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Eye development: Notch lends a handedness
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Eye development: Notch lends a handedness Seth S. Blair
The compound eyes of arthropods are subdivided into a large number of repeating units, termed ommatidia. Each ommatidium is formed by a stereotypical latticework of pigment cells, lens-forming cone cells, bristle cells and a central cluster of eight different photoreceptors (R1–R8). Each type of photoreceptor occupies a stereotyped position within the cluster. In the mature ommatidia of Drosophila, R7 and R8 are found centrally, while R1–R6 are arranged around them in a roughly trapezoidal fashion, with R3 defining the peak of the trapezoid (Figure 1; reviewed in [6]). Neighboring ommatidia have identical polarity along the anteroposterior axis; their chirality, however, is reversed at the boundary between the dorsal and ventral domains, the eye’s equator (Figure 1). Thus, within each ommatidium the R3 photoreceptor lies further anterior than R4, and is the furthest from the dorsoventral equator, pointing towards either the dorsal or ventral poles of the eye.
The arrangement of photoreceptors in the ommatidia of the Drosophila compound eye is polarized, having a handedness or chirality. Notch signalling helps determine this handedness, first by establishing a signalling center at the eye equator, and second by mediating a choice between two photoreceptor fates. Address: Department of Zoology, University of Wisconsin, 250 N Mills Street, Madison, Wisconsin 53706, USA. Current Biology 1999, 9:R356–R360 http://biomednet.com/elecref/09609822009R0356 © Elsevier Science Ltd ISSN 0960-9822
Many developing epithelia become not simply subdivided, but polarized in the plane of the epithelium. This planar polarity can be expressed within single cells or, in those tissues containing repeating, multicellular units, in the arrangement of cell types within each unit. One of the most dramatic examples of multicellular polarity is found in the arrangement of photoreceptors in the compound eyes of arthropods. This polarity exists in not just one but two axes, so that the tissue has a handedness or chirality. Drosophila genetics has provided powerful tools for investigating the signalling mechanisms that establish this polarity. Two sets of recent studies have demonstrated previously unsuspected roles for the transmembrane receptor Notch in the establishment of Drosophila eye polarity. Notch signalling has been found to act in two quite different ways to help establish eye polarity, the first by specifying a signalling center at the equator of the eye [1–3], and the second by mediating a choice between two critical cell types, the photoreceptors R3 and R4 [4,5].
The photoreceptors are formed during late larval and early pupal stages from the eye portion of the eye-antennal imaginal disc (Figure 1; reviewed in [6]). During these stages, a morphological furrow sweeps across the eye epithelium from posterior to anterior. Cells just posterior to the furrow form small clusters of photoreceptor precursors. Initially the posterior-most cell of the cluster is the R8 precursor, followed by the R2/R5 pair and the R3/R4 pair. R7 and the R1/R6 pair then join the cluster. The more polar cells of each pair become R4–R6, and the more equatorial R1–R3. Finally, the clusters rotate 90°, in opposite directions in dorsal and ventral halves of the eye, and the photoreceptors take up their mature positions.
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Morphogenetic furrow
The development of photoreceptor polarity in the eye-antennal imaginal disc of Drosophila. (See text for details.)
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Figure 2 A model of equator specification in the developing eye. (a) A number of signals — Unpaired (Upd) from the posterior equator, Wingless (Wg) from the poles and unknown factors from the ventral pole — combine to determine the extent of dorsal Iroquois complex expression [9,10]. (b) Iroquois complex expression controls the dorsal production of Delta and the ventral production of Serrate and Fringe. Fringe blocks Serrate reception ventrally, but potentiates Delta reception; thus ventral Serrate signals to adjacent dorsal cells, and dorsal Delta signals to adjacent ventral cells. (c) This signalling induces the production of a dorsoventral ‘Factor X’ from the equator.
