Delta/Notch and Boss/Sevenless signals act combinatorially to specify the Drosophila R7 photoreceptor.

The R7 photoreceptor, a unique cell type within the Drosophila ommatidium, was initially proposed to be specified by two distinct signals from neighboring cells, one from the R8 photoreceptor and another from the R1/6 photoreceptor pair. The R8-to-R7 signal is the transmembrane ligand Bride of Sevenless (Boss), which is received by the receptor tyrosine kinase Sevenless (Sev) and transduced via Ras activation within the presumptive R7 cell. However, the identity of the R1/6-to-R7 signal has remained elusive. Here, we present evidence that the transmembrane ligand Delta (Dl), expressed by the R1/6 pair, activates the receptor Notch (N) in the presumptive R7 cell and constitutes the postulated R1/6-to-R7 signal required in combination with the Boss/Sev signal to specify the R7 fate.     

Neuronal development in the Drosophila compound eye: Photoreceptor cells R1, R6, and R7 fail to differentiate in the retina aberrant in pattern (rap) mutant

The compound eye of Drosophila is a reiterated pattern of 800 unit eyes known as ommatidia. In each ommatidium there are eight photoreceptor neurons (R1–R8) and an invariant number of accessory cells organized in a precise manner. In the developing eye, specification of cell fates is triggered by sequential inductive events mediated by cell-cell interactions. The R8 photoreceptor neuron is the first cell to differentiate and is thought to play a central role in the recruitment of the remaining photoreceptor cells. Our previous work demonstrated that mutations in the retina aberrant in pattern (rap) locus lead to abnormal pattern formation in the compound eye. Genetic mosaic experiments demonstrated that for normal retinal patterning to occur, rap gene function is required only in the photoreceptor cell R8. In this study we analyzed the R cell composition of developing as well as the adult eyes of rap mutants employing a variety of R cell specific markers. We show that in rap mutants, although some of the R8-specific markers show normal expression patterns, other aspects of the R8 cell differentiation are abnormal. In addition, the cells R1, R6, and R7 fail to differentiate properly in rap mutants. These results suggest that the rap gene encodes an R8-specific function that plays a role in the determination of the photoreceptor cells R1, R6, and R7. © 1996 John Wiley & Sons, Inc.     

Ligand-independent activation of the sevenless receptor tyrosine kinase changes the fate of cells in the developing Drosophila eye

Cell fate in the developing eye is determined by a cascade of inductive interactions. In this process, the sevenless protein--a receptor tyrosine kinase--is required for the specification of the R7 photoreceptor cell fate. We have constructed a gain-of-function sevenless mutation (SevS11) by overexpressing a truncated sevenless protein in the cells where sevenless is normally expressed. In SevS11 mutant flies, all sevenless-expressing cells initiate neural development. This results in the formation of multiple R7-like photoreceptors per ommatidium. Therefore, sevenless activity appears to be necessary and sufficient for the determination of R7 cell fate. These results illustrate the central role receptor tyrosine kinases can play in the specification of cell fate during development.     

Neuronal differentiation in the Drosophila ommatidium

Using monoclonal and polyclonal antibodies as differentiation markers, we have found that the eight photoreceptors of the Drosophila ommatidium differentiate in a fixed sequence. The foundation photoreceptor, R8, expresses neural antigens first. The paired photoreceptors R2/5 are next to express, followed by the pair R3/4, followed by the pair R1/6; R7 is the final photoreceptor to differentiate. From previous studies it is known that Drosophila photoreceptors use local, positional cues to select their identities. Together with the morphological picture of ommatidial development, the sequential order of photoreceptor differentiation demonstrated here suggests that these cues may be encoded in the particular combination of cells an undetermined cell finds itself in contact with.     

