc-derived inductive potential of dharma-overexpressing yolks, it has been suggested that dharma could function in a non-cell-autonomous manner (figure 3b) [97]. Thus, taken together, the zebrafish experiments seem to indicate that an autonomous molecular mechanism responsible for the specification of future dorsal prospective cells is initiated within the blastoderm, partly supported by maternally derived material, and that the selected site is subsequently imprinted on the YSL. Once this occurs, it appears that the specified dorsal YSL is capable of instructing ‘naive’ dorsal blastodermal cells. This conclusion could provide a rational explanation for the transplantation experiments performed in rainbow trout (Salmo gairdneri) [99]. Donor blastoderms separated from the yolk prior to the formation of the embryonic shield were transplanted into older embryos in which the blastoderm was removed leaving the YSL intact but in which the positions of the shield were previously recorded using chalk particles. In these cases, the position of the shield in the host blastoderm almost always coincided with the chalk particles within the yolk, strongly suggesting that some ‘dorsal substance’ emanating from the YSL might influence the position of the new embryonic organizer [99]. Indeed, in zebrafish, it was observed that yolk material, in the form of vesicles or ‘particles’ of different size and shape, probably containing ribosomes and ribonucleoproteins of maternal origin, was continuously transferred to the blastoderm from the cleavage stages until late epiboly [88].

Figure 3.

Proposed roles of the extraembryonic YSL and EVL in D-V patterning and mesendoderm induction. (a) Schematic of the zebrafish embryo at the early blastula stage (128- to 256-cell stage,) showing how maternal signalling activities related to non-canonical Wnt (purple 7-transmembrane receptor and bound ligand) and Ccr7 chemokine receptor (light brown 7-transmembrane receptor and bound ligand) promote Ca²⁺ transients in EVL progenitors (upper green cells). Calcium release from endoplasmic reticulum is proposed to inhibit ß-catenin-dependent transcription (and axis formation) in all EVL progenitors excepting those located at the dorsal Side, where elevated ß-catenin protein levels are maintained by activation of canonical Wnt signalling. The proposed cell autonomous effect of Ca²⁺-dependent activity is shown, although the existence of a still uncharacterized non-cell autonomous mechanism is possible (i.e. from EVL to DCL). (b) Schematic of the zebrafish embryo at the 512-1 K cell stage showing the mechanisms that initiate D-V patterning in the blastoderm (DCL and EVL). Maternal squint mRNA within the blastoderm becomes asymmetric in prospective dorsal blastomeres from the 4–8 cell stage (orange cells, also present in a). It has been suggested that Squint may function as a scaffold capable of recruiting dorsal–mesodermal promoter factors that trigger, among others, the activation of canonical Wnt signalling pathway. At this stage, nuclear ß-catenin (red circles and nuclei) is enhanced in prospective dorsal blastomeres by a Huluwa-dependent mechanism (blue bottles and arrows) that stabilizes the cytoplasmic ß-catenin pool by inducing degradation of the inhibitory protein Axin. Target genes of ß-catenin-dependent co-transcriptional activity, including dharma and squint, are expressed and co-localize with ß-catenin-positive dorsal cells. Nuclear accumulation of ß-catenin is further detected in the most dorsal nuclei within the YSL and a positive feedback loop is evident at the dorsal site. (c) Schematic of the zebrafish embryo at the dome/sphere stage showing the mesendodermal inducing role of the YSL. Shortly after epiboly begins, nuclear accumulation of ß-catenin expands to cover the ring of nuclei within the YSL and two Nodal-related proteins (Squint and Cyclops), function in a positive feedback loop within the YSL and DCL to induce mesendodermal fate (orange cells in right panel). For further details and references, see text.

(b) . Yolk syncytial layer and mesendodermal induction

A more conciliatory view on an essential property of the YSL in teleosts involves its mesendodermal fate-inducing function. In cyprinid fish, including zebrafish, prospective mesodermal and endodermal cells remain intermingled until the early gastrula stage, but preferentially occupying the middle and lower (i.e. overlying the YSL) positions within the DCL, respectively [24]. In these embryos, two ligands of the highly conserved TGFß Nodal signalling pathway [100–103] are expressed during early morphogenesis and have been demonstrated to induce mesendodermal fate. Nodal-related 1, commonly known as squint (sqt), is encoded by a maternally derived mRNA and detected asymmetrically in prospective dorsal blastomeres from the 4–8 cell stage (figure 3a,b) [92,104]. Maternal sqt seems to function as a scaffolding mRNA capable of recruiting or sequestering dorsal–mesodermal promoter factors, probably related to the canonical Wnt signalling pathway [105]. Disruption of its asymmetric localization and/or overexpression of a non-coding mutant mRNA results in dorsal expansion of gsc expression and nuclear accumulation of b-catenin [105]. Interestingly, both the dorsal mesodermal-inducing role and the asymmetric localization of maternal sqt involve conserved sequences within its mRNA 3′ untranslated region [92,105,106]. Zygotic expression of sqt starts shortly after MBT under the control of b-catenin and, therefore, initially covers a sparse cluster of dorsal blastomeres that progressively extends to include a cluster of underlying dorsal eYSL nuclei (figure 3b) [95,106,107]. At late blastula stages, a peak of sqt expression is observed extending along the blastoderm margin, including both mesendodermal cells and the e- and iYSL (figure 3c). Thereafter, at the shield stage, the intensity of sqt expression fades, being detectable only in DFCs [106,108,109]. Nodal-related 2, commonly known as cyclops (cyc), although not maternally inherited, shares a highly comparable expression pattern with sqt. The slight differences in the early stages lie mainly in that cyc initially covers a symmetrical ring of expression along the blastoderm margin of the late blastula, including the YSL, becoming restricted towards dorsal hypoblastic blastomeres at shield stage (figure 3c) [109]. The requirement of sqt and cyc for mesendodermal specification in teleosts has been consistently demonstrated in zebrafish by using mutant lines. Similar phenotypes, including deficiencies in the formation of the dorsal mesendoderm, prechordal plate and ventral nervous system, have been observed in sqt and cyc mutants, suggesting that the functions of both genes partially overlap [108,110,111]. As expected, cyc;sqt double mutants show stronger phenotypes than single mutants, including severe impairments of mesendoderm gene expression, failure of morphogenetic movements and morphological defects [108]. Notably, the specific disruption of sqt and cyc in the YSL, by injecting fluorescently labelled antisense morpholinos against both genes directly into the YSL shortly after it is formed, not only causes severe reduction in the expression of sqt and cyc in the blastoderm but also seems to partially recapitulate the phenotype of the cyc;sqt double mutant [112]. This finding strongly suggests that YSL-derived Nodal-related signals greatly contribute to the severe mesendodermal phenotypes observed in the mutant lines. But how to reconcile this major mesendodermal inductive role of the YSL with a recent report in zebrafish suggesting that mesendoderm induction still operates in blastula-derived explants lacking this extraembryonic structure? [113]. Closer inspection of embryonic explants revealed that, in agreement with [112], the specification of the two cell types that require the highest levels of Nodal signalling, i.e. dorsal mesoderm and endoderm, becomes highly variable and random in position in the explants. Consequently, not only is phosphorylation of the Nodal-effector proteins Smad2/3 (pSMAD2/3) severely reduced in blastodermal cells, but explants also show a temporal shift in pSMAD2/3 positive nuclei [112,113]. So, it appears that although not essential, the inductive role of the YSL is to stabilize and reinforce a Nodal signalling network at the embryonic/extraembryonic interface that, in turn, ensures precise spatio-temporal control in subsequent mesendodermal specification (figure 3c) [112–115].

