Lab Meeting #1

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 Generate adapter- ligated cDNA Isolate RNA Fragment RNA

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

5. Strong GFP expression in midline No (or very little) GFP expression outside of midline Expressed early, stays on late Quest for a midline specific driver 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 3.7sim > LL5 3.7sim > LL5

7. 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 @ 25oC 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 Specific, but weak expression Ectopic expression outside midline Strong and specific

14. 3.7sim > LL6

15. 3.7sim > LL6

16. 3.7sim-GAL4/CyO ; UAS-mCD8-GFP-LL6 17-21 hrs @ 25oC: 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. Cell Dissociation With 3.7sim > tau-GFP 2 hr collection  ~75 – 100 uL of embryos ~20,000 cells / uL or ~20 x 106 cells / collection

19. Fluorescence Activated Cell Sorting(FACS)

20. w1118 3.7sim > tau-GFP presort Presort with PI Sorted into FACS buffer Sorted into PBS FACS 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)   Nkx6.1 (HGTX) Lmx1a Msx1  (indirectly) Ngn2, (tap) TH, (pale) Nurr1, (Hr38) Pitx3 (Ptx1)

25. Runt T1 T2 CG32105 3.7sim > tauGFP * 1 1 * * *

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

27. T2 T3 Runt CG32105 * * 1 1 1 * 1 1 * * A1 A2 T2 A1 A2 T3

28. A1 A2 Runt CG32105 1 1 1 1 * 1 * * * * A1 A2 A1 A2 A1 A2

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

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

31. * * * * * * * * * * A7 A8 A7 A8 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. w1118

35. w1118

36. w1118

37. elav-GAL4 > UAS-wds