You will be presented with the abstract, introduction, and discussion section from a biomedical research article. Your task is to create a summary that a layperson can understand, capturing the essence of the research article. Keep essential scientific terms, but ensure that the language remains clear and the concepts are explained in an uncomplicated manner.

Title: EphrinB/EphB Signaling Controls Embryonic Germ Layer Separation by Contact-Induced Cell Detachment}
Article:
Abstract:
The primordial organization of the metazoan body is achieved during gastrulation by the establishment of the germ layers. Adhesion differences between ectoderm, mesoderm, and endoderm cells could in principle be sufficient to maintain germ layer integrity and prevent intermixing. However, in organisms as diverse as fly, fish, or amphibian, the ectoderm-mesoderm boundary not only keeps these germ layers separated, but the ectoderm also serves as substratum for mesoderm migration, and the boundary must be compatible with repeated cell attachment and detachment. We show that localized detachment resulting from contact-induced signals at the boundary is at the core of ectoderm-mesoderm segregation. Cells alternate between adhesion and detachment, and detachment requires ephrinB/EphB signaling. Multiple ephrinB ligands and EphB receptors are expressed on each side of the boundary, and tissue separation depends on forward signaling across the boundary in both directions, involving partially redundant ligands and receptors and activation of Rac and RhoA. This mechanism differs from a simple differential adhesion process of germ layer formation. Instead, it involves localized responses to signals exchanged at the tissue boundary and an attachment/detachment cycle which allows for cell migration across a cellular substratum.
Introduction:
When Townes and Holtfreter[1] observed the sorting of mixed embryonic ectoderm, mesoderm, and endoderm cells, they proposed that this segregation of germ layers and the consequent self-assembly of the basic body structure was based on mutual “tissue affinities. ” This concept was later refined into Steinberg's[2] Differential Adhesion Hypothesis, which posited that simple adhesion differences between cell populations are sufficient for their separation and their positioning relative to each other, explaining, for example, the arrangement of germ layers in amphibian embryos[3],[4]. However, if the boundary between two germ layers also serves for tissue translocation, more specialized tissue separation mechanisms may be required. In the Xenopus gastrula, mesoderm translocates across the ectodermal blastocoel roof( BCR), and the boundary between these two germ layers, Brachet's cleft, must permit this movement yet prevent invasion of the BCR by the migratory mesoderm( Figure 1A). Interaction with a sparse network of fibronectin fibrils controls the motility of mesoderm cells, but their adhesion to the BCR is fibronectin-independent[5]. In fact, BCR and mesoderm cells are in direct contact[6], and the same adhesion molecules, C- and XB/U-cadherin, are expressed in both tissues( reviewed in[7]). This ectoderm-mesoderm boundary has been established as a model for tissue separation. Properties of the mesoderm and the BCR that underlie their separation can be studied in an in vitro assay( Figure 1A)[5],[8]. The molecular control of separation behavior is partly known. Non-canonical Wnt signaling downstream of the Wnt receptor Xfz7[9], interaction of Xfz7 with paraxial protocadherin( PAPC) and with the ankyrin repeat domain protein 5( xANR5), and activation of RhoA and Rho kinase[10]–[12] are involved in the implementation of a proximal yet still unknown cellular mechanism that actually generates the boundary. A candidate for this proximal mechanism is ephrin/Eph signaling. Eph receptor tyrosine kinases are subdivided into EphA and EphB subclasses and their membrane-linked ephrin ligands correspondingly into ephrinAs and ephrinBs. Within subclasses, binding appears promiscuous, although some Eph receptors have a higher affinity for specific ephrins. Receptor ligation and clustering initiates “forward signaling, ” but receptor-ligand interaction can also stimulate “reverse signaling” downstream of the ephrin ligand. Ephrin/Eph signaling has been implicated in boundary formation under conditions where receptor and ligand are expressed in complementary patterns[13]–[18]. For example, a model was proposed for rhombomere-boundary formation based on repulsive Eph-ephrin signaling, acting in parallel with an Eph-dependent regulation of adhesion within the rhombomeres[19],[20]. However, direct observation of repulsive behavior at the boundary, similar to what is classically seen during neuron guidance, has not been attempted. In the early Xenopus embryo, expression of several Eph receptors and ephrins has been reported( reviewed in[21]). Their in vivo functions during gastrulation have not yet been established, although gain-of-function experiments indicate that they can regulate cell adhesion, migration, and sorting[22]–[24]. Thus, ephrin signaling is a prime candidate to mediate tissue separation in the early Xenopus embryo.
