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Fig. 1. Monolayered versus multi-layered development. A) The typical invertebrate blastula consists of a hollow ball made of an epithelial monolayer surrounding a cavity called the blastocoel. Gastrulation usually occurs by simple invagination of a portion of the monolayer. B) The cleavage pattern of vertebrate embryos has been modified by the introduction of asymmetric divisions that produce superficial and deep cells, resulting in a multi-layered blastula. Gastrulation typically involves an inward flow of cell mass.
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Box 1. Cortical tension, contact tension, tissue surface tension and epithelial coating. A,B) Cortical and contact tensions. A) Cell and tissue geometry reflect underlying forces. For a simple cell doublet, the angles at vertices are determined by the equilibrium between cortical tensions Ct and contact tension T. Contact tension is mainly dictated by the local cortical contractility along the contact, which is reduced under the influence of adhesive interactions relative to the free surface tension. This reduction defines the relative adhesiveness α = (T-Ct)/Ct, and is directly related to the angle θ by the relation α = 1-cos(θ) [14,136]. A’) Doublet asymmetry indicates a difference in Ct: The cell with the lowest Ct, here B, tends to engulf the other cell. The same relationship applies to groups of cells and tissues, replacing Ct and T with “tissue surface tensions” γ [13,16]. B) Example of two doublets with high and low θ, indicative of high and low adhesiveness, respectively. C,D) Heterotypic contacts and boundary formation. C) For cell types with different homotypic tension TAA < TBB, heterotypic tension TAB is predicted to be intermediate, consistent with the interfacial tension hypothesis (DITH). Under these conditions, cells can cluster but do not efficiently segregate. D) Higher contact tension can be triggered by local stimulation of contractility and/or decrease in adhesion. High heterotypic interfacial tension (HIT) creates a smooth boundary. E,F) Tissue positioning and epithelial coating. E) In reaggregation or tissue fusion experiments, tissues with the highest tissue surface tension γ are surrounded by cells with lower γ. Thus, ectoderm typically ends up at the centre, surrounded by mesoderm and endoderm. Coating the tissue aggregate with the superficial epithelial layer (ep) reverses the position of the tissues, reproducing the normal organization in the embryo. This is due to the non-adhesive apical surface (dark red), which forces the superficial position of polarized cells, which in turn preferentially attract the deep ectoderm cells [16]. F) Epithelial coating also explains tissue positioning in linear patterns, for instance for anterior “A” and posterior “B” mesoderm. In vitro, “A” engulfs “B” due to its lower γ. Upon inclusion of the ectoderm epithelial layer, which mimics the natural situation, the system is only subjected to the tissue to tissue tensions, which are much weaker than the surface tensions of the tissues exposed to the medium. This allows the mesoderm regions to sort based on other parameters, such as specific adhesion [13,16].
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Fig. 2. Segregation in early Xenopus development. A–D) Cleavage and formation of the blastula. A) The egg membrane has apical properties (dark red). During cleavage, the furrows (blue) are formed by delivery of new membrane through exocytosis. This membrane has basolateral characteristics. The boundary between the old apical membrane and the new basolateral membrane is delimited by accumulation of tight junction components (orange spots). B) The morula (32 cells) is composed of a single polarized epithelium sealed by tight junctions (orange spots). C) The two subsequent divisions display variable cleavage planes. Division planes perpendicular to the apical surface (dark green arrows) produce two identical daughter cells. Division planes parallel to the apical surface (purple arrows) produce two dissimilar daughter cells, one superficial and one internal. The latter is not polarized (light blue). In the case of oblique planes, the result depends on whether the egg membrane is inherited by both cells (light green arrows), or only one cell (light purple arrows). The latter case yields a superficial and a deep cell. D) The early blastula (128 cells) is composed of a polarized outer layer enclosing non-polarized deep cells. E–F) Organization of the late blastula (E) and early gastrula (F), with the position of the germ layers and regionalization of the mesoderm. The dorsal mesoderm is subdivided into leading mesendoderm (ME), anterior (AM) and posterior (PM) mesoderm. The red line highlights the boundary between the involuting mesoderm and the ectoderm. The interface between the endoderm and the mesoderm does not form a visible boundary (dotted red line). af, archenteron floor; ar, archenteron roof; bc, bottle cells; bp, blastopore; sf, superficial layer. G) Enlargement of the dorsal region of the early gastrula, highlighting the ectoderm-mesoderm boundary. The mesendoderm and the mesoderm form a single unit in terms of separation behaviour. At the blastopore lip (bl), the mesoderm progressively acquires its separation capacity (hatched), its posterior end remaining continuous with the ectoderm. Dashed arrows: direction of involution. G’) Detail showing the superficial ectoderm and the deep layers of the ectoderm, mesoderm and endoderm. H) Molecular control of ectoderm-mesoderm separation. Top: Simplified diagram of the ephrin-Eph network. The red double arrows symbolize repulsive signals. The asymmetric expression of the ephrinB3-EphA4 and the ephrinB2-EphB4 pairs are crucial to produce a stronger repulsive signal across the boundary. Weaker activity is detected in the mesoderm. In the ectoderm, the weak ephrin-Eph interactions positively impact on cell adhesion (green double arrow). Bottom: Differential action of the protocadherin PAPC, which favours adhesion at homotypic contacts in the mesoderm but decreases it at the heterotypic contacts across the boundary. I) Separation behaviour at the ectoderm-mesoderm boundary. Heterotypic contacts undergo cycles of repulsion due to ephrin-Eph-induced contractility (red lines) and re-adhesion (cadherins in green). This mechanism is well-suited to maintain separation, while the mesoderm crawls using the ectoderm as an adhesive substrate. Arrow: direction of mesoderm migration.
