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Lab Meeting 10-4-10. Generation of CNS Neuronal and Glial Diversity Immature CNS Cells Interneurons Motorneurons Neurosecretory cells Glia.

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Presentation on theme: "Lab Meeting 10-4-10. Generation of CNS Neuronal and Glial Diversity Immature CNS Cells Interneurons Motorneurons Neurosecretory cells Glia."— Presentation transcript:

1 Lab Meeting 10-4-10

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

3 H-Cell: Dopaminergic midline neuron tyrosineL-DOPAdopamine TH (ple)Ddc VMAT DAT

4 Control of Neurotransmitter Choice MP1 H-cell sib mVUMiVUM AMG MP3 PMG MNB PMG MP4MP5MP6MP1AMG DAGluGABAOct Notch ?? What is controlling gene expression in H-cell and mVUMs?

5 Proneural Genes of the AS-C 1020300405060708090100110120 kb acsc l(1)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 Reviewer: would misexpression using a different driver increase frequency of phenotype? Phenotype observed 14-24%

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

9 AS-C Mutants Df(1)sc 10-1 Df(1)sc B57 Df(1)ase 1 l(1)sc ase acsc l(1)sc acsc l(1)sc ase

10 ac, sc and ase are Not Required for H-Cell Gene Expression Df(1)ase 1 Df(1)sc 10-1 ple Reviewer: why is only a subset of H-cell genes looked at in sc 10-1 ? single mutant for sc: EMS allele sc m6

11 l(1)sc Regulates H-Cell Neural Function Gene Expression Additional H-cell neural function genes analyzed: DAT, Vmat l(1)sc DAT NPFR1 ple Vmat Wild -type Df(1)sc B57 NPFR1ple 5-HT1A

12 l(1)sc Regulates H-Cell Transcription Factor Expression BarH1 Wild -type Df(1)sc B57 SoxN pdm l(1)sc BarH1 SoxN Tup CG13565 28%72%

13 Is l(1)sc solely responsible for controlling H-cell gene expression? Is there another gene missing from sc B57 deletion that contributes to phenotype? Df(1)sc 19 ase Wild-type Df(1)sc 19 ple 1A8-1B4 scB57:1B7-1B4

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 1.Knock down l(1)sc by using RNAi: mtd-gal4 x UAS-l(1)sc RNAi 2.Rescue sc B57 with UAS-l(1)sc

15 l(1)sc Regulation of H-Cell Gene Expression Neural Function Genes ple, DAT, NPFR1, Vmat Dopaminergic cell fate BarH1SoxN ? l(1)sc tup ?

16 Tup is Required for ple and DAT Expression Wild-type ple NPFR1 DAT NPFR1Vmat NPFR1 tup

17 Relationship between Tup and L(1)sc 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 Tup pdmL(1)sc pdm

18 SoxNeuro Regulates H-Cell Neurotransmitter Receptor Gene Expression Wild-type Tup NPFR1 SoxN Reviewer: Could SoxN be regulating other H-cell genes?

19 SoxNeuro is required for NPFR1, but not for ple and DAT Expression Wild-type SoxN pleNPFR1

20 l(1)sc Regulation of H-Cell Gene Expression Dopaminergic cell fate BarH1 tup SoxN ? ple DAT NPFR1Vmat ? ?? l(1)sc

21 Multiple Pathways Regulate H-Cell and H-Cell Sib Gene Expression Dopaminergic cell fate tup ? MP3 H-cell sib MP3 H-cell l(1)scNotch H-cell sib genes H-cell genes Common genes 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 ElavL(1)sc Eve Mid s11 late s11 GMC 4-2a RP2-notch independent RP2sib-notch dependent

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? 0 DAT + cells 7/12 hemisegments DAT TupDAT

25 Things left to do 1.l(1)sc RNAi: absence or reduction of ple 2.sc B57 rescue with UAS-l(1)sc 3.Co-misexpress l(1)sc, tup and SoxN: increase in ple 4.sc m6 : confirm presence of ple, DAT, Vmat, Tup, NPFR1, BarH1, SoxN 5.Tup mutant: is l(1)sc affected? 6.Confirm SoxN mutant via fluorescence with SoxN Ab

26

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