Three distinct roles for notch in Drosophila R7 photoreceptor specification

Receptor tyrosine kinases (RTKs) and Notch (N) proteins are different types of transmembrane receptors that transduce extracellular signals and control cell fate. Here we examine cell fate specification in the Drosophila retina and ask how N acts together with the RTKs Sevenless (Sev) and the EGF receptor (DER) to specify the R7 photoreceptor. The retina is composed of many hundred ommatidia, each of which grows by recruiting surrounding, undifferentiated cells and directing them to particular fates. The R7 photoreceptor derives from a cohort of three cells that are incorporated together following specification of the R2-R5 and R8 photoreceptors. Two cells of the cohort are specified as the R1/6 photoreceptor type by DER activation. These cells then activate N in the third cell (the R7 precursor). By manipulation of N and RTK signaling in diverse combinations we establish three roles for N in specifying the R7 fate. The first role is to impose a block to photoreceptor differentiation; a block that DER activation cannot overcome. The second role, paradoxically, is to negate the first; Notch activation up-regulates Sev expression, enabling the presumptive R7 cell to receive an RTK signal from R8 that can override the block. The third role is to specify the cell as an R7 rather than an R1/6 once RTK signaling has specified the cells as a photoreceptor. We speculate why N acts both to block and to facilitate photoreceptor differentiation, and provide a model for how N and RTK signaling act combinatorially to specify the R1/6 and R7 photoreceptors as well as the surrounding non-neuronal cone cells.     

R7 photoreceptor specification requires Notch activity

The eight photoreceptors in each ommatidium of the Drosophila eye are assembled by a process of recruitment [1,2]. First, the R8 cell is singled out, and then subsequent photoreceptors are added in pairs (R2 and R5, R3 and R4, R1 and R6) until the final R7 cell acquires a neuronal fate. R7 development requires the Sevenless receptor tyrosine kinase which is activated by a ligand from R8 [3]. Here, we report that the specification of R7 requires a second signal that activates Notch. We found that a Notch target gene is expressed in R7 shortly after recruitment. When Notch activity was reduced, the cell was misrouted to an R1/R6 fate. Conversely, when activated Notch was present in the R1/R6 cells, it caused them to adopt R7 fates or, occasionally, cone cell fates. In this context, Notch activity appears to act co-operatively, rather than antagonistically, with the receptor tyrosine kinase/Ras pathway in R7 photoreceptor specification. We propose two models: a ratchet model in which Notch would allow cells to remain competent to respond to sequential rounds of Ras signalling, and a combinatorial model in which Notch and Ras signalling would act together to regulate genes that determine cell fate.     

Specification of cell fate in the developing eye of Drosophila.

Determination of cell fate in the developing eye of Drosophila depends on a precise sequence of cellular interactions which generate the stereotypic array of ommatidia. In the eye imaginal disc, an initially unpatterned epithelial sheath of cells, the first step in this process may be the specification of R8 photoreceptor cells at regular intervals. Genes such as Notch and scabrous, known to be involved in bristle development, also participate in this process, suggesting that the specification of ommatidial founder cells and the formation of sensory organs in the adult epidermis may involve a similar mechanism, that of lateral inhibition. The subsequent steps of ommatidial assembly, following R8 assignment, involve a different mechanism: Undetermined cells read their position based on the contacts they make with neighbors that have already begun to differentiate. The development of the R7 photoreceptor cell, one of the eight photoreceptor cells in the ommatidium, is best understood. An important role seems to be played by sevenless, a receptor tyrosine kinase on the surface of the R7 precursor. It transmits the positional information--most likely encoded by the boss protein on the neighboring R8 cell membrane--into the cell via its tyrosine kinase, which activates a signal transduction cascade. Constitutive activation of the sevenless kinase by overexpression of an N-terminally truncated form results in the diversion of other ommatidial cells into the R7 pathway suggesting that activation of the sevenless signalling pathway is sufficient to specify R7 development. Genetic dissection of this pathway should therefore identify components of a signalling cascade activated by a tyrosine kinase.     

Induction in the developing compound eye of Drosophila: multiple mechanisms restrict R7 induction to a single retinal precursor cell.

The development of the Drosophila R7 photoreceptor cell is determined by a specific inductive interaction between the R8 photoreceptor cell and a single neighboring precursor cell. This process is mediated by bride of sevenless (boss), a cell-surface bound ligand, and the sevenless (sev) tyrosine kinase receptor. The boss ligand is expressed specifically on the surface of the R8 cell, whereas the sev receptor is expressed on 5 cells contacting the developing R8 cell and other cells not in contact with R8. By altering the spatial and temporal expression of boss, we demonstrate that sev-expressing cells that do not contact R8 can assume an R7 cell fate. By contrast, the sev-expressing precursor cells to the R1-R6 photoreceptor cells that do contact R8 are nonresponsive to the inductive cue. Using the rough and Nspl mutations, we demonstrate that an early commitment to an R1-R6 cell fate blocks the pathway of sev activation in these cells.     

