A cell-based model of Nematostella vectensis gastrulation including bottle cell formation, invagination and zippering

The gastrulation of Nematostella vectensis, the starlet sea anemone, is morphologically simple yet involves many conserved cell behaviors such as apical constriction, invagination, bottle cell formation, cell migration and zippering found during gastrulation in a wide range of more morphologically complex animals.In this article we study Nematostella gastrulation using a combination of morphometrics and computational modeling. Through this analysis we frame gastrulation as a non-trivial problem, in which two distinct cell domains must change shape to match each other geometrically, while maintaining the integrity of the embryo. Using a detailed cell-based model capable of representing arbitrary cell-shapes such as bottle cells, as well as filopodia, localized adhesion and constriction, we are able to simulate gastrulation and associate emergent macroscopic changes in embryo shape to individual cell behaviors.We have developed a number of testable hypotheses based on the model. First, we hypothesize that the blastomeres need to be stiffer at their apical ends, relative to the rest of the cell perimeter, in order to be able to hold their wedge shape and the dimensions of the blastula, regardless of whether the blastula is sealed or leaky. We also postulate that bottle cells are a consequence of cell strain and low cell–cell adhesion, and can be produced within an epithelium even without apical constriction. Finally, we postulate that apical constriction, filopodia and de-epithelialization are necessary and sufficient for gastrulation based on parameter variation studies.     

FGF signalling through RAS/MAPK and PI3K pathways regulates cell movement and gene expression in the chicken primitive streak without affecting E-cadherin expression

Background  

FGF signalling regulates numerous aspects of early embryo development. During gastrulation in amniotes, epiblast cells undergo an epithelial to mesenchymal transition (EMT) in the primitive streak to form the mesoderm and endoderm. In mice lacking FGFR1, epiblast cells in the primitive streak fail to downregulate E-cadherin and undergo EMT, and cell migration is inhibited. This study investigated how FGF signalling regulates cell movement and gene expression in the primitive streak of chicken embryos.     

Primary Invagination of the Vegetal Plate During Sea Urchin Gastrulation

The initial phase of echinoid gastrulation, primary invagination,involves an inpocketing of a monolayered epithelium. To gaininformation about the nature of the mechanical forces that areresponsible for primary invagination, several experimental approacheshave been taken, using the transparent embryos of the sea urchin,Lytechinus pictus, as the principal material. Vegetal platesisolated microsurgically well before the onset of gastrulationwill invaginate normally, demonstrating that the forces responsiblefor primary invagination are generated by the cells in the vegetal to of the embryo. As shown by serial reconstructions of L.pictus embryos, relatively few cells (about 100) take part inprimary invagination. Both the number of cells and the totalvolume of tissue in the wall of the archenteron increase withtime. Even so, it can be shown that very little movement ofcells over the lip of the blastopore takes place during primaryinvagination, and this process is best viewed as a simple inpocketingof the vegetal epithelium. The cells in the wall of the archenteronhave a distinctive shape; they are elongated along their apico-basalaxes and frequently have enlarged, rounded, basal ends. However,they do not undergo any dramatic changes in shape during primaryinvagination. In particular, there is only a slight decreasein the height of the cells (length along the apico-basal axis),a result that is inconsistent with the hypothesis that invaginationis due to cell rounding (Gustafson and Wolpert, 1967). Examinationof L. pictus and Strongylocentrotus purpuratus gastrulae bytransmission electron microscopy reveals that cells in the wallof the archenteron continue to be joined by typical junctionalcomplexes during primary invagination. In addition, the morphologyof the junctional complex at the gastrula stage is more elaboratethan previously described. Sparse bands of micronlaments areassociated with the plasma membrane at the level of the junctionalcomplexes in both endodermal and ectodermal cells. These andother relevant data on early echinoid gastrulation are discussedin relation to several possible mechanisms of epithelial morphogenesis.     

Epithelial to mesenchymal transition during gastrulation: An embryological view

Gastrulation is a developmental process to generate the mesoderm and endoderm from the ectoderm, of which the epithelial to mesenchymal transition (EMT) is generally considered to be a critical component. Due to increasing evidence for the involvement of EMT in cancer biology, a renewed interest is seen in using in vivo models, such as gastrulation, for studying molecular mechanisms underlying EMT. The intersection of EMT and gastrulation research promises novel mechanistic insight, but also creates some confusion. Here we discuss, from an embryological perspective, the involvement of EMT in mesoderm formation during gastrulation in triploblastic animals. Both gastrulation and EMT exhibit remarkable variations in different organisms, and no conserved role for EMT during gastrulation is evident. We propose that a ‘broken-down’ model, in which these two processes are considered to be a collective sum of separately regulated steps, may provide a better framework for studying molecular mechanisms of the EMT process in gastrulation, and in other developmental and pathological settings.     

