BIOLOGICAL OVERVIEW

There are eight photoreceptor cells (R-cells) in each ommatidium (facet) of the Drosophila compound eye. The first cell to develop is the R8 cell, followed by R1 through R6, with R7 following these. The determination of R7 photoreceptor fate depends on the expression of boss in the neighboring R8 cell. What is the origin of this observation? How can the identity and genotype of each cell be determined in the developing retina?

Mosaic analysis has been used to investigate the cellular requirements for R7 development. The chaoptic (chp) mutation which, as a homozygote mutant, gives an altered cell morphology was linked to the boss mutation. Flies heterozygous for the linked mutations boss and chp were then subject to X-rays to induced mitotic recombination. Mitotic recombination gives some cells of altered morphology identifiable as boss negative cells (on the basis of their altered morphology induced by the recessive chp mutation.

About one in a hundred flies exhibits mosaic retinas, that is, some cells inherit wild type chromosomes and other cells inherit recombinante chromosomes homozygous for chp and boss mutations. The double mutant cells exist as clones in the developing retina; the cells of particular interest are found at the border of mutant and normal clones, in ommatidia that possess cells of mixed ancestory. R7 development is independent of boss expression in R1 through R6 cells in mixed ommatidia but the genotype of R8 cells has to be wild type for bossif R7 cells are to develop. Since wild type R8 could not rescue an R7 in adjacent ommatidia but only within an ommatidium, it is concluded that BOSS can act non-cell autonomously as a ligand to induce the R7 fate only over a short distance (Reinke, 1988).

The target of BOSS is the receptor Sevenless. Studies reveal that interaction of cells bearing the BOSS ligand can aggregate with cells bearing the Sevenless receptor. There is considerable interest in the fate of receptor and ligand when ligand induces receptor signaling. In the case of the BOSS-Sevenless interaction, BOSS is internalized by sev expressing cells. BOSS immunoreactivity is not found on the surface of Sevenless bearing cells in the developing retina, but rather in vesicles inside the cell. This vesicular BOSS depends on endocytosis. When endocytosis is prevented in shabire mutants (shabire codes for the Drosophila homolog of vertebrate Dynamin, involved in endocytosis), BOSS protein is not apparent in R7 cells (Krämer, 1993).

Binding of boss-expressing cells to sev-expressing cells results in a rapid increase in phosphorylation of the Sevenless receptor tyrosine kinase. A BOSS amino-terminal peptide containing only the large extracellular domain was found to act as an inhibitor of BOSS-Sevenless directed cell adhesion, preventing SEV from undergoing phosphorylation (Hart, 1993). These experiments unequivocally show that Boss, as a ligand, binds and activates SEV (Yamamoto, 1994).

BOSS structure is that of a seven pass transmembrane G protein coupled receptor. Does BOSS actually function as a G protein coupled receptor, and if so, what is its function? Could BOSS be signaling back to the cell bearing it that an R7 fate has been determined in an adjacent cell? There will be other instances of G protein coupled receptors behaving as ligands during development.

Evidence for signaling from R7 to R8 photoreceptors (reverse information flow) comes from a study of rhodopsin expression in the two cell populations. The outer (R1-R6) and inner (R7, R8) photoreceptors represent two physiologically distinct systems with two different projection targets in the brain. All cells of the primary system, R1-R6, express the same rhodopsin and are functionally identical. In contrast, the R7 and R8 photoreceptors are different from each other. They occupy anatomically precise positions, with R7 on top of R8. In fact, there are several classes of R7/R8 pairs, which differ morphologically and functionally and are characterized by the expression of one of two R7-specific opsins: rh3 or rh4. A new opsin gene has been identified, rhodopsin 5, expressed in one subclass of R8 cells. Interestingly, this subclass represents R8 cells that are directly underneath the R7 photoreceptors expressing rh3, but are never under those expressing rh4. These results confirm the existence of two subpopulations of R7 and R8 cells, which coordinate the expression of their respective rh genes. Thus, developmental signaling pathways between R7 and R8 lead to the exclusive expression of a single rhodopsin gene per cell and to the coordinate expression of another one in the neighboring cell. Consistent with this, rh5 expression in R8 disappears when R7 cells are absent (in sevenless mutant) (Papatsenko, 1997).

