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Eph Receptors: Two Ways to Sharpen Boundaries

2005; Elsevier BV; Volume: 15; Issue: 6 Linguagem: Inglês

10.1016/j.cub.2005.03.013

1879-0445

Dalit Sela‐Donenfeld, David G. Wilkinson,

Wnt/β-catenin signaling in development and cancer

Eph receptors and ephrins can sharpen domains within developing tissues by mediating repulsion at interfaces. An Eph receptor has now been shown also to regulate cell adhesion within tissue subdivisions. Eph receptors and ephrins can sharpen domains within developing tissues by mediating repulsion at interfaces. An Eph receptor has now been shown also to regulate cell adhesion within tissue subdivisions. An important problem in developmental biology is to understand how precise spatial patterns of cell types are maintained despite the potential for rearrangement by cell division and intercalation. This question applies to a key phase in the patterning of many tissues, in which they are subdivided into domains, each with a distinct regional identity that specifies a particular set of cell types. One way in which the precision of regional patterning can be maintained is by the inhibition of cell mixing between domains [1Lumsden A. Krumlauf R. Patterning the vertebrate neuraxis.Science. 1996; 274: 1109-1115Crossref PubMed Scopus (947) Google Scholar, 2Pasini A. Wilkinson D.G. Stabilizing the regionalisation of the developing vertebrate central nervous system.Bioessays. 2002; 24: 427-438Crossref Scopus (50) Google Scholar]. Two types of mechanism have been found to restrict cell intermingling. First, cells can be confined within a regional domain because they preferentially adhere to each other, for example by homophilic adhesion via cadherins. A classical demonstration of this is the sorting that occurs when cells with different affinities are mixed, driven by cells minimising contact with others that have different adhesive properties [3Steinberg M.S. Takeichi M. Experimental specification of cell sorting, tissue spreading, and specific spatial patterning by quantitative differences in cadherin expression.Proc. Natl. Acad. Sci. USA. 1994; 91: 206-209Crossref PubMed Scopus (490) Google Scholar]. A second mechanism involves the mutual inhibition of cell invasion via bidirectional activation of Eph receptors and ephrins at the interface of domains [4Kullander K. Klein R. Mechanisms and functions of Eph and ephrin signalling.Nat. Rev. Mol. Cell Biol. 2002; 3: 475-486Crossref PubMed Scopus (941) Google Scholar, 5Poliakov A. Cotrina M. Wilkinson D.G. Diverse roles of Eph receptors and ephrins in the regulation of cell migration and tissue assembly.Dev. Cell. 2004; 7: 465-480Abstract Full Text Full Text PDF PubMed Scopus (358) Google Scholar]. In this issue, Cooke et al. [6Cooke J.E. Kemp H.A. Moens C.B. EphA4 is required for cell adhesion and rhombomere boundary formation in the zebrafish.Curr. Biol. 2005; (this issue)Google Scholar] now report the important finding that an Eph receptor regulates cell affinities within tissue subdivisions, and so may sharpen boundaries by modulating cell adhesion both at interfaces and within regional domains. Interactions between Eph receptor tyrosine kinases and ephrins mediate cell-contact-dependent signalling in which both components can transduce signals that trigger cell responses [4Kullander K. Klein R. Mechanisms and functions of Eph and ephrin signalling.Nat. Rev. Mol. Cell Biol. 2002; 3: 475-486Crossref PubMed Scopus (941) Google Scholar]. Binding between members of the vertebrate Eph receptor and ephrin families falls largely into two classes: GPI-anchored ephrinAs bind to EphA receptors, and transmembrane ephrinBs bind to EphB receptors and to EphA4. Collectively, Eph and ephrin proteins are expressed in many, perhaps all, tissues during development, and have key roles in the repulsive guidance of migrating cells and axonal growth cones at