The cessation of gastrulation: BMP signaling and EMT during and at the end of gastrulation

An integral component of gastrulation in all organisms is epithelial to mesenchymal transition (EMT), a fundamental morphogenetic event through which epithelial cells transform into mesenchymal cells. The mesenchymal cells that arise from epithelial cells during gastrulation contribute to various tissue rudiments during subsequent development, including the notochord, somites, heart, gut, kidney, body wall and lining of the coelom. The process of gastrulation has been the subject of several hundred scientific papers. Despite all that has been written, it is likely that what we currently know about gastrulation is still considerably less than what remains to be learned. One critical remaining question that we consider here is how does gastrulation cease at the right place along the body axis, and at the right time? In this commentary, we focus on the molecular mechanism for the cessation of gastrulation, using the chick embryo as a model system.Key words: epithelial to mesenchymal transition (EMT), gastrulation, basal membrane, tail bud, ventral ectodermal ridge (VER), BMP, noggin, E-cadherin, chick embryo     

An amicable separation: Chick’s way of doing EMT

Epithelial to mesenchymal transition (EMT) is a morphogenetic process in which cells lose their

epithelial characteristics and gain mesenchymal properties, and is fundamental for many tissue

remodeling events in developmental and pathological conditions. Although general cell biology of

EMT has been well-described, how it is executed in diverse biological settings depends largely on

individual context, and as a consequence, regulatory points for each EMT may vary. Here we discuss

developmental and cellular events involved in chick gastrulation EMT. Regulated disruption of

epithelial cell/basement membrane (BM) interaction is a critical early step. This takes place after

molecular specification of mesoderm cell fate, but before the disruption of tight junctions. The

epithelial cell/BM interaction is mediated by small GTPase RhoA and through the regulation of basal

microtubule dynamics. We propose that EMT is not regulated as a single morphogenetic event.

Components of EMT in different settings may share similar regulatory mechanisms, but the sequence

of their execution and critical regulatory points vary for each EMT.     

Transitions between epithelial and mesenchymal states and the morphogenesis of the early mouse embryo

Multicellular organisms arise from the generation of different cell types and the organization of cells into tissues and organs. Cells of metazoa display two main phenotypes, the ancestral epithelial state and the recent mesenchymal derivative. Epithelial cells are usually stationary and reside in twodimensional sheets. By contrast mesenchymal cells are loosely packed and can move to new positions, thereby providing a vehicle for cell rearrangement, dispersal and novel cell-cell interactions. Transitions between epithelial and mesenchymal states drive key morphogenetic events in the early vertebrate embryo, including gastrulation, germ layer formation and somitogenesis. The cell behaviors and molecular mechanisms promoting transitions between these two states in the early mouse embryo are discussed in this review.Key words: mouse embryo, EMT, MET, morphogenesis, gastrulation, somitogenesis, epiblast, mesoderm, endoderm, primitive streak, paraxial mesoderm     

Gastrulation in the sea urchin embryo is accompanied by the rearrangement of invaginating epithelial cells

The second phase of gastrulation in the sea urchin embryo, secondary invagination, involves a dramatic elongation of the tube-like gut rudiment. The cells in the wall of the rudiment, which are organized as a monolayered epithelium, change their arrangement during this process. The number of cells in the wall of the gut rudiment at any given level along its long axis decreases markedly as determined by light microscopy of serial cross sections and by scanning electron microscopy, an observation that can be accounted for only if some of the cells exchange nearest neighbors during secondary invagination. Transmission electron microscopy reveals that cell rearrangement takes place despite the continued presence of typical intercellular junctional complexes. In addition to undergoing rearrangement, the cells in the wall of the gut rudiment change their shape during secondary invagination, becoming more flattened. These data raise the possibility that mechanisms other than the contraction of the filopodia of the presumptive secondary mesenchyme cells contribute to the second phase of invagination in the sea urchin embryo. In addition, the observation that cells in the wall of the gut rudiment undergo rearrangement during secondary invagination provides additional evidence that epithelial sheets can exhibit fluid-like properties during morphogenesis.     

