Enhancer geneticsProblem set EHow are specific cell types generated during development? There arethought to be 10,000 different types of neurons in the human nervoussystem. How are all of these neural types specified? One approach to thisproblem has been to use genetics in simple invertebrates. Drosophila eyedevelopment, for example, is being studied to understand how neuronsand support cells are specified. The fly eye consists of approximately 80020-cell repeating units known as ommatidia. Each ommatidium consists ofeight photoreceptor neurons (R1-R8), four lens secreting cone cells andeightadditionalaccessorycells. Theommatidiaarise from anundifferentiated epitheliumby a series ofcellinteractions.We will onlyconsider aninteractionbetween theR8 andpresumptiveR7 cells thatdeterminesthe fate of R7.The R7photoreceptordetects lightin the UVrange.Screens formutants thatfailed todetect UVlightidentified2genes that were necessary for formation of R7 cells: mutants failed todetect UV because they had ommatidia that lacked R7 cells. Two of thegenes were sevenless (sev) and bride of sevenless (Boss). Adult flieshomozygous for mutations in these genes have ommatidia that lack an R7cell and contain an additional cone cell. In the absence of R7differentiation, the presumptive R7 cell becomes a cone cell. sev is areceptor tyrosine kinase, and it acts in R7 to specify R7's fate. boss encodesthe ligand for the Sev receptor tyrosine kinase, and in contrast to sev, actsin R8 cell to specify R7's fate.R8SinaSevBossR7?Model for interactions between R8 and the presumptive R7 cell to specifythe fate of R7BUT, investigators knew that because Sev is a receptor tyrosinekinase, it must signal to regulate R7 fate. Why weren’t genes involved inthis signaling identified in the screens for UV blind mutants. There areseveral reasons why a screen does not identify genes involved in aparticular process. First, a trivial reason is that the screens are notsaturated. This is a statistical argument that says if the screen werecontinued additional genes would be identified. Usually, if only singlemutant alleles exist for genes identified in a screen, that means that if thescreen is continued, other genes will probably be identified. However, ifmultiple mutant alleles have been identified for each gene, continuing thescreen will be less likely to uncover new genes. Second, when two genescan provide the same function, mutating only one of the genes will notlead to a phenotype. This type of redundancy is often a concern forgeneticists. Finally, if the genes involved are essential for viability, then thescreens may not identify mutants because the mutants will die before theyexpress the phenotype of interest. This is a big concern when studying thefly eye because it is an adult structure. If genes involved in Sev signalingare essential for earlier aspects of development, they won’t be easilyisolated in UV screens.3This latter concern pops up a lot in developmental genetics. Onesolution is to identify hypomorphic alleles that don’t completely disruptfunction, allowing enough animals to survive to analyze the phenotypesthat occur later in development. It turns out that the Ras pathway is usedby the both the Sev receptor tyrosine kinase in R7 development and by theEGF receptor tyrosine kinase in C. elegans vulvval development. Mutationsin Ras pathway genes in both Drosophila and C. elegans result in recessivelethality and preclude isolation of amorphic or null alleles that affect R7 orvulval development because the mutants die before these structuresdevelop. In C. elegans, investigators were able to identify hypomorphicalleles of Ras pathway genes that allowed mutants to survive and havevulval phenotypes for which they were screening. Drosophilainvestigators took a different approach.With the objective of identifying genes that encode components thatfunction downstream of the receptor Sev tp regulate R7 fate, an elegantscreen was designed by Mike Simon and Gerry Rubin in the MCBDepartment at Berkeley to identify mutations that disrupt this signaltransduction pathway. F1 animals were screened for dominant enhancersand suppressors of a temperature-sensitive sev mutant.A temperature-sensitive sev receptor tyrosine kinaseTemperature-sensitive missense mutations in regions of the viral src genethat are conserved between the src and sev tyrosine kinases made itpossible to generate ts alleles of sev. Mutations were made in vitro in sev,and the mutant sev genes were reintroduced into a sev mutant by Pelement mediated transposition. (This mutant gene now encoded a Sevprotein that had the same changed amino acid that was in thetemperature-sensitive Src protein.) Lines carrying the mutant allele weregenerated and tested for rescue and temperature sensitivity for theSevenless phenotype. The mutant conferred a ts sev phenotype and wasused to isolate enhancers of the Sevenless phenotype. A fly carrying onecopy of the mutant gene was wild type at 22.7o C (R7 was present) butmutant at 24.3o C (R7 was absent).4R7 presentR7 absentR7 absentsev/sev; +/+; P[sev-ts]/0 @ 22.7sev/sev; */+; P[sev-ts]/0 @ 22.7look for mutation (*) that confersdominant enhancement of sev phenotypesev/sev; +/+; P[sev-ts]/0 @ 24.3Strategy for isolating dominant enhancers of sev. When light is projectedthough the back of a fly eye, the individual photoreceptor cells act as lighttubes and seven light transmitting spots, represented by dots in thediagram above, can be seen in each wild-type ommatidium. The sevenspots are R1-R7 photoreceptor cells. R8 is located deeper in the eye, anddoesn't show up in this assay.Although this strain is wild type when raised at 22.7o C, it ispresumably near the threshold were Sev activity becomes limiting. Therationale for the screen is that a 50% reduction in a gene product playing arole in the sev pathway may tilt the balance to the Sev mutant phenotype.Loss-of-function mutations in genes in the sev pathway would act asdominant enhancers of the ts sev mutant.sev/Y; +/+; +/+ sev/sev; +/+; P[sev-ts]/ balancersev; */+; P[sev-ts]/0 screen for absence of R7Figure 2. Screen for enhancers of sev [E(sev)]This approach identified mutations in seven genes. Mutations in allof these genes result in a recessive lethal phenotype, precluding theanalysis of fly eye phenotypes in homozygous mutants. The screen thusidentified genes that it was designed to detect.5Two of these genes were earmarked for further study because
View Full Document
Unlocking...