Discussion:
Previous attempts to explain the separation of ectoderm and mesoderm[3],[4],[36] have been based on a thermodynamic model involving the minimization of tissue surface free energy. It assumes that two respective cell types are intrinsically different in terms of cell adhesiveness or cortical tension, and that this difference can drive cell sorting, boundary formation, and tissue positioning, analogous to the phase separation of immiscible fluids[2],[36]–[38]. Here we have shown evidence for a different model, in which signaling across the ectoderm-mesoderm boundary is crucial to locally regulate cell detachment and eventually tissue separation. Thus, although all cells of the two populations have the potential to form a cleft-like boundary if juxtaposed, acute separation behavior is not based on permanent adhesion differences between cells, but on transient contact-dependent reactions. The resulting, alternating phases of adhesion and repulsion appear to be part of a self-regulating loop( Figure 9). The spreading of an adhesion zone brings ephrins and Ephs in contact, inducing a repulsion signal. Once cells are apart, the signal decays, cells start to explore the intercellular space created at the boundary, and eventually re-establish adhesion. This mechanism prevents mesoderm cells from intruding into the BCR while providing necessary substratum contacts for migration. Cell detachment is restricted to the tissue interface, and although various ephrins and their cognate receptors are coexpressed within each tissue, they do not lead to overall mutual cell repulsion and hence tissue dissociation. One possible explanation is suggested by our reaggregation experiments which show that EphB and ephrinB are required for normal ectoderm cohesion. This result is consistent with the fact that Eph/ephrin interaction between cells can mediate not only repulsion but also adhesion. The switch between these opposite responses depends on ephrin/Eph density, with adhesion being generally favored at low levels[39],[40], reviewed by Poliakov et al.[41]. In addition to expression levels, the different affinities for each other of the various Eph and ephrin subtypes[16] are likely to influence the strength of the response. Coactivation of EphB and ephrinB within the same cell can also induce an adhesive instead of a repulsive response[42], a mechanism that may likewise vary for different subtypes. In fact, our observation that overexpression of the mesodermally enriched ephrinB2 in the ectoderm is sufficient to trigger separation, but that ephrinB1 is unable to do so, suggests a surprising degree of specificity. This leads us to propose that the subtypes and levels of ephrins and Eph receptors expressed in each of the two tissues may be the main determinant for tissue separation. Since the ectoderm and the mesoderm express similar yet distinctly different mixtures of receptors and ligands, it is conceivable that the sum of interactions within each tissue is adhesive, while interactions across the boundary result in repulsion. However, in contrast to the ectoderm, no clear effect of Eph/ephrin function on tissue cohesion was observed for the mesoderm in our assay; possibly, the tissue is close to the transition point between adhesion and repulsion. Parallel mechanisms could further attenuate EphB/ephrinB signals within the mesoderm or enhance repulsion at the boundary. A candidate would be Xfz7/PAPC signaling, which is sufficient to induce separation in the ectoderm[11], and functions upstream or in parallel to EphB. Importantly, regulation of tissue cohesion by EphB/ephrinB function seems to be independent of the control of tissue separation. In the ectoderm, ephrinB2 overexpression which triggers separation and ephrin/Eph depletion which inhibits it both decreased reaggregation, and in the mesoderm, inhibition of separation behavior is not associated with a change in overall cell-cell adhesion. This is consistent with other results indicating that changes in global adhesive strength do not necessarily affect tissue separation[43],[44]. The results indicate also that ectoderm-mesoderm separation is sensitive to the detailed expression pattern of Eph receptors and ligands, which is more complex than the classical complementary pattern, but may determine boundary formation in an essentially similar manner. Ephrin/Eph-mediated boundary formation often involves the complementary expression of receptors and ligands in adjacent tissues and bidirectional signaling( e. g.,[28]). This leads to rapid, large-scale changes in downstream pathways which differ in forward- and reverse-signaling cells[45]. Nevertheless, unidirectional signaling can be sufficient for cell segregation. Thus, in the zebrafish embryo, formation of the gap between adjacent somites depends on Eph