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Polarity mutations and axis specification
A number of mutations have been isolated that disrupt the polarity of photoreceptors within each ommatidium, randomizing the polarity in one or both axes. Simultaneous changes in orientation along both axes do not alter chirality; such defects could be accounted for by changes in initial cell specification within the photoreceptor cluster, or simply by changes in the subsequent rotation of the cluster. However, changes in one axis alone do alter the chirality of the cluster, and must result from changes in the specification of cell fates within each cluster. That changes in chirality occur suggests that photoreceptor specification is controlled separately along each axis of polarity. Indeed, the anterior–posterior component of cell specification depends in some manner on the movement or placement of the morphogenetic furrow (reviewed in [7]). The anterior progression of the furrow requires the expression of the secreted morphogen Hedgehog within the furrow itself, and creating ectopic sites of Hedgehog signalling just anterior to the furrow can initiate an ectopic furrow moving posteriorly across the eye. This backwards moving furrow can reverse the anterior–posterior polarity of the resultant ommatidia without affecting the dorsoventral component. Similarly, the equator of the eye helps establish the dorsoventral component of polarity in the eye. A number of genes have been found that are expressed specifically along the equator, or in a broad region centered on the equator. Manipulations that induce ectopic equator-like gene expression reverse the chirality of ommatidia on the equatorial side of the new equator, as might be expected if clusters were orienting with reference to a signal from the ectopic equator [1–3,7,8]. Specifying the equator
How is the equator region of the eye specified? Equatorspecific gene expression can be observed in the early eye
disc prior to the formation of the morphogenetic furrow: three recent studies [1–3] have shown that Notch signalling between dorsal and ventral cells of the eye is critical for this specification. First, however, the eye must be divided into dorsal and ventral regions. This is likely established by the dorsal-specific expression of three members of the Iroquois complex — mirror, araucan and caupolican — in the early disc, each of which encodes a related transcription factor [1–3,8]. The boundary of Iroquois complex expression lies at the equator, and creating ectopic mirror boundaries can induce ectopic equators [8]. It is not entirely clear how the dorsal domain of Iroquois complex gene expression is established (Figure 2a). The secreted morphogen encoded by wingless is expressed at the poles of eye and appears to play a role in this process, as reducing wingless expression levels reduces the dorsal expression of mirror [9]. The JAK/STAT kinase signalling pathway also plays a role; unpaired, which encodes a ligand that activates the JAK/STAT pathway in target cells, is expressed at the posterior end of the equator, and reducing its expression levels expands mirror expression into ventral regions [10]. It is unclear, however, how Wingless signalling from both poles and Unpaired signalling from the equator can combine to generate a dorsal pattern of gene expression; this suggests that other, as yet unknown, factors are also likely to be involved. The dorsal expression of the Iroquois complex is followed by ventral expression of the Notch ligand Serrate, dorsal expression of Notch ligand Delta, and ventral expression of the secreted molecule Fringe [1–3] (Figure 2b). Ectopic caupolican expression can repress fringe expression [3], and thus the Iroquois complex may be acting as a dorsal ‘selector’ to establish these dorsoventral differences. This is very similar to what occurs in the developing wing imaginal disc, except that in the wing the Apterous transcription
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choice between alternate developmental fates ([4,5] and A. Tomlinson and G. Struhl, personal communication). In essence, the two members of the R3/R4 pair define an equivalence group, each precursor having an equivalent developmental potential and each competing for the ‘primary’ R3 fate. The cell with the highest levels of Notch activity is inhibited from adopting this fate, and so takes on the ‘secondary’ R4 fate.
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Changes in photoreceptor specification induced by the loss or gain of Notch activity. (See text for details.)
factor acts as a dorsal selector, and the dorsal and ventral domains of Serrate, Delta and Fringe expression are reversed (reviewed in [11]). Serrate, Delta and Fringe then establish the eye equator in a process that is almost identical to the formation of dorsoventral boundary cells in the developing wing ([1–3], reviewed in [11]). Notch activity along the equator of the eye rises, as evidenced by the expression of Notch-driven reporter genes, and is necessary and sufficient for the expression of these and other equatorial markers. It is the boundary of fringe expression that makes this possible. Ventrally expressed fringe prevents the reception of Serrate-based signalling in ventral cells, but increases the sensitivity of these cells to Delta-based signals. Thus, ventral Serrate signals to adjacent dorsal cells, and dorsal Delta signals to adjacent ventral cells (Figure 2b,c). As in the wing, heightened Notch activity increases ligand expression via a positive feedback loop, likely reinforcing signalling at the boundary.
Two different results hinted at the involvement of Notch: Notch mutations were isolated as dominant enhancers of the frizzled mutant eye polarity phenotype [4]; and the expression of a promoter–lacZ construct that is known to be sensitive to Notch signalling was observed very early in development in the polar member of the R3/R4 pair [5]. Subsequent experiments yielded the predicted results: reductions in Notch activity result in the duplication of R3 within each cluster, while gains in Notch activity result in the duplication of R4 (Figure 3). How is this competitive interaction normally mediated? The R3/R4 decision is sensitive to levels of the Notch ligand Delta, and indeed higher levels of Delta expression are observed in the initially equatorial member of the pair [4,5]. Thus, it seems likely that a bias develops between polar and equatorial cells; the equatorial cell produces more Delta signal and may also become less sensitive to Notch activity and thus becomes an R3, while the polar cell produces less Delta, receives more Notch signal and thus becomes R4. A negative feedback loop between Notch activity and Delta expression amplifies any initial bias [5]. This mechanism is similar to that thought to operate during another Notch-mediated process — the selection of neuronal precursors from proneural equivalence groups.
