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Lab Meeting 10-4-10

Lab Meeting 10-4-10. Generation of CNS Neuronal and Glial Diversity. Interneurons. Motorneurons. Neurosecretory cells. Immature CNS Cells. Glia. H-Cell: Dopaminergic midline neuron. TH ( ple ). Ddc. VMAT. tyrosine. L-DOPA. dopamine. DAT. Control of Neurotransmitter Choice. AMG.

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Lab Meeting 10-4-10

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  1. Lab Meeting 10-4-10

  2. Generation of CNS Neuronal and Glial Diversity Interneurons Motorneurons Neurosecretory cells Immature CNS Cells Glia

  3. H-Cell: Dopaminergic midline neuron TH (ple) Ddc VMAT tyrosine L-DOPA dopamine DAT

  4. Control of Neurotransmitter Choice AMG AMG AMG AMG PMG PMG PMG PMG AMG AMG MP1 MP3 MP4 MP5 MP6 MNB H-cell sib H-cell MP1 MP1 mVUM iVUM ? Notch ? Notch DA Glu Oct GABA What is controlling gene expression in H-cell and mVUMs?

  5. kb 0 10 20 30 40 50 60 70 80 90 100 110 120 Proneural Genes of the AS-C l(1)sc ac sc ase • Chromosomal complex • bHLH transcription factors • Control neuroblast formation and cell fate in the lateral CNS

  6. AS-C Genes are Present in MPs and their Neuronal Progeny L(1)sc Sc Ac

  7. l(1)sc Misexpression Expands H-Cell Gene Expression sim-Gal4 UAS-l(1)sc sim-Gal4 UAS-sc Wild-type l(1)sc Transcription Factors BarH1 SoxN tup Neural Function genes DAT NPFR1 ple Vmat Phenotype observed 14-24% Reviewer: would misexpression using a different driver increase frequency of phenotype?

  8. l(1)sc Misexpression with elav-Gal4 does not Expand H-Cell Gene Expression Wild-type elav-Gal4 UAS-l(1)sc sim-Gal4 UAS-l(1)sc ple Analyzed 36 embryos Could be due to: Levels: sim-Gal4 vs elav-Gal4 Timing: sim-Gal4 vs elav-Gal4

  9. AS-C Mutants Df(1)scB57 l(1)sc ac sc ase l(1)sc ase Df(1)sc10-1 l(1)sc ac sc Df(1)ase1

  10. ac, sc and ase are Not Required for H-Cell Gene Expression ple Df(1)sc10-1 Df(1)ase1 Reviewer: why is only a subset of H-cell genes looked at in sc10-1? single mutant for sc: EMS allele scm6

  11. l(1)sc Regulates H-Cell Neural Function Gene Expression ple5-HT1A NPFR1 Additional H-cell neural function genes analyzed: DAT, Vmat Wild -type l(1)sc DAT NPFR1 ple Vmat Df(1)scB57

  12. l(1)sc Regulates H-Cell Transcription Factor Expression BarH1 SoxN pdm Tup CG13565 Wild -type Df(1)scB57 72% 28% BarH1 SoxN l(1)sc

  13. Is l(1)sc solely responsible for controlling H-cell gene expression? Is there another gene missing from scB57 deletion that contributes to phenotype? ase Df(1)sc19 1A8-1B4 scB57:1B7-1B4 Wild-type Df(1)sc19 ple ple

  14. Is l(1)sc solely responsible for controlling H-cell gene expression? • Does l(1)sc play a redundant role with other proneural genes? • Used indirect ways of analyzing l(1)sc function • Knock down l(1)sc by using RNAi: mtd-gal4 x UAS-l(1)sc RNAi • Rescue scB57 with UAS-l(1)sc

  15. l(1)sc Regulation of H-Cell Gene Expression ? l(1)sc ? tup SoxN BarH1 Neural Function Genes ple, DAT, NPFR1, Vmat Dopaminergic cell fate

  16. Tup is Required for ple and DAT Expression pleNPFR1 DATNPFR1 VmatNPFR1 Wild-type tup

  17. Relationship between Tup and L(1)sc Tup pdm L(1)sc pdm Reviewer: tup LOF phenotype seems stronger than scB57 (100% vs 78%) Is tup upstream of l(1)sc? L(1)sc phenotype is greater/affects all H-cell genes vs tup affects a subset Tup could be causing l(1)sc to degrade. Will look @ l(1)sc expression in tup

  18. SoxNeuro Regulates H-Cell Neurotransmitter Receptor Gene Expression TupNPFR1 Wild-type SoxN Reviewer: Could SoxN be regulating other H-cell genes?

  19. SoxNeuro is required for NPFR1, but not for ple and DAT Expression ple NPFR1 Wild-type SoxN

  20. l(1)sc Regulation of H-Cell Gene Expression ? l(1)sc ? ? SoxN BarH1 tup ple DAT ? NPFR1 Vmat Dopaminergic cell fate

  21. Multiple Pathways Regulate H-Cell and H-Cell Sib Gene Expression MP3 MP3 H-cell sib H-cell H-cell ? l(1)sc Notch tup H-cell sib genes H-cell genes Common genes Dopaminergic cell fate How common is this mechanism?

  22. Does l(1)sc act in neurons in lateral CNS and in other Notch independent cells? L(1)sc Elav L(1)sc Eve late s11 Mid s11 RP2-notch independent RP2sib-notch dependent GMC 4-2a

  23. Does l(1)sc act to specifically control development of dopaminergic neurons? TH (ple) islet (Tup) Dorsolateral neurons Stage late 15- e16

  24. Does l(1)sc act to specifically control development of dopaminergic neurons? DATTup DAT 0 DAT+ cells 7/12 hemisegments

  25. Things left to do • l(1)sc RNAi: absence or reduction of ple • scB57 rescue with UAS-l(1)sc • Co-misexpress l(1)sc, tup and SoxN: increase in ple • scm6: confirm presence of ple, DAT, Vmat, Tup, NPFR1, BarH1, SoxN • Tup mutant: is l(1)sc affected? • Confirm SoxN mutant via fluorescence with SoxN Ab

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