boundaries, or along gradients, of complementary expression [4Kullander K. Klein R. Mechanisms and functions of Eph and ephrin signalling.Nat. Rev. Mol. Cell Biol. 2002; 3: 475-486Crossref PubMed Scopus (941) Google Scholar, 5Poliakov A. Cotrina M. Wilkinson D.G. Diverse roles of Eph receptors and ephrins in the regulation of cell migration and tissue assembly.Dev. Cell. 2004; 7: 465-480Abstract Full Text Full Text PDF PubMed Scopus (358) Google Scholar]. Similarly, Eph–ephrin signalling can restrict the intermingling of cells across interfaces within tissues, which studies in the hindbrain have suggested may involve bi-directional cell responses. The hindbrain is transiently subdivided during development into repeated segments called rhombomeres, each expressing a distinct set of transcription factors that underlie regional specification [1Lumsden A. Krumlauf R. Patterning the vertebrate neuraxis.Science. 1996; 274: 1109-1115Crossref PubMed Scopus (947) Google Scholar]. The precision of this segmental organisation involves the formation of sharp interfaces by restriction of cell intermingling between adjacent rhombomeres [7Fraser S. Keynes R. Lumsden A. Segmentation in the chick embryo hindbrain is defined by cell lineage restrictions.Nature. 1990; 344: 431-435Crossref Scopus (564) Google Scholar]. Several ephrinBs and interacting EphA4 and EphB receptors are segmentally expressed in the hindbrain, largely (but not entirely) in a complementary manner, such that Eph–ephrinB interactions and bi-directional signalling could occur at the interface of segments. The results of dominant-negative blocking [8Xu Q. Alldus G. Holder N. Wilkinson D.G. Expression of truncated Sek-1 receptor tyrosine kinase disrupts the segmental restriction of gene expression in the Xenopus and zebrafish hindbrain.Development. 1995; 121: 4005-4016Google Scholar], mosaic overexpression [9Xu Q. Mellitzer G. Robinson V. Wilkinson D.G. In vivo cell sorting in complementary segmental domains mediated by Eph receptors and ephrins.Nature. 1999; 399: 267-271Crossref PubMed Scopus (372) Google Scholar] and cell transplantation [10Cooke J. Moens C. Roth L. Durbin L. Shiomi K. Brennan C. Kimmel C. Wilson S. Holder N. Eph signalling functions downstream of Val to regulate cell sorting and boundary formation in the caudal hindbrain.Development. 2001; 128: 571-580Google Scholar] experiments suggest that Eph–ephrinB signalling at interfaces underlies the restriction of mixing across hindbrain boundaries. Furthermore, bi-directional Eph–ephrinB signalling at the interface of in vitro cell aggregates was found to bi-directionally restrict cell invasion [11Mellitzer G. Xu Q. Wilkinson D.G. Eph receptors and ephrins restrict cell intermingling and communication.Nature. 1999; 400: 77-81Crossref PubMed Scopus (412) Google Scholar]. These findings suggest that localised activation of Eph receptors and ephrinBs at the interface of complementary expression domains underlies a mutual repulsion that prevents intermingling. Cooke et al. [6Cooke J.E. Kemp H.A. Moens C.B. EphA4 is required for cell adhesion and rhombomere boundary formation in the zebrafish.Curr. Biol. 2005; (this issue)Google Scholar] further examined the role of Eph–ephrin interactions in the hindbrain in loss-of-function 'knockdown' experiments by injecting antisense morpholino oligonucleotides into zebrafish embryos. As anticipated, knockdown of EphA4 — which is expressed in rhombomeres r3 and r5 — resulted in fuzzy interfaces between r3/r5 and adjacent segments. However, mosaic knockdown experiments in which EphA4-knockdown cells were transplanted into an uninjected embryo, or vice versa, led to an unexpected result: that cell sorting occurred within r3 and r5, with EphA4-knockdown cells segregating to the edges and uninjected cells to the centre of these segments (Figure 1A,B ). Detection of r3/r5-specific gene expression revealed that the segregated EphA4-knockdown