Genetic Studies in Drosophila Identified Key Signal-Transducing Proteins in the Ras/MAP Kinase Pathway

Our knowledge of the proteins involved in the Ras/MAP kinase pathway came principally from genetic analyses of mutant fruit flies (Drosophila) and roundworms (C. elegans) that were blocked at particular stages of differentiation. To illustrate the power of this experimental approach, let’s consider the development of a particular type of cell in the compound eye of Drosophila.

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The compound eye of the fruit fly is composed of some 800 individual eyes called ommatidia (Figure 16-19a). Each ommatidium consists of twenty-two cells, eight of which are photosensitive neurons called retinula, or R cells, designated R1–R8 (Figure 16-19b). An RTK called Sevenless (Sev) is specifically expressed in, and is essential for development of, the R7 cell; Sev is not required for any other known function. In flies with a mutant sevenless (sev) gene, the R7 cell in each ommatidium fails to form (Figure 16-19c, bottom). But since the R7 photoreceptor is necessary for flies to see only in ultraviolet light, mutants that lack functional R7 cells but are otherwise normal are easily isolated. Therefore, fly R7 cells are an ideal genetic system for studying signal transduction downstream of an RTK.

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FIGURE 16-19 The compound eye of Drosophila melanogaster. (a) Scanning electron micrograph showing the individual ommatidia that compose the fruit fly eye. (b) Longitudinal and cutaway views of a single ommatidium. Each of these tubular structures contains eight photoreceptors, designated R1–R8, which are long, cylindrically shaped light-sensitive cells. R1–R6 (yellow) extend throughout the depth of the retina, whereas R7 (brown) is located toward the surface of the eye and R8 (blue) toward the back side, where the axons exit. (c) Comparison of eyes from wild-type and sevenless mutant flies viewed by a special technique that can distinguish the photoreceptors in an ommatidium. The plane of sectioning is indicated by the blue arrows in (b), and the R8 cell is out of the plane of these images. The seven photoreceptors in this plane are easily seen in the wild-type ommatidia (top), whereas only six are visible in the mutant ommatidia (bottom). The eyes of flies with the sevenless mutation lack the R7 cell.
[Part (a) Cheryl Power/Science Source; part (c) courtesy of Utpal Banerjee, UCLA.]

During the development of each ommatidium, a protein called Boss (Bride of Sevenless) is expressed on the surface of the R8 cell. The extracellular domain of this membrane-tethered protein is the ligand for the Sev RTK on the surface of the neighboring R7 precursor cell, signaling it to develop into a photosensitive neuron (Figure 16-20a). In mutant flies that do not express a functional Boss protein or Sev RTK, interaction between the Boss and Sev proteins cannot occur, and no R7 cells develop (Figure 16-20b); this result is the origin of the name “Sevenless” for the RTK in the R7 cells.

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EXPERIMENTAL FIGURE 16-20 Genetic studies reveal that activation of Ras induces development of R7 photoreceptors in the Drosophila eye. (a) During larval development of wild-type flies, the R8 cell in each developing ommatidium expresses a cell-surface protein, called Boss, which binds to the Sev RTK on the surface of its neighboring R7 precursor cell. This interaction induces changes in gene expression that result in differentiation of the precursor cell into a functional R7 neuron. (b) In fly embryos with a mutation in the sevenless (sev) gene, R7 precursor cells cannot bind Boss and therefore do not differentiate normally into R7 cells. Rather, the precursor cell enters an alternative developmental pathway and eventually becomes a cone cell. (c) Double-mutant larvae (sev–/–; RasD) express a constitutively active Ras (RasD) in the R7 precursor cell, which induces differentiation of R7 precursor cells in the absence of the Boss-mediated signal. This finding shows that activated Ras is sufficient to mediate induction of an R7 cell. See M. A. Simon et al., 1991, Cell 67:701, and M. E. Fortini et al., 1992, Nature 355:559.

To identify intracellular signal-transducing proteins in the Sev RTK pathway, investigators produced mutant flies expressing a temperature-sensitive Sev protein. When these flies were maintained at a permissive temperature, all their ommatidia contained R7 cells; when they were maintained at a nonpermissive temperature, no R7 cells developed. At a particular intermediate temperature, however, just enough of the Sev RTK was functional to mediate normal R7 development. The investigators reasoned that at this intermediate temperature, the signaling pathway would become defective (and thus no R7 cells would develop) if the level of another protein involved in the pathway was reduced, thereby reducing the activity of the overall pathway below the level required to form an R7 cell. A recessive mutation affecting such a protein would have this effect because, in diploid organisms such as Drosophila, a heterozygote containing one wild-type and one mutant allele of a gene will produce half the normal amount of the gene product; hence, even if such a recessive mutation is in an essential gene, the organism will usually be viable. However, a fly carrying a temperature-sensitive mutation in the sev gene and a second mutation affecting another protein in the signaling pathway would be expected to lack R7 cells at the intermediate temperature.

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By use of this screen, researchers identified three genes encoding important proteins in the Sev pathway: an SH2-containing protein exhibiting 64 percent amino acid sequence identity to human GRB2 (growth factor receptor–bound protein 2), a GEF called Sos (Son of Sevenless) exhibiting 45 percent identity with its mouse counterpart, and a Ras protein exhibiting 80 percent identity with its mammalian counterparts. These three proteins were later found to function in other signaling pathways initiated by ligand binding to other RTKs and to be used at different times and places in the developing fly.

In subsequent studies, researchers introduced a mutant rasD gene into fly embryos carrying a mutation in the sevenless gene. As noted earlier, the rasD gene encodes a constitutively active Ras protein that is present in the active GTP-bound form even in the absence of a hormone signal. Although no functional Sev RTK was expressed in these double mutants (sev−/−; rasD), R7 cells formed normally, indicating that the presence of an activated Ras protein is sufficient for induction of R7-cell development (Figure 16-20c). This finding, which is consistent with the results of experiments with cultured fibroblasts described earlier, supports the conclusion that activation of Ras is a principal step in intracellular signaling by most, if not all, RTKs and cytokine receptors.