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Box 2. Experimental demonstration of cell autonomous inheritance of the apical domain. A) A single blastomere dissociated from a 64-cell stage embryo can divide along various cleavage planes. Daughter cells that inherit the apical egg membrane (light purple) will remain superficial cells (dark green), and develop into spheroids expressing the bHLH gene ESR6e, while those lacking the apical domain will become deep cells (light green), and develop into ESR6e-negative spheroids [21]. B) A single blastomere from a labelled embryo is grafted inside the blastocoel of a host embryo. Descendant blastomeres that have inherited of the original egg membrane form an ectopic, inverted, polarized layer with the apical domain facing the inside of the blastula. Other descendants lacking this apical domain integrate into the deep layer [23]
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Box 3. Ectoderm-mesoderm separation as an experimental model for cell sorting at embryonic boundaries. A) The classical sorting assay [4] involves dissection of tissue explants and their dissociation in an alkaline, calcium-free buffer. Cell populations are mixed and left to reaggregate in a physiological buffer. Wild type ectoderm and mesoderm cells sort into small clusters that progressively merge into larger groups delimited by smooth boundaries [19]. Sorting is quantified in terms of clustering and interface smoothness. B) The tissue separation assay [56] uses a large piece of ectoderm as cellular substrate, on which tissue aggregates are laid. Wild type ectoderm aggregates sink and mix within the ectoderm substrate, while mesoderm aggregates remain separated, thus reconstituting the endogenous boundary. Both assays can be used to test any recombination of tissue
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Box 4. The ephrin code. Ephrin and Eph receptors are cell surface molecules involved in repulsive reactions. They have been extensively studied in the nervous system, where they function as contact guidance cues, but are also widely expressed in other tissues, in adults and embryos. Ephrin-Eph interaction can trigger signalling both in the Eph-expressing cell (forward signalling) and in the ephrin-expressing cell (reverse signalling). Their activation leads to local remodelling of the actin cytoskeleton, mainly via Rho-dependent actomyosin contractility. A) Ephrins and Eph receptors are classified in A and B subfamilies. Traditionally, ephrin-Eph interactions are considered to be promiscuous within each subfamily, ephrinAs reacting with all EphAs, and ephrinBs with all EphBs (top panel). One exception is EphA4, which can interact with ephrins of both subfamilies. However, this assumed promiscuity is at odds with widely different binding affinities, even within the same subfamilies [64,65,137]. Furthermore, it could not account for the case of the early vertebrate gastrula, where multiple ephrins and Eph receptors are expressed in all tissues, yet overt repulsion is restricted at the tissue boundaries. We demonstrated a strong functional selectivity for ephrinB1,2,3 and EphB2, B3 and A4 receptors in the physiological context for early embryonic boundaries, resulting in a network of interactions (bottom panel): Some members bind multiple partners (e.g. ephrinB2 binds all three receptors), while others can only interact with a single partner (e.g. ephrinB3 with EphA4, ephrinB1 with EphB2). B) Simplified diagram presenting the major ephrin and Eph receptors expressed in the dorsal ectoderm and mesoderm (ephrins and Ephs presented in separate cells for clarity’s sake). The output of these complex systems relies on three simple principles, selectivity, complementary expression, and balance with adhesive forces (cadherins in green): Repulsion at a given contact results from the combined action of multiple ephrin-Eph pairs (red double arrows, thickness symbolizes the relative intensity). The partially complementary expression of some key ephrin-Eph pairs serves as a “code” that discriminates between homotypic contacts, where repulsion is weak (ectoderm) to moderate (mesoderm), and heterotypic contacts, where it is sufficiently strong to overcome adhesion. In addition to the dorsal ectoderm-mesoderm boundary, this code also accounts for ventral ectoderm-mesoderm separation (not shown), as well as for the notochord boundary (Fig. 3). The existence of weaker repulsive signals at homotypic contacts likely contributes to dynamic adhesion within the tissues [37]. Similar systems with multiple ephrins and Eph receptors are frequently observed, which may be explained based on the same principles [63,65,138,139].
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