cells remained within r3/r5, rather than mixing into adjacent territory, which can most easily be explained by continued expression of other Eph receptors that mediate repulsion by ephrin at segment interfaces. Indeed, there is a stronger disruption of the interfaces between r3/r5 and r4 when both EphA4 and ephrinB2a — expressed in r1/r4/r7 — are knocked down. The sorting of EphA4-knockdown cells shows that EphA4 regulates cell–cell affinity within r3/r5, which the results of time-lapse experiments suggest contributes to the normal restriction of cell mixing between segments. Taken together with previous findings, Eph–ephrin interactions thus contribute to the sharpening of segments by regulating both repulsion at interfaces and cell affinity within rhombomeres (Figure 1C). The results of the mosaic EphA4 knockdown experiments [6Cooke J.E. Kemp H.A. Moens C.B. EphA4 is required for cell adhesion and rhombomere boundary formation in the zebrafish.Curr. Biol. 2005; (this issue)Google Scholar] imply that EphA4 regulates cell affinity, and thus is being activated, throughout r3/r5. Indeed, although most functional studies have focussed on complementary Eph–ephrin expression, overlaps in expression occur in many tissues, including the hindbrain. Furthermore, there has been growing evidence that Eph receptor activation can trigger alternative adhesion or repulsion responses that might be regulated by overlapping expression with ephrins [5Poliakov A. Cotrina M. Wilkinson D.G. Diverse roles of Eph receptors and ephrins in the regulation of cell migration and tissue assembly.Dev. Cell. 2004; 7: 465-480Abstract Full Text Full Text PDF PubMed Scopus (358) Google Scholar]. An intriguing feature of the Eph–ephrin system is that the nature of the cell response may depend upon the degree of clustering and activation [5Poliakov A. Cotrina M. Wilkinson D.G. Diverse roles of Eph receptors and ephrins in the regulation of cell migration and tissue assembly.Dev. Cell. 2004; 7: 465-480Abstract Full Text Full Text PDF PubMed Scopus (358) Google Scholar]. Low level Eph receptor clustering or activation promotes cytoskeletal assembly, invasion and adhesion, whereas increased clustering or activation triggers cytoskeletal disassembly and repulsion (for example [12Hansen M.J. Dallal G.E. Flanagan J.G. Retinal axon response to ephrin-as shows a graded, concentration-dependent transition from growth promotion to inhibition.Neuron. 2004; 42: 717-730Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar]). Overlapping expression of Eph receptors and ephrins suppresses cell repulsion responses [13Hornberger M.R. Dutting D. Ciossek T. Yamada T. Handwerker C. Lang S. Weth F. Huf J. Wessel R. Logan C. et al.Modulation of EphA receptor function by coexpressed ephrinA ligands on retinal ganglion cell axons.Neuron. 1999; 22: 731-742Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar] and promotes adhesion [14Dravis C. Yokoyama N. Chumley M.J. Cowan C.A. Silvany R.E. Shay J. Baker L.A. Henkemeyer M. Bidirectional signaling mediated by ephrin-B2 and EphB2 controls urorectal development.Dev. Biol. 2004; 271: 272-290Crossref PubMed Scopus (193) Google Scholar]. A biochemical basis for this suppression is suggested by the finding that co-expressed ephrin interacts in cis with Eph receptor to form inactive complexes, and inhibits trans Eph–ephrin interactions between cells [15Yin Y. Yamashita Y. Noda H. Okafuji T. Go M.J. Tanaka H. EphA receptor tyrosine kinases interact with co-expressed ephrin-A ligands in cis.Neurosci. Res. 2004; 48: 285-296Crossref Scopus (83) Google Scholar]. Consequently, overlapping Eph–ephrin expression may cause low-level activation that promotes an adhesive response. These findings suggest a model in which EphA4 has overlapping expression with some ephrins within r3/r5 that underlies low level activation and cell adhesion, and complementary expression with other ephrins that