Photoreceptor specification along the dorsoventral axis
Factor(s) X and the bias in Notch signalling
The equator of the eye has long-range effects on eye polarity, but the nature of the equator signal is unknown. However, the polarity defects that result from mutations in frizzled and dishevelled indicate that Wingless/Wnt-like signalling is critical in this process [7,12]; frizzled encodes a transmembrane protein capable of transducing Wingless signal, and dishevelled encodes a downstream component of the Wingless pathway. The analysis of loss-of-function and gain-of-function mosaics indicated that Frizzled is required in the R3/R4 pair of photoreceptor precursors ([7,12] and A. Tomlinson and G. Struhl, personal communication). But while frizzled mutant ommatidia have randomized chirality, they almost always contain both an R3 and an R4 cell [12]. R3 is often formed by the wrong, initially polar member of the R3/R4 pair, but only rarely does this result in the formation of two R3’s.
As discussed above, it is possible that the equator cells produce a single ‘Factor X’ and that this is a Wnt-like signal. It would certainly make sense if the Notch activity is lowered in the equatorial cells by higher levels of a Wingless/Wnt-like signal, as Wingless signalling often inhibits Notch activity and provides a spatial bias to Notch-dependent equivalence groups. Moreover, polarity need not depend on an absolute level of Factor X signalling, as any difference in Factor X reception between the polar and equatorial cell in each cluster would be amplified by Notch. Several different mechanisms have been proposed for the inhibition of Notch by Wingless, including a direct physical interaction between Notch and Dishevelled proteins [13] or, more provocatively, the direct binding of Wingless to Notch itself [14].
The fate decisions in the R3/R4 pair are thus linked. Recent studies have shown that Notch signalling provides this link; as in many other situations Notch mediates a
Factor X signalling differs, however, from classic Wingless signalling. While Frizzled and Dishevelled are common to both pathways, the interpretation of the equatorial signal does not depend on further downstream
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Figure 4 The development of eye polarity might involve graded production of a hypothetical ‘Factor X’ across the dorsovental axis. It is assumed that Factor X is an inhibitor of Notch signalling, with higher expression at the equator. (a) Equatorial expression of the JAK/STAT ligand and polar expression of Wingless (Wg) promote and inhibit, respectively, the expression of the Factor X gene (blue) and protein (red). (b) A local reduction in Factor X gene expression (green) — induced by lowered JAK/STAT or heightened Wg reception — creates a local sink for the diffusible Factor X protein, reversing the slope of the Factor X gradient on the polar side of the mutant clone, in both wild-type (red) and mutant (purple) cells. (c) A local heightening of Factor X gene expression — induced by heightened JAK/STAT or lowered Wg reception — creates a local source of the Factor X protein,
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reversing the slope of the Factor X gradient on the equatorial side of the mutant clone, in both
members of the Wingless pathway, such as Armadillo [7]. In fact, recent results have shown that polarity requires different parts of the Dishevelled protein than those required for Wingless signalling, and that the polarity signal may be mediated by JNK or other mitogen-activated protein (MAP) kinase pathways [15,16]. Interestingly, overexpression of the known Drosophila Wnts does not induce polarity defects in the eye [7]. It may also be wrong to think of a single Factor X produced exclusively by equatorial cells. Rather, Factor X signal may be produced in a graded fashion across the dorsoventral axis of the eye, in response to other equatorial and polar signals. In genetic mosaics, cells unable to receive either Wingless [7] or JAK/STAT [10] signals can induce chirality defects in neighboring wild-type cells. Because both signalling pathways can influence the dorsal expression of the Iroquois complex (see above), one possible explanation for these ‘non-autonomous’ effects is that mutant cells induce local changes in Iroquois complex expression; this, in turn, would juxtapose Iroquois complex expressing and nonexpressing cells, creating an ectopic equator. However, changes in JAK/STAT reception do not change expression of the Iroquois complex member mirror [10]. It has therefore been suggested that an equatorial JAK/STAT signal combines with a polar Wingless signal to create a gradient of Factor X production [7,10] (Figure 4). Assuming that Factor X is a negative regulator of Notch signalling, it would have high expression at the equator and lowered expression at the poles. Local gains or losses in Factor X production would affect the chirality of neighboring wild-type cells by creating local sources or sinks of the short-range signal, reversing the direction of the Factor X gradient within and outside the clone. As chirality depends on the direction of the Factor X gradient,
wild-type and mutant cells. (Red arrows indicate polarity of ommatidia.)
