Lab Meeting 10-4-10
Generation of CNS Neuronal and Glial Diversity
ImmatureCNS Cells
Interneurons
Motorneurons
Neurosecretory cells
Glia
H-Cell: Dopaminergic midline neuron
tyrosine L-DOPA dopamineTH (ple) Ddc VMAT
DAT
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
AS-C Genes are Present in MPs and their Neuronal Progeny
L(1)
scSc
Ac
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
AS-C Mutants
Df(1)sc 10-1
Df(1)sc B57
Df(1)ase1
l(1)sc ase
ac sc l(1)sc
ac sc l(1)sc ase
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
?
Tup is Required for ple and DAT Expression
Wild
-typ
eple NPFR1 DAT NPFR1 Vmat NPFR1
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?
SoxNeuro is required for NPFR1, but not for ple and DAT Expression
Wild
-typ
eSo
xN
ple NPFR1
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