1. Lab Meeting #1 9-13-2010

2. Expression profiles and transcriptional networks in the CNS midline Wheeler et al., 2006

3. High-throughput sequencing of the transcriptome Sequence all fragments Isolate RNAFragment RNA Generate adapter- ligated cDNA

4. Steps to RNA-seq Isolate Cells  Purify RNA  Prepare cDNA  Sequence  Analyze Find midline specific GAL4 driver Stage embryos @ 25 o C Dissociate cells Sort cells via FACS

5. Quest for a midline specific driver Strong GFP expression in midline No (or very little) GFP expression outside of midline Expressed early, stays on late 3.7sim-GAL4 > UAS-mCD8-GFP (LL5) 1.0sim-GAL4 > UAS-mCD8-GFP (LL5) sema1b-I1F3sC-GAL4 > UAS-mCD8-GFP (LL5) 3.7sim-GAL4 > UAS-tau-GFP on Chromosome II

6. 3.7sim > LL5

8. 1.0sim > LL5

9. sema1b > LL5

10. 3.7sim > tauGFP on II

11. Unforeseen problem with 3.7sim > tau-GFP on II Embryos @ 25 o C for 14 -16 hours : all should be stage 16 – 17 Actual:56%  Stage 16+ 14%  Stage 13-16 9%  Stage 11-12 21%  Dead N=132 Possible quick fix : 3.7sim > tau-GFP on III Problem: Flies do not lay very well on plates

12. Lysosomal dysfunction promotes cleavage and neurotoxicity of tau in vivo. Phosphorylation differentiates tau-dependent neuronal toxicity and dysfunction. Differential effects of Tau on the integrity and function of neurons essential for learning in Drosophila. Tau and tau reporters disrupt central projections of sensory neurons in Drosophila.

13. Alternatives to tau-GFP 3.7sim-GAL4 UAS-mCD8-GFP (LL4) : on X UAS-mCD8-GFP (LL5) : on II UAS-mCD8-GFP (LL6) : on III Ectopic expression outside midline Specific, but weak expression Strong and specific

14. 3.7sim > LL6

16. 3.7sim-GAL4/CyO ; UAS-mCD8-GFP-LL6 17-21 hrs @ 25 o C: all should be stage 17 or later 2%  Stage 13-14 84%  Stage 17+ 14%  Dead N = 120 3.7sim-GAL4 ; UAS-mCD8-GFP-LL6 16-18 hrs @ 25oC: all should be stage 17+ 97%  Stage 17+ 3%  Dead N = 91

17. Cell Dissociation

18. With 3.7sim > tau-GFP 2 hr collection  ~75 – 100 uL of embryos ~20,000 cells / uL or ~20 x 10 6 cells / collection

19. Fluorescence Activated Cell Sorting (FACS)

20. w 1118 3.7sim > tau-GFPpresort Presort with PI Sorted into FACS bufferSorted into PBSFACS buffer

21. Current Status Isolate Cells  Purify RNA  Prepare cDNA  Sequence  Analyze Find midline specific driver Stage embryos Dissociate cells Sort cells via FACS

22. Future Direction 3.7sim-QF > QUAS-mtdTomato-3xHA CoolEnhancer-GAL4 > UAS-GFP

23. CG32105  Otx (orthodenticle, ocelliless) Lmx1a  Msx1 Nkx6.1 (HGTX)  (indirectly) Ngn2, (tap) TH, (pale) Nurr1, (Hr38) Pitx3 (Ptx1)

25. * * * * 11 Runt CG32105 3.7sim > tauGFP T1T2

26. 1 1 * * * * Runt CG32105 3.7sim > tauGFP Abdominal segments

27. A1A2T2T3 Runt CG32105 * T2 1 T3 * 1 A1A2 * * * 1 1 1

28. A1A2A1A2 Runt CG32105 A1A2 * * 1 1 A1A2 * * * 1 1 1

29. A3A4A5 Runt CG32105 * * A3 1 * * * A4 1 * * (*) A5 1

30. Stage 11 * * * * * * * * * Stage 12-anterior segments zfh1 CG32105

31. A7 A8 * * * * * * * * * zfh1 CG32105 *

32. Summary of CG32105 expression Stage 11 – not expressed Stage 12 – expressed in mVUMs of all segments looked at mVUM4 and 5 early mVUM4, 5, and 6 later Stage 15-16 – not expressed in thoracic mVUMs may be expressed in 1-2 thoracic MNBp expressed in abdominal mVUMs -maybe not all mVUMs in anterior abdominal segments

33. will die slowly (wds)

34. w 1118

37. elav-GAL4 > UAS-wds