rather than absolute levels, gains in Factor X expression would affect chirality on the equatorial side and losses on the polar side of the mutant cells (Figure 4b,c). In this, losses of Wingless reception resemble gains in Factor X expression, and losses of JAK/STAT reception resemble losses of Factor X expression [7,10]. Even this model, however, is likely to be too simplistic; it assumes that the difference in Factor X reception between R3 and R4 acts purely by regulating a Notchbased cell interaction between the cells of a single ommatidial cluster. However, ommatidia lacking frizzled, which encodes the presumed Factor X receptor, also affect the chirality of ommatidia polar to the mutant cells [12]. It seems unlikely that this non-autonomous effect is due to Notch signalling, as it would have to act over several cell diameters. Moreover, frizzled mutations cause similar nonautonomous effects in the developing wing, where frizzled is required for the orientation of epithelial hairs (reviewed in [17]); Notch has no known role in wing-hair polarity. One possible explanation for the non-autonomy of frizzled mutations is that Frizzled signalling affects the expression of long-range polarity signals, such as Wnts or JAK/STAT ligands. Alternatively, it has been suggested that Frizzled is involved not only in the reception of a polarizing signal but also in the polarised transmission of that signal to adjacent cells [17]. The latter is a particularly intriguing idea, as it would provide yet another mechanism for the coordination of planar polarity within an epithelium. Acknowledgements I thank Bill Feeny for help with the figures. This work was supported by grants from the NSF (IBN-9723564) and NIH (R01-NS28202).
References 1. Papayannopoulos V, Tomlinson A, Panin VM, Rauskolb C, Irvine KD: Dorsal-ventral signaling in the Drosophila eye. Science 1998,
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281:2031-2034. 2. Choi K-O, Choi KW: Fringe is essential for mirror symmetry and morphogenesis in the Drosophila eye. Nature 1998, 396:272-276. 3. Dominguez M, de Celis JF: A dorsal/ventral boundary established by Notch controls growth and polarity in the Drosophila eye. Nature 1998, 386:276-278. 4. Fanto M, Mlodzik M: Asymmetric Notch activation specifies photoreceptors R3 and R4 and planar polarity in the Drosophila eye. Nature 1999, 397:523-526. 5. Cooper MTD, Bray SJ: Frizzled regulation of Notch signalling polarizes cell fate in the Drosophila eye. Nature 1999, 397:526-530. 6. Wolff T, Ready, DF: Pattern formation in the Drosophila retina. In The Development of Drosophila melanogaster. Edited by Bate M, Martinez Arias A. Cold Spring Harbor: Cold Spring Harbor Laboratory Press; 1993:1277-1325. 7. Wehrli M, Tomlinson A: Independent regulation of anterior/posterior and equatorial/polar polarity in the Drosophila eye; evidence for the involvement of Wnt signaling in the equatorial/polar axis. Development 1998, 125:1421-1432. 8. McNeill H, Yang CH, Brodsky M, Ungos J, Simon MA: Mirror encodes a novel PBX-class homeoprotein that functions in the definition of the dorsal-ventral border in the Drosophila eye. Genes Dev 1997 11:1073-1082 9. Heberlein U, Borod ER, Chanut FA: Dorsoventral patterning in the Drosophila retina by wingless. Development 1998, 125:567-577. 10. Zeidler MP, Perrimon N, Strutt DI: Polarity determination in the Drosophila eye: a novel role for Unpaired and JAK/STAT signalling. Genes Dev 1999, in press. 11. Blair SS: Limb development: Marginal Fringe benefits. Curr Biol 1997, 7:R686-R690. 12. Zheng L, Zhang J, Carthew RW: Frizzled regulates mirrorsymmetric pattern formation in the Drosophila eye. Development 1995, 121:3045-3055. 13. Axelrod JD, Matsuno K, Artavanis-Tsakonas S, Perrimon N: Interaction between Wingless and Notch signaling pathways mediated by Dishevelled. Science 1996, 271:1826-1832. 14. Couso JP, Martinez Arias A: Notch is required for wingless signaling in the epidermis of Drosophila. Cell 1994 79:259-272 15. Axelrod JD, Miller JR, Shulman JM, Moon RT, Perrimon N: Differential recruitment of Dishevelled provides signaling specificity in the planar cell polarity and Wingless signaling pathways. Genes Dev 1998, 12:2610-2622. 16. Boutros M, Paricio N, Strutt DI, Mlodzik M: Dishevelled activates JNK and discriminates between JNK pathways in planar polarity and wingless signaling. Cell 1998, 94:109-119. 17. Adler PN, Krasnow RE, Liu J: Tissue polarity points from cells that have higher frizzled levels towards cells that have lower frizzled levels. Curr Biol 1997, 7:940-949.
