Ipek Subay

RNAseq Analysis for b1 and b3 motor neuron receptors

I selected the cells with different combination of markers and different levels of expression. In addition to split-gal4 driver markers I also added FoxP, aPKC and VGlut to get more specific results. gene names on graph legends are not correct and will be fixed

IS38497 b1 and b3 motor driver line

AD: beat-IIIB DBD: bab1

SS98650 b3 driver

AD: ct DBD: Ubx

IS69306 b1 mn driver line

AD: CG14388 DBD: CG31345

SS48311 b3 mn driver

AD: Ubx DBD: NetB

SS40980 b1 mn driver line

AD: CG12680 DBD: CG10137

Control imaging for confocal

As I was seeing some structures that were not supposed to be there, first I tested if my antibodies has problems so I ran the experiment with WTB flies and primary antibodies with corresponding 2nd antibody

I used 3 predefined laser settings with minor changes. Alexa 488 uses laser at 488nm, Alexa 555 uses laser at 514 nm, Alexa 647 uses laser at 633nm

nc82 antibody (anti-mouse) and anti-mouse 650 ab. Image taken on green channel (488 excitation, 493-779 emission)

same sample but image taken on 555 wavelength:

same sample but image taken on 647 wavelength where I was supposed see the background staining. Its not clear because I did not adjust the laser power:

anti-RFP (rat) ab, anti-rat 555 staining:

image taken on 555 wavelength:

same sample but image taken on 647 wavelength:

Since the structure were visible with both of the antibodies, I suspected the reagents that I have been using might have contaminated so I prepared 4 different setups with no antibody staining at all:

Preparation 1: Includes prefixing the samples and blocking them with my existing NGS

Image taken with 488 laser and emission 493-737nm:

Same sample with 488 laser and emission 491-736nm

Same sample with laser 555 and emission 535-779:

Same sample with laser 647 and emission 638-779:

Preparation 2: Includes prefixing the samples and blocking them with aliquot from a new batch of NGS

Image taken with 488 laser and emission 493-744nm:

Image taken with 488 laser and emission 492-744nm:

Image taken with 555 laser and emission 535-756nm:

Image taken with 647 laser and emission 638-779nm:

Preparation 3: Includes prefixing the samples and no blocking with NGS:

Image taken with 488 laser and emission 493-739nm:

Image taken with 555 laser and emission 535-756nm:

Image taken with 647 laser and emission 638-779nm:

Preparation 4: No fixing and no blocking with NGS

Image taken with 488 laser and emission 493-744nm:

Image taken with 488 laser and emission 492-744nm:

Image taken with 555 laser and emission 535-779nm:

Image taken with 647 laser and emission 638-779nm:

Trans-tango repeat from previous week

I was not able get any trans-tango signal from my reciprocal MB399B crosses so I repeated the experiment with anti-RFP antibody instead of anti-HA antibody

77124xMB399B (MBON02 reciprocal cross) stained with rabbit anti-GFP (2nd: anti-rabbit 488), rat anti-RFP (2nd: anti-rat 555), nc82 (2nd: anti-mouse 650)

MBON02 trans-tango images from Kaun Lab as reference:

Tango imaging

99661xSS98650 (retro tango imaging for b3 mn) stained with rabbit anti-GFP (2nd: anti-rabbit 488), rat anti-RFP (2n: anti-rat 555), nc82 (2nd: anti-mouse 650)

GFP channel:

somehow my antibodies decided to work on retro tango flies even though I did not change anything in the protocol

retro-tango channel:

b3 presynaptic neurons from BANC dataset:

99661xSS48311 (b3 mn) stained with rabbit anti-GFP (2nd: anti-rabbit 488), rat anti-RFP (2n: anti-rat 555), nc82 (2nd: anti-mouse 650)

GFP:

Retro-tango:

99661xSS40980 (b1 mn) stained with rabbit anti-GFP (2nd: anti-rabbit 488), rat anti-RFP (2n: anti-rat 555), nc82 (2nd: anti-mouse 650)

GFP:

retro-tango:

b1 presynaptic neurons from BANC dataset:

77124xMB399B (MBON02 reciprocal cross) stained with rabbit anti-GFP (2nd: anti-rabbit 488), rat anti-HA (2n: anti-rat 555), nc82 (2nd: anti-mouse 650)

GFP:

trans-tango:

trans-tango maximum intensity projection:

Habit Formation Wildtype Test 02.03-06.03

I wanted to try Habit formation with wildtype and tested 4 wheels of flies (48) over the last week. Unfortunately, I did a mistake when setting the timeslots file for right torque so it had an additional testing period at P13. So analyzed the left and right individually:

For Punishment on Left Torque:

Optomotor traces (right/left) at start of experiment:

Performance Index box&dotplot without notches:

For Punishment on Right Torque:

Optomotor traces (right/left) at start of experiment:

Performance Index box&dotplot without notches:

WTB Test for Torquemeter February 19-20

10 flies completed out of 36 flies.

Optomotor traces (right/left) at start of experiment

Optomotor traces (right/left) at end of experiment

Performance Index bar plot with SEM:

Performance Index bar plot – preference subtracted:

Performance Index box&dotplot without notches:

Statistical tests of single groups against zero

Retro-Tango endogenous GFP Expression

On the right: Endogenous GFP expression (without antibody) from retro tango flies (BDSC: 99661) crossed with GF-Gal4 (BDSC: 79602) driver line.

On the left: GFP expression in retro-tango x GF-Gal4 flies from retro-tango publication ( https://doi.org/10.7554/eLife.85041 )

Same image but in retro-tango signal channel:

Control Experiments for Driver Line Expression

Giant fiber Gal4 driver (BDSC 79602), green channel shows giant fiber neurons, magenta channel shows background staining with nc82:

Giant fiber neurons on Codex BANC v626 dataset:

b1 mn driver (SS40980, also targets iii1 mn, iii3 mn):

Janelia image for SS40980:

b3 mn driver (SS48311, also targets hg1 MN, XBI002, WBI009):

Janelia image for SS48311:

b3 mn driver (SS98650):

Janelia image for SS98650:

Control Experiments for Driver Line Expression

SS45779 – Targeting b3 MN, iii3 MN, hg1 MN

Our line:

Janelia image:

IS69306 – b1 mn driver line

Janelia image:

Retro-Tango control crossed with GF-Gal4 (BDSC: 79602), presynaptic neurons in magenta, giant fiber neurons in cyan and background staining in gray

Retro-Tango x GF-Gal4 from Sorkac et. al (