1. 1Biol 466Toll-7 Project Determining the role of Toll-7 in Drosophila melanogaster through RNAi Biol466, Spring 2004 Cassandra Kleve

2. 2Biol 466Toll-7 Project What is a Toll gene? -Toll-related receptors are named for their sequential and structural similarity with Toll -Toll was discovered during a mutagenesis screen and found to have a role in dorsal ventral patterning of the embryo -It was later learned that it had a role responding to infection.

3. 3Biol 466Toll-7 Project Mammalian TLRs (Toll-like receptors) -The most conserved region of the genes is the TIR domain -Stands for Toll/interleukin-1 receptor -Mammalian Toll-like receptors respond to distinct microbial patterns -TLR4: bacterial cell-wall LPS -TLR3: viral dsRNA

4. 4Biol 466Toll-7 Project Extra-cellular Domain Cytosol TIR domain Tauszig et al. 2000

5. 5Biol 466Toll-7 Project Why Toll-7? - Mutations in 18-wheeler cause death during larval development with no obvious morphological defect -Sequence similarity and close proximity to 18- wheeler on the chromosome make Toll-7 a candidate for a compensating gene.

6. 6Biol 466Toll-7 Project Determining the Role of Toll-7 -A convenient method for creating “knockdown” mutants of Toll-7 is through RNAi -RNAi is thought to work through the processing of dsRNA into 21-23 bp fragments of small interfering RNA (siRNA) -Catalyze the cleavage of the complementary mRNA -Cause a functional loss of Toll-7

7. 7Biol 466Toll-7 Project Preliminary Data -The GAL4/UAS system can be utilized to control the production of the dsRNA -Three lines of flies with the P-element vector, pWIZ, with the Toll-7 insertion will be used for these experiments UAS Promoter intron GAL4 Toll 7

8. 8Biol 466Toll-7 Project Project goals: -1- Demonstrate the production of siRNA in the presence of GAL4 -2- Show Toll-7 mRNA degradation in tissues producing GAL4 -3- Examine embryos for a defect present in the absence of Toll-7

9. 9Biol 466Toll-7 Project -The RNAi will become activated when flies with the Toll-7 construct are crossed with GAL4 lines - Using flies that produce GAL4 in specific tissues will allow us to cause Toll-7 deficiencies in specific tissues Problem with these crosses: both the GAL4 and Toll-7 insertions are labeled with red eyes!

10. 10Biol 466Toll-7 Project Balancer Chromosomes -1- Have a dominant morphological mutation -2- Have a number of inversions to prevent recombination -3- Are homozygous lethal

11. 11Biol 466Toll-7 Project Sample Cross: - A minimum of 10 crosses will be set - This will be repeated with a second insertion of the Toll-7 construct

12. 12Biol 466Toll-7 Project Toll-7 RNA probes - Toll-7 mRNA will be detected using digoxygenin labeled RNA probes - These will be synthesized as run off transcripts from the pSPT19 vector with a Toll-7 insertion - Two different fragments of Toll-7 will be used - an extracellular fragment that was inserted into pWIZ - a fragment from the TIR domain

13. 13Biol 466Toll-7 Project Toll-7 insertion SP6 T7 Using the SP6 or T7 promoters on either end of the Toll-7 insertion will allow us to make sense and antisense probes For the transcription of run-off transcripts the vector is digested on either end of the insertion

14. 14Biol 466Toll-7 Project The antisense probes will hybridize to Toll-7 mRNA produced in the cell Toll-7 insertion SP6 T7 Cleaved by restriction enzyme

15. 15Biol 466Toll-7 Project Sense probes will be a control for nonspecific staining Toll-7 insertion SP6 T7 Cleaved by restriction enzyme

16. 16Biol 466Toll-7 Project (1) Test for the production of siRNA - Cross the Toll-7 construct into the da-GAL4 line - daughterless is expressed ubiquitously throughout development -A northern blot analysis should reveal the presence of 21-23 bp fragments that hybridize to both sense and antisense probes Roignant et al.

17. 17Biol 466Toll-7 Project (2) Toll-7 mRNA degradation - For flies that emerge from the da-GAL4 cross we will use northern blots to measure the level of Toll-7 mRNA present -We would expect to see less Toll-7 mRNA in flies with the Toll-7/da-GAL4 combination than without - We can also use tissue specific GAL4 lines -For these we would perform in situ hybridizations to evaluate Toll-7 mRNA levels

18. 18Biol 466Toll-7 Project Toll-7 appears to be expressed in the CNS - We will cross the Toll-7 construct into flies that produce GAL4 in the CNS - If Toll-7 has a role in the the development of the CNS we would expect to see developmental abnormalities Kambris et al.

19. 19Biol 466Toll-7 Project Tissue specific Toll-7 mRNA degradation - GAL4 can be located using anti-GAL4 antibodies - Toll-7 will be expressed normally in tissues that do not produce GAL4 - Toll-7 should not be present in tissues producing GAL4 Kambris et al.

20. 20Biol 466Toll-7 Project (3) Do we see a defect? - Lethality of flies with both the Toll-7 construct and GAL4 driver could be one of the first signs of a defect - Embryos resulting from the CNS GAL4 crosses will be stained with antibodies to label the CNS to screen for developmental abnormalities - The strongest phenotype would be present in flies crossed with the da-GAL4 driver - Abnormalities may be more apparent in flies with multiple copies of the Toll-7 construct or GAL4 insertion

21. 21Biol 466Toll-7 Project Acknowledgements: - Dr. Eldon and the lab - Dr. Carthew’s lab at Northwestern University for pWIZ - Dr. Marsh’s lab at UCI for help with the embryo injections - Howard Hughes Medical Institute