If you found this dispatch interesting, you might also want to read the April 1999 issue of
Current Opinion in Cell Biology which included the following reviews, edited by Joan S Brugge and Frank McCormick, on Cell regulation: Regulation of tyrosine kinase cascades by G-protein coupled receptors Louis M Lutrell, Yehia Daaka and Robert J Lefkowitz Epidermal growth factor receptors: critical mediators of multiple receptor pathways Peter O Hackel, Esther Zwick, Norbert Prenzel and Axel Ullrich Multiple positive and negative regulators of signaling by the EGF receptor Nadeem Moghal and Paul W Sternberg Interactions between mitogenic stimuli, or, a thousand and one connections Martin A Schwartz and Veronique Baron Costimulatory regulation of T cell function Cynthia A Chambers and James P Allison Organization and regulation of mitogen-activated protein kinase signaling pathways Timothy P Garrington and Gary L Johnson Signalling through phosphatidylinositol 3-kinases: the lipids take centre stage Sally J Leevers, Bart Vanhaesebroeck and Michael D Waterfield κB Multiple signals converging on NF-κ Frank Mercurio and Anthony M Manning Regulation of LEF-1/TCF transcription factors by Wnt and other signals Quinn Eastman and Rudolf Grosschedl In or out? Regulating nuclear transport Jennifer K Hood and Pamela A Silver Organization and regulation of proteins at synapses Jee Hae Kim and Richard L Huganir Apoptosis control by death and decoy receptors Avi Ashkenazi and Vishva M Dixit Deciphering the pathways of life and death Honglin Li and Junying Yuan The full text of Current Opinion in Cell Biology is in the BioMedNet library at http://BioMedNet.com/cbiology/cel
Dispatch
Eye development: Notch lends a handedness Seth S. Blair
The compound eyes of arthropods are subdivided into a large number of repeating units, termed ommatidia. Each ommatidium is formed by a stereotypical latticework of pigment cells, lens-forming cone cells, bristle cells and a central cluster of eight different photoreceptors (R1–R8). Each type of photoreceptor occupies a stereotyped position within the cluster. In the mature ommatidia of Drosophila, R7 and R8 are found centrally, while R1–R6 are arranged around them in a roughly trapezoidal fashion, with R3 defining the peak of the trapezoid (Figure 1; reviewed in [6]). Neighboring ommatidia have identical polarity along the anteroposterior axis; their chirality, however, is reversed at the boundary between the dorsal and ventral domains, the eye’s equator (Figure 1). Thus, within each ommatidium the R3 photoreceptor lies further anterior than R4, and is the furthest from the dorsoventral equator, pointing towards either the dorsal or ventral poles of the eye.
The arrangement of photoreceptors in the ommatidia of the Drosophila compound eye is polarized, having a handedness or chirality. Notch signalling helps determine this handedness, first by establishing a signalling center at the eye equator, and second by mediating a choice between two photoreceptor fates. Address: Department of Zoology, University of Wisconsin, 250 N Mills Street, Madison, Wisconsin 53706, USA. Current Biology 1999, 9:R356–R360 http://biomednet.com/elecref/09609822009R0356 © Elsevier Science Ltd ISSN 0960-9822
Many developing epithelia become not simply subdivided, but polarized in the plane of the epithelium. This planar polarity can be expressed within single cells or, in those tissues containing repeating, multicellular units, in the arrangement of cell types within each unit. One of the most dramatic examples of multicellular polarity is found in the arrangement of photoreceptors in the compound eyes of arthropods. This polarity exists in not just one but two axes, so that the tissue has a handedness or chirality. Drosophila genetics has provided powerful tools for investigating the signalling mechanisms that establish this polarity. Two sets of recent studies have demonstrated previously unsuspected roles for the transmembrane receptor Notch in the establishment of Drosophila eye polarity. Notch signalling has been found to act in two quite different ways to help establish eye polarity, the first by specifying a signalling center at the equator of the eye [1–3], and the second by mediating a choice between two critical cell types, the photoreceptors R3 and R4 [4,5].
The photoreceptors are formed during late larval and early pupal stages from the eye portion of the eye-antennal imaginal disc (Figure 1; reviewed in [6]). During these stages, a morphological furrow sweeps across the eye epithelium from posterior to anterior. Cells just posterior to the furrow form small clusters of photoreceptor precursors. Initially the posterior-most cell of the cluster is the R8 precursor, followed by the R2/R5 pair and the R3/R4 pair. R7 and the R1/R6 pair then join the cluster. The more polar cells of each pair become R4–R6, and the more equatorial R1–R3. Finally, the clusters rotate 90°, in opposite directions in dorsal and ventral halves of the eye, and the photoreceptors take up their mature positions.