promote high-level activation and repulsion across boundaries. It will be interesting to uncover whether ephrins regulate cell affinity within even-numbered segments, or if their only role is to participate in bi-directional repulsion The findings of Cooke et al. [6Cooke J.E. Kemp H.A. Moens C.B. EphA4 is required for cell adhesion and rhombomere boundary formation in the zebrafish.Curr. Biol. 2005; (this issue)Google Scholar] also raise the question of whether Eph receptors and ephrins have additional roles at segment interfaces. An important mechanism for patterning of some tissues is the formation of a signalling centre at the interface of regional domains. Distinct boundary cells form at the interface of hindbrain segments [16Lumsden A. Keynes R. Segmental patterns of neuronal development in the chick hindbrain.Nature. 1989; 337: 424-428Crossref PubMed Scopus (706) Google Scholar, 17Trevarrow B. Marks D.L. Kimmel C.B. Organisation of hindbrain segments in the zebrafish embryo.Neuron. 1990; 4: 669-679Abstract Full Text PDF Scopus (447) Google Scholar], and recent work suggests that expression of Wnt signals and Notch activation at hindbrain boundaries in zebrafish regulates neurogenesis and the localisation of boundary cells [18Cheng Y.C. Amoyel M. Qiu X. Jiang Y.J. Xu Q. Wilkinson D.G. Notch activation regulates the segregation and differentiation of rhombomere boundary cells in the zebrafish hindbrain.Dev. Cell. 2004; 6: 539-550Abstract Full Text Full Text PDF Scopus (102) Google Scholar, 19Amoyel M. Cheng Y.C. Jiang Y.J. Wilkinson D.G. Wnt1 regulates neurogenesis and mediates lateral inhibition of boundary cell specification in the zebrafish hindbrain.Development. 2005; 132: 775-785Crossref PubMed Scopus (90) Google Scholar]. As blocking or knockdown of EphA4 leads to a depletion of boundary cells [6Cooke J.E. Kemp H.A. Moens C.B. EphA4 is required for cell adhesion and rhombomere boundary formation in the zebrafish.Curr. Biol. 2005; (this issue)Google Scholar, 8Xu Q. Alldus G. Holder N. Wilkinson D.G. Expression of truncated Sek-1 receptor tyrosine kinase disrupts the segmental restriction of gene expression in the Xenopus and zebrafish hindbrain.Development. 1995; 121: 4005-4016Google Scholar], Eph–ephrin signalling could have a direct role in boundary cell specification. This is an attractive possibility since boundary cells are induced by interactions between adjacent segments [20Guthrie S. Lumsden A. Formation and regeneration of rhombomere boundaries in the developing chick hindbrain.Development. 1991; 112: 221-229Google Scholar], at the site of bi-directional Eph–ephrinB activation. On the other hand, there may be an indirect relationship in which boundary cell formation requires a stable interface between segments that is disrupted by blocking of Eph–ephrin signalling. In view of evidence that local interactions switch the segmental identity of any isolated cells that move into an adjacent segment (reviewed in [2Pasini A. Wilkinson D.G. Stabilizing the regionalisation of the developing vertebrate central nervous system.Bioessays. 2002; 24: 427-438Crossref Scopus (50) Google Scholar]), there may be more intermingling occurring than suggested by the fuzzy interfaces of r3/r5 gene expression following loss of EphA4 function [6Cooke J.E. Kemp H.A. Moens C.B. EphA4 is required for cell adhesion and rhombomere boundary formation in the zebrafish.Curr. Biol. 2005; (this issue)Google Scholar, 8Xu Q. Alldus G. Holder N. Wilkinson D.G. Expression of truncated Sek-1 receptor tyrosine kinase disrupts the segmental restriction of gene expression in the Xenopus and zebrafish hindbrain.Development. 1995; 121: 4005-4016Google Scholar]. Further studies will thus be required to address the relative contribution of the regulation of cell movement and identity to the formation of sharp interfaces, and the potential role of Eph-ephrin signalling in boundary cell specification.