Download 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