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Lin et al., 2014 (unpublished data)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

5

• Li4 (NP1582)

Otsuna et al., 2014 Frontiers in Neural Circuits

6

• 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

8

Single cell morphology – Li4 (R89D06)

Mi T/Y?

10μm24B

10Fas

IIIGFP

9

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

10

Single cell morphology – (Li3/4/5)

Li3 Li4 Li5

11

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

12

• Li5 (R84C01)• Li3 (R43E05)

Gal4 expression in the larval CNS

Janelia Farm, FlyLight

13

Whole pattern – (Li3/4/5)

Li4 (NP1582)

GFP24B10FasIII

Lin et al., 2014 (unpublished data)

GFP24B10

Pupae

Single cell morphology

14

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

15

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

16

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 ?

17

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

18

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

19

• 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

21

Mutants from Yuh Nung Jan’s Lab that affect dendritic development of PNS

neurons

22

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