(c) . Enveloping layer and dorsoventral patterning

The EVL also appears to influence cell fate decisions in the embryonic DCL. This idea stems from studies in early zebrafish embryos showing that intracellular Ca²⁺ transients appearing exclusively in the forming EVL are somehow linked to the establishment of the embryonic D-V axis (reviewed in [116,117]). These Ca²⁺ transients are observed from around 32- to 128-cell stage in single or small groups of forming EVL cells and follow stereotyped changes throughout development, being uniform across the EVL before the MBT, then showing a transient dorsal bias after MBT, and finally becoming uniform again at gastrula stages [73,74,118,119]. Ca²⁺ transients in the blastula are generated by mobilization of intracellular Ca²⁺ stores by upstream activation of non-canonical Wnt/Ca²⁺ [120,121] and chemokine G-Protein coupled receptor [122] signalling, both promoting increased levels of inositol 1,4,5-trisphosphate and the opening of calcium channels mainly in the endoplasmic reticulum. It has been proposed that Ca²⁺ derived transients present in the forming EVL prior to MBT have an antagonistic effect on the canonical Wnt/ß-catenin signalling pathway [120–125], which would help restrict nuclear localization of ß-catenin and axis formation to the dorsal side, where canonical Wnt signalling is high (figure 3a). Accordingly, overexpression of non-canonical Wnt5A or chemokine receptor Ccr7 increases Ca²⁺ transients in the EVL and blocks the ability of canonical Wnt ligands to induce ectopic axes [119,122,123], whereas abrogation of Wnt-5A, Ccr7, inositol 1,4,5-trisphosphate or endogenous Ca²⁺ modulation results in expanded dorsal signalling centres and ectopic axes [121–124]. The mechanisms underlying the antagonistic role of Ca²⁺-dependent activity in the EVL in embryonic axis formation, and how they interact with the proposed role of the YSL (see above, §5a), are still unknown [74,118], but are thought to be part of negative control mechanisms that predominate in early axis formation [122]. However, it is intriguing that Ca²⁺ transients show a dorsal bias at later stages of the blastula [73,74], which is counterintuitive given the proposed role for them in antagonizing axis formation, so it has been speculated that this would imply a change in Wnt/ß-catenin regulation associated with MBT [73,117] after which dorsal Ca²⁺ transients would take on a role in the establishment and/or maintenance of the dorsal organizer [74].

(d) . Enveloping layer and left–right patterning

Although teleost extraembryonic tissues are not directly involved in the regulation of left–right pattering, studies in zebrafish show that they are key players in the origin and shaping of a transient epithelial structure that is necessary for left–right axis specification. Among vertebrates, a key conserved mechanism of left–right axis determination is driven by transient epithelial structures located in the posterior part of the gastrula/neurula embryo, which through the activity of motile cilia generate or refine genetic cascades confined to one side of the embryo (reviewed in [126–130]). In teleosts, this structure corresponds to Kupffer's vesicle (KV) [131–133], a ciliated epithelial vesicle often referred to as laterality organ, organ of asymmetry or left–right organizer, whose origin, shaping and function has been closely related to extraembryonic domains [76–78]. In zebrafish, KV progenitors arise at the blastoderm margin of the late blastula by a delamination process mediated by TGFß Nodal signalling that transforms a group of 20–30 superficial epithelial cells of the dorsal EVL into deep mesenchymal-like cells (figure 4b) [76–78]. EVL cells fated to become KV have a unique lineage history. They derive at the 512-cell stage from dorsal marginal blastomeres, before they coalesce to form the YSL (see above, §4b), and retain cytoplasmic connections with the dorsal edge of the yolk that are thought to provide essential maternal determinants for KV specification (figure 4a) [77]. Once KV progenitors arise by delamination, extraembryonic tissues provide the substrate and driving forces to guide their movements to the site of differentiation. In fact, KV progenitors are pulled towards the vegetal pole by their apical attachments to the EVL and eYSL [78], which spread autonomously to the vegetal pole to cover the embryo in the movement of epiboly (see below, §6a and 6b) (figure 4b). Concomitant with mechanical traction by extraembryonic tissues [78], clustering mechanisms dependent on contact-mediated interactions between progenitor cells [78,134–137], and with the YSL [138] and DCL [139], ensure progenitor allocation as a compact cluster at the posterior end of the notochord, a prerequisite for differentiating as a functional epithelial vesicle (figure 4c). Consistently, defects in the specification, pulling, clustering or compaction in zebrafish lead to defective KV organogenesis and left–right asymmetry defects, having a detrimental effect on embryo development [78,134–136,140,141]. Near the end of epiboly, the tight cluster of KV progenitors detaches from the extraembryonic domains and begins a mesenchymal-to-epithelial transformation leading to the formation of an epithelial vesicle with motile monocilia lining its interior [76,135]. In the early stages of somitogenesis, cilia rotation generates an asymmetric fluid flow within the KV cavity which through as yet unclear mechanisms is sensed to drive asymmetric gene expression around the KV and the subsequent establishment of embryo chirality (reviewed in [129,142]). A key component of this asymmetric regulation in zebrafish and medaka is Charon, a Cerberus/Dan-family Nodal pathway repressor, which as a consequence of flow is downregulated on the left side promoting left-sided activation of the Nodal signalling pathway [143,144].

