1. Transcriptional regulation of tracheal fusion cell gene expression Isolate enhancer of three fusion cell expressed genes: dys, CG15252, CG13196 Identify CRM required for fusion cell gene expression Transcriptional pathways in terminally differentiating neurons Glutamatergic (H-cell sib )and GABAergic (iVUM) neurons Transcriptional regulation of vglut expression in H-cell sib Transcriptional regulation gene expression in iVUM neurons

2. Identify dys Fusion cell Enhancer B2.7 C1.7 P155 T523 dys early enhancer (S12-S17) dys late enhancer (S14-S17)

3. Sequence alignment of dys155

4. GATCGG TTAAATCAAA TGCCTGAC Ets GACCCAAAAC GCGCG POU 1 2 3 4 5 6 7 1 2 3 4 5 6 7 Motifs Required for dys Activation 12 3 456 7 dys155

5. WTpnt Dys Ets protein Pointed is required for dys expression in fusion cells

6. Sequence of alignment of dys523 late enhancer

7. dys late enhancer is auto-regulated by Dys protein dys P155C1.7

8. D92 CG13196 Identify CG13196 Fusion cell Enhancer

9. Sequence alignment of CG16196 enhancer

10. GFPDysMerge Unmutated D92muC D92muD D92muAAG D92muMotif8 TCGTG-DTCGTG-CAAG Motif 8 Motifs Required for CG13196 Expression

11. CG15252 C100 Identify CG15252 Fusion cell Enhancer

12. Sequence alignment of CG15252 enhancer

13. CG15252 C100 TCGTG-DTCGTG-C AAGMotif 7AAG GFPDysMerge Unmutated C100muC C100muD C100muAAG C100muMotif9 Motifs Required for CG15252 Activation

14. xx A350muBC CG13196 C100 CG15252 C100muCD CG15252 A350 CG13196 Co-activator sequences required for CG13196 expression

15. Transcriptional pathways in terminally differentiating neurons Sim Sim + En

16. Gene expression porfile in Glutamatergic and GABAergic interneurons TF gene expression H-cell sib sim per fkh nvy Lim1 iVUM sim per fkh nvy Lim1 * en NF gene expression H-cell sib Vglut GluR1 5-HT 1A CG13565 iVUM Gad1 VIAAT(CG8394) CG15236

17. vglut expression in sim, fkh, Lim1, nvy, per mutants? vglut H-cell sib enhancer, identify potential transcription factor binding sites? Transcriptional regulation of vglut expression in H-cell sib

18. Lim1 does not regulate glutamatergic phenotype of H-cell sib Lim1/FM7 Lim1 VGlut Lim1 VGlut/CG13565/En H-cell sib sim per fkh nvy Lim1

19. vglut expression in Forkhead mutant WT fkh SimvglutMerge H-cell sib sim per fkh nvy Lim1

20. VGlut Genomic Locus 14.5 kb 2.4 kb 2.5 kb 2.8 kb 3.7 kb 2.7 kb 4.8 kb 0.8 kb 0.6 kb 0.45 kb 0.47 kb 4.4 kb 0.3 kb CG3557

21. 0.6 kb VGlut intron 2 contains H-cell sib, iVUM mVUM

22. CG3557 VGlut intron 2-b fragment contains H-cell sib enhancer Intron 2-b 0.3kb

23. Future Direction on vglut expression in H-cell sib Role of Fkh in H-cell sib Cell death in H-cell sib? caspase-3 staining in sim-tau GFP embryos Morphology of H-cell sib in sim-tau GFP embryos H-cell sib gene expression in H99 mutant embryos vglut expression in sim, nvy and per mutants Further identify vglut H-cell sib enhancer mutate potential Fkh binding site in intron2b

24. Transcriptional regulation of gene expression in iVUM TF gene expression H-cell sib sim per fkh nvy Lim1 iVUM sim per fkh nvy Lim1 * en NF gene expression H-cell sib Vglut GluR1 5-HT 1A CG13565 iVUM Gad1 VIAAT(CG8394) CG15236

25. Gad1 expression is not regulated by Fkh Gad1enSimMerge WT Fkh