Transcript l(1)sc
Lab Meeting 10-4-10 Generation of CNS Neuronal and Glial Diversity Interneurons Motorneurons Immature CNS Cells Neurosecretory cells Glia H-Cell: Dopaminergic midline neuron TH (ple) tyrosine Ddc L-DOPA VMAT dopamine DAT Control of Neurotransmitter Choice MP1 AMG AMG AMG AMG PMG PMG PMG PMG AMG AMG MP1 MP3 MP4 MP5 MP6 MNB MP1 H-cell H-cell sib mVUM iVUM ? Notch ? Notch DA Glu Oct GABA What is controlling gene expression in H-cell and mVUMs? Proneural Genes of the AS-C kb 0 10 20 ac 30 40 sc 50 l(1)sc 60 70 80 90 ase • Chromosomal complex • bHLH transcription factors • Control neuroblast formation and cell fate in the lateral CNS 100 110 120 Ac Sc L(1)sc AS-C Genes are Present in MPs and their Neuronal Progeny sim-Gal4 UAS-sc sim-Gal4 UAS-l(1)sc Wild-type l(1)sc Misexpression Expands H-Cell Gene Expression 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? l(1)sc Misexpression with elav-Gal4 does not Expand H-Cell Gene Expression elav-Gal4 UAS-l(1)sc ple Wild-type Analyzed 36 embryos Could be due to: Levels: sim-Gal4 vs elav-Gal4 Timing: sim-Gal4 vs elav-Gal4 sim-Gal4 UAS-l(1)sc AS-C Mutants ac sc l(1)sc ase l(1)sc ase Df(1)sc B57 Df(1)sc 10-1 Df(1)ase1 ac sc l(1)sc ac, sc and ase are Not Required for H-Cell Gene Expression Df(1)ase1 Df(1)sc10-1 ple Reviewer: why is only a subset of H-cell genes looked at in sc10-1? single mutant for sc: EMS allele scm6 l(1)sc Regulates H-Cell Neural Function Gene Expression Df(1)scB57 Wild -type ple 5-HT1A NPFR1 Additional H-cell neural function genes analyzed: DAT, Vmat l(1)sc DAT NPFR1 ple Vmat l(1)sc Regulates H-Cell Transcription Factor Expression SoxN pdm Tup CG13565 Df(1)scB57 Wild -type BarH1 l(1)sc BarH1 SoxN 72% 28% 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 Wild-type ple Df(1)sc 19 Df(1)sc 19 ple 1A8-1B4 scB57:1B7-1B4 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 scB57 with UAS-l(1)sc l(1)sc Regulation of H-Cell Gene Expression ? tup l(1)sc SoxN BarH1 ? Neural Function Genes ple, DAT, NPFR1, Vmat Dopaminergic cell fate Tup is Required for ple and DAT Expression tup Wild-type ple NPFR1 DAT NPFR1 Vmat NPFR1 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 pdm Tup pdm 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 SoxNeuro Regulates H-Cell Neurotransmitter Receptor Gene Expression SoxN Wild-type Tup NPFR1 Reviewer: Could SoxN be regulating other H-cell genes? SoxNeuro is required for NPFR1, but not for ple and DAT Expression SoxN Wild-type ple NPFR1 l(1)sc Regulation of H-Cell Gene Expression ? tup l(1)sc ? ple DAT SoxN BarH1 ? NPFR1 ? Vmat Dopaminergic cell fate Multiple Pathways Regulate H-Cell and H-Cell Sib Gene Expression MP3 MP3 H-cell H-cell tup l(1)sc Dopaminergic cell fate H-cell sib ? Notch H-cell sib genes How common is this mechanism? H-cell genes Common genes Does l(1)sc act in neurons in lateral CNS and in other Notch independent cells? late s11 L(1)sc Eve L(1)sc Elav Mid s11 RP2-notch independent RP2sib-notch dependent GMC 4-2a Does l(1)sc act to specifically control development of dopaminergic neurons? TH (ple) islet (Tup) Dorsolateral neurons Stage late 15- e16 Does l(1)sc act to specifically control development of dopaminergic neurons? DAT DAT Tup 0 DAT+ cells 7/12 hemisegments Things left to do 1. l(1)sc RNAi: absence or reduction of ple 2. scB57 rescue with UAS-l(1)sc 3. Co-misexpress l(1)sc, tup and SoxN: increase in ple 4. scm6: 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