Figure 4.

Role of extraembryonic domains in the origin and shaping of Kupffer's vesicle, the laterality organ or left–right organizer of teleosts. Diagrams are arranged to show zebrafish embryos at different stages at the top (a, early blastula; b, 60% epiboly; c, tail bud), with the EVL labelled in white and the dorsal EVL-derived KV progenitors in red, and below the cellular events associated with the role of extraembryonic domains in the origin and shaping of the KV in this species. (a) In the early blastula, dorsal maternal determinants for the specification of KV are transferred from the yolk to the marginal cells of the blastoderm (red arrow), from which KV progenitors will later arise after formation of the YSL (red). (b) Dorsal EVL cells fated to be become KV undergo delamination under the control of TGFß Nodal signalling, and during epiboly are pulled toward the vegetal pole by the EVL and YSL (red arrows) through apical junctions (yellow circles) that these cells maintain with both extraembryonic tissues. (c) Concomitantly, cell clustering mechanisms allow KV progenitors to progressively form a compact cluster, which is a prerequisite for differentiation into an epithelial vesicle at the posterior end of the notochord. For further details and references, see text.

(e) . The peculiar case of annual killifishes

A mode of embryonic development that contrasts with that of most teleosts is found along annual killifishes, a group of small, sexually dimorphic Cyprinodontiformes fishes inhabiting shallow, relatively small, temporary water bodies of variable size in the floodplains of Africa and South America [145–147]. Two major changes during early embryonic development have been correlated with annual life cycles, namely the occurrence of a unique dispersion/re-aggregation (D/R) phase, which physically and temporally separates epiboly from gastrulation, and the occurrence of up to three reversible arrests of development and metabolism known as diapauses [21,146–153]. Although the evolutionary origin of the D/R phase remains speculative, it has been suggested that delayed gastrulation in annual killifishes is the result of the low cell number that characterize early developmental stages in these fish [154] (for review, see [153]). Although the temporal extent of the D/R phase throughout annual killifishes is quite variable, most published papers agree that the majority of embryonic cells at this stage remain undifferentiated and only sexual-somatic separation has been reported from the epiboly stage in the South American killifish Austrolebias Charrua [153,155]. Accordingly, PCR-mediated analysis of temporal expression of several pluripotency-inducing genes including Pou5fI/Oct4 and Sox2/3 demonstrate detectable levels up to late epiboly and throughout the D/R phase in the annual killifish Austrofundulus limnaeus [156]. Although a shift in timing, i.e. heterochrony in the specification of the D-V axis towards the end of the D/R phase in the seasonal killifish, is a tantalizing idea, several lines of evidence based on molecular data suggest that this scenario might be an oversimplification. First, in A. limnaeus embryos, high expression of chordin has been detected since fertilization, suggesting a maternal origin, in contrast to the rather conserved zygotic temporal expression of this gene and its functionality associated with D-V patterning starting just before gastrulation in other teleosts such as zebrafish, medaka and Astyanax mexicanus [156–159]. Second, nuclear accumulation of b-catenin, the hallmark of prospective dorsal side in teleosts, has been observed to symmetrically cover most of the blastoderm from early blastula stages in the killifish Nothobranchius furzeri [160]. Finally, the cyc homologue in the annual killifish appears not to be restricted to a particular population within the blastoderm until the re-aggregation stages and its early functionality has been linked to the coordination of cell migration within the blastoderm and the subsequent axis formation [160]. Therefore, the influence of maternal pre-patterning, extraembryonic domains as well as the spatio-temporal control of conserved molecular networks related to early cell specification and axis formation remain mostly unknown in annual killifishes. Intriguingly, a recent study reported that the first cells forming the re-aggregate, i.e. the annual killifish deep cell structure from which the embryonic axis develops, move later laterally and, thus, do not take part of the embryo proper, raising the possibility that a new extraembryonic cellular domain, previously uncharacterized in teleosts, might have evolved in annual killifish, playing a guiding role in the first steps of re-aggregation and embryo formation [152].

6. Teleost extraembryonic domains and embryo morphogenesis

The early events of embryo morphogenesis in teleosts have been extensively studied and reviewed elsewhere [161–164]. Here we focus on describing how extraembryonic cellular domains (YSL and EVL) interact with each other and with the embryonic DCL to guide early embryo morphogenesis. In teleosts, early morphogenesis begins with massive spread of tissues from all cellular domains of the blastula in a process known as epiboly, which ultimately results in engulfment of the yolk and closure of the blastopore at the vegetal pole. In parallel, movements of internalization, convergence and extension gives rise to the germ layers and provide the characteristic elongated shape of the embryo. In each of these morphogenetic movements, critical cellular behaviours and molecular mechanisms within specific embryonic and extraembryonic domains have been described as major providers of the driving forces of morphogenesis. However, an emerging message of recent studies is that, despite the prominence of specific cellular domains as drivers of morphogenesis, global coordination and synchronization among all domains is central, a phenomenon that is still largely unexplored.

