building a functional nervous system
Post on 07-Feb-2016
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Building a functional nervous system
Immature CNS Cells
Motorneurons
Neurosecretory cells
Glia
Interneurons• Cell fate determination• Cell migration• Apoptosis• Axonogenesis• Synapse formation• Ensheathment
Big questions
1. How do MG arise?a. transcriptional controlb. formation/ fate (hh signaling)
2. What are MG doing?
Notch signaling is required for MG cell fate
wra
pper
Dl3 / Dl3Sim-Gal4>
UAS-Su(H).VP16wild type
MP
MG
MG MG
MGMG
MG
Delta Notch Su(H) MG gene expression
What is the glial gene expression hierarchy?
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?
MG gene expression
Notch
Su(H)
Delta
?????D VvlSim
When does MG gene expression begin?
s9 s10 s11 s12 >s12CG32244 - P A/P A/P A/Pwrapper - A A/P A/P A/P
epac - - A/P A/P A/PCG31145 - - A A NAargos - - P A/P A/PCG31116 - - P P NACG32030 - - - A ACG33275 - - - A ACG7271 - - - A ACG8776 - - - A AnetA NAnetB NApqbp-1 NAshep NAsim (2.8kb frag) - - ? A/P A/Pslit-lacZ - - ? A/P A/Ptsl NAw NA
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 fragmentsa. Test the requirement for binding sites by site directed
mutagenesisb. 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 controlb. formation/ fate (hh signaling)
2. What are MG doing?
Hh signaling, in brief
Ptc
Hh
Smo
CiAct CiRep
PtcSmo
Hh
CiAct CiRep
Other Proteins Other Proteins
Nucleus
Cytoplasm
hh directs midline neuronal fate
wild
type
hh-
AA142-lacZ (MG)
Hummell et al., 1999
X55-lacZ(MN)
ptc-
16 cells/seg 2-4 cells/seg
0-1 cell/seg ># of cell/seg
constitutive repression of hh targets.
constitutive activation of hh targets
hh specifies posterior cell fates
Bossing and Brand., 2006in 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
Model 1: hh specifies posterior cell fates
anterior fates posterior fates hh
Outcome of hh mutant:
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 neuronsand ~ 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
PMG
PMG
Outcome of hh mutant:
In the absence of hh function, all MPs are transformed into MG).
~16 MG/ segment
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.
AMG PMGPMGAMG
Model 3: hh regulates posterior cell formation
PMG
PMG
PMG
Outcome of hh mutant:
In the absence of hh function, MP4-6, MNB would not develop properly (maybe transformed into PMG).~13 MG/ segment
MP1,3,4 would be present.
This is sort of consistent with Hummell data.
> MG< MN
Experiments
1. loss of functiona. 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-tauGFPa. UAS-hh – activate hh targets in all midline cellsb. UAS-hhN – activate hh targets in all midline cellsc. UAS-Ci[76] – represses hh targets in all midline cells
3. Reporter expressiona. ptc-lacZ – activated in response to hh signaling
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