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Morphogenetic furrow
The development of photoreceptor polarity in the eye-antennal imaginal disc of Drosophila. (See text for details.)
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Figure 2 A model of equator specification in the developing eye. (a) A number of signals — Unpaired (Upd) from the posterior equator, Wingless (Wg) from the poles and unknown factors from the ventral pole — combine to determine the extent of dorsal Iroquois complex expression [9,10]. (b) Iroquois complex expression controls the dorsal production of Delta and the ventral production of Serrate and Fringe. Fringe blocks Serrate reception ventrally, but potentiates Delta reception; thus ventral Serrate signals to adjacent dorsal cells, and dorsal Delta signals to adjacent ventral cells. (c) This signalling induces the production of a dorsoventral ‘Factor X’ from the equator.
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Polarity mutations and axis specification
A number of mutations have been isolated that disrupt the polarity of photoreceptors within each ommatidium, randomizing the polarity in one or both axes. Simultaneous changes in orientation along both axes do not alter chirality; such defects could be accounted for by changes in initial cell specification within the photoreceptor cluster, or simply by changes in the subsequent rotation of the cluster. However, changes in one axis alone do alter the chirality of the cluster, and must result from changes in the specification of cell fates within each cluster. That changes in chirality occur suggests that photoreceptor specification is controlled separately along each axis of polarity. Indeed, the anterior–posterior component of cell specification depends in some manner on the movement or placement of the morphogenetic furrow (reviewed in [7]). The anterior progression of the furrow requires the expression of the secreted morphogen Hedgehog within the furrow itself, and creating ectopic sites of Hedgehog signalling just anterior to the furrow can initiate an ectopic furrow moving posteriorly across the eye. This backwards moving furrow can reverse the anterior–posterior polarity of the resultant ommatidia without affecting the dorsoventral component. Similarly, the equator of the eye helps establish the dorsoventral component of polarity in the eye. A number of genes have been found that are expressed specifically along the equator, or in a broad region centered on the equator. Manipulations that induce ectopic equator-like gene expression reverse the chirality of ommatidia on the equatorial side of the new equator, as might be expected if clusters were orienting with reference to a signal from the ectopic equator [1–3,7,8]. Specifying the equator
How is the equator region of the eye specified? Equatorspecific gene expression can be observed in the early eye
disc prior to the formation of the morphogenetic furrow: three recent studies [1–3] have shown that Notch signalling between dorsal and ventral cells of the eye is critical for this specification. First, however, the eye must be divided into dorsal and ventral regions. This is likely established by the dorsal-specific expression of three members of the Iroquois complex — mirror, araucan and caupolican — in the early disc, each of which encodes a related transcription factor [1–3,8]. The boundary of Iroquois complex expression lies at the equator, and creating ectopic mirror boundaries can induce ectopic equators [8]. It is not entirely clear how the dorsal domain of Iroquois complex gene expression is established (Figure 2a). The secreted morphogen encoded by wingless is expressed at the poles of eye and appears to play a role in this process, as reducing wingless expression levels reduces the dorsal expression of mirror [9]. The JAK/STAT kinase signalling pathway also plays a role; unpaired, which encodes a ligand that activates the JAK/STAT pathway in target cells, is expressed at the posterior end of the equator, and reducing its expression levels expands mirror expression into ventral regions [10]. It is unclear, however, how Wingless signalling from both poles and Unpaired signalling from the equator can combine to generate a dorsal pattern of gene expression; this suggests that other, as yet unknown, factors are also likely to be involved. The dorsal expression of the Iroquois complex is followed by ventral expression of the Notch ligand Serrate, dorsal expression of Notch ligand Delta, and ventral expression of the secreted molecule Fringe [1–3] (Figure 2b). Ectopic caupolican expression can repress fringe expression [3], and thus the Iroquois complex may be acting as a dorsal ‘selector’ to establish these dorsoventral differences. This is very similar to what occurs in the developing wing imaginal disc, except that in the wing the Apterous transcription
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choice between alternate developmental fates ([4,5] and A. Tomlinson and G. Struhl, personal communication). In essence, the two members of the R3/R4 pair define an equivalence group, each precursor having an equivalent developmental potential and each competing for the ‘primary’ R3 fate. The cell with the highest levels of Notch activity is inhibited from adopting this fate, and so takes on the ‘secondary’ R4 fate.