(a) . Epiboly initiation

The first morphological hallmark of epiboly initiation in zebrafish, often referred to as doming, involves an upward bending of the blastoderm–yolk interface, resulting in a marked reduction in blastoderm height at the centre and an extension of its length at the periphery (figure 5a). This critical event involves the coordinated biomechanical interaction of autonomous mechanisms developed in the three cellular domains of the blastula: (i) a local reduction of surface tension within the EVL epithelium [165]; (ii) a fluidization of the DCL mediated by dynamic changes in cell–cell adhesion [166,167] and, (iii) a contractile wave along the eYSL [161,168]. It is conceivable that contraction of the eYSL could be the initial instructive stimulus that triggers both the vegetal-directed traction of the peripheral ring of EVL cells attached to the YSL and the net flow of yolk granules in the opposite direction in the center of the blastoderm (figure 5a) [164,168,169]. Interestingly, the yolk granules elongate slightly along the radial axis during doming, suggesting the presence of radial tension within the yolk [165].

Figure 5.

Roles of teleost extraembryonic domains in early morphogenesis and organogenesis. (a) Epiboly initiation. In zebrafish, epiboly begins with upward bending of the blastoderm–yolk interface, commonly known as doming. This event involves (i) a contractile wave (black arrows, left panel) along the eYSL (purple dots) responsible for the appearance of the iYSL (dark blue dots in the middle and right panels), (ii) a local reduction of surface tension within the EVL epithelium (orange arrows in the middle and right panels) and (iii) a fluidization of the central part of the DCL mediated by dynamic changes in cell–cell adhesion (light blue gradient in the middle and right panels) reducing the height of the blastoderm in the center and extending its length in the periphery (vertical and horizontal green arrows, respectively, in the middle and right panels). (b,c) Epiboly progression. (b) The vegetal movement of nuclei within the eYSL (arrows) in zebrafish is mediated by a kinesin-dependent transport mechanism along a highly ordered microtubule network present in the yolk cell (green filaments). (c) Actomyosin-mediated contraction and flows, and localized endocytosis within the YSL and the yolk cytoplasmic layer are critical for the progression of epiboly in zebrafish. The top panel shows how a supramolecular actomyosin ring organizes as a remarkable circumferential band around 70–80% epiboly within the eYSL (red filaments) triggering contractile activity that helps to reduce the width of the blastoderm margin (vertical black arrows) while pulling the overlying EVL towards the vegetal pole (red arrows). The lower panel shows how throughout the process, the EVL remains tightly bound to the eYSL by adhesive structures (yellow dots). Thus, the circumferential tension generated by actomyosin cortex activity in the eYSL pulls the EVL margin toward the vegetal pole (outer black arrows). A retrograde flow of both actin and zonula adherens proteins (upward arrow, and red and yellow dots in the left panel) in combination with endocytosis localized within the yolk cytoplasmic layer (black vesicles and curved arrows in the right panel) are required for epiboly progression. (d) Convergence and extension. In zebrafish, the movements of convergence and extension of the iYSL require E-Cadherin-dependent adhesion with the overlying mesendoderm. Cortical flows (white arrows) generated within the iYSL are probably induced by cell movements of the overlying mesendoderm and transmitted through E-Cadherin–actin complexes (pink ovals and red filaments). (e,f) Epibolic spreading of the DCL. The establishment of E-Cadherin-dependent contacts between the DCL and EVL (pink ovals in e and f) are critical for the vegetal spreading of the DCL during epiboly in both zebrafish (e) and annual killifish (f). In zebrafish, as the EVL increases it surface area during epiboly, adhesive interactions between the DCL and EVL result in spreading of the most superficial layer of DCL cells (blue cells) and subsequent radial intercalation of the deeper DCL layers (green cells) directed towards the surface (red horizontal arrow in (e)). In annual killifish, preferential adhesive contacts between DCL cells and the EVL are observed at EVL–EVL cell junctions, which is mediated by increased tension at these sites (f). (g,i) The YSL functions as a platform for migratory events of myocardial progenitors (h) and hepatoblasts (i) during organogenesis. (g) Schematic of the 19–20 hpf zebrafish embryo showing in red and blue, the territories of presumptive myocardial progenitor and hepatoblast migration, respectively. (h,i) Myocardial progenitors derive from the lateral mesodermal plate (cells with pink gradient) while hepatoblasts arise from endodermal cells (yellow line). Both progenitors are generated on both sides of the embryo and migrate toward the midline (black arrows) using an extracellular matrix derived substrate that is proposed to be produced and/or organized by the YSL. Defective migration often results in organ duplications. For further details and references, see text.

A critical aspect of effective doming is related to the EVL cell-autonomous decreases in surface tension [165] (figure 5a). The balance of forces is equilibrated just before epiboly and a slight reduction in EVL surface tension is sufficient for triggering the upward bulk of blastoderm-to-yolk interface and radial intercalation behaviour within the DCL [165]. Interestingly, disruption of genes that participate in EVL differentiation result in epiboly delay [56,60,170,171]. Therefore, it appears that EVL differentiation is required to initiate its epibolic spreading and, although the reasons are still unclear, the construction of a mature intermediate filament-based cytoskeleton appears to be mandatory, based on the pronounced upregulation of the expression of several keratin genes during EVL differentiation [64,66,172].

