BIOLOGICAL OVERVIEW

R7 is the last of eight photoreceptor cells to differentiate in the fly's compound eye. The regulation of R7 fate determination is one of the best studied differentiation processes in Drosophila or in any developmental system. This is due to the many advantages it affords the researcher. First, it is a process clearly localized in time and space. Second, it takes place post mitotically, thus distinctly separating fate determination from cell cycle control. Finally, the inputs are relatively well defined and a precise geometry is involved in the cell-cell interactions.

sevenless expression is not limited to R7 cells: it has been detected in the R1 and R6 pair, the R3 and R4 pair, four cone cell precursors and mystery cells (cells that are left out of the differentiation process and ultimately under programmed cell death). Some sevenless expressing cells such as cone cell precursors are competent to become R7 cells but fail to do so since they do not contact R8 cells (expressing the ligand for Sevenless known as BOSS). In addition, there is a refractoriness of R1 and R6 cells in response to BOSS (Bride of Sevenless) signals; neither R1 nor R6 internalize BOSS. R1 and R6 can, however, respond to Sevenless signals when the SEV tyrosine kinase is activated constitutively (Basler, 1991) The refractoriness of R1 and R6 is relieved in Notch and rough mutant backgrounds, where multiple cells in individual clusters internalize Boss (Van Vactor, 1991). Furthermore, the spatiotemporal pattern of seven-up expression, confined as it is to the R1/R6 and R3/R4 fate, plays an essential role in controlling the number and cellular origin of the R7 neuron in the ommatidium (Hiromi, 1993).

Activation of Sevenless by BOSS results in the phosphorylation of Sevenless and the transduction of the Sevenless signal, through the ras pathway, into the nucleus. The docking protein DRK (Downstream of receptor kinase) binds to phosphorylated Sevenless, and participates in building a protein complex associated with Sevenless that includes the SOS (Son of sevenless) and Ras1 proteins. RAS (a GTP exchange factor) and its target RAF (a serine/threonine kinase) initiate a phosphorylation cascade that transduces and amplifies the Sevenless signal, resulting in activation of pathway targets.

What are the targets of the ras pathway? The list is long and varied. JUN cooperates with the ETS domain transcription factor Pointed to induce R7 fate (Treier, 1995). Acting counter to this activation is Yan, a negative regulator of R7 fate. Two novel proteins, Seven in absentia (SINA) (Carthew, 1994) and Phyllopod (Chang, 1995) are downstream targets of the ras pathway, but neither required the ras pathway for expression. Activation of the ras pathway also activates a gene coding for a basal transcription factor (dTFIIA-S, the small subunit of TFIIA) (Zeidler, 1996). Also involved in Sevenless signaling are Hsp83 (a Drosophila chaperone protein) and cdc37, which interacts with cell cycle genes (Cutforth, 1994). Cdc37, in concert with the mammalian homolog of Hsp83, has been shown to be required for Raf-1 function (Grannatikakis, 1999).

The Sevenless signaling which initiates R7 photoreceptor maturation, triggers a phosphorylation cascade that activates transcription factors, modifies cell cycle regulation, affects basal transcription factors and involves protein chaperones. Even the Notch pathway is brought into play to restrict the effects of a ligand-receptor interaction. The R7 pathway continues to yield new information in what has become an important model for cell differentiation (Yamamoto, 1994).

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. In this study Lz was ectopically supplied to precluster cells, and the various RTK/N codes that specify each of three second wave cell fates were concomitantly supplied. This protocol reproduced molecular markers of each of the second wave cell types in first wave precluster cells. Three inferences were drawn; (1) it was confirmed that Lz provides key intrinsic information to second wave cells, and this can now be combined with the RTK/N signaling to provide a cell fate specification code that entails both extrinsic and intrinsic information. (2) the reproduction of each second wave cell type in the precluster confirms the accuracy of the RTK/N signaling code, and (3) RTK/N signaling and Lz need only be presented to the cells for a short period of time in order to specify their fate (Mavromatakis, 2013).

This paper explored three inter-related themes bearing on the nature of the signals that specify the cell types in the Drosophila ommatidium. The ability of a transcription factor to predispose the cellular responses to developmental signals was examined, the accuracy of the signaling code that represents these developmental signals was validated, and it was inferred that both the intrinsic and extrinsic aspects are only required for a brief period of time (Mavromatakis, 2013).

