lab meeting 10-4-10. generation of cns neuronal and glial diversity immature cns cells interneurons...
TRANSCRIPT
Generation of CNS Neuronal and Glial Diversity
ImmatureCNS Cells
Interneurons
Motorneurons
Neurosecretory cells
Glia
Control of Neurotransmitter Choice
MP1MP1 H-cell H-cellsib mVUM iVUM
AMG AMG AMG AMG
AMG MP3
PMG PMG
MNB
PMGPMG
MP4 MP5 MP6MP1AMG
DA Glu GABAOct
Notch Notch? ?
What is controlling gene expression in H-cell and mVUMs?
Proneural Genes of the AS-C
10 20 300 40 50 60 70 80 90 100 110 120kb
ac sc l(1)sc ase
• Chromosomal complex• bHLH transcription factors• Control neuroblast formation and cell fate in the lateral CNS
l(1)sc Misexpression Expands H-Cell Gene Expression
sim
-Gal
4U
AS-l(
1)sc
sim
-Gal
4U
AS-s
c
Wild
-typ
e
l(1)sc
Transcription FactorsBarH1SoxNtup
Neural Function genesDAT
NPFR1ple
Vmat
Reviewer: would misexpression using a different driver increase frequency of phenotype?
Phenotype observed 14-24%
Wild-type elav-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-Gal4Timing: sim-Gal4 vs elav-Gal4
ple
Analyzed 36 embryos
ac, sc and ase are Not Required for H-Cell Gene Expression
Df(1
)ase
1D
f(1)s
c10-1
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
Additional H-cell neural function genes analyzed: DAT, Vmat
l(1)sc
DATNPFR1pleVmat
Wild
-typ
eD
f(1)s
cB57
NPFR1ple 5-HT1A
l(1)sc Regulates H-Cell Transcription Factor Expression
BarH1
Wild
-typ
eD
f(1)s
cB57
SoxN pdm
l(1)sc BarH1SoxN
Tup CG13565
28%72%
Is l(1)sc solely responsible for controlling H-cell gene expression?
Is there another gene missing from scB57 deletion that contributes to phenotype?
Df(1)sc 19
ase
Wild
-typ
e
Df(
1)sc
19
ple ple1A8-1B4scB57:1B7-1B4
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
Neural Function Genesple, DAT, NPFR1, Vmat
Dopaminergic cell fate
BarH1SoxN ?
l(1)sc
tup
?
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 subsetTup could be causing l(1)sc to degrade. Will look @ l(1)sc expression in tup
Tup
pdm
L(1)
sc p
dm
SoxNeuro Regulates H-Cell Neurotransmitter Receptor Gene Expression
Wild
-typ
e
Tup NPFR1
SoxN
Reviewer: Could SoxN be regulating other H-cell genes?
l(1)sc Regulation of H-Cell Gene Expression
Dopaminergic cell fate
BarH1tup SoxN
?
pleDAT NPFR1 Vmat?
??
l(1)sc
Multiple Pathways Regulate H-Cell and H-Cell Sib Gene Expression
Dopaminergic cell fate
tup ?
MP3
H-cell H-cellsib
MP3
H-cell
l(1)sc Notch
H-cell sibgenes
H-cellgenes
Commongenes
How common is this mechanism?
Does l(1)sc act in neurons in lateral CNS and in other Notch independent cells?
L(1)
sc E
lav
L(1)
sc E
ve
Mid s11 late s11
GMC 4-2a
RP2-notch independent
RP2sib-notch dependent
Does l(1)sc act to specifically control development of dopaminergic neurons?
TH (ple) islet (Tup)
Dorsolateral neuronsStage late 15- e16
Does l(1)sc act to specifically control development of dopaminergic neurons?
0 DAT+ cells 7/12 hemisegments
DAT TupDAT
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