The possible contribution of the DCL to the overall process of doming and epiboly progression (see below, §6b) has historically been neglected. During doming, DCL cells elongate and intercalate radially [173]. However, radial intercalation appears to be only critical for homogeneous blastoderm thinning during doming but dispensable for epiboly of the EVL and YSL [81,165,174]. By contrast, EVL spreading has been shown to be instructive for DCL epiboly and coupling of spreading between these two cellular domains requires Cadherin-mediated EVL-DCL adhesions (figure 5e,f). In the annual killifish Austrolebias nigripinnis, functional impairment of E-Cadherin induces epiboly arrest of the DCL without any notable effect on the vegetal spreading of the EVL and YSL [81]. Similarly, zebrafish embryos defective in E-Cadherin also exhibit epiboly delay that primarily affects the DCL [166,175–177]. Epiboly of the DCL appears to require a temporal shift in E-Cadherin cellular localization from the plasma membrane towards intracellular vesicles, which is triggered by Pou5f1/Oct4, and the transition from a highly adherent non-motile cell state seen in the early blastula stages to a motile cell state [166]. The internalization of cell–cell adhesion molecules within the DCL could, in principle, favour a change in the mechanical properties of the tissue. Indeed, it has recently been shown that an active fluidization of the central domain of deep cells within the DCL is necessary for efficient doming [167]. Just before doming, mitotic events are equally distributed throughout the DCL, but only deep cells located in the central regions fail to reestablish cell–cell contacts after completion of cell division. Instead, active cell cohesion is maintained at the margin by local activation of non-canonical Wnt signalling [167].

(b) . Epiboly progression

Studies in zebrafish show that epiboly progression beyond the embryo equator is strictly dependent on the function of a supramolecular actomyosin ring that assembles just below the blastoderm margin in the eYSL (figure 5c) [178–181]. A vegetal-to-animal flow of actomyosin starts just before 50% epiboly and coalesces as a notable circumferential band around 70–80% [180]. Throughout the process, the EVL remains tightly bound to the eYSL by adhesive structures that increase in number and complexity as epiboly progresses [79,182–184]. Thus, circumferential tension generated by actomyosin cortex activity in the eYSL pulls the EVL margin toward the vegetal pole (figure 5c) [180]. Two lines of evidence suggest that the eYSL and the EVL epithelium are at least partially coupled during epiboly. First, partial or complete detachment of the EVL margin not only produces an immediate retraction response confirming that the epithelial tissue is under tension, but also accelerates eYSL epiboly [185]. Interestingly, shortly after this retraction-induced response, epithelial cells move using the iYSL as a substrate to eventually reach the leading edge [185], suggesting that even if the EVL is normally pulled by the eYSL, it also has the ability to move autonomously. Accordingly, filopodial protrusions have been described extending from the EVL into the eYSL [57] and the DCL [186], which could account for this migratory ability, but other functions for EVL filopodia including cell signalling, membrane repository for cell expansion and innate immune response have been proposed [57,186,187] (see below, §7a). Second, the tight junction components Zonula Occludens-1 (ZO-1) and Claudin-E are required to maintain EVL–eYSL coupling, as impairment of their function results in epiboly delay [79,183–185]. Remarkably, the tight junctions formed at the EVL/eYSL interface function as mechanosensitive elements that grow in response to increased circumferential actomyosin tension in the eYSL during epiboly (figure 5c) [184]. Such mechanosensation mechanism is necessary for EVL/eYSL vegetal spreading and relies on the retrograde flow of actomyosin within the eYSL that transports non-junctional ZO-1 clusters to tight junctions as a function of tension [184]. Epiboly progression also requires removal of the inner membrane of the yolk cell as the eYSL expands (figure 5c), as first observed in Fundulus embryos [168]. In zebrafish, a mechanism of localized endocytosis, dependent on the activity of the small GTPase Rab5ab, has recently been reported [163,169]. Embryos impaired in Rab5ab function show delayed epiboly associated with defects is retrograde actomyosin flow and contractility [169]. Therefore, it appears that membrane tension at the eYSL–yolk interface is maintained by a feedback loop between actomyosin contractility and endocytic machineries [169,180,181].

A second cytoskeletal mechanism based on a microtubule network located in the yolk cell has been associated with eYSL epiboly (figure 5b) [188]. Embryos treated with microtubule depolymerizing or stabilizing agents show epiboly delay that primarily affects the movement of YSL nuclei [188,189]. Recently, a motor-dependent cargo transport involving the function of Kinesin-1 and the outer nuclear membrane LINC complex protein Syne2 has been proposed as a possible microtubule-dependent mechanism for eYSL nuclear movements (figure 5b) [190]. What triggers epiboly in eYSL nuclei remains largely elusive, but appears to depend critically on changes in the dynamics of the microtubule network. Microtubules are highly dynamic during the onset and up to 50% of epiboly [191] and become more stabilized during epiboly progression, which likely enables or supports the transmission of mechanical forces [191,192]. Although the mechanisms behind these changes remain obscure, the function of the steroid hormones Pregnenolone and Progesterone are required for microtubule stability and epiboly progression in zebrafish [191,193–195]. Interestingly, supportive production of extraembryonic tissue-derived steroids has been demonstrated also in mouse and Drosophila [196,197].

(c) . Convergence and extension movements

The extensive movements of convergence, internalization and medio-lateral cell intercalation that occur at the dorsal side of the embryo characterize gastrulation in teleosts (reviewed in [162,198,199]). Unlike the eYSL, most iYSL nuclei do not undergo epiboly but show movements that resemble those of overlying mesendodermal cells [17,200–203]. Morphogenetic coupling between mesendodermal and iYSL movements require E-Cadherin-mediated adhesive contacts between these domains (figure 5d), although the underlying mechanism remains elusive [204]. Furthermore, convergence of iYSL nuclei is almost abolished in mutant embryos lacking most mesendodermal progenitors, [205], strongly suggesting that the embryonic mesoderm guides the convergence movement of the extraembryonic iYSL. Interestingly, fluorescent microspheres injected into the YSL show convergence movements similar to those of the neighbouring iYSL, suggesting that cortical flows are generated within the iYSL, which are likely induced by cell movements of the overlying mesendoderm and transmitted through the E-Cadherin–actin complexes (figure 5d) [200,204].

The presence of a morphogenetic coupling between the convergence and extension movements of the iYSL and the DCL, and between the epiboly movements of the eYSL and the EVL, is striking. In the case of the eYSL/EVL, these structures share a common origin, surface position and a polarized cytoskeletal organization and dynamics that is intimately connected. Although the origin of iYSL remains unclear, its cytoskeletal organization differs from that of the eYSL raising the possibility that there may be a more intimate connection between the iYSL and DCL that deserves to be explored in the future.

