CNS midline sim expression: early vs. late

2. midline neurons and glia s12 CNS midline sim expression: early vs. late midline precursors s6 s8 s11

3. s17 WT Lim1 Sim En + simtaugfp Late sim midline expression is restricted to glia, H-cell sib, iVUMs and MNB progeny s13 WT zfh1 En VGlut/En/Sim zfh1/En/Sim What is the function of late Sim in mature midline neurons and glia? What are late Sim direct target genes? Does Sim interact with other TFs to activate late gene expression? If so,which ones?

4. sim contains two alternative promoters CNS midline neurons and glia Central brain neurons Optic lobes Mesectodermal precursors CNS midline primordium

5. Sim regulates axonogenesis in central brain neurons WT Sim DAPI sim² D. Lau

6. Testing function of late Sim in midline development and transcription sim locus del Df(3R) ry75 simH9 * • Df(3R) ry75 removes Sim late promoter leaving early sim expression intact • simH9 is a null point mutation that introduces an early stop codon in the protein • simH9/TM3 twi-GFP X Df(3R) ry75/TM3 twi-GFP • --assay GFP-negative embryos using AP ISH • simH9/TM3 twi-GFP X w; {3.7sim-gal4,w+}, {UAS-tau::gfp,w+}; Df(3R) ry75/TM3 ftz-lacZ • --assay twi-GFP- and lacZ-negative embryos using FISH/antibody staining • --3.7sim-Gal4<UAS-tau::gfp marks all midline cells

7. ry75/simH9 embryos do not express Sim protein after mid-embryogenesis s16 s16 ry75/simH9 simH9/TM3 Sim sim-Gal4; UAS-taugfp Sim sim-Gal4; UAS-taugfp ry75/simH9 = simlate

8. Late Sim is not required for wrapper expression, but midline glia have mild positioning defects s11 twi-GFP control simlate s11 wrapper + gfp simlate s17 s17 simlate twi-GFP control s17 s16 simlate

9. Late Sim is not required for sim transcription simlate simlate s17 s13 sim sim

10. Embryos lacking late sim have a midline axonogenesis defect simlate simH9/TM3 simlate

11. Sim+ CNS midline neurons are GABAergic or Glutamatergic Wt Sim En/Sim/VGlut Gad1 En En/Sim/Gad1 Gad1: GABAergic neuronal marker VGlut: Glutamatergic neuronal marker iVUMs/pMNB: En+, Sim+, Gad1+ H-cell sib: En-, Sim+, VGlut+

12. Late sim does not regulate Lim1 or Gad1 expression in iVUMs/MNB progeny s15 simlate Gad1/Lim1/simtaugfp Gad1 Lim1

13. Late sim regulates VGlut expression in H-cell sib simlate simlate 30% 70% VGlut/simtau VGlut/simtau simlate simlate s16 s16 CG13565 CG13565

14. en mutants have ectopic CNS midline VGlut expression Wt Sim VGlut Gad1 en Sim VGlut Tbh en mutants: 2-4 VGlut+ Sim+ neurons/segment

15. Misexpression of En in H-cell sib represses VGlut and does not activate Gad1 VGlut PerGal4<tau En Wt H-sib axon H-sib axon Gad1/VGlut/Per CG13565/En/Per Per-Gal4, UAS-tau::gfp; UAS-engrailed

16. Proposed mechanism for midline VGlut transcriptional regulation glutamatergic H-cell sib RNA pol II Sim VGlut H-sib CRM mVUM CRM • H-cell sib and mVUMs exhibit different levels of VGlut expression • H-sib VGlut expression can be repressed by en • H-sib VGlut expression requires late Sim

17. Proposed mechanism for midline VGlut transcriptional regulation glutamatergic H-cell sib RNA pol II X Sim VGlut H-sib CRM mVUM CRM • Late Sim is co-expressed in H-cell sib with at least 4 other TFs (Per, Lim1, Fkh, Nvy)

18. En Proposed mechanism for midline VGlut transcriptional regulation GABAergic iVUM/pMNB RNA pol II Sim VGlut H-sib CRM mVUM CRM • En mutants exhibit ectopic VGlut expression in Sim+ neurons • En misexpression in H-cell sib represses VGlut

19. En Proposed mechanism for midline VGlut transcriptional regulation GABAergic iVUM/pMNB RNA pol II Sim VGlut H-sib CRM mVUM CRM This model presumes En and Sim directly regulates VGlut Testable via identification of midline CRMs and En/Sim binding sites

20. Sim Sim En En En En En En Sim Sim VGlut genomic locus 14.5 kb 10 kb 2.5 kb 6 putative En binding sites (YAATYANB) identical from melanogaster to virilis 3 putative Sim binding sites (ACGTG) identical from melanogaster to virilis Genomic fragments cloned into GFP expression P-element for injection

21. En En En En En En Transgenic expression analysis Sim Sim Sim Sim

22. En En En En En En Transgenic expression analysis Sim Sim Sim Sim 2 independent insertions No embryonic expression

23. En En En En En En Transgenic expression analysis Sim Sim Sim Sim CNS: 2 cells/ hemisegment in thoracic segments 1 cell/hemisegment in abdominal segments Outside CNS during mid-embryogenesis Only 1 insertion line

24. En En En En En En Transgenic expression analysis Sim Sim Sim Sim CNS: 2 cells/ hemisegment in thoracic segments 1 cell/hemisegment in abdominal segments Outside CNS during mid-embryogenesis Only 1 insertion line

25. En En En En En En Transgenic expression analysis Sim Sim Sim Sim 2 independent insertions Expression not determined

26. En En En En En En Transgenic expression analysis Sim Sim Sim Sim TA cloned Not subcloned into stinger

27. ry75/simH9 allelic combination eliminates late sim expression in midline glia and iVUMs, H-cell sib, and pMNB simlate mutants express wrapper, but may have a MG defect. simlate mutants exhibit ectopic midline axonal projections to the segmental nerve root (likely iVUM axons) simlate mutants have normal midline Gad1 expression, but fail to express VGlut in H-cell sib (~70% segments) En misexpression in H-cell sib phenocopies simlate H-cell sib phenotype En represses VGlut in GABAergic midline neurons, preventing activation by a TF complex containing Sim. Manipulate late Sim function using misexpression of dominant-negative sim constructs; Per-Gal4 mediated expression could test neuron specific Sim function en; simlate double mutant to test requirement of sim on formation of ectopic glutamatergic Sim+ neurons ( or use expression of sim dominant-negative constructs in en mutant) Identify H-cell sib CRM in VGlut locus and test dependence on Sim and En Test panel of neuronal/glial markers to identify additional late Sim targets Test other H-cell sib TF mutants for VGlut expression Conclusions Future Directions