Determining the role of Toll-7 in Drosophila melanogaster through RNAi Biol466, Spring 2004

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

2. 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. Toll-7 Project

3. 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 Toll-7 Project

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

5. 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. Toll-7 Project

6. 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 Toll-7 Project

7. GAL4 Toll 7 Toll 7 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 Toll-7 Project

8. 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 Toll-7 Project

9. 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! Toll-7 Project

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

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

12. 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 Toll-7 Project

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

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

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

16. (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. Toll-7 Project

17. (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 Toll-7 Project

18. Toll-7 appears to be expressed in the CNS Kambris et al. - 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 Toll-7 Project

19. 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. Toll-7 Project

20. (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 Toll-7 Project

21. 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 Toll-7 Project