An SEM study of cellular morphology,contact, and arrangement,as related to gastrulation inXenopus laevis

Summary Cellular morphology, contact, and arrangement in the late blastula and in various stages of gastrulation ofXenopus were examined by SEM of specimens dissected after fixation or fractured in amyl acetate. The prospective ectoderm of the blastocoel roof consists of several layers of interdigitating cells connected by numerous small protrusions which may function in the decrease in number of cell layers observed during ectodermal epiboly. During gastrulation, prospective mesoderm is regionally differentiated by cellular morphology and arrangement into preinvolution mesoderm, the mesodermal involution zone, and involuted mesoderm. The involuted anterodorsal (head), lateral, and ventral mesoderm consists of a stream of loosely-packed, irregularly shaped cells having large extensions of the cell body attached locally to other cells by small protrusions. Involuted posterodorsal mesoderm (chordamesoderm) consists of elongated cells arranged in palisade fashion and connected by similar protrusions. Involuted mesodermal cells in all regions are attached to the overlying prospective ectodermal cells by numerous small protrusions along the entire interface between the two cell layers. Suprablastoporal endodermal cells involute as an epithelial sheet, changing in shape in the process, to form the roof of the archenteron. Bottle cell morphology, arrangement, and position with respect to the mesodermal cell stream is described. Evidence presented here and elsewhere suggests that involution of mesoderm and of the archenteron roof inXenopus is dependent primarily upon the relative movement of the mesodermal cell stream and of the overlying ectoderm.     

Fast Neurotransmission Related Genes Are Expressed in Non Nervous Endoderm in the Sea Anemone Nematostella vectensis

Cnidarian nervous systems utilize chemical transmission to transfer signals through synapses and neurons. To date, ample evidence has been accumulated for the participation of neuropeptides, primarily RFamides, in neurotransmission. Yet, it is still not clear if this is the case for the classical fast neurotransmitters such as GABA, Glutamate, Acetylcholine and Monoamines. A large repertoire of cnidarian Fast Neurotransmitter related Genes (FNGs) has been recently identified in the genome of the sea anemone, Nematostella vectensis. In order to test whether FNGs are localized in cnidarian neurons, we characterized the expression patterns of eight Nematostella genes that are closely or distantly related to human central and peripheral nervous systems genes, in adult Nematostella and compared them to the RFamide localization. Our results show common expression patterns for all tested genes, in a single endodermal cell layer. These expressions did not correspond with the RFamide expressing nerve cell network. Following these results we suggest that the tested Nematostella genes may not be directly involved in vertebrate-like fast neurotransmission.     

Jun N‐terminal kinase activity is required for invagination but not differentiation of the sea urchin archenteron

Although sea urchin gastrulation is well described at the cellular level, our understanding of the molecular changes that trigger the coordinated cell movements involved is not complete. Jun N‐terminal kinase (JNK) is a component of the planar cell polarity pathway and is required for cell movements during embryonic development in several animal species. To study the role of JNK in sea urchin gastrulation, embryos were treated with JNK inhibitor SP600125 just prior to gastrulation. The inhibitor had a limited and specific effect, blocking invagination of the archenteron. Embryos treated with 2 μM SP600125 formed normal vegetal plates, but did not undergo invagination to form an archenteron. Other types of cell movements, specifically ingression of the skeletogenic mesenchyme, were not affected, although the development and pattern of the skeleton was abnormal in treated embryos. Pigment cells, derived from nonskeletogenic mesenchyme, were also present in SP600125‐treated embryos. Despite the lack of a visible archenteron in treated embryos, cells at the original vegetal plate expressed several molecular markers for endoderm differentiation. These results demonstrate that JNK activity is required for invagination of the archenteron but not its differentiation, indicating that in this case, morphogenesis and differentiation are under separate regulation. genesis 53:762–769, 2015. © 2015 Wiley Periodicals, Inc.     

