Enhancer genetics Problem set E How are specific cell types generated during development There are thought to be 10 000 different types of neurons in the human nervous system How are all of these neural types specified One approach to this problem has been to use genetics in simple invertebrates Drosophila eye development for example is being studied to understand how neurons and support cells are specified The fly eye consists of approximately 800 20 cell repeating units known as ommatidia Each ommatidium consists of eight photoreceptor neurons R1 R8 four lens secreting cone cells and eight additional accessory cells The ommatidia arise from an undifferentiat ed epithelium by a series of cell interactions We will only consider an interaction between the R8 and presumptive R7 cells that determines the fate of R7 The R7 photoreceptor detects light in the UV range Screens for mutants that failed to detect UV light identified genes that were necessary for formation of R7 cells mutants failed to detect UV because they had ommatidia that lacked R7 cells Two of the genes were sevenless sev and bride of sevenless Boss Adult flies homozygous for mutations in these genes have ommatidia that lack an R7 cell and contain an additional cone cell In the absence of R7 differentiation the presumptive R7 cell becomes a cone cell sev is a receptor tyrosine kinase and it acts in R7 to specify R7 s fate boss encodes the ligand for the Sev receptor tyrosine kinase and in contrast to sev acts in R8 cell to specify R7 s fate R7 R8 Sina Sev Boss Model for interactions between R8 and the presumptive R7 cell to specify the fate of R7 BUT investigators knew that because Sev is a receptor tyrosine kinase it must signal to regulate R7 fate Why weren t genes involved in this signaling identified in the screens for UV blind mutants There are several reasons why a screen does not identify genes involved in a particular process First a trivial reason is that the screens are not saturated This is a statistical argument that says if the screen were continued additional genes would be identified Usually if only single mutant alleles exist for genes identified in a screen that means that if the screen is continued other genes will probably be identified However if multiple mutant alleles have been identified for each gene continuing the screen will be less likely to uncover new genes Second when two genes can provide the same function mutating only one of the genes will not lead to a phenotype This type of redundancy is often a concern for geneticists Finally if the genes involved are essential for viability then the screens may not identify mutants because the mutants will die before they express the phenotype of interest This is a big concern when studying the fly eye because it is an adult structure If genes involved in Sev signaling are essential for earlier aspects of development they won t be easily isolated in UV screens 2 This latter concern pops up a lot in developmental genetics One solution is to identify hypomorphic alleles that don t completely disrupt function allowing enough animals to survive to analyze the phenotypes that occur later in development It turns out that the Ras pathway is used by the both the Sev receptor tyrosine kinase in R7 development and by the EGF receptor tyrosine kinase in C elegans vulvval development Mutations in Ras pathway genes in both Drosophila and C elegans result in recessive lethality and preclude isolation of amorphic or null alleles that affect R7 or vulval development because the mutants die before these structures develop In C elegans investigators were able to identify hypomorphic alleles of Ras pathway genes that allowed mutants to survive and have vulval phenotypes for which they were screening Drosophila investigators took a different approach With the objective of identifying genes that encode components that function downstream of the receptor Sev tp regulate R7 fate an elegant screen was designed by Mike Simon and Gerry Rubin in the MCB Department at Berkeley to identify mutations that disrupt this signal transduction pathway F1 animals were screened for dominant enhancers and suppressors of a temperature sensitive sev mutant A temperature sensitive sev receptor tyrosine kinase Temperature sensitive missense mutations in regions of the viral src gene that are conserved between the src and sev tyrosine kinases made it possible 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 P element mediated transposition This mutant gene now encoded a Sev protein that had the same changed amino acid that was in the temperature sensitive Src protein Lines carrying the mutant allele were generated and tested for rescue and temperature sensitivity for the Sevenless phenotype The mutant conferred a ts sev phenotype and was used to isolate enhancers of the Sevenless phenotype A fly carrying one copy of the mutant gene was wild type at 22 7o C R7 was present but mutant at 24 3o C R7 was absent 3 R7 present sev sev P sev ts 0 22 7 R7 absent sev sev P sev ts 0 24 3 R7 absent sev sev P sev ts 0 22 7 look for mutation that confers dominant enhancement of sev phenotype Strategy for isolating dominant enhancers of sev When light is projected though the back of a fly eye the individual photoreceptor cells act as light tubes and seven light transmitting spots represented by dots in the diagram above can be seen in each wild type ommatidium The seven spots are R1 R7 photoreceptor cells R8 is located deeper in the eye and doesn t show up in this assay Although this strain is wild type when raised at 22 7o C it is presumably near the threshold were Sev activity becomes limiting The rationale for the screen is that a 50 reduction in a gene product playing a role 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 as dominant enhancers of the ts sev mutant sev Y sev sev P sev ts balancer sev P sev ts 0 screen for absence of R7 Figure 2 Screen for enhancers of sev E sev This approach identified mutations in seven genes Mutations in all of these genes result in a recessive lethal phenotype precluding the analysis of fly eye phenotypes in homozygous mutants The screen thus identified genes that it was designed to detect 4 Two of these genes were earmarked for further study because they acted in a second receptor tyrosine kinase
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