Determining the cause of defective reproductive tract development and retained Drosophila melanogaster
Download ReportTranscript Determining the cause of defective reproductive tract development and retained Drosophila melanogaster
Determining the cause of defective reproductive tract development and sterility in retained mutant female Drosophila melanogaster Jennifer Coppersmith Dr. Barbara Taylor Department of Zoology Oregon State University “Why can’t a woman be more like a man?... Why can’t a woman behave like a man?” – Rex Harrison, My fair lady (1964) What makes males and females look and behave differently? ♀ ♂ ♂ ♀ ♂ ♀ How do reproductive tracts develop in females and how does improper development affect the sterility and behavior of that species. Why Fruit Flies? Easy maintenance Fast life cycle Entire genome is well known Model Organism Mutations How Mutations are Created P-transposable elements (P-elements): are DNA sequences, which can insert randomly into chromosomes Requires enzyme transposase for removal and reinsertion. Can cause mutations that allow us to make genomic clones to study the expression pattern of that gene P-element Normal Gene Insertion Disrupted Gene retained Gene Development 132, 155-164 (2005) Published by The Company of Biologist 2005 P-Element Strong mutations, prevents the gene from transcription, therefore no expression. Results in embryonic lethal Weak mutations, a single amino acid change (missense). Adults viable, but female sterile with abnormalities in courtship behavior. Female Reproductive System Genital Disc Keisman, E.L., Christiansen, A.E., and Baker, B.S. (2001). Stages and development of the reproductive system with in the female fruit fly Ovaries Oviducts Uterus Volker Hartenstein, published by Cold Spring Harbor Laboratory Press, 1993 Determine the cause of sterility in retn mutants How is the development of the reproductive tract different in the retn mutants compared to wild type? Methods and materials Dissect out reproductive tract or genital disc, then stain for expression pattern using immunohistochemistry. Dissection After staining, analyze and compare normal vs. mutant using CCD Camera to capture images. Analyze Method (cont’d) Immunohistochemistry Useful in determining pattern of expression of genes Fluorescent/staining tag Secondary antibody Primary antibody CELL Tissue antigen Gene Expression retnDac Hth Abd-B Genital Disc Reproductive difference between Wild Type and mutant genotypes Wild Type retn- dac- AbdB0569 Fertility Fertile Not fertile ----- Not Fertile Courtship / Copulation Normal Abnormal ------ Abnormal Reproductive tract Normal Abnormal Abnormal structure structure Marker Staining Fas III + Abd B + Dac + Fas III +/Abd B + Dac + ------------- Normal Fas III +/ Abd B + ----- Comparing Wild Type vs. retn Mutant Average Surface Area Oviduct Ovaries 800 700 Mcd8 CSA R044/dri8 z2426/dri8 R044 Ovaries Surface Area 600 Oviduct 500 400 300 200 100 0 1 2 3 4 5 Oviduct Genotype Uterus Normal (Wild Type) fruit fly reproductive tract Uterus Retn mutant fruit fly reproductive tract Fas III staining Wild type B0569 Calyx retn- Abd- Diagram of oviducts and egg laying retn- Wild Type EGG Calyx Abd-B0569 Genetic Crosses retn+ retn+ XX+ retn+ X retnretn+ retn- (punnett squares) X+ X+ Genetic interaction? Fertile and normal reproductive tract retnX+ X- X- retn- X- Sterile and abnormal reproductive tract Determining possible Genetic interactions retndri8/ dac4 Fertility Courtship / Copulation Reproductive tract retnR044 /dac retnR044/ rk364 Hth retnR044 /Abd-B05659 Fertile Low Fertility Fertile Normal Normal Normal Fertile Normal Normal Normal Normal Normal Summery of Data •Has an abnormal reproductive structure and Fas III staining •Suggest that Abd B and Dac are not regulated by retn •Possible genetic interaction with Dac rk364 Determine the cause of sterility in retn mutants Can we recreate this oviduct abnormality and sterility by removing retn function by using selective RNA interfere in the female reproductive tract or through genetic interaction studies? retn RNAi target the retn transcription, and destroys it. Future Work Perform more Fas III staining to verify results Waiting for flies to emerge from crosses to further test sterility and observe possible defects in reproductive tract Setting up crosses with RNAi’s to recreate Sterility and abnormal reproductive tracts. Dr. Barbara Taylor (department of Zoology) Howard Hughes Medical Institute Dr. Kevin Ahern (department of biochemistry/biophysics) Everyone working in the Taylor Lab! "Time's fun when you're having flies." -- Kermit the Frog'