Decoding vectorial information from a gradient: sequential roles of the receptors Frizzled and Notch in establishing planar polarity in the Drosophila eye

The Drosophila eye is composed of several hundred ommatidia that can exist in either of two chiral forms, depending on position: ommatidia in the dorsal half of the eye adopt one chiral form, whereas ommatidia in the ventral half adopt the other. Chirality appears to be specified by a polarizing signal with a high activity at the interface between the two halves (the 'equator'), which declines in opposite directions towards the dorsal and ventral poles. Here, using genetic mosaics, we show that this polarizing signal is decoded by the sequential use of two receptor systems. The first depends on the seven-transmembrane receptor Frizzled (Fz) and distinguishes between the two members of the R3/R4 pair of presumptive photoreceptor cells, predisposing the cell that is located closer to the equator and having higher Fz activity towards the R3 photoreceptor fate and the cell further away towards the R4 fate. This bias is then amplified by subsequent interactions between the two cells mediated by the receptor Notch (N) and its ligand Delta (Dl), ensuring that the equatorial cell becomes the R3 photoreceptor while the polar cell becomes the R4 photoreceptor. As a consequence of this reciprocal cell fate decision, the R4 cell moves asymmetrically relative to the R3 cell, initiating the appropriate chiral pattern of the remaining cells of the ommatidium.     

Localization of the sevenless protein, a putative receptor for positional information, in the eye imaginal disc of Drosophila

The Drosophila gene sevenless encodes a putative trans-membrane receptor required for the formation of one particular cell, the R7 photoreceptor, in each ommatidium of the compound eye. Mutations in this gene result in the cell normally destined to form the R7 cell forming a non-neuronal cell type instead. These observations have led to the proposal that the sevenless protein receives at least part of the positional information required for the R7 developmental pathway. We have generated antibodies specific for sevenless and have examined expression of the protein by light and electron microscopy. sevenless protein is present transiently at high levels in at least 9 cells in each developing ommatidium and is detectable several hours before any overt differentiation of R7. The protein is mostly localized at the apices of the cells, in microvilli, but is also found deeper in the tissue where certain cells contact the R8 cell. This finding suggests that R8 expresses a ligand for the sevenless protein.     

The sevenless signaling pathway: variations of a common theme

Many developmental processes are regulated by intercellular signaling mechanisms that employ the activation of receptor tyrosine kinases. One model system that has been particular useful in determining the role of receptor tyrosine kinase-mediated signaling processes in cell fate determination is the developing Drosophila eye. The specification of the R7 photoreceptor cell in each ommatidium of the developing Drosophila eye is dependent on activation of the Sevenless receptor tyrosine kinase. This review will focus on the genetic and biochemical approaches that have identified signaling molecules acting downstream of the Sevenless receptor tyrosine kinase which ultimately trigger differentiation of the R7 photoreceptor cell.     

Asymmetric localization of frizzled and the determination of notch-dependent cell fate in the Drosophila eye

BACKGROUND: During patterning of the Drosophila eye, a critical step is the Notch-mediated cell fate decision that determines the identities of the R3/R4 photoreceptor pair in each ommatidium. Depending on the decision taken, the ommatidium adopts either the dorsal or ventral chiral form. This decision is directed by the activity of the planar polarity genes, and, in particular, higher activity of the receptor Frizzled confers R3 fate. RESULTS: We present evidence that Frizzled does not modulate Notch activity via Rho GTPases and a JNK cascade as previously proposed. We find that the planar polarity proteins Frizzled, Dishevelled, Flamingo, and Strabismus adopt asymmetric protein localizations in the developing photoreceptors. These protein localizations correlate with the bias of Notch activity between R3/R4, suggesting that they are necessary to modulate Notch activity between these cells. Additional data support a mechanism for regulation of Notch activity that could involve direct interactions between Dishevelled and Notch at the cell cortex. CONCLUSIONS: In the light of our findings, we conclude that Rho GTPases/JNK cascades are not major effectors of planar polarity in the Drosophila eye. We propose a new model for the control of R3/R4 photoreceptor fate by Frizzled, whereby asymmetric protein localization is likely to be a critical step in modulation of Notch activity. This modulation may occur via direct interactions between Notch and Dishevelled.     

Neuronal differentiation in Drosophila ommatidium

Using monoclonal and polyclonal antibodies as differentiation markers, we have found that the eight photoreceptors of the Drosophila ommatidium differentiate in a fixed sequence. The foundation photoreceptor, R8, expresses neural antigens first. The paired photoreceptors R2/5 are next to express, followed by the pair R3/4, followed by the pair R1/6; R7 is the final photoreceptor to differentiate. From previous studies it is known that Drosophila photoreceptors use local, positional cues to select their identities. Together with the morphological picture of ommatidial development, the sequential order of photoreceptor differentiation demonstrated here suggests that these cues may be encoded in the particular combination of cells an undetermined cell finds itself in contact with.     

The color-vision circuit in the medulla of Drosophila

BACKGROUND: Color vision requires comparison between photoreceptors that are sensitive to different wavelengths of light. In Drosophila, this is achieved by the inner photoreceptors (R7 and R8) that contain different rhodopsins. Two types of comparisons can occur in fly color vision: between the R7 (UV sensitive) and R8 (blue- or green sensitive) photoreceptor cells within one ommatidium (unit eye) or between different ommatidia that contain spectrally distinct inner photoreceptors. Photoreceptors project to the optic lobes: R1-R6, which are involved in motion detection, project to the lamina, whereas R7 and R8 reach deeper in the medulla. This paper analyzes the neural network underlying color vision into the medulla. RESULTS: We reconstruct the neural network in the medulla, focusing on neurons likely to be involved in processing color vision. We identify the full complement of neurons in the medulla, including second-order neurons that contact both R7 and R8 from a single ommatidium, or contact R7 and/or R8 from different ommatidia. We also examine third-order neurons and local neurons that likely modulate information from second-order neurons. Finally, we present highly specific tools that will allow us to functionally manipulate the network and test both activity and behavior. CONCLUSIONS: This precise characterization of the medulla circuitry will allow us to understand how color vision is processed in the optic lobe of Drosophila, providing a paradigm for more complex systems in vertebrates.     

Genetic mosaic analysis of the equatorial-less mutation in Drosophila mezunogaster

eql (equatorial-less) is a recessive lethal mutation on the second chromosome of Drosophila melanogasfer. J. Campos-Ortega found that eql clones in somatic mosaic flies have reduced numbers of photoreceptor cells, and he suggested that only the R1, R6, and R7 photoreceptor cells were missing in this mutant. These photoreceptor cells help to define the inverted orientation of ommatidial facets along the equatorial midline of the fly eye, hence the mutation was named “equatorial-less”. We have conducted a detailed analysis of the eql mutation, by serial section reconstruction of eql clones marked with bw or w^? in somatic mosaic flies. We found that all photoreceptor cell types (Rl–R8) could be deleted by the eql mutation, and in rare cases the number of photoreceptor cells was increased. The apparent lack of photoreceptor cell type specificity was confirmed by our analysis of genetically mosaic facets, which indicated that no single photoreceptor cell, or subset of photoreceptor cells, was uniquely required to express eql Rather, eql appears to function in all photoreceptor cells, and possibly in all eye precursor cells. The distribution of photoreceptor cell numbers in w eql facets was consistent with the hypothesis that each photoreceptor cell was deleted independently of the others. The eql gene is located on the right arm of chromosome 2 at map location 2 ? 104.5 ± 0.7 and lies between the polytene chromosome bands 59D8 and 60A7. © 1995 Wiley-Liss, Inc.     

Developing compound eye in lozenge mutants of Drosophila: lozenge expression in the R7 equivalence group

 The lozenge locus is genetically complex, containing two functionally distinct units, cistrons A and B, that influence the structure of the compound eye. Extreme mutations of either cistron produce adult phenotypes that share similarities and that have striking differences. We have analyzed the expression of several developmentally important eye genes including boss, scabrous, rhomboid, seven-up, and Bar in lozenge mutant backgrounds representing both cistrons. This analysis follows the progressive recruitment of photoreceptor neurons during eye development and has confirmed that the initial development of photoreceptors is normal up to the five cell precluster stage (R8, R2/5 and R3/4). However, when lozenge is mutant, further eye development is perturbed. As cells R1, R6 and R7 are recruited, patterns of gene expression for seven-up and Bar become abnormal. We have also characterized the expression of two different enhancer trap alleles of lozenge. The lozenge product(s) appear to be first expressed in the eye disc in undifferentiated cells shortly after the five cell precluster forms. Then, as distinct cells are recruited to a fate, lozenge expression persists and is refined in those cells. Our data suggests that lozenge functions in cone cells and pigment cells as well as in specific glia. With respect to photoreceptor neurons, lozenge biases the developmental potential of cells R1, R6 and R7, by directly influencing the expression of genes important for establishing cell fate. Received: 26 July 1996 / Accepted: 6 January 1997