(d) . Organogenesis

In contrast to the extensive knowledge on the role of the YSL during early morphogenesis, only a few studies have addressed the role of this extraembryonic domain in organogenesis (figure 5g–i). In zebrafish, myocardial progenitors derived from bilateral mesodermal populations in the anterior lateral plate migrate between the pharyngeal endoderm and the YSL towards the midline to fuse and form a single heart tube (figure 5h) (reviewed in [206]). Failure in this migration often results in the formation of two separate hearts, a phenotype commonly known as cardia bifida. Several mix family genes are expressed in the YSL, including mxtx1, mxtx2 and mixl1 [140,207,208], and functional abrogation of mxtx1 specifically in the YSL can induce cardia bifida [209]. This phenotype appears to result from defective migration of myocardial progenitors due to reduced fibronectin deposition around myocardial progenitors and in the basal part of pharyngeal endodermal cells [209], which is controlled non-cell-autonomously by the YSL, likely in an indirect manner [210–212]. Similar to heart formation, the liver in zebrafish arises, at least in part, from different groups of endodermal cells that are initially located in bilateral regions on either side of the midline (figure 5i) [202,213]. The retinol transporter Rbp4 is expressed in the YSL, hypochord and skin, and specific disruption of the function of this transporter in the YSL results in liver duplication [214,215]. Although the underlying mechanism remain unclear, failure in the migration of liver progenitors cells is thought to depend, at least in part, in the formation of a defective fibronectin 1 network in the ventrolateral yolk [215]. Interestingly, expression of Rbp4 is observed in the extraembryonic visceral endoderm in rodents and chicks, suggesting a conserved function [216–218]. Therefore, given that many embryonic cell types, including endodermal and myocardial cells, are able to migrate over the YSL, it is possible that this extraembryonic structure functions as a migratory scaffold operating at different developmental stages.

7. Nutrition and protective functions of teleost extraembryonic structures

(a) . Nutrition of the embryo and larva

Teleost fish eggs are typically closed, shell-covered living systems (i.e. cleidoic) and therefore rely on endogenous yolk reserves accumulated in the oocyte as a source of nutrients and energy for early development and growth, prior to the onset of feeding. These yolk reserves are transferred to embryonic and larval tissues by a process of resorption and represent the sole source of nutrients during the endotrophic phase, which extends from fertilization to the onset of feeding, and can last several days depending on the species (e.g. about 5, 8, 20 and 47 days in zebrafish, turbot, Atlantic cod and Atlantic halibut, respectively [219,220]). When the larva begins to feed, exogenous nutrients from the diet are added to endogenous yolk reserves in the endo-exotrophic phase until the yolk is completely resorbed and the larva is nourished exclusively by exogenous nutrients in the exotrophic phase [219,221,222]. The process of yolk resorption in teleosts has been described as occurring in four phases [223]. The initial phase, which occurs in the blastula, is rather quiescent and involves very little transfer of yolk material to the embryo through the cytoplasmic contacts connecting the yolk to the basal region of the blastomeres (see above, §4a and 4b) [20,42]. Subsequently, once the extraembryonic YSL is formed (see above, §4b) and envelops the yolk mass after completion of epiboly (see above, §6a and 6b), the yolk reserves are actively resorbed through the YSL (see below). In a third phase, the emergence of systemic circulation facilitates YSL-mediated nutrient resorption. The systemic circulation develops from the splanchnic lateral plate mesoderm prior to hatching and enables the massive distribution of nutrients from the yolk to nascent tissues and organs, as well as other components such as gases, osmolytes, metabolites and signalling molecules [224]. In zebrafish and other teleosts (e.g. medaka, Atlantic cod, orange roughy), venous blood returning through the posterior cardinal vein empties into the large duct of Cuvier and flows into the anterior subdermal space coming into direct contact with the YSL of the yolk sac allowing the transfer of nutrients to the blood before it re-enters the venous sinus of the heart [28,225–229]. In species such as Atlantic salmon, trout and Nile tilapia, yolk transfer is enhanced by the presence of an elaborate yolk sac vasculature that is established early in development [230–232]. As development proceeds, in the fourth phase of resorption, nutrients from the yolk pass through the YSL and the liver anlage. These two tissues appear to show a close structural and functional connection, with the YSL facilitating certain functions of the liver prior to the onset of its activity and participating in its development (see above, §6d) [215,233], while the liver participates in yolk metabolism and reabsorbs the YSL when the yolk is depleted [234].

The yolk of teleost eggs is composed of different elements including, in decreasing order, proteins, amino acids, lipids, inorganic ions and glycogens [219,234]. The relative proportion of these components depends on the species [219], and within species varies according to maternal age, spawning season, and embryo batch [235–237]. Yolk proteins and lipids are deposited into oocytes by way of vitellogenin, a high-density lipoprotein synthesized by the maternal liver [238] that is then proteolytically cleaved to generate phosvitin and lipovitellin, and stored in the yolk for later use as nutrients by the developing embryo and larva [222]. Ultrastructural, immunocytochemical, biochemical and genetic studies demonstrate that the teleost YSL plays an active role in yolk resorption, aiding nutrient uptake and transport to embryonic and larval tissues [88,221,222,227,239–256], a function that is reminiscent of the yolk sac endoderm and placenta of higher vertebrates [257,258]. The YSL exports free amino acids [219] and manufactures several enzymes involved in early metabolism and nutrition-related functions, such as creatine metabolism [250], steroidogenesis [251], iron transport [242,243] and lipid metabolism [243–247,249]. It also synthesizes lipoproteins, which are macromolecular protein-containing complexes that serve the function of packaging and transporting yolk lipids [222,227,241]. Studies in zebrafish show that during embryogenesis, yolk lipids undergo lipolysis and re-esterification and are then packaged into lipoproteins in the endoplasmic reticulum of the YSL. Then they are secreted into the perisyncytial space and distributed through the systemic circulation to provide energy and building blocks for the developing embryo. Consistent with a vital role of the YSL in lipid package and transport, genetic conditions with defective lipoprotein function result in trapping of lipids in the yolk, and often the inability of larvae to survive [247,248,252,253,256].