The most striking observation reported here is that rh5 is expressed in a subset of R8 cells, which corresponds exactly to the R7 cells expressing rh3. This makes it very likely that there is a mechanism for synchronization of rh gene expression between the two cells. Two models are suggested:

(1) Two subclasses of ommatidia may be specified early, at the time of the determination of the eight photoreceptor clusters. Later on, they may express either the rh3/rh5 pair, or the rh4/putative rh6 pair. It must be noted that rh gene expression occurs at least 4 days later than photoreceptor determination. In favor of this model, enhancer trap lines have been identified that are expressed in a subset of R8 cells just posterior to the morphogenetic furrow. Unfortunately, their expression does not last long enough to overlap rh expression. This may either represent an early distinction of different R8 cells during photoreceptor differentiation, or may be simply due to variable expression of a weakly expressing line.
(2) The distinction between the two types of R7/R8 pairs appears late, when the rh genes are first transcribed. Expression of a specific rh gene (in R7 or in R8) may be a stochastic event, which is reinforced by an unknown mechanism, while expression of the other rh gene would be repressed in this cell. Communication between the R7 and R8 cells expressing specific rh pairs (at least one signal going from R7 to R8, as shown by loss of rh5 expression in sev mutants) would permit coordination of expression of the rh genes. It is possible that the Rhodopsin molecules themselves mediate this communication, in a manner that is similar to what has been observed for the olfactory system. The olfactory receptor molecules serve as guidance signals for projection to specific targets in the olfactory bulb (Papatsenko,1997).

Although the absence of rh5 in sevenless mutants does not provide evidence in favor of either model, it may reflect the existence of a positive feedback loop in the interaction between the R8 and R7 cells. Since the R8 cells do not develop properly (rh5 is not expressed) in the absence of R7, a signal from R7 must be necessary to complete retinal development and for the formation of the different subclasses of ommatidia. The sevenless-boss pathway, or some part of it, may be involved in this interaction. Alternatively, a totally distinct pathway may lead to coordination of rh gene expression in R8. It is interesting to mention that physiological data indicate that there are still functional R8 cells in sevenless mutants. Indeed, a photopigment could be detected in the retina of flies lacking both functional R1-6 cells (ninaE mutants) and R7 cells (sev mutants). Thus, an unknown rhodopsin (rh6?), which is predicted to be expressed in the remaining subset of R8 cells (R8y; in concert with rh4 in R7y), could be expressed in all R8 cells in sevenless flies. If this is true, the R7 cell controls the expression of rh5 in R8, and thus the determination of the particular subclass of ommatidia. In other words, the decision for transcriptional exclusion between rh3/5 and rh4/6 would occur in R7, downstream of the boss-sev signaling and upstream of the suggested feedback control from R7 to R8. It must be noted that expression of rh3 in R8 cells of the dorsal margin is not affected in sevenless mutants. This indicates that at least some of the R8 development is not affected by R7 cells. In conclusion, the discrimination between rh3 and rh4 (and between rh5 and putative rh6) expression, and the coordination between pairs of rh genes in neighboring photoreceptors provides a powerful paradigm for studying not only the mechanism of transcriptional exclusion often found in sensory systems, but also for studying the concerted evolution of rhodopsin genes in achieving specific functions, such as color vision (Papatsenko, 1997).

GENE STRUCTURE

Base pairs in 3' UTR - 213

PROTEIN STRUCTURE

Amino Acids - 896

Structural Domains

The boss gene encodes a protein of 896 amino acids with a putative amino-terminal signal sequence, a large extracellular region of 498 amino acids, and seven potential transmembrane domains followed by a carboxy-terminal cytoplasmic tail of 115 amino acids. The putative membrane localization of the BOSS protein is consistent with a model in which direct interaction between the BOSS and Sevenless proteins specify R7 cell fate. No homology to known G protein-linked receptors was found (Hart, 1990).

date revised: 20 June 98

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