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Lab Meeting 10-4-10. Generation of CNS Neuronal and Glial Diversity. Interneurons. Motorneurons. Neurosecretory cells. Immature CNS Cells. Glia. H-Cell: Dopaminergic midline neuron. TH ( ple ). Ddc. VMAT. tyrosine. L-DOPA. dopamine. DAT. Control of Neurotransmitter Choice. AMG.

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Lab meeting 10 4 10

Lab Meeting 10-4-10


Generation of cns neuronal and glial diversity
Generation of CNS Neuronal and Glial Diversity

Interneurons

Motorneurons

Neurosecretory cells

Immature

CNS Cells

Glia


H cell dopaminergic midline neuron
H-Cell: Dopaminergic midline neuron

TH (ple)

Ddc

VMAT

tyrosine

L-DOPA

dopamine

DAT


Control of neurotransmitter choice
Control of Neurotransmitter Choice

AMG

AMG

AMG

AMG

PMG

PMG

PMG

PMG

AMG

AMG

MP1

MP3

MP4

MP5

MP6

MNB

H-cell

sib

H-cell

MP1

MP1

mVUM

iVUM

?

Notch

?

Notch

DA

Glu

Oct

GABA

What is controlling gene expression in H-cell and mVUMs?


kb

0

10

20

30

40

50

60

70

80

90

100

110

120

Proneural Genes of the AS-C

l(1)sc

ac

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
AS-C Genes are Present in MPs and their Neuronal Progeny

L(1)sc

Sc

Ac


L 1 sc misexpression expands h cell gene expression
l(1)sc Misexpression Expands H-Cell Gene Expression

sim-Gal4

UAS-l(1)sc

sim-Gal4

UAS-sc

Wild-type

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
l(1)sc Misexpression with elav-Gal4 does not Expand H-Cell Gene Expression

Wild-type

elav-Gal4

UAS-l(1)sc

sim-Gal4

UAS-l(1)sc

ple

Analyzed 36 embryos

Could be due to:

Levels: sim-Gal4 vs elav-Gal4

Timing: sim-Gal4 vs elav-Gal4


AS-C Mutants

Df(1)scB57

l(1)sc

ac

sc

ase

l(1)sc

ase

Df(1)sc10-1

l(1)sc

ac

sc

Df(1)ase1


Ac sc and ase are not required for h cell gene expression
ac, sc and ase are Not Required for H-Cell Gene Expression

ple

Df(1)sc10-1

Df(1)ase1

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
l(1)sc Regulates H-Cell Neural Function Gene Expression

ple5-HT1A

NPFR1

Additional H-cell neural function

genes analyzed: DAT, Vmat

Wild -type

l(1)sc

DAT

NPFR1

ple

Vmat

Df(1)scB57


L 1 sc regulates h cell transcription factor expression
l(1)sc Regulates H-Cell Transcription Factor Expression

BarH1

SoxN pdm

Tup CG13565

Wild -type

Df(1)scB57

72%

28%

BarH1

SoxN

l(1)sc


Is l 1 sc solely responsible for controlling h cell gene expression
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

Df(1)sc19

1A8-1B4

scB57:1B7-1B4

Wild-type

Df(1)sc19

ple

ple


Is l 1 sc solely responsible for controlling h cell gene expression1
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

  • Knock down l(1)sc by using RNAi: mtd-gal4 x UAS-l(1)sc RNAi

  • Rescue scB57 with UAS-l(1)sc


L 1 sc regulation of h cell gene expression
l(1)sc expression? Regulation of H-Cell Gene Expression

?

l(1)sc

?

tup

SoxN

BarH1

Neural Function Genes

ple, DAT, NPFR1, Vmat

Dopaminergic cell fate


Tup is required for ple and dat expression
Tup is Required for expression?ple and DAT Expression

pleNPFR1

DATNPFR1

VmatNPFR1

Wild-type

tup


Relationship between tup and l 1 sc
Relationship between Tup and L(1)sc expression?

Tup pdm

L(1)sc pdm

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 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
SoxNeuro expression? Regulates H-Cell Neurotransmitter Receptor Gene Expression

TupNPFR1

Wild-type

SoxN

Reviewer: Could SoxN be regulating other H-cell genes?


Soxneuro is required for npfr1 but not for ple and dat expression
SoxNeuro expression? is required for NPFR1, but not for ple and DAT Expression

ple

NPFR1

Wild-type

SoxN


L 1 sc regulation of h cell gene expression1
l(1)sc expression? Regulation of H-Cell Gene Expression

?

l(1)sc

?

?

SoxN

BarH1

tup

ple

DAT

?

NPFR1

Vmat

Dopaminergic cell fate


Multiple pathways regulate h cell and h cell sib gene expression
Multiple Pathways Regulate H-Cell and H-Cell Sib Gene Expression

MP3

MP3

H-cell

sib

H-cell

H-cell

?

l(1)sc

Notch

tup

H-cell sib

genes

H-cell

genes

Common

genes

Dopaminergic

cell fate

How common is this mechanism?


Does l 1 sc act in neurons in lateral cns and in other notch independent cells
Does l(1)sc act in neurons in lateral CNS and in other Notch independent cells?

L(1)sc Elav

L(1)sc Eve

late s11

Mid s11

RP2-notch independent

RP2sib-notch dependent

GMC 4-2a


Does l 1 sc act to specifically control development of dopaminergic neurons
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 neurons1
Does l(1)sc act to specifically control development of dopaminergic neurons?

DATTup

DAT

0 DAT+ cells

7/12 hemisegments


Things left to do
Things left to do dopaminergic neurons?

  • l(1)sc RNAi: absence or reduction of ple

  • scB57 rescue with UAS-l(1)sc

  • Co-misexpress l(1)sc, tup and SoxN: increase in ple

  • scm6: confirm presence of ple, DAT, Vmat, Tup, NPFR1, BarH1, SoxN

  • Tup mutant: is l(1)sc affected?

  • Confirm SoxN mutant via fluorescence with SoxN Ab


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