(b) . Immune defence

Teleost extraembryonic tissues play a role in immunity in the early stages of embryonic development, protecting the vulnerable offspring against developmental abnormalities and pathogen attack, before specific embryonic tissues and organs assume this role normally after hatching. This function involves both aiding the transfer of maternally derived immune factors and providing cell-mediated innate immune responses. As in other vertebrates that develop as eggs, maternally derived immune factors in teleosts are transferred from mother to offspring via the egg yolk, and include, among others, members of the adaptive (e.g. IgM) and innate (e.g. complement component C3, lysozymes and cathelicidin) immune responses (reviewed in [259–261]). Furthermore, the vitellogenin-derived proteins phosvitin and lipovitellin, in addition to their role as nutrients (see above, §7a) also have an immunological role in the embryo and larva [262–265]. The YSL, in turn, besides transferring immune factors from the egg to the embryo and larva, also synthesizes innate immune components at embryonic stages, such as complement component C3 [266], although its significance is still unknown.

Teleost extraembryonic tissues also provide innate immune cellular protection to the embryo. Recent studies in zebrafish show that the EVL epithelium plays an active role in the scavenging of damaged embryonic cells destined for apoptosis during the early stages of development [187]. At the blastula stage, the EVL captures apoptotic embryonic cells across its basal face by a phagocytic mechanism that relies on cooperative mechanical interactions between epithelial cells [187]. This clearance capacity of the EVL is though to prevent the accumulation of errors that could lead to developmental defects or compromise embryonic survival, and appears to be an evolutionarily conserved feature of surface extraembryonic epithelia lining the early embryo, as it is also present in the mouse trophoblast [187].

(c) . Mechanical protection and osmoregulation

Teleost fish occupy a wide range of ecological niches and natural waters ranging from low ionic strength (near distilled) to high salinity waters, so their eggs are exposed to a variety of environmental threats, including mechanical insults and osmotic stresses [267,268]. In these varied environments, protection of the embryo and larvae is achieved through a series of mechanisms involving structural elements surrounding the egg, extraembryonic structures and embryonic tissues/organs. Prior to hatching, the first layer of protection is provided by the chorion and the PVF. The chorion is a thick acellular envelope outside the egg, composed mainly of proteins and glycoproteins, which is assembled during oogenesis and hardens after egg activation [25]. It shows great variability of resistance, thickness and layering between species as a form of adaptation to the environmental conditions in which the eggs develop [25,269]. In addition to its primary mechanical protective function, the chorion also regulates the water balance of the egg, is a partial barrier against microorganisms and environmental pollutants and, in many fish species ensures anchoring of the egg to the substrate [25,270]. The PVF is a macromolecule-rich fluid that forms during egg activation following the release of hydrophilous colloids into the perivitelline space that suck water from the outside [271]. Owing to its osmotic properties, the PVF generates a high hydrostatic pressure that keeps the chorion under tension, thus creating a buffered aqueous environment that protects the embryo from dehydration, mechanical injury and osmotic stress [268].

A second layer of mechanical and osmotic protection for the embryo is provided by the extraembryonic EVL. As a surface epithelium, the EVL functions as a primary ‘skin’ that protects the underlying embryonic cells from environmental trauma. This function is present from the earliest stages of development in the blastula until at least the larval period, after which the periderm (derived from the EVL) is thought to be replaced by the definitive epidermis derived from basal keratinocytes of ectodermal origin [24,60,69,70]. In addition to its mechanical protective function, the EVL also protects the embryo from excessive hydro-mineral imbalance, acting as a high-resistance permeability barrier for water and ions [268,272–278]. This function is important in the regulation of osmotic pressure and tissue surface tension especially in the early stages of development, before integrated ion and water transport mechanisms develop in the integument, gills, kidney and gut around hatching [268,279–281]. The osmotic seal provided by the EVL helps to establish intra-embryonic conditions distinct from the surrounding environment [274,278] and is therefore essential for normal development. This protective function depends on the close lateral apposition of epithelial cells mediated by circumapical tight junctions, adherens junctions, desmosomes and gap junctions [59,177,178,182,282–286], with the former playing the most critical permeability barrier role [182,282,283,286]. Indeed, studies in zebrafish demonstrate that disruption of tight junctions affects the barrier function of the EVL, as revealed by increased surface permeability of low molecular weight tracers, and compromised osmoregulation leading to increased sensitivity to osmotic stress and embryo rupture [286]. Similarly, in zebrafish embryos with EVL differentiation defects, the cytoskeleton or adherens junctions often show impaired epithelial integrity [56,58–60,287–289], sometimes leading to embryo rupture before gastrulation [56,58,60,178,289]. Thus, having a protective and permeability barrier on the surface of the teleost embryo has significant advantages for early development, and it has been proposed that this may have played an important role in the evolutionary origin of the EVL (see below, §8), also allowing teleosts to occupy a wide diversity of ecological niches [15].

(d) . Embryo lodging into external host environments

A remarkable evolutionary specialization of extraembryonic tissues that allows teleosts to establish a symbiotic relationship with invertebrate animals is observed in the bitterling, a small cyprinid fish that has a particular life history and an atypical development. Bitterling fish show a unique reproductive strategy where eggs are deposited and fertilized inside freshwater mussels. Within the host and thanks to their elliptical shape and the development of elongated lateral processes of the extraembryonic yolk sac, the large eggs of bitterling become anchored within the interlamellar spaces of the mussels' gills where they obtain oxygen and develop protected from predators until larval stages. At this stage, the yolk sac is reabsorbed and juvenile fish are released to continue their development outside the mussel [290]. The specialization of extraembryonic yolk sac thus allows developing bitterling to delay the exposure to predation until juvenile stages which increase the probability of survival.

