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