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March 15 th , 2010 - Lab Meeting midline cell fate

March 15 th , 2010 - Lab Meeting midline cell fate. Outline. Hh and its role in midline cell fate MG Neurons Wg and its potential role (or lack thereof) in midline cell fate. hh activates PMG specific gene expression patterns. hh[AC]. WT. UAS-hh. hh activate midline En in Tribolium.

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March 15 th , 2010 - Lab Meeting midline cell fate

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  1. March 15th, 2010 - Lab Meetingmidline cell fate

  2. Outline • Hh and its role in midline cell fate • MG • Neurons • Wg and its potential role (or lack thereof) in midline cell fate

  3. hh activates PMG specific gene expression patterns hh[AC] WT UAS-hh

  4. hh activate midline En in Tribolium

  5. hh represses AMG specific gene expression patterns hh[AC] WT UAS-hh

  6. What might be happening? Hh Hh Hh Hh Hh en expression • hh from row G, H and the lateral CNS activate en expression in rows G?, H, A, B • From rows G-B, the MP4-6, MNB, and PMG arise • En in PMG represses AMG gene expression and promotes PMG gene expression

  7. What might be happening? Hh Hh Hh Hh Runt Runt Hh en expression • Runt represses en expression and keeps it out of rows C-E • Does Hh direct gene expression in row F, or even more anterior? What about neurons?

  8. hh effects MP3 formation/fate Tup Elav GFP hh[AC] WT ple GFP hh[AC] WT ptc GFP

  9. What kind of cells are made in hh mutants? Cas Elav GFP Runt GFP En GFP The simplest explanation is that there are 4 neurons, 2 MP1 and 2 MP4/5. (Cas is also on in lots of MG, so Cas may be effected in a more complex way) Conclusion: the effect of hh extends past the En+ cells

  10. Outline • Hh and its role in midline cell fate • MG • Neurons • Wg and its potential role (or lack thereof) in midline cell fate

  11. Reasons to think that wg is involved in midline cell fate acquisition • Its wg, it is involved in lots of cell patterning and cell fate • pubmed search for ‘wingless signaling’ yields 980 papers and 164 reviews • ‘wnt signaling’ yields 7,848 papers and 1,493 reviews • During segmentation, wg is in a regulatory loop with en and hh to producing a signaling center en hh ptc wg

  12. Reasons to think that wg is involved in midline cell fate acquisition • wg function regulates anterior cell fates in the epidermis and lateral CNS • in wg mutants, naked cuticle (anterior epidermal cell fate) is lost • in wg mutants, NB4-2 (anterior cell) cell fate is lost

  13. Reasons to think that wg is involved in midline cell fate acquisition • wg is expressed in the midline cells early in their development stage 8 stage 9 wg En stage 10 stage 11 stage 12

  14. Reasons to think that wg is involved in midline cell fate acquisition Ectopic wg and wg mutantsshow alterations in l(1)sc expression stage 10 Bossing and Brand, 2006

  15. Reasons to think that wg is NOT involved in midline cell fate acquisition • misexpression of wg in the midline has no effect on Runt/En/morphology Bossing and Brand, 2006: The earliest time we can express Wingless in all midline cells is stage 10. At this stage, ectopic Wingless no longer interferes with midline cell differentiation. stage 11 stage 15 Runt En GFP

  16. Reasons to think that wg is NOT involved in midline cell fate acquisition misexpression of Wg downstream effectors in the midline has no effect on Runt/En/morphology UAS-arm[s10] UAS-tcfΔN stage 13 stage 15 stage 11 stage 11 Runt En GFP

  17. Reasons to think that wg is NOT involved in midline cell fate acquisition wg mutants still show Runt+ Sim+ cells and En+ posterior cells wt wg[1-12] stage 13 stage 13 Sim Runt En GFP

  18. Reasons to think that wg is NOT involved in midline cell fate acquisition Ectopic wg and wg mutantsshow alterations in l(1)sc expression….but we dont know what is being assayed. stage 10 where is the midline? can wg turn off l’sc in non-midline cells, it looks like it. Is this then related to segmentation? Bossing and Brand, 2006

  19. Reasons to think that wg is NOT involved in midline cell fate acquisition In wg mutants ‘early’ en is absent, but ‘late’ en is not effected Loss of ‘early’ En is likely due to a loss of the regulatory loop wgenhhwg the presence of en seems due to the fact that wg is not regulating midline gene expression Bossing and Brand, 2006

  20. So, what do I think wg is probably NOT involved in midline cell cell fate/ differentiation

  21. So, what do I think • Epidermal patterning and segmentation: • There is an early role for wg in establishing en and hh expression in the posterior of the segment • wg regulates anterior cell fate by repressing the expression of genes that in the wg mutant are activated ectopically in the anterior. • l(1)sc is expressed in the anterior of the segment due to segmentation genes (which ones I don’t know). l(1)sc wg En/hh

  22. So, what do I think • Midline cell patterning and cell fate: • Misexpression of wg or downstream components does not alter midline cell fate whereas hh and ci can. So, wg regulation of l’sc is probably not occurring at the same time as hh regulation (probably earlier, during segmentation). • The presence of late en and absence of early en is good proof that wg plays no role in hh regulation of en (and probably l’sc). • This likely means that wg repression of gene expression in the anterior of the segment is not present. This might allow hh signaling to effect gene expression in anterior cells (ie MP1, MP3), which it does in MP3. • Something makes the midline cells different from lateral CNS and epidermis, a good candidate is single-minded. How does it do this?

  23. How can one nail down the role of wg? • reporters of wg activity • Top-Flash (not tried in flies, just s2 cells, works in mice) • nkd-GFP • verify the expression of en and l’sc in wg mutants. If they are present in the expected cells than wg plays no role.

  24. What might be happening? Hh Hh Hh Hh Runt Hh Hh en expression Runt in Row C, D, E opposes Hh activity and keeps en expression off. Evidence comes from Josephs work on Runt and En cross-repression

  25. What might be happening? Hh Hh Hh Hh Runt Hh Hh MP1 en expression MP1 arises from Rows C, D, E (Runt+ cells). odd expression in Rows A and B is unrelated to MP1 , odd expression is intiated de novo in MP1

  26. What might be happening? Hh Hh Hh Hh Runt Hh Hh MP4 MP3 MP1 MP5 MP6 en expression MNB MP3 arises from row F MP4-MNB and PMG arise from rows G-B AMG arise from rows C-F

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