Lz had long been assumed to be a key factor that distinguishes how second wave cells differ from the precluster cells in their response to developmental signals. This paper rigorously tested this concept and reproduced features typical of the three second wave cell types in the R3/4 precluster cells by supplying ectopic Lz along with the appropriate RTK/N cell fate code. It is thus inferred that the presence of Lz in R3/4 precluster cells is sufficient to endow them with the second wave cell fate response repertoire. A number of issues related to these observations and their interpretations are discussed (Mavromatakis, 2013).

Normal R3/4 precursors undergo an N-Dl interaction that results in the R4 precursor experiencing much higher levels of N activity than the R3 precursor. When Lz was supplied to R3/4 precursors (sev.lz), the cell in the R4 position frequently transformed into an R7, consistent with the requirement of high N for R7 specification. Less frequently, both R3/4 precursors adopted the R7 fate, and sometimes it was the cell in the R3 position alone that generated an ectopic R7. These results suggest that in the sev.lz flies the R3/R4 N-Dl interaction does not occur correctly. When the mδ0.5.lacZ reporter line was used as a reporter of N activity (which in wild-type larvae is robustly upregulated in R4 precursors) an erratic pattern was observed, sometimes showing the wild-type pattern, sometimes showing both R3/4 cells with high levels of lacZ expression, and sometimes showing R3 alone with high levels. Hence, by expressing Lz in the R3/4 precursors the cells were not only endowed with second wave response abilities but also they were prevented from executing their N-Dl interactions properly. Indeed, it was only when N was artificially activated to a high level with activated Notch (sev.lz; sev.N^**) that the R7 fate was potently induced in both R3/4 precursors (Mavromatakis, 2013).

Native R7s critically require sev gene function; in its absence, they differentiate as cone cells. However, some ectopic R7s were able to differentiate when Lz was provided to the R3/4 precursors, even in the absence of sev (sev⁰; sev.lz), suggesting that normal R7 specification was not fully reiterated here. Examination of these eye discs suggested that some R3/4 precursors differentiated as R7s whereas others became cone cells. Thus, these cells appear to be on the cusp of the R7/cone cell fate choice, and some cells expressing markers for both cell types were observed. In the cells that became R7s, the presence was inferred of sufficient RTK activity, which was likely to have been supplied by endogenous DER signaling active in the precluster cells. Only when N activity was raised in these cells (sev⁰; sev.lz; sev.N^*) did their full sev dependence for the R7 fate emerge, when all R3/4 precursors differentiated as cone cells (Mavromatakis, 2013).

The sev.N^* construct is a very useful activator of the N pathway in developing eye cells. Since N activity drives sev expression, the sev.N^* transgene feeds back on itself and promotes its own expression, and by subsequent iterations of this effect the cells are left with potent N activity. This level is still within the physiological range, unlike that produced using Gal4/UAS techniques, and is therefore the choice method for activating the N pathway. The transgene that was routinely use to knock down N activity [sev.Su(H)EnR] has the opposite effect; it reduces its own expression, and mildly compromises N activity. This level of reduction in N activity is usually sufficient to trigger major effects without the disadvantage of the severe downregulation that can accompany the use of Gal4/UAS technology. Since the sev.lz construct would also be downregulated by sev.Su(H)EnR, it was necessary to ectopically express Lz in the precluster using another enhancer element, and to this end the ro.Gal4 line was generated. When UAS.lz was expressed under ro control, the R3/4 precursors frequently differentiated as R7s, and crucially, when N activity was concomitantly reduced [ro.Gal4; UAS.lz; sev.Su(H)EnR] cells displaying R1/6 molecular features were now detected in the R3/4 precursors (Mavromatakis, 2013).

The cells in the R2/5 positions in ro.G4; UAS.lz developing ommatidia appear to develop normally; they express Elav and Ro, but none of the other fate markers. This suggests that R2/5 cells are insensitive to the presence of Lz, and argues that there is a major molecular difference between these cells and the R3/4 precursors. Also noteworthy is the transformation of all lz mutant second wave cells into R3/4 types characterized by the expression of Svp (a marker that is not expressed in R2/5 precursors) and Elav. Thus, it appears that ectopic Lz selectively transforms R3/4 precursors of the precluster to the second wave fate, and second wave cells lacking Lz adopt the R3/4 fate. Hence, it is suspected that Lz might not provide the intrinsic information that distinguishes the second wave cells from precluster cells per se, but rather distinguishes second wave cells from R3/4 types. Experiments to evaluate this view are currently being undertaken (Mavromatakis, 2013).

