Transcriptional regulation of tracheal fusion cell gene expression

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Transcriptional regulation of tracheal fusion cell gene expression • Isolate enhancer of three fusion

Transcriptional regulation of tracheal fusion cell gene expression • Isolate enhancer of three fusion cell expressed genes: dys, CG 15252, CG 13196 • Identify CRM required for fusion cell gene expression Transcriptional pathways in terminally differentiating neurons • Glutamatergic (H-cell sib )and GABAergic (i. VUM) neurons • Transcriptional regulation of vglut expression in H-cell sib • Transcriptional regulation gene expression in i. VUM neurons

Identify dys Fusion cell Enhancer B 2. 7 P 155 C 1. 7 T

Identify dys Fusion cell Enhancer B 2. 7 P 155 C 1. 7 T 523 dys early enhancer (S 12 -S 17) dys late enhancer (S 14 -S 17)

Sequence alignment of dys 155

Sequence alignment of dys 155

Motifs Required for dys Activation 1 2 3 4 5 6 7 dys 155

Motifs Required for dys Activation 1 2 3 4 5 6 7 dys 155 1 4 7 2 5 3 6 1 GATCGG 2 TTAAATCAAA 3 GCGCG 4 TGCCTGAC 5 Ets 6 GACCCAAAAC 7 POU

Ets protein Pointed is required for dys expression in fusion cells Dys WT pnt

Ets protein Pointed is required for dys expression in fusion cells Dys WT pnt

Sequence of alignment of dys 523 late enhancer

Sequence of alignment of dys 523 late enhancer

dys late enhancer is auto-regulated by Dys protein P 155 C 1. 7 dys

dys late enhancer is auto-regulated by Dys protein P 155 C 1. 7 dys

Identify CG 13196 Fusion cell Enhancer CG 13196 D 92

Identify CG 13196 Fusion cell Enhancer CG 13196 D 92

Sequence alignment of CG 16196 enhancer

Sequence alignment of CG 16196 enhancer

Motifs Required for CG 13196 Expression Motif 8 TCGTG-C GFP Unmutated D 92 mu.

Motifs Required for CG 13196 Expression Motif 8 TCGTG-C GFP Unmutated D 92 mu. C D 92 mu. D D 92 mu. AAG D 92 mu. Motif 8 AAG TCGTG-D Dys Merge

Identify CG 15252 Fusion cell Enhancer CG 15252 C 100

Identify CG 15252 Fusion cell Enhancer CG 15252 C 100

Sequence alignment of CG 15252 enhancer

Sequence alignment of CG 15252 enhancer

Motifs Required for CG 15252 Activation Motif 7 AAG C 100 CG 15252 TCGTG-C

Motifs Required for CG 15252 Activation Motif 7 AAG C 100 CG 15252 TCGTG-C GFP Unmutated C 100 mu. C C 100 mu. D C 100 mu. AAG C 100 mu. Motif 9 Dys TCGTG-D Merge

Co-activator sequences required for CG 13196 expression x A 350 mu. BC CG 13196

Co-activator sequences required for CG 13196 expression x A 350 mu. BC CG 13196 x C 100 CG 15252 C 100 mu. CD CG 15252 A 350 CG 13196

Transcriptional pathways in terminally differentiating neurons Sim + En

Transcriptional pathways in terminally differentiating neurons Sim + En

Gene expression porfile in Glutamatergic and GABAergic interneurons TF gene expression H-cell sib sim

Gene expression porfile in Glutamatergic and GABAergic interneurons TF gene expression H-cell sib sim per fkh nvy Lim 1 * i. VUM sim per fkh nvy Lim 1 en NF gene expression H-cell sib Vglut Glu. R 1 5 -HT 1 A CG 13565 i. VUM Gad 1 VIAAT(CG 8394) CG 15236

Transcriptional regulation of vglut expression in H-cell sib vglut expression in sim, fkh, Lim

Transcriptional regulation of vglut expression in H-cell sib vglut expression in sim, fkh, Lim 1, nvy, per mutants? vglut H-cell sib enhancer, identify potential transcription factor binding sites?

Lim 1 does not regulate glutamatergic phenotype of H-cell sib sim per fkh nvy

Lim 1 does not regulate glutamatergic phenotype of H-cell sib sim per fkh nvy Lim 1/FM 7 VGlut/CG 13565/En Lim 1

vglut expression in Forkhead mutant Sim WT fkh vglut Merge H-cell sib sim per

vglut expression in Forkhead mutant Sim WT fkh vglut Merge H-cell sib sim per fkh nvy Lim 1

VGlut Genomic Locus 14. 5 kb CG 3557 4. 8 kb 2. 5 kb

VGlut Genomic Locus 14. 5 kb CG 3557 4. 8 kb 2. 5 kb H-cell sib, i. VUM, m. VUM 3. 7 kb m. VUM 2. 7 kb 0. 8 kb 0. 6 kb 0. 3 kb 4. 4 kb 2. 4 kb 0. 47 kb 0. 45 kb

VGlut intron 2 contains H-cell sib, i. VUM m. VUM CG 3557 0. 6

VGlut intron 2 contains H-cell sib, i. VUM m. VUM CG 3557 0. 6 kb H-cell sib, i. VUM, m. VUM Sim i. VUMH H-cell sib m. VUM GFP En Merge

VGlut intron 2 -b fragment contains H-cell sib enhancer CG 3557 Intron 2 -b

VGlut intron 2 -b fragment contains H-cell sib enhancer CG 3557 Intron 2 -b 0. 3 kb Sim GFP En Merge

Future Direction on vglut expression in H-cell sib Role of Fkh in H-cell sib

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 H 99 mutant embryos vglut expression in sim, nvy and per mutants Further identify vglut H-cell sib enhancer • mutate potential Fkh binding site in intron 2 b

Transcriptional regulation of gene expression in i. VUM TF gene expression H-cell sib sim

Transcriptional regulation of gene expression in i. VUM TF gene expression H-cell sib sim per fkh nvy Lim 1 * i. VUM sim per fkh nvy Lim 1 en NF gene expression H-cell sib Vglut Glu. R 1 5 -HT 1 A CG 13565 i. VUM Gad 1 VIAAT(CG 8394) CG 15236

Gad 1 expression is not regulated by Fkh Gad 1 WT Fkh en Sim

Gad 1 expression is not regulated by Fkh Gad 1 WT Fkh en Sim Merge

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