8. Evolutionary origin of teleost extraembryonic structures

From a phylogenetic point of view, although the extraembryonic yolk, YSL and EVL are highly derived structures, they are proposed to have evolved from cellular/tissue domains present in the eggs of ancient actinopterygians, which are thought to be similar to those observed in present-day sturgeons, bichirs, and amphibians (e.g. [17,291–293]). For example, several lines of evidence support the endodermal characteristics of the teleost yolk and its associated YSL. Indeed, it has been proposed that the yolk evolved from a fusion event of endoderm-derived blastomeres present in an ancestral chordate [15,203,294,295]. The YSL, in turn, has been related to a thin sheet of hypoblastic cells located at the floor of the segmentation cavity in the bowfin Amia calva [296] and, at the molecular level, it expresses several amphibian endodermal homologous including gata5, hhex, mixl1 and forkhead 2 [140,297,298]. The EVL, on the other hand, is an outer epithelium that is proposed to have evolved as a permeability barrier (see above, §7c) derived from the surface epithelial layer of an ancestral actinopterygian, that is observed in present-day amphibians [15,17]. In support of this idea, the EVL of zebrafish and the surface epithelial layer of Xenopus, which have been studied in some detail, both derive from the outermost layer of the blastoderm and form distinct outer epithelial sheets joined by tight junctions that serve a primarily protective function [70,299]. However, the EVL surrounds the entire embryo at all stages and, with few exceptions (see above, §5d), forms the periderm which functions as a primitive skin [24,60,69,70]. By contrast, in Xenopus only the ventral part of the epithelial layer gives rise to the larval epidermis, which covers the entire embryo from the neural stage onwards, while its dorsal part participates in neurulation and gives rise to the ependymal layer in the neural tube and the non-neural cement gland [300–302]. In addition, prior to gastrulation, the embryonic epithelium is continuous with an epithelial layer of endodermal and mesodermal cells that involute during gastrulation to form the lining of the archenteron and the gastrocoel roof plate (GRP), respectively [303,304]. The endodermal epithelium has organizing properties and participates in embryo patterning [305] while the GRP is an epithelial plate containing motile cilia that is required for left–right axis determination [306]. Interestingly, the zebrafish EVL appears to play a role in D-V patterning of the embryo (see above, §5c) and contains a subpopulation of dorsal cells that internalizes to form the laterality organ or KV (see above, §5d), an epithelial vesicle homologous to the GRP of Xenopus in terms of its ability to control left–right patterning [132,133]. Together, these findings suggests that if the teleost EVL did indeed evolve from an outer epithelial layer similar to that found in present-day amphibians, it must have lost during evolution its potential to form most of the dorsal embryonic structures, but retained some organizing properties and the ability to internalize and form epithelial structures involved in left–right patterning.

9. Concluding remarks

In teleosts, three zygote-derived lineages are recognized as extraembryonic: the acellular yolk, the syncytial YSL and the epithelial EVL. These lineages diverge during early ontogeny and play critical roles throughout development. Nutrition and protection of the developing embryo and larva are essential functions of teleost extraembryonic structures that, while shared with all other vertebrates, are particularly relevant in this group of animals due to the cleidoic nature of their eggs. Almost all teleost fishes are strictly dependent on maternally derived factors and extraembryonic structures for early development, nutrition, osmoprotection and immunity until specialized tissues take over these functions and the larva begins to feed. But the extraembryonic–embryonic relationship is a highly dynamic co-construction that is remodelled at each stage of development. Consequently, the emerging concept now places the YSL in a close molecular and mechanical feedback interaction with embryonic lineages, and with the EVL, resulting in a mutual reinforcement of the gene regulatory networks and cell/tissue-level mechanical processes responsible for embryonic patterning and morphogenesis. Despite technological advances and evolving ideas, our understanding of how this precise and coordinated phenomenon occurs is far from elucidated and many questions remain unanswered. One of these concerns the specific aspects of early development for which the YSL is indispensable, as well as the details of the molecular and mechanical interactions that the YSL has with the DCL and EVL. Furthermore, while the YSL has been studied in some detail, there is a considerable gap in the knowledge of the patterning functions of the EVL and the role that differentiation of this epithelial tissue plays in patterning and in the acquisition of mechanical properties relevant to embryonic morphogenesis. Another limitation in the field is the lack of detailed information beyond model species, such as zebrafish, which raises the question of how universal the functions of extraembryonic structures are within the teleost group. The peculiarities of annual killifish development is an example that makes us think about alternative developmental trajectories, and offers the opportunity to address the conserved versus derived nature and roles of teleost extraembryonic domains in embryo patterning and morphogenesis, broadening and enriching our biological perspectives. Future research should address these and other questions by taking advantage of the unique possibility offered by teleost eggs to easily combine in vivo, ex vivo and synthetic stem cell-based embryonic approaches, with ease of visualization and genetic manipulation. These approaches promise to shed new light on how teleost extraembryonic domains provide the boundary conditions (genetic, geometric, mechanical) necessary to channel the self-organizing patterning properties of early embryonic cells and translate them into the generation of species-specific form and shape.

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Authors' contributions

M.L.C.: conceptualization, methodology, visualization, writing—original draft, writing—review and editing; G.R.: conceptualization, methodology, visualization, writing—original draft, writing—review and editing.

All authors gave final approval for publication and agreed to be held accountable for the work performed therein.

Conflict of interest declaration

We declare we have no competing interests.

Funding

M.L.C. is supported by the Chilean National Agency for Research and Development (ANID) projects grant nos: ICN09_015; ACE210007; FONDAP 15150012; FONDECYT 1190806; PIA ACT192015; REDES170212; and the ‘Millennium Nucleus Physics of Active Matter’. G.R. is supported by Climat-AmSUD grant no. CLI2020004.