A counter-argument emerges from the fate of the majority of cells in the R3 positions in sev.lz eyes, which do not switch their fate. Only when N activity is activated or reduced in these cells is a change seen in their fates, and to be sure that the R2/5 cells are insensitive to Lz expression, it would also need to correspondingly vary N activity in them. Experiments to do this using ro.Gal4 produced severely disrupted preclusters, presumably as a result of interference with N function at earlier stages of precluster formation. Since these clusters were largely uninterpretable, the issue of whether R2/5 cells are insensitive to Lz expression remains unresolved (Mavromatakis, 2013).

For many years, the role of N in photoreceptor specification was confusing. In some contexts N appeared to oppose photoreceptor specification and in others N seemed to promote it, and this confusion prevented substantial progress in defining the fate codes that specified the different cell types. In recent work three distinct roles for N in this process were identified, and with that information the cell fate codes for R1/6, R7 and the cone cells were inferred. A major goal of this current work has to been to test this code by its reiteration in the R3/4 cells of the precluster using Lz expression to endow them with second wave cell qualities. In these experiments, each of the cell codes induced the expected cell fates, providing cogent support for the validity of the code (Mavromatakis, 2013).

DER is assumed to be ubiquitously expressed in the eye disc tissue, and its ligand, Spitz, diffusing from precluster cells, is thought to reach more distant cells with time. But the N and Sev signals are regulated in a different manner. Both their ligands are membrane bound, and their receptor activations only occur in immediate neighbors. Dl, the ligand for N, is expressed transiently by differentiating cells, and, accordingly, activates N in neighboring cells for only short periods of time (a few hours). sev is an N response gene and, in consequence, Sev is only expressed in cells for a short period of time. By contrast, Boss, the Sev ligand, is expressed for a prolonged period by the R8 precursor. Thus, both the N and Sev signaling systems are only available to the cells for restricted periods, with this restriction controlled by ligand expression in the N system and by receptor expression in the Sev system (Mavromatakis, 2013).

Although Lz is expressed in the second wave cells in a persistent manner in the eye disc, the experiments suggest that it, like the extrinsic signals, is required only for a brief developmental window. Consider the transformation of sev.lz R3/4 cells to the R7 fate. The sev enhancer is only active in these cells for a few hours and yet a complete transformation of the cells is achieved. The expression of specific cell type markers in the eye disc might erroneously indicate the transformation of a cell when only a transient effect occurs, but the presence of ectopic R7s in the adult retina argues otherwise and suggests that the transformations are potent and permanent. This view is further validated by the rescue of lz mutant second wave cells by the sev.lz transgene. This rescue is complete and is evident by the molecular markers expressed in the disc and by the morphology of the adult cells. Thus, it is inferred that Lz is only required during the same time window when the RTK/N signals are transduced, and it is further inferred that the combined activities of the RTK and N pathways, in concert with Lz, function in a short-lived manner to lock in the fate of the cells. How the presence of ephemeral extrinsic and intrinsic information is molecularly 'remembered' by the cells to allow their appropriate differentiation over a prolonged developmental period remains an intriguing question (Mavromatakis, 2013).

GENE STRUCTURE

Bases in 5' UTR -798

Exons - 12

Bases in 3' UTR - 322

PROTEIN STRUCTURE

Amino Acids - 3554

Structural Domains

The transmembrane domain stretches from residues 2042-2051. The C-terminal region shows a high degree of homology to all known protein tyrosine kinase domains (Bowtell, 1988).

Sevenless protein possesses protein tyrosine kinase activity. The protein is first synthesized as a 280-kDa glycoprotein precursor. This subsequently cleaves into two subunits that remain associated by noncovalent interations: a 220-kDa amino-terminal and a 58-kDa carboxyl-terminal. The 220-kDa subunit is glycosylated and contains most of the extracellular portion of the protein, and the 58-kDa subunit is composed of a small portion of the extracellular sequences and the intracellular protein tyrosine kinase domain, that includes an ATP binding site (Mullins, 1991). This complex is subsequently cleaved into either 49- or 48-kDa carboxyl-terminal fragments with concomitant degradation of the rest of the protein (Simon, 1989).

EVOLUTIONARY HOMOLOGS

The DNA sequence of the sevenless gene from Drosophila melanogaster has been compared with that of D. virilis. The two species diverged approximately 60 million years ago. There is 60% conservation in the extracellular domain and complete homology in the transmembrane domain. The kinase domains are 83% identical. 42 of the 43 cysteines in DM are conserved in DV, while DV has an additional 9 cysteine residues (Michael, 1990)

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