Jiangnan Luo Lee Lab 30 Dec 2014 1 Lab meeting. Ongoing projects 2 1.Single cell morphology of...
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Transcript of Jiangnan Luo Lee Lab 30 Dec 2014 1 Lab meeting. Ongoing projects 2 1.Single cell morphology of...
1
Jiangnan LuoLee Lab
30 Dec 2014
Lab meeting
Ongoing projects
2
1. Single cell morphology of lobular neurons
• Screening Gal4 lines (Janelia Farm & Kyoto DGRC)• Cell types and numbers labeled by Gal4 lines• Dendritic morphology of single cell (lobula layer, dendritic field,
orientation…)
2. Morphology of Tm5c is activity-dependent on photoreceptors? (R1-6/7-8)
3. Screening Mutants that affect dendrites of Tm20 neurons
3
Downstream targets for trans-medulla neurons (Tm5/20)
Lin et al., 2014 (unpublished data)
A
P
Li
Ventral view
• Layer• Dendritic field• Orientation
4
Janelia Farm, FlyLightNC82GFP
D
V
~ 12/lobe
• Li3 (R43E05)
~30 / lobe (Clustered)
• Li4 (R89D06)
Anterior view
Anterior view
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• Li4 (NP1582)
Otsuna et al., 2014 Frontiers in Neural Circuits
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• Li5 (R84C01) Janelia Farm, FlyLight
Li5: ~20 / lobe
NC82GFP
Ongoing project 1
7
• Single cell morphology of lobular neurons
Li3/4/5-Gal4 > hsp1-FLP; UAS-FRT-CD2-y+-FRT-mCD8-GFP
3rd instar larvae (mid- / late-)
Adult (2-4 days old)
Heat shock (2-3 min, 37°C)
Dissect
Li3
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Single cell morphology – Li4 (R89D06)
Mi T/Y?
10μm24B
10Fas
IIIGFP
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Single cell morphology – (Li3/4/5)
Li3
ConGFP
34 12
Li4: Lo4, 5
20μm
Li5Ventral view
A
P
Li3: Lo5,6
Li4 (NP1582)
Li5: Lo4, 5,6
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Single cell morphology – (Li3/4/5)
Li3 Li4 Li5
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Whole pattern – (Li3/4/5) Li3 Li5
1234
Fas III Con GFP 20μm
#1
#2
#3
#1
#2
N=12
N=11
N=13
N=19
N=21
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• Li5 (R84C01)• Li3 (R43E05)
Gal4 expression in the larval CNS
Janelia Farm, FlyLight
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Whole pattern – (Li3/4/5)
Li4 (NP1582)
GFP24B10FasIII
Lin et al., 2014 (unpublished data)
GFP24B10
Pupae
Single cell morphology
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Gal4 linesSample Numbe
r
Positive hits
Percentage
Cell type flipped out
Heat shock time (min)
• Li3 (R43E05)
27 4 15% Li 2
40 6 15% Li 2.5
59 5 8% Li 3
• Li4 (R89D06)17 0 0% Mi 2
12 0 0% Mi, T? 2.5
• Li5 (R84C01)
28 2 7% Li 2
72 5 7% Li 2.5
21 1 5% Li 3
• Li4 (NP1582) 30 10 33% Li 2
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Future work – Project 1
1. Behavior study
2. Check Li3-Gal4 expression under developmental stages
• Manipulation of Li3 and test UV preference
Ongoing project 2
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1. Single cell morphology of lobular neurons
2. Morphology of Tm neurons is activity-dependent? (on R1-6/7-8) De- or hyper- polarization of photoceceptors -> Dendrites of Tm5/20 neurons
3. Screening Mutants that affect dendrites of Tm neurons
L-GMR-Gal4; NaChBac orL-GMR-Gal4; Kir2.1
> hsp1-FLP; C1-LexA; pLots2-stop-cd2-cherry, pLots2-stop-cd8-gfp
Morphology of Tm neurons are activity dependent on R7-8 ?
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Increase sodium conductance• Depolarization (NaChBac, bacterial sodium channel)
Flies ready, soon have the results
• Hyperpolarization (Kir2.1, Inward-rectifying potassium channel)
Prevent membrane polarization
Genetics almost done
L-GMR-Gal4; NaChBac> hsp1-FLP; C1-LexA; pLots2-s-cd2-cherry, pLots2-s-cd8-gfp
L-GMR-Gal4; Kir2.1 > hsp1-FLP; C1-LexA; pLots2-s-cd2-cherry, pLots2-s-cd8-gfp
Ongoing project 3
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1. Single cell morphology of lobular neurons
2. Morphology of Tm neurons is activity-dependent? (on R1-6/7-8)
3. Screening mutants that affect dendrites of Tm20 neurons
Ongoing project 3
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• Screening Mutants that affect dendrites of Tm20 neurons
Mutant -FRT > hsp122-FLP; FRT40,42,G13-Gal80; ortC1-gal4, UAS-cd8-cherry
Late 3rd instar larvae
Adult (2-4 days old)
Heat shock (1 min, 37°C)
Dissect
20
Screening Mutants that affect dendrites of Tm neurons
Sema1a -/-
Tm20
Tm20
Tm5(a/b)
24B10RFP
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Mutants from Yuh Nung Jan’s Lab that affect dendritic development of PNS
neurons
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Future work – project 2 & 3
1. Dendritic study
2. Activity dependent studyHyper- or de-polarization of photoceceptors ---> Dendrites of Tm5c
• Request / search mutants (Jan lab & Kyoto stock
center)• Test the effects of mutations on Tm20 neurons