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Changes in photoreceptor specification induced by the loss or gain of Notch activity. (See text for details.)
factor acts as a dorsal selector, and the dorsal and ventral domains of Serrate, Delta and Fringe expression are reversed (reviewed in [11]). Serrate, Delta and Fringe then establish the eye equator in a process that is almost identical to the formation of dorsoventral boundary cells in the developing wing ([1–3], reviewed in [11]). Notch activity along the equator of the eye rises, as evidenced by the expression of Notch-driven reporter genes, and is necessary and sufficient for the expression of these and other equatorial markers. It is the boundary of fringe expression that makes this possible. Ventrally expressed fringe prevents the reception of Serrate-based signalling in ventral cells, but increases the sensitivity of these cells to Delta-based signals. Thus, ventral Serrate signals to adjacent dorsal cells, and dorsal Delta signals to adjacent ventral cells (Figure 2b,c). As in the wing, heightened Notch activity increases ligand expression via a positive feedback loop, likely reinforcing signalling at the boundary.
Two different results hinted at the involvement of Notch: Notch mutations were isolated as dominant enhancers of the frizzled mutant eye polarity phenotype [4]; and the expression of a promoter–lacZ construct that is known to be sensitive to Notch signalling was observed very early in development in the polar member of the R3/R4 pair [5]. Subsequent experiments yielded the predicted results: reductions in Notch activity result in the duplication of R3 within each cluster, while gains in Notch activity result in the duplication of R4 (Figure 3). How is this competitive interaction normally mediated? The R3/R4 decision is sensitive to levels of the Notch ligand Delta, and indeed higher levels of Delta expression are observed in the initially equatorial member of the pair [4,5]. Thus, it seems likely that a bias develops between polar and equatorial cells; the equatorial cell produces more Delta signal and may also become less sensitive to Notch activity and thus becomes an R3, while the polar cell produces less Delta, receives more Notch signal and thus becomes R4. A negative feedback loop between Notch activity and Delta expression amplifies any initial bias [5]. This mechanism is similar to that thought to operate during another Notch-mediated process — the selection of neuronal precursors from proneural equivalence groups.
Photoreceptor specification along the dorsoventral axis
Factor(s) X and the bias in Notch signalling
The equator of the eye has long-range effects on eye polarity, but the nature of the equator signal is unknown. However, the polarity defects that result from mutations in frizzled and dishevelled indicate that Wingless/Wnt-like signalling is critical in this process [7,12]; frizzled encodes a transmembrane protein capable of transducing Wingless signal, and dishevelled encodes a downstream component of the Wingless pathway. The analysis of loss-of-function and gain-of-function mosaics indicated that Frizzled is required in the R3/R4 pair of photoreceptor precursors ([7,12] and A. Tomlinson and G. Struhl, personal communication). But while frizzled mutant ommatidia have randomized chirality, they almost always contain both an R3 and an R4 cell [12]. R3 is often formed by the wrong, initially polar member of the R3/R4 pair, but only rarely does this result in the formation of two R3’s.
As discussed above, it is possible that the equator cells produce a single ‘Factor X’ and that this is a Wnt-like signal. It would certainly make sense if the Notch activity is lowered in the equatorial cells by higher levels of a Wingless/Wnt-like signal, as Wingless signalling often inhibits Notch activity and provides a spatial bias to Notch-dependent equivalence groups. Moreover, polarity need not depend on an absolute level of Factor X signalling, as any difference in Factor X reception between the polar and equatorial cell in each cluster would be amplified by Notch. Several different mechanisms have been proposed for the inhibition of Notch by Wingless, including a direct physical interaction between Notch and Dishevelled proteins [13] or, more provocatively, the direct binding of Wingless to Notch itself [14].
