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1/25/10

runt and engrailed -dependent specification of midline glia. 1/25/10. Outline. runt and engrailed specify AMG and PMG, respectively. Runt and the midline. The role of hunchback and runt in the specification of MP1. Establishing early midline fate. Downstream targets of runt.

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1/25/10

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  1. runt and engrailed-dependent specification of midline glia 1/25/10

  2. Outline runt and engrailed specify AMG and PMG, respectively Runt and the midline The role of hunchback and runt in the specification of MP1 Establishing early midline fate Downstream targets of runt

  3. Key developmental time points – AMG and PMG

  4. Runt En AMG PMG PMG AMG AMG PMG AMG AMG MP1 PMG MP1 AMG

  5. Distinct expression patterns in AMG vs PMG

  6. Runt and Engrailed are spatially restricted to either AMG or PMG

  7. runt and engrailed control AMG and PMG gene expression, respectively Wildtype UAS-runt UAS-en Wildtype UAS-runt UAS-en

  8. Previous work runt expression engrailed expression WT hs-Runt Klinger and Gergen, 1993 Aronson, et al, 1997

  9. Runt and Engrailed cross-repress each other in order to generate AMG and PMG wildtype

  10. When are expression domains established? 13 sag 13 sag 13 sag 13 sag RuntEnGFP RuntEn Runt Wrapper

  11. Consequences of converting AMG to PMG or PMG to AMG wildtype late 16 GFPwrapper wrapper UAS-runt UAS-en late 16 late 16 GFP GFPwrapper

  12. ?

  13. L(1)sc en l(1)sc runt?

  14. Does engrailed regulate l(1)sc? UAS-en enE/enE wildtype L(1)scStg runt negatively regulates l(1)sc UAS-runt y1 w1 f1 run3 L(1)scStg 12/1 L(1)scStgodd L(1)scStg

  15. L(1)sc ? en L(1)sc runt

  16. Remaining experiments • Determine the spatial and temporal expression of Runt and Engrailed • Determine role of L(1)sc • Quantify glial cells in UAS-runt and UAS-en backgrounds • Finish images for runt and engrailed mutant backgrounds

  17. The specification of neuron and glial cell fate by the transcription factor Runt • Mechanisms used to generate MP1s • Establishment of early midline fate • Transcriptional regulation of AMG gene expression Due April 8th (end of March for UNC?)

  18. Runt En AMG PMG PMG AMG AMG PMG AMG AMG MP1 PMG MP1 AMG

  19. runt hb lim3 HGTX pdf odd AMG PMG PMG AMG AMG PMG AMG AMG MP1 PMG MP1 AMG

  20. hb PMG PMG ? AMG PMG ? ? MP1 PMG MP1 AMG ? Odd, HGTX, pdf, lim3?

  21. sim::Gal4 driving uas-hb generates extra lim-3 HGTX + cells WT WT control (no HGTX staining) stg, uas-hb UAS-hb Lim3GFPHGTX Lim3HGTX

  22. Wrapper and lim3 co-localize in stg uas-hb embryos Lim-3 + wrapper + neurons Lim-3 + wrapper - neuron lim3 stg wrapper

  23. Stop codon deletion

  24. Lim3 and odd are both expressed in hb12 alleles lim3 stg odd

  25. runt hb lim3 HGTX pdf odd AMG PMG PMG AMG AMG PMG AMG AMG MP1 PMG MP1 AMG

  26. Ectopic Runt expression does not affect Lim3 expression Stg uas-runt WT stage 13 GFPEn Lim3 GFPEn Lim3

  27. WT stg, uas-hb stg, uas-runt

  28. Driving engrailed in the midline reduces odd expression GFPoddRunt GFPoddRunt

  29. runt mutants lack odd staining 12/0 12/0 GFPodd GFPodd

  30. Hunchback and Runt? HB Runt odd HGTX pdf lim3

  31. MP1 transcription factors Lim3 ectopic expression? X Uas-odd Uas-ftz X Uas-exex X exex- Lim3 normal

  32. Aim 1 Methods and goals: • Test available mutants and uas lines for altered MP1 fate • Morphology • gene expression • Identify MP1 enhancer • Hunchback, Runt, and Engrailed binding sites • Mutate sites and identify • Microarray MP1 neurons • Isolate MP1s using glec enhancer and sim-GAL4UAS-TAU-GFP (2-color sorting) • Amplify and hybridize to affy microarray chip or use chip seq

  33. 1 kb Runt binding site Sim binding site Hb binding site

  34. Aim 2 – Early specification of midline by runt and additional transcription factors

  35. eve ftz en wingless wingless slp wingless en gooseberry-neuro – MP3s patched

  36. Establishing early midline identity A, B C D, E F G H l(1)sc runt runt, slp1 and 2 slp1 and 2, gsb-n, l(1)sc eve, en en en, PMG MP1, hb, odd AMG MP3 MNB, Vums

  37. Aim 2: Establishing early midline identity • Test available mutants and uas lines for altered midline • Morphology • Gene expression • Based on results, determine which transcription factors specify each cell type

  38. Aim 3: Identify the transcriptional mechanisms used to generate AMG and PMG

  39. Goals: Identify Runt binding sites within AMG cis-regulatory regions Identify Engrailed binding sites Determine if Engrailed and Runt function in conjunction with Sim to specify AMG and PMG cell fate

  40. CG8776 1 kb Sim binding site Runt binding site

  41. Summary runt and engrailed co-repress each other to specify AMG and PMG fates, respectively runt is necessary to generate MP1s, hunchback is sufficent Further clues pertaining to midline development should arise from uas lines and genetic analysis

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