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Building a functional nervous system

Building a functional nervous system. Immature CNS Cells. Interneurons. • Cell fate determination • Cell migration • Apoptosis • Axonogenesis • Synapse formation • Ensheathment. Motorneurons. Neurosecretory cells. Glia. Big questions. How do MG arise? transcriptional control

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Building a functional nervous system

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  1. Building a functional nervous system Immature CNS Cells Interneurons • Cell fate determination • Cell migration • Apoptosis • Axonogenesis • Synapse formation • Ensheathment Motorneurons Neurosecretory cells Glia

  2. Big questions • How do MG arise? • transcriptional control • formation/ fate (hh signaling) • What are MG doing?

  3. 2 types of MG are present at stage 10

  4. Notch signaling is required for MG cell fate wild type MG MG wrapper MG MP MG MG MG Sim-Gal4> UAS-Su(H).VP16 Dl3 / Dl3 Delta  Notch  Su(H)  MG gene expression

  5. What is the glial gene expression hierarchy? Delta Notch Sim MG gene expression ????? Su(H) D Vvl Are there multiple mechanisms for MG gene expression? How are alternate mechanisms used? shadow enhancers? as parts of an overall expression pattern? How are PMG and AMG specified differentially?

  6. When does MG gene expression begin? s9 s10 s11 s12 >s12 CG32244 - P A/P A/P A/P wrapper - A A/P A/P A/P epac - - A/P A/P A/P CG31145 - - AA NA argos - - PA/PA/P CG31116 - - P P NA CG32030 - - - A A CG33275 - - - AA CG7271 - - - A A CG8776 - - - A A netANA netBNA pqbp-1 NA shepNA sim (2.8kb frag) - - ? A/P A/P slit-lacZ - - ?A/P A/P tslNA w NA

  7. MG gene expression argos PMG only AMG only CG31145 AMG and PMG epac

  8. summary • The timing of the initiation of MG gene expression suggests that there may be multiple mechanisms for turning on MG gene expression.

  9. Future direction • Examine the expression of additional MG expressed genes during stages 10-12 • Test enhancer fragments for MG gene expression • Identify potential transcription factor binding sites in MG enhancer fragments • Test the requirement for binding sites by site directed mutagenesis • Examine enhancer fragment expression in mutants • How does the regulation of the de novo set of genes compare with the non de novo set identified by Joe Pearson

  10. Big questions • How do MG arise? • transcriptional control • formation/ fate (hh signaling) • What are MG doing?

  11. Hh signaling, in brief Hh Hh Ptc Smo Smo Ptc Other Proteins Other Proteins CiAct CiRep CiAct CiRep Cytoplasm Nucleus

  12. hh directs midline neuronal fate AA142-lacZ (MG) X55-lacZ (MN) wild type hh- constitutive repression of hh targets. 16 cells/seg 2-4 cells/seg constitutive activation of hh targets ptc- ># of cell/seg 0-1 cell/seg Hummell et al., 1999

  13. Where is hh expressed?

  14. hh specifies posterior cell fates Bossing and Brand., 2006 in hh mutant: at stage 10: en and l(1)sc expression are lost in the midline (~2 en+ cells/seg) at stage 13: en expression is lost (i.e. VUM neurons are missing) sim-gal4>UAS-en results in a loss of MP1 neurons Therefore: hh en in the posterior which induces posterior cell fate

  15. How does this correspond to what we know?

  16. Model 1: hh specifies posterior cell fates Outcome of hh mutant: In the absence of hh function, posterior fates (MP4-6, MNB, and PMG) are transformed into anterior fates (MP1, MP3, AMG). Would see increase in MP1 and MP3 neurons and ~ 10 MG per segment This is inconsistent with Hummell data where there is a reduction of X55+ neurons and increase in AA142+ MG. hh anterior fates posterior fates

  17. Model 2: hh regulates all MP formation Outcome of hh mutant: In the absence of hh function, all MPs are transformed into MG). ~16 MG/ segment This is consistent with Hummell data where there an increase to 16 AA142 cells per segment. Reports of loss of sim expression in hh-. Maybe because there is not Notch signaling from MPs. AMG PMG PMG AMG PMG PMG

  18. Model 3: hh regulates posterior cell formation Outcome of hh mutant: In the absence of hh function, MP4-6, MNB would not develop properly (maybe transformed into PMG). ~13 MG/ segment MP1,3,4 would be present. This is sort of consistent with Hummell data. > MG < MN PMG PMG PMG

  19. Experiments • loss of function • hh mutants (AC: p-element deletion, 13C: strong ems allele) • cross into sim-Gal4 UAS-tauGFP background (use to count midline cells) • examine markers for midline cell fate at stage 10-17 (use to identify cell types) • misexpression using sim-Gal4, UAS-tauGFP • UAS-hh – activate hh targets in all midline cells • UAS-hhN – activate hh targets in all midline cells • UAS-Ci[76] – represses hh targets in all midline cells • Reporter expression • a. ptc-lacZ – activated in response to hh signaling

  20. A tiny bit of preliminary data Sim stage 15 sagittal hh[AC]/ + Sim stage 15 sagittal hh[AC]/ hh[AC]

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