Reinke, R. and S. L. Zipursky. 1988. Cell-cell interaction in the Drosophila retina: the bride of sevenless gene is required in photoreceptor cell R8 for R7 cell development. Cell 55: 321-330. The authors identify a new gene, boss, required for R7 cell development. Mosaic analysis demonstrates the function of this gene in the neighboring R8 cell, revealing a signaling pathway central for ommitidial assembly.

Questions for Thought

1. Briefly describe the mutagenesis screen used to identify boss. Why was boss the only gene identified in this screen? Is 10,000 a lot of flies? Why didn't the authors obtain mutations in sevenless? What other types of genes could have been identified by such a screen? How would you distinguish those genes from genes involved in this signaling cascade?

Why did they make so many additional alleles of boss?

Why did the authors make double mutants with sev? That is, what is the logic behind making double mutants with mutations that cause the same phenotype?

2. We will go through Figure 4 in detail. How are mosaic patches generated in the eye? When are clones induced? Why irradiate at that time? Why do the authors use two markers, chaoptic and white?

Figure 4C and Table 1: How far can boss diffuse? Which ommitidia demonstrate this principle? How would the data in Table 1 appear if boss could be expressed in R8 or R1 or R6?

If suitable markers existed, could this method be used in C. elegans? Why or why not?

3. Calvin Bridges started out in Thomas Hunt Morgan's lab as chief bottle washer. When he began to do research, he did all of his experiments in a big way (GO BIG OR STAY HOME!). In fact, while studying the inheritance pattern of the white gene, he was able to prove the chromosome theory of inheritance because he looked at so many flies and was able to find those rare few whose pattern of inheritance differed from the rest. These "exceptions" proved the rule.

Calvin's grandson got a job in Larry Zipursky's lab. He decided to repeat Rosemary Reinke's mosaic analyses, but following in his grandfather's footsteps, he wanted to look at lots of flies. He used the exact same protocol that Reinke used, but looked at 30,000 flies. Using the w+ marker, he identified 300 flies that exhibited mosaic patches. Most of these flies gave the same result that Reinke saw, that is, if R8 was mutant for chaoptic, R7 failed to develop. Six flies, however, did not show the same phenotype. Instead, ommitidia with chaoptic R8 cells still had clear R7 cells. Explain this paradoxical result. (Hint: not the same reason that Bridges saw exceptions.)

4. You have cloned the boss gene and have both an RNA probe and an antibody to Boss protein. Would you still do mosaic analysis, or would you simply look at the RNA and protein expression patterns? Think about the work involved to do transmission EMs, as done here. In situ hybridization and immunohistochemistry can be performed on whole tissues, not sections, and would take about a week total. Does that change your mind?

5. Here is a gene, boss, that must be expressed in R8 for R7 to develop properly. R8 touches all of the photoreceptor cells and sev is expressed not only in R7 but in all other cells except R2, R5 and R8. Why don't all the cells that express sev become R7 cells? Suggest experiments to test your hypotheses.