Download sim

Stephanie Freer
Lab Meeting 7.6.09
Post-embryonic expression of sim
Hartenstein (1993)
D
OL
Posterior
B
T
Anterior
Sim
•
•
•
•
3 anterior Sim+ clusters
Optic lobes
Midline of the VNC
Posterior clusters
Analysis of sim-GAL4 enhancer lines
To visualize the specific projection pattern for each enhancer-GAL4 line:
1. Cross enhancer-GAL4 to UAS-GFP.nls or UAS tau-GFP
2. Analyze embryonic and larval brains for GFP-expression in sim+ cells
3. Further dissect fragments not restricted to central brain clusters
sim transgenic lines
Construct
Embryonic Expression
Larval Expression
1.0 kb
2.8 kb
3.1 kb
5.5 kb
3.7 kb
sandman
all midline cells
midline glia, muscle precursors
hindgut
foregut, pharynx
all midline cells
midline glia, muscle precursors
none
small posterior clusters
none
none
ventral anterior clusters (FLP out)
small posterior clusters
Frag 1
Frag 2
Frag 3
Frag 4
Frag 5
Intron 3
Intron 7
3’sim F1
3’sim F2
3’sim F3
3’sim F4
coming soon
midline glia, muscle precursors
CNS
none
gut
CNS
CNS, PNS
none
CNS
none
laminar cells (OL), small post. clusters
central brain NBs, GMCs
none
none
medullar cells (OL), ant./post. clusters
CNS
none
anterior lateral clusters (timeout?)
none
Optic lobe expression
sim
GFP
Laminar OL Neurons
Medullar OL Neurons
Posterior central brain clusters
sim
GFP
Small Posterior Cluster
Lateral Posterior Cluster
Expression in the anterior clusters
BP106
GFP
Intron 3-GAL4 x UAS mCD8-GFP
Intron 3 contains enhancer elements responsible for sim expression in all 3 of the
anterior central brain clusters
Additional intronic enhancers
3 Intron 3 500bp fragments
Intron 3 Frag 1 – posterior clusters
- subset of anterior clusters?
- optic lobes
Intron 3 Frag 2 – no expression
Intron 3 Frag 3 – Optic lobes (medullar subset)
New fragment spanning intron 4-6 to be injected
Intron 3 Frag 2 Anterior
Intron 3 Frag 3
Intron 3 F1-GAL4 x UAS mCD8-GFP
nc82
GFP
Anterior
Posterior
GFP expression - a subset of anterior central brain cells
- medullar optic lobe expression (subset)
- posterior clusters
Reduction of late sim leads to structural
defects in the adult
simJ1-47
WT
• simJ1-47 - temperature sensitive allele
• Neuropil disorganization in the adult
• Sterile with behavioral defects
Rein et al., 2002
Central complex structure
Neckameyer and Matsuo, 2008
Fan-shaped body – visual pattern learning
Ellipsoid body, noduli (ebo) – decay in walking activity
Protocerebral bridge (nob) – deficits in step length
Abnormal turning behavior, “left-right bargaining” in mutants of both eb and pb
 defects typically at the midline
Intron 3-GAL4 expression in the adult brain
nc82
GFP
Expression in a subset of fan-shaped body axons
Lack of expression in the protocerebral bridge
and ellipsoid body
In general, lots of axons stained  specificity?
Intron 3-GAL4 expression in the optic lobe
nc82
GFP
Expression in the SOG
nc82
GFP
Intron 3 F1-GAL4 x UAS mCD8-GFP
Expression in the anterior subeosphogeal ganglion, send projections dorsally
Corresponds to Trdm?
Summary of adult expression
nc82
GFP
Intron 3 F1-GAL4 x UAS mCD8-GFP
Expression in the SOG, optic lobes, PNs in the AL, fan-shaped body, MBs, superior
medial protocerebrum, superior lateral protocerebrum
Further dissection of Intron 3
1.4kb
F1
F3
F2
F1a
F1b
F1+ F2
1. Separate optic lobe expression from central brain expression
2. Capture all central brain neurons within one line
Another possibility: Mutate potential optic lobe elements
Transmitter phenotype of sim-expressing midline
cells in the embryo
***
Sim
VGlut/Sim
En/Sim
Gad1/Sim
Posterior VGlut expression
VGlut
Sim
VGlut RNA co-localizes with Sim protein
Neurotransmitter phenotype in posterior simpositive neurons  VGlut
VGlut Fragment 4-6 in the 3rd instar brain
En
En Sim
intron 2
Sim
SimSim
En
En
En
En
intron 1
Sim
GFP
intron 3
CG3557
4.8 kb
Frag 8
2.8 kb
Frag 1
2.5 kb
Frag 2
3.7 kb
Frag 4
2.7 kb
Frag 5
0.8 kb 0.6 kb
Frag 7 Frag
9
2.4 kb
Frag 6
VGlut Antibody in 3rd Instar Larvae
VGlut
Tau
No apparent protein co-localization at the cell bodies
 VGlut protein in transit to the synapse
VGlut staining absent in secondary lineages labeled by BP106 (Daniels et al. 2008)
Transmitter phenotype in anterior simpositive central brain clusters
Medial Cluster
Sim
Gad1
VGlut
•
1-2 VGlut+, sim+ cells observed for each cluster
•
No Gad1+, sim+ cells observed
Two options:
1. Cells not differentiated yet
 Stain pupal and adult brains
2. Another transmitter involved
- monoamines: dopamine, serotonin, octopamine
- neuropeptides: neuropeptide F
Summary
Post-embryonic expression within Intron 3
•
Expression from intron 3-GAL4 in the adult
- Subset in the fan-shaped body
- Structures other than the CC
•
•
Posterior clusters are glutamatergic
Analyze additional fragments
- Frag 1, other introns, 3’ sim F2
- Intron 3 dissection
Neurotransmitter staining in pupae
and adults
Behavioral Analysis
- Larval and adult locomotion