Urodeles remove mesoderm from the superficial layer by subduction through a bilateral primitive streak

Urodeles begin gastrulation with much of their presumptive mesoderm in the superficial cell layer, all of which must move into the deep layers during development. We studied the morphogenesis of superficial mesoderm in the urodeles Ambystoma maculatum, Ambystoma mexicanum, and Taricha granulosa. In all three species, somitic, lateral, and ventral mesoderm move into the deep layer during gastrulation, ingressing through a "bilateral primitive streak" just inside the blastopore. The mesodermal epithelium appears to slide under the endodermal epithelium by a mechanism we term "subduction." Subduction removes the large expanse of superficial presumptive somitic and lateral-ventral mesoderm that initially separates the sub-blastoporal endoderm from the notochord, leaving the endoderm bounding the still epithelial notochord along the gastrocoel roof. Subduction may be a common feature of urodele gastrulation, differing in this regard from anurans. Subducting cells constrict their apices and become bottle-shaped as they approach the junction of the mesodermal and endodermal epithelia. Subducting bottle cells endocytose apical membrane and withdraw the tight junctional component cingulin from the contracting circumferential tight junctions. Either in conjunction with or immediately after subducting, the mesodermal cells undergo an epithelial-to-mesenchymal transition. The mechanism by which epithelial cells release their apical junctions to become mesenchymal, without disrupting the integrity of the epithelium, remains mysterious, but this system should prove useful in understanding this process in a developmental context.     

Ultraviolet irradiation of eggs and blastomere isolation experiments suggest that gastrulation in the direct developing ascidian, Molgula pacifica, requires localized cytoplasmic determinants in the egg and cell signaling beginning at the two-cell stage

Gastrulation in the maximum direct developing ascidian Molgula pacifica is highly modified compared with commonly studied "model" ascidians in that endoderm cells situated in the vegetal pole region do not undergo typical invagination and due to the absence of a typical blastopore the involution of mesoderm cells is highly modified. At the gastrula stage, embryos are comprised of a central cluster of large yolky cells that are surrounded by a single layer of ectoderm cells in which there is only a slight indication of an inward movement of cells at the vegetal pole. As a consequence, these embryos do not form an archenteron. In the present study, ultraviolet (UV) irradiation of fertilized eggs tested the possibility that cortical cytoplasmic factors are required for gastrulation, and blastomere isolation experiments tested the possibility that cell signaling beginning at the two-cell stage may be required for the development of the gastrula. Irradiation of unoriented fertilized eggs with UV light resulted in late cleavage stage embryos that failed to undergo gastrulation. When blastomeres were isolated from two-cell embryos, they developed into late cleavage stage embryos; however, they did not undergo gastrulation and subsequently develop into juveniles. These results suggest that cytoplasmic factors required for gastrulation are localized in the egg cortex, but in contrast to previously studied indirect developers, these factors are not exclusively localized in the vegetal pole region at the first stage of ooplasmic segregation. Furthermore, the inability of embryos derived from blastomeres isolated at the two-cell stage to undergo gastrulation and develop into juveniles suggests that important cell signaling begins as early as the two-cell stage in M. pacifica. These results are discussed in terms of the evolution of maximum direct development in ascidians.     

Gastrulation in the sea urchin embryo: a model system for analyzing the morphogenesis of a monolayered epithelium

Processes of gastrulation in the sea urchin embryo have been intensively studied to reveal the mechanisms involved in the invagination of a monolayered epithelium. It is widely accepted that the invagination proceeds in two steps (primary and secondary invagination) until the archenteron reaches the apical plate, and that the constituent cells of the resulting archenteron are exclusively derived from the veg2 tier of blastomeres formed at the 60-cell stage. However, recent studies have shown that the recruitment of the archenteron cells lasts as late as the late prism stage, and some descendants of veg1 blastomeres are also recruited into the archenteron. In this review, we first illustrate the current outline of sea urchin gastrulation. Second, several factors, such as cytoskeletons, cell contact and extracellular matrix, will be discussed in relation to the cellular and mechanical basis of gastrulation. Third, differences in the manner of gastrulation among sea urchin species will be described; in some species, the archenteron does not elongate stepwise but continuously. In those embryos, bottle cells are scarcely observed, and the archenteron cells are not rearranged during invagination unlike in typical sea urchins. Attention will be also paid to some other factors, such as the turgor pressure of blastocoele and the force generated by blastocoele wall. These factors, in spite of their significance, have been neglected in the analysis of sea urchin gastrulation. Lastly, we will discuss how behavior of pigment cells defines the manner of gastrulation, because pigment cells recently turned out to be the bottle cells that trigger the initial inward bending of the vegetal plate.