The fate decisions in the R3/R4 pair are thus linked. Recent studies have shown that Notch signalling provides this link; as in many other situations Notch mediates a
Factor X signalling differs, however, from classic Wingless signalling. While Frizzled and Dishevelled are common to both pathways, the interpretation of the equatorial signal does not depend on further downstream
Dispatch
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Figure 4 The development of eye polarity might involve graded production of a hypothetical ‘Factor X’ across the dorsovental axis. It is assumed that Factor X is an inhibitor of Notch signalling, with higher expression at the equator. (a) Equatorial expression of the JAK/STAT ligand and polar expression of Wingless (Wg) promote and inhibit, respectively, the expression of the Factor X gene (blue) and protein (red). (b) A local reduction in Factor X gene expression (green) — induced by lowered JAK/STAT or heightened Wg reception — creates a local sink for the diffusible Factor X protein, reversing the slope of the Factor X gradient on the polar side of the mutant clone, in both wild-type (red) and mutant (purple) cells. (c) A local heightening of Factor X gene expression — induced by heightened JAK/STAT or lowered Wg reception — creates a local source of the Factor X protein,
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reversing the slope of the Factor X gradient on the equatorial side of the mutant clone, in both
members of the Wingless pathway, such as Armadillo [7]. In fact, recent results have shown that polarity requires different parts of the Dishevelled protein than those required for Wingless signalling, and that the polarity signal may be mediated by JNK or other mitogen-activated protein (MAP) kinase pathways [15,16]. Interestingly, overexpression of the known Drosophila Wnts does not induce polarity defects in the eye [7]. It may also be wrong to think of a single Factor X produced exclusively by equatorial cells. Rather, Factor X signal may be produced in a graded fashion across the dorsoventral axis of the eye, in response to other equatorial and polar signals. In genetic mosaics, cells unable to receive either Wingless [7] or JAK/STAT [10] signals can induce chirality defects in neighboring wild-type cells. Because both signalling pathways can influence the dorsal expression of the Iroquois complex (see above), one possible explanation for these ‘non-autonomous’ effects is that mutant cells induce local changes in Iroquois complex expression; this, in turn, would juxtapose Iroquois complex expressing and nonexpressing cells, creating an ectopic equator. However, changes in JAK/STAT reception do not change expression of the Iroquois complex member mirror [10]. It has therefore been suggested that an equatorial JAK/STAT signal combines with a polar Wingless signal to create a gradient of Factor X production [7,10] (Figure 4). Assuming that Factor X is a negative regulator of Notch signalling, it would have high expression at the equator and lowered expression at the poles. Local gains or losses in Factor X production would affect the chirality of neighboring wild-type cells by creating local sources or sinks of the short-range signal, reversing the direction of the Factor X gradient within and outside the clone. As chirality depends on the direction of the Factor X gradient,
wild-type and mutant cells. (Red arrows indicate polarity of ommatidia.)
rather than absolute levels, gains in Factor X expression would affect chirality on the equatorial side and losses on the polar side of the mutant cells (Figure 4b,c). In this, losses of Wingless reception resemble gains in Factor X expression, and losses of JAK/STAT reception resemble losses of Factor X expression [7,10]. Even this model, however, is likely to be too simplistic; it assumes that the difference in Factor X reception between R3 and R4 acts purely by regulating a Notchbased cell interaction between the cells of a single ommatidial cluster. However, ommatidia lacking frizzled, which encodes the presumed Factor X receptor, also affect the chirality of ommatidia polar to the mutant cells [12]. It seems unlikely that this non-autonomous effect is due to Notch signalling, as it would have to act over several cell diameters. Moreover, frizzled mutations cause similar nonautonomous effects in the developing wing, where frizzled is required for the orientation of epithelial hairs (reviewed in [17]); Notch has no known role in wing-hair polarity. One possible explanation for the non-autonomy of frizzled mutations is that Frizzled signalling affects the expression of long-range polarity signals, such as Wnts or JAK/STAT ligands. Alternatively, it has been suggested that Frizzled is involved not only in the reception of a polarizing signal but also in the polarised transmission of that signal to adjacent cells [17]. The latter is a particularly intriguing idea, as it would provide yet another mechanism for the coordination of planar polarity within an epithelium. Acknowledgements I thank Bill Feeny for help with the figures. This work was supported by grants from the NSF (IBN-9723564) and NIH (R01-NS28202).
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If you found this dispatch interesting, you might also want to read the April 1999 issue of
Current Opinion in Cell Biology which included the following reviews, edited by Joan S Brugge and Frank McCormick, on Cell regulation: Regulation of tyrosine kinase cascades by G-protein coupled receptors Louis M Lutrell, Yehia Daaka and Robert J Lefkowitz Epidermal growth factor receptors: critical mediators of multiple receptor pathways Peter O Hackel, Esther Zwick, Norbert Prenzel and Axel Ullrich Multiple positive and negative regulators of signaling by the EGF receptor Nadeem Moghal and Paul W Sternberg Interactions between mitogenic stimuli, or, a thousand and one connections Martin A Schwartz and Veronique Baron Costimulatory regulation of T cell function Cynthia A Chambers and James P Allison Organization and regulation of mitogen-activated protein kinase signaling pathways Timothy P Garrington and Gary L Johnson Signalling through phosphatidylinositol 3-kinases: the lipids take centre stage Sally J Leevers, Bart Vanhaesebroeck and Michael D Waterfield κB Multiple signals converging on NF-κ Frank Mercurio and Anthony M Manning Regulation of LEF-1/TCF transcription factors by Wnt and other signals Quinn Eastman and Rudolf Grosschedl In or out? Regulating nuclear transport Jennifer K Hood and Pamela A Silver Organization and regulation of proteins at synapses Jee Hae Kim and Richard L Huganir Apoptosis control by death and decoy receptors Avi Ashkenazi and Vishva M Dixit Deciphering the pathways of life and death Honglin Li and Junying Yuan The full text of Current Opinion in Cell Biology is in the BioMedNet library at http://BioMedNet.com/cbiology/cel