forward signaling, while the ephrin reverse signal is dispensable[46]. Surprisingly, the same process using the same receptor and ligand isoforms requires ephrin reverse signaling, but not an Eph forward signal, in the chick embryo[47]. We found that similar to zebrafish somite segmentation, forward signaling is essential for ectoderm-mesoderm separation, and that two antiparallel forward signals are sufficient for complete repulsion at the boundary. The demonstrated requirement for both RhoA and Rac in tissue separation is consistent with their known functions in Eph/ephrin mediated repulsion. Thus, the termination of EphB-ephrinB interaction, required for repulsion, involves endocytosis of receptor and ligand[48], which in turn depends on Rac function downstream of EphB[49]. RhoA and Rho kinase activation downstream of an EphB receptor can be mediated by the adaptor protein, Dishevelled, and underlies experimentally induced cell sorting of Xenopus gastrula ectoderm cells[24]. The local activation of Rac and RhoA at the ectoderm-mesoderm interface agrees with the notion that Eph/ephrin signaling is activated strongly at the boundary only, despite the widespread co-expression of receptors and ligands. It is worth noting that despite the very low levels used in our experiments, each of the constitutively active RhoA and Rac construct could efficiently rescue separation. While this may be due in part to the potency of these forms, the cellular phenotypes produced by their overexpression appeared relatively mild. This was illustrated by the fact that even though the boundaries often appeared locked in a separated state upon rescue, attachment/detachment cycles were still observed occasionally. We hypothesize that in our inhibition experiments, ephrin/Eph signaling is decreased below the threshold required to maintain separation, but not completely abolished. Low amounts of active GTPases are then sufficient to boost the process and restore repulsion, while still allowing subsequent re-attachment. Alternatively, low levels of residual ephrin/Eph signaling may be sufficient to deliver exogenous active RhoA and Rac to their respective targets, though not to activate the endogenous GTPases sufficiently. The weak rescuing effects of wild type Rac and RhoA are consistent with both possibilities. In any case, the process of tissue separation appears particularly sensitive to small positive or negative changes in RhoGTPase activity, while larger alterations are required to affect other processes known to depend on such activities, in particular cell-cell adhesion. Cleft-like boundaries based on attachment/repulsion cycles could generally be a requirement for the migration of cells across the surface of an adjacent tissue. A cleft-like ectoderm-mesoderm boundary is also seen in the zebrafish gastrula[50], and migration of mesoderm cells across the ectodermal layer occurs in Drosophila gastrulation[51]. It would be interesting to see whether the ephrinB/EphB mechanism of tissue separation is conserved in these cases. In contrast to the sparse, cell-permeable network of fibronectin fibrils that covers the ectodermal BCR of Xenopus, mouse or chicken gastrulae possess a well-developed basal lamina which physically separates ectoderm from mesoderm, potentially rendering germ layer separation independent of a cell repulsion mechanism[52]. Indeed, mouse ephrinB1, ephrinB2, and EphB4 null mutants apparently gastrulate normally[53]–[56]. After gastrulation, numerous mass cell migration events take place, for example neural crest migration, which in principle could also employ the mechanism presented here. Finally, deposition of an extracellular matrix seems to require free tissue surface[57],[58], and initially separating rhombomeres or somites by a repulsion cleft could be the first step in the boundary maturation process which ends with a matrix-filled space between tissue blocks.
The formation and maintenance of tissue boundaries is an essential feature of multicellular animals , including humans . Using the frog embryo as a model system , we describe a mechanism of tissue separation that involves repeated cycles of cell attachment and detachment at the boundary between two adjacent tissues . Molecularly , this mechanism is based on a signal exchange across the boundary by a system of receptors and ligands—EphB receptors and ephrinB ligands—that are both integral cell membrane proteins , and thus require direct cell contact for signaling . In this way , cell attachment-dependent contact induces signaling which leads to a temporary detachment , followed by reattachment and a next round of signaling . Such an attachment-detachment mechanism allows for cell migration along the boundary , while at the same time preventing invasion of the stationary tissue by the migrating one .