1. A modest collection of Midline CRMs Joe Pearson Lab Meeting 8/25/08

2. Does common expression indicate common regulation? A B C D CD AB A B CD CDAB wxyz

3. Goals Clone minimal CRMs that drive midline primordium expression Dissect and analyze midline primordium CRMs Determine mechanisms governing common gene expression patterns

4. CG13333 Midline primordium genes argosab bibbnb btl cdicenB1ACG31145 CG32594CG3409CG7224 CG8291 ctdve glec hbsHGTXmfasoc rhoSema-1b sog sty vvl Tkr CG9634 rstsimTl

5. Gene# fragments (transformed) Gene# fragments (transformed) argos5(4)CG72241(0) bib3(2)CG82911(1) bnb4(0)CG96342(0) cdi2(0)glec5(4) cenb1a2(1)HGTX8(2) CG133332(2)mfas3(3) CG325944(3)Sema-1b1* CG34094(0)sty6(4) Midline Fragment Cloning Progress

6. Methods Lines tested: Fragments with potential midline expression (live GFP, GFP in situ, or  -GFP histochemical) Fluorescent antibody detection: – Rabbit  -GFP(a488) – Guinea Pig  -Sim(Cy3) – Mouse  -EN or Rat  -ELAV(a647)

7. argos Expression St. 11 midline expression (subset) St. 13+ midline glial expression (strong AMG) Locus 37 kb noncoding locus (18.5 kb tested) 5 Fragments cover 5’ + Introns 4 transformed, 3 tested

8. argos 5’ Fragments 5’-1 5’-2 5’Fragments contain no midline CRMs.

9. argos Intron1-1 St. 13: Sporadic PMG expression St. 14+: Expression in 1-2 AMG, PMG fade into the ether GFPSIM

10. CG13333 Expression St. 11 midline expression (all) St. 13+ midline glial expression St. 15 midline expression weakens Locus 1.5 kb noncoding locus 2 Fragments cover 5’ + 3’ 2 transformed, 2 tested

11. CG13333 5’ 5’ St. 13: AMG, PMG, and iVUMs GFPSIM GFP SIM

12. CG13333 5’ motifs AGGTAG AGGTAC AGGTAG AGGTAC Dmel Dsim Dsec Dyak Dere Dana Dpse Dper Dwil Dgri Dmoj Dvir AGGTGG AGGTAG AGGTAA AGGTAG CACGT CTCGT CACGT AGGTAG AGGAAG PhyloGibbs identified AGGTRG as a repeated, conserved motif.

13. CG13333 3’ 3’ 3’Fragment drives no midline expression. Off-target integration? Additional regions to test?

14. glec Expression St. 8 midline expression (all) refines to 6-7 cells by St. 11 St. 13+ midline glial expression Locus 16.5 kb noncoding locus 5 Fragments cover 5’, Intron1, 3’ 4 transformed, 4 tested

15. glec Intron1 Intron1 contains no midline CRMs

16. glec 5’-2 St. 10 St. 10: 2-3 cells express GFP St. 11: 2-3 cells (AMG?) express strongly, others may be expressing weakly St. 13: AMG, MP1, H-cell/Sib, and variable VUMs St. 16: MG, VUMs, MNB?, H-Cell/Sib, MP1 GFPSIM GFPSIM GFPSIM GFPSIM EN

17. glec 5’-3 St. 11: MP1 (variable weak other cells) St. 13/14: MP1, variable VUMs, AMG, H-Cell sib St. 16: MP1, weak H-Cell/Sib, some AMG GFPSIM GFP SIMGFPSIM GFP SIM ELAV

18. glec 3’ St. 11: 2-3 cells per midline segment St. 13: AMG, weak PMG GFPSIM GFPSIM EN

19. mfas Expression St. 10 midline expression (all) St. 12+ midline glia Locus 7.2 kb noncoding locus 3 Fragments cover 5’ & Intron1 3 transformed, 2 tested

20. St. 12/13:VUMs, moving ventrally St. 16: VUMs mfas 5’frag/Intron1 St. 11: 4 basal, and 2 apical cells (what are they?) GFPSIM GFP SIM GFPSIM EN

21. Conclusions/Lingering Questions “Midline primordium” expression may be amalgam of multiple CRMs Multiple regulatory modes involved Can these elements be “cleaned up”? Have I captured full elements?

22. Next Steps More enhancer screening (10+12+20+10) Identify “minimal” (1kb) midline CRMs (20) Computationally analyze, test motif function Compare co-expressed CRMs