Download 11/3/08 Scott

Building a functional nervous system
Immature CNS Cells
Interneurons
Motorneurons
Neurosecretory cells
Glia
• Cell fate determination
• Cell migration
• Apoptosis
• Axonogenesis
• Synapse formation
• Ensheathment
Big questions
1.
How do MG arise?
a. transcriptional control
b. formation/ fate (hh signaling)
2.
What are MG doing?
2 types of MG are present at stage 10
Notch signaling is required for MG cell fate
Sim-Gal4>
UAS-Su(H).VP16
Dl3 / Dl3
wrapper
wild type
MG
MG
MG
MP
MG
MG
MG
Delta  Notch  Su(H)  MG gene expression
What is the glial gene expression hierarchy?
Delta
Notch
Su(H)
Sim
D Vvl
?????
MG gene expression
Are there multiple mechanisms for MG gene expression?
How are alternate mechanisms used?
shadow enhancers?
as parts of an overall expression pattern?
How are PMG and AMG specified differentially?
When does MG gene expression begin?
s9
CG32244
wrapper
epac
CG31145
argos
CG31116
CG32030
CG33275
CG7271
CG8776
netA
NA
netB
NA
pqbp-1
NA
shep
NA
sim (2.8kb frag) slit-lacZ
tsl
NA
w
NA
s10
P
A
-
s11
A/P
A/P
A/P
A
P
P
-
s12
A/P
A/P
A/P
A
A/P
P
A
A
A
A
>s12
A/P
A/P
A/P
NA
A/P
NA
A
A
A
A
-
?
?
A/P
A/P
A/P
A/P
MG gene expression
argos
CG31145
epac
PMG only
AMG only
AMG and PMG
summary
1. The timing of the initiation of MG gene expression suggests that
there may be multiple mechanisms for turning on MG gene
expression.
Future direction
1.
Examine the expression of additional MG expressed genes during
stages 10-12
2.
Test enhancer fragments for MG gene expression
3.
Identify potential transcription factor binding sites in MG enhancer
fragments
a. Test the requirement for binding sites by site directed
mutagenesis
b. Examine enhancer fragment expression in mutants
How does the regulation of the de novo set of genes compare with the
non de novo set identified by Joe Pearson
Big questions
1.
How do MG arise?
a. transcriptional control
b. formation/ fate (hh signaling)
2.
What are MG doing?
Hh signaling, in brief
Hh
Hh
Ptc
Smo
Smo
Ptc
Other Proteins
CiAct
CiRep
Other Proteins
CiAct
Cytoplasm
Nucleus
CiRep
hh directs midline neuronal fate
X55-lacZ
(MN)
hh-
wild type
AA142-lacZ
(MG)
constitutive repression
of hh targets.
16 cells/seg
2-4 cells/seg
ptc-
constitutive activation
of hh targets
0-1 cell/seg
># of cell/seg
Hummell et al., 1999
Where is hh expressed?
hh specifies posterior cell fates
Bossing and Brand., 2006
in hh mutant:
at stage 10: en and l(1)sc expression are lost in the midline (~2 en+ cells/seg)
at stage 13: en expression is lost (i.e. VUM neurons are missing)
sim-gal4>UAS-en results in a loss of MP1 neurons
Therefore: hh en in the posterior which induces posterior cell fate
How does this correspond to what we know?
Model 1: hh specifies posterior cell fates
Outcome of hh mutant:
anterior fates
posterior fates
hh
In the absence of hh
function,
posterior fates (MP4-6,
MNB, and PMG) are
transformed into
anterior fates (MP1,
MP3, AMG).
Would see increase in
MP1 and MP3 neurons
and ~ 10 MG per
segment
This is inconsistent with
Hummell data where
there is a reduction of
X55+ neurons and
increase in AA142+ MG.
Model 2: hh regulates all MP formation
Outcome of hh mutant:
In the absence of hh
function,
all MPs are transformed
into MG).
~16 MG/ segment
AMG
AMG
PMG
PMG
PMG
PMG
This is consistent with
Hummell data where
there an increase to 16
AA142 cells per
segment.
Reports of loss of sim
expression in hh-. Maybe
because there is not
Notch signaling from
MPs.
Model 3: hh regulates posterior cell formation
Outcome of hh mutant:
In the absence of hh
function,
MP4-6, MNB would not
develop properly (maybe
transformed into PMG).
~13 MG/ segment
PMG
PMG
PMG
MP1,3,4 would be
present.
This is sort of consistent
with Hummell data.
> MG
< MN
Experiments
1. loss of function
a. hh mutants (AC: p-element deletion, 13C: strong ems allele)
b. cross into sim-Gal4 UAS-tauGFP background (use to count midline cells)
c. examine markers for midline cell fate at stage 10-17 (use to identify cell types)
2. misexpression using sim-Gal4, UAS-tauGFP
a. UAS-hh – activate hh targets in all midline cells
b. UAS-hhN – activate hh targets in all midline cells
c. UAS-Ci[76] – represses hh targets in all midline cells
3. Reporter expression
a. ptc-lacZ – activated in response to hh signaling
A tiny bit of preliminary data
Sim stage 15 sagittal hh[AC]/ +
Sim stage 15 sagittal hh[AC]/ hh[AC]