Biol 466 Toll-7 Project 1

Determining the role of Toll-7 in Drosophila melanogaster through RNAi

Biol466, Spring 2004

Cassandra Kleve

Biol 466 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.

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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

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Extra-cellular

Domain

Cytosol

TIR

domainTauszig et al. 2000

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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.

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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

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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 Toll 7

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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

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-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!

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Balancer Chromosomes

-1- Have a dominant morphological mutation

-2- Have a number of inversions to prevent recombination

-3- Are homozygous lethal

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Sample Cross:

- A minimum of 10 crosses will be set

- This will be repeated with a second insertion of the Toll-7 construct

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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

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Toll-7 insertionSP6 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

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The antisense probes will hybridize to Toll-7 mRNA produced in the cell

Toll-7 insertionSP6 T7

Cleaved by restriction enzyme

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Sense probes will be a control for nonspecific staining

Toll-7 insertionSP6 T7

Cleaved by restriction enzyme

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(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.

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(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

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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.

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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.

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(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

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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