The role of Sevenless in Drosophila R7 photoreceptor specification.

Sevenless (Sev) is a Receptor Tyrosine Kinase (RTK) that is required for the specification of the Drosophila R7 photoreceptor. Other Drosophila photoreceptors are specified by the action of another RTK; the Drosophila EGF Receptor (DER). Why Sev is required specifically in the R7 precursor, and the exact role it plays in the cell's fate assignment have long remained unclear. Notch (N) signaling plays many roles in R7 specification, one of which is to prevent DER activity from establishing the photoreceptor fate. Our current model of Sev function is that it hyperactivates the RTK pathway in the R7 precursor to overcome the N-imposed block on photoreceptor specification. From this perspective DER and Sev are viewed as engaging the same transduction machinery, the only difference between them being the level of pathway activation that they induce. To test this model, we generated a Sev/DER chimera in which the intracellular domain of Sev is replaced with that of DER. This chimerical receptor acts indistinguishably from Sev itself; a result that is entirely consistent with the two RTKs sharing identical transduction abilities. A long-standing question in regard to Sev is the function of a hydrophobic domain some 60 amino acids from the initiating Methionine. If this represents a transmembrane domain, it would endow Sev with N-terminal intracellular sequences through which it could engage internal transduction pathways. However, we find that this domain acts as an internal signal peptide, and that there is no Sev N-terminal intracellular domain. phyllopod (phyl) is the target gene of the RTK pathway, and we show that R7 precursors are selectively lost when phyl gene function is mildly compromised, and that other photoreceptors are removed when the gene function is further reduced. This result adds a key piece of evidence for the hyperactivation of the RTK pathway in the R7 precursor. To facilitate the hyperactivation of the RTK pathway, Sev is expressed at high levels. However, when we express DER at the levels at which Sev is expressed, strong gain-of-function effects result, consistent with ligand-independent activation of the receptor. This highlights another key feature of Sev; that it is expressed at high levels yet remains strictly ligand dependent. Finally, we find that activated Sev can rescue R3/4 photoreceptors when their DER function is abrogated. These results are collectively consistent with Sev and DER activating the same transduction machinery, with Sev generating a pathway hyperactivation to overcome the N-imposed block to photoreceptor specification in R7 precursors.

Parsimony and complexity: Cell fate assignment in the developing Drosophila eye.

The specification of the R7 photoreceptor in the Drosophila eye has become a classic model for understanding how cell fates are assigned in developing systems. R7 is derived from a group of cells that also gives rise to the R1/6 photoreceptor class and the non-photoreceptor cone cells. Our studies examine the signals and cellular information that direct each of these cell types. The cell fates are directed by the combined actions of the Receptor Tyrosine Kinase (RTK) and Notch (N) signaling pathways. The RTK pathway acts to remove the transcription factor Tramtrack (Ttk) which represses the photoreceptor fate. If a cell receives an RTK signal sufficient to remove Ttk then the photoreceptor fate is specified; if not, the cone cell fate results. If Ttk is removed from a cell and its N activity is high then it is specified as an R7, but if its N activity is low then it becomes an R1/6 class photoreceptor. Thus, a remarkably simple molecular code underlies the specification of the fates: 1. Ttk degraded or not: 2. N activity high or low. In the R1/6 and cone cell precursors the molecular codes are achieved with relative simplicity but in the R7 precursor, manifold interactions occur between the RTK and N pathways, and to-date we have identified 4 distinct roles played by N in R7 fate specification. In this review we detail this molecular complexity, and describe how the RTK/N pathway crosstalk eventually leads to the simple molecular code of Tramtrack removed and N activity high. Furthermore, we describe the role played by the transcription factor Lozenge (Lz) in directing retinal precursor fates, and how the RTK/N signals specify different retinal cell types depending on the presence or absence of Lz.

R7 Photoreceptor Specification in the Developing Drosophila Eye: The Role of the Transcription Factor Deadpan.

As cells proceed along their developmental pathways they make a series of sequential cell fate decisions. Each of those decisions needs to be made in a robust manner so there is no ambiguity in the state of the cell as it proceeds to the next stage. Here we examine the decision made by the Drosophila R7 precursor cell to become a photoreceptor and ask how the robustness of that decision is achieved. The transcription factor Tramtrack (Ttk) inhibits photoreceptor assignment, and previous studies found that the RTK-induced degradation of Ttk was critically required for R7 specification. Here we find that the transcription factor Deadpan (Dpn) is also required; it is needed to silence ttk transcription, and only when Ttk protein degradation and transcriptional silencing occur together is the photoreceptor fate robustly achieved. Dpn expression needs to be tightly restricted to R7 precursors, and we describe the role played by Ttk in repressing dpn transcription. Thus, Dpn and Ttk act as mutually repressive transcription factors, with Dpn acting to ensure that Ttk is effectively removed from R7, and Ttk acting to prevent Dpn expression in other cells. Furthermore, we find that N activity is required to promote dpn transcription, and only in R7 precursors does the removal of Ttk coincide with high N activity, and only in this cell does Dpn expression result.

Switching cell fates in the developing Drosophila eye.

The developing Drosophila ommatidium is characterized by two distinct waves of pattern formation. In the first wave, a precluster of five cells is formed by a complex cellular interaction mechanism. In the second wave, cells are systematically recruited to the cluster and directed to their fates by developmental cues presented by differentiating precluster cells. These developmental cues are mediated through the receptor tyrosine kinase (RTK) and Notch (N) signaling pathways and their combined activities are crucial in specifying cell type. The transcription factor Lozenge (Lz) is expressed exclusively in second wave cells. Here, we ectopically supply Lz to precluster cells and concomitantly supply the various RTK/N codes that specify each of three second wave cell fates. We thereby reproduce molecular markers of each of the second wave cell types in precluster cells and draw three inferences. First, we confirm that Lz provides key intrinsic information to second wave cells. We can now combine this with the RTK/N signaling to provide a cell fate specification code that entails both extrinsic and intrinsic information. Second, the reproduction of each second wave cell type in the precluster confirms the accuracy of the RTK/N signaling code. Third, RTK/N signaling and Lz need only be presented to the cells for a short period of time in order to specify their fate.

Stop and go: antagonistic signals in the specification of the Drosophila R7 photoreceptor viewed from an evolutionary perspective.

The Drosophila R7 photoreceptor precursor is directed to its fate by signals from adjacent cells that activate its Receptor Tyrosine Kinase (RTK) and Notch (N) signaling pathways. Counter-intuitively, the N activity both promotes and inhibits the photoreceptor fate in the R7 precursor. We offer an evolutionary perspective for this in which earlier ommatidia had fewer photoreceptors and used N to inhibit the addition of any more. When additional photoreceptors were added by evolution, an RTK signal was used to overcome the N inhibition in these cells, and these new additions potently activated N in their neighboring cells, preventing them from also responding to the RTK signal. The R7 precursor also receives this block, and requires robust RTK activation for it to become a photoreceptor. This is achieved by N transcriptionally activating a new RTK, one that is potently activated in the R7 precursor and sufficing to overcome the N inhibition. The unusually high RTK signal in R7 requires additional transduction components not needed when the signal is mild; in R7 the small GTPases Ras and Rap are both required to transduce the signal, but in other photoreceptors Ras alone suffices.

The role of the small GTPase Rap in Drosophila R7 photoreceptor specification.

The Drosophila R7 photoreceptor provides an excellent model system with which to study how cells receive and "decode" signals that specify cell fate. R7 is specified by the combined actions of the receptor tyrosine kinase (RTK) and Notch (N) signaling pathways. These pathways interact in a complex manner that includes antagonistic effects on photoreceptor specification: RTK promotes the photoreceptor fate, whereas N inhibits. Although other photoreceptors are subject to only mild N activation, R7 experiences a high-level N signal. To counter this effect and to ensure that the cell is specified as a photoreceptor, a high RTK signal is transduced in the cell. Thus, there are two levels of RTK transduction in the photoreceptors: in R7 it is high, whereas in others it is low. Here, we address how this high-level RTK signal is transduced in R7 and find that, in addition to Ras, another small GTPase, Rap, is also engaged. Thus, when N activity is high, a robust RTK signal operates that uses both Ras and Rap, but when N activity is low, only a mild RTK signal is transduced and Ras alone suffices for the purpose.

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.