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A modest collection of Midline CRMs

A modest collection of Midline CRMs. Joe Pearson Lab Meeting 8/25/08. Does common expression indicate common regulation?. A. B. C. A. B. D. C. D. A. C. D. B. w. x. y. z. A. B. C. D. Goals. Clone minimal CRMs that drive midline primordium expression

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A modest collection of Midline CRMs

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

  2. Does common expression indicate common regulation? A B C A B D C D A C D B w x y z A B C D

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

  4. Midline primordium genes ab argos bib bnb cdi cenB1A CG13333 CG31145 CG8291 CG32594 CG3409 CG7224 ct dve CG9634 glec hbs HGTX mfas oc vvl sty Tkr sog Sema-1b btl rho rst sim Tl

  5. Midline Fragment Cloning Progress

  6. Methods • Lines tested: Fragments with potential midline expression (live GFP, GFP in situ, or a-GFP histochemical) • Fluorescent antibody detection: • Rabbit a-GFP(a488) • Guinea Pig a-Sim(Cy3) • Mouse a-EN or Rat a-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’-2 5’-1 5’Fragments contain no midline CRMs.

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

  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’ GFP SIM St. 13: AMG, PMG, and iVUMs GFP SIM

  12. CG13333 5’ motifs Dmel Dsim Dsec Dyak Dere Dana Dpse Dper Dwil Dgri Dmoj Dvir AGGTAG AGGTAG AGGTAG AGGTAG AGGTAG AGGTAG AGGTAG AGGTAG AGGTAG AGGTAC AGGTAG AGGTAC AGGTGG AGGTGG AGGTGG AGGTGG AGGTGG AGGTGG AGGTGG AGGTGG AGGTAG AGGTAG AGGTAG AGGTAG AGGTAG AGGTAG AGGTAG AGGTAG AGGTAG AGGTAG AGGTAG AGGTAA AGGTAG CACGT CACGT CACGT CACGT CTCGT CACGT CACGT CACGT AGGTAG AGGAAG 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 GFP SIM St. 10: 2-3 cells express GFP St. 10 GFP SIM St. 11: 2-3 cells (AMG?) express strongly, others may be expressing weakly GFP SIM St. 13: AMG, MP1, H-cell/Sib, and variable VUMs EN GFP SIM SIM St. 16: MG, VUMs, MNB?, H-Cell/Sib, MP1

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

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

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

  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

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