1. rhomboid MLE regulation by Sim:Tgo and Su(H) 8/24/09

2. Evidence for Sim Target Regulation expression is lost in sim mutants Sim:Tgo sites are required for midline expression Sim over-expression expands target expression in neuroectoderm – What restricts to neuroectoderm? – Maybe Su(H), says Scott Nambu, 1991 heat-shock Sim control sim rho cdi sli

3. Circumstantial evidence for Sim-Su(H) cooperation sim initation requires Su(H) sim, m5/8 (mesectoderm), rst F6d (midline) expression is expanded with Su(H) over-expression Su(H)/Notch required for multiple stages of midline development (Wheeler et al. ’08) Frequent Su(H) binding sites in midline primordium elements ElementSim:TgoSu(H) sim 3.7pE4(5)6 (10) sim 1.0pL21 sim Sandmann30 rho E-Ss21 tl 95042 rst F6d22 ElementSim:TgoSu(H) btl 2330 Sema1b I1F3sC32 hlh m5/814 Kr StH0.610 Kr PP3.032 Sim:Tgo=ACGTG Su(H)=T/C A/G TG A/G/T GAA

4. Test cases for Sim-Su(H) cooperation rho MLE Sim Su(H) Sim sema1b I1F3sC Sim Su(H) Sim sim1.0pL Sim Su(H) Sim Su(H)

5. rho embryonic expression st. 5st. 6 st. 9 st. 11 st. 13 st. 17 Initially broad neuroectodermal expression Refines to mesectoderm before gastrulation In all midline thru st. 11

6. rho cis-regulatory locus Ip ‘92- 2.2 kb upstream controls neuroectoderm and midline expression – NEE activated by Dorsal, Twist, repressed by Snail – MLE contains 2 Sim:Tgo binding sites “1- to 2-hr delay in onset of (rho 2.2 -lacZ) expression in the mesectoderm relative to the endogenous rho gene”

7. rho embryonic cis-regulation Ip, 1992 Zelzer, 2000

8. rho MLE (Midline Element) motifs Sim/SuH sites Repeats 1/2

9. rho MLE expression S U S w.t. S U S SimMut X X S U S Su(H)Mut X St. 11 St. 13 GFPSimGFPSimGFP

10. 12xSu(H)-LacZ expression Wheeler et al.,2008 Expression in AMG, PMG, MP5, MP6, MNB VUM expression gradient Similar to rho MLE SimMut

11. How is rho MLE regulated? Sim enhances AMG, MP1, MP4 expression – Sim not required for posterior midline expression – Sim not required for midline restriction Activated Su(H) enhances AMG, MP1 (With Sim) PMG and Posterior neurons (without Sim) – Inactive Su(H) represses MP3/4? rho MLE is not mesectoderm element – Where is it?

12. sim 6(10)Su(H), 4(5) Sim:Tgo sites sim3.7 modified from genome.ucsc.edu

13. hlh m5/8 5/8 modified from genome.ucsc.edu 4 Su(H), 1 Sim:Tgo sites

14. Sema-1b 3 Su(H), 2 Sim:Tgo sites I1F3sC modified from genome.ucsc.edu

15. Where’s the rho mesectoderm element? Multiple regions bound by Dl, Sna, Twi Upstream region I : 1 conserved Su(H), 3 Sim:Tgo sites (0 conserved Upstream region II : 0 Su(H), 4 Sim:Tgo sites (3 conserved) Upstream region III : 1 Su(H), 2 Sim:Tgo sites IIIIII modified from genome.ucsc.edu

17. Midline Primordum Elements with Sim:Tgo and Su(H) consensus sites ElementSim:TgoSu(H) sim3.746 sim1.021 simSandmann30 rho E-Ss21 tl95042 rst F6d22 ElementSim:TgoSu(H) btl 2330 Sema1b I1F3sC32 hlh m5/800 Kr StH0.610 Kr PP3.032 Sim:Tgo=ACGTG Su(H)=T/C A/G TG A/G/T GAA

18. Sim-Su(H) hypothesis Sim requires a co-activator in neuroectoderm Su(H) activation (from Notch) detected by Su(H)-LacZ expantion thru neuroectoderm Su(H) required for Sim initiation in mesectoderm Many Sim target elements have conserved Su(H) sites Sim and Su(H) sites can overlap, evolutionary tuning mechanism?

19. rho MLE expression: St. 13 S U S S U S S U S w.t. SimMut Su(H)Mut X X X

20. rho MLE expression: St. 14

21. Where’s the rho mesectoderm enhancer?

22. Sema-1b

23. sim

24. hlh m5/8