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Genetics discussion “Sex in the Brain” 11/02/2011 Drosophila/O’Farrell Hotta and Benzer published a beautiful and classical analysis in 1972 in which they made mosaic flies that were half male and half female (attached). These are gynandromorphs. They examined their behavior in mating assays and were able to “map” the behaviors to areas in the brain. While really elegant and highly recommended, I didn’t use this paper for the discussion because it is very much dated. Nonetheless, the assigned paper tackles a similar issue at much higher resolution – can we define the neuronal and genetic basis of specific behaviors? Kimura K, Hachiya T, Koganezawa M, Tazawa T, Yamamoto D. (2008) Fruitless and doublesex coordinate to generate male-specific neurons that can initiate courtship. Neuron 59: 759-69 I have included a review (Siwicki and Kravitz, 2009) that might help get you in gear to read this paper and I suggest that you look at the first two pages of the review before tackling the paper. Also the paper uses a fancy version of mosaics (clonal analysis) in which things are turned around so that the clones specifically activate expression of a gene. This is call the MARCM technique. I am planning on presenting it in lecture but realized that this comes after your discussion (I might try an advance it, but right now I don’t see how it fits). I have attached a brief technical account of the method. It is based on clonal loss of GAL80, which is a repressor of GAL4. The authors mention in an easily overlooked spot an important detail in their descriptions of their genotypes. The thing they describe as a fruNP21reporter has GAL4 under the control of the fru promoter. It is not directly a reporter, but it induces genes that are placed under UAS control (in flies when we say UAS it is short for UASGAL4), and of course only induces these when the fruNP21-GAL4 is expressed and when the GAL4 is active. Importantly, the GAL4 is only active when clones, induced by action of heat shock induced Flip recombinase expression, remove the GAL80 repressor (tub in tub-GAL80 refers to the tubulin promoter which is ubiquitously expressed – e.g GAL80 is everywhere unless a mitotic clone removes the chromosome arm carrying this transgene). The UAS construct that they use to make the cells green is called UAS-mCD8-GFP. The mCD8 fusion makes it membrane bound so that it marks the full path of the neurons. It sounds pretty complicated and they don’t really say why they are doing all this just to define the cells that express fru. The reason is that so many cells express fru that the brain would be an indiscernible mass of stain if they were to all express at the same time. By making clones they can see individual cells and their processes in each clone and they add up the result from many clones to define the whole. I’ve appended the supplement to the PDF of the paper. In addition there was a movie that I’ve include as separate quicktime file if all the stuff gets uploaded. The movie is nice but just for fun. The text describing it was: Movie S1. Male-Type Sexual Behavior Exhibited by a tra1 Mosaic Female. A tra1 mosaic female with red eyes follows a wild-type female with white eyes and occasionally extends one of her wings. Embryos of the y hs-flp/w; G13 UAS-mCD8-GFP; tra1 ri FRT2A fruNP21/tub-Gal80 FRT2A genotype were exposed to heat shock at 37°C for 1 hr to induce somatic recombination. The last frame of the video shows the brain from the same mosaic female whose courtship behavior was recorded in the video. Double staining of the brain with the anti-GFP antibody and nc82 Mab showed that the female fly had P1 neurons as a result of the tra− mediated masculinization, as shown by GFP expression. I hope this is enough for you go with. Try and understand how the genetic tools were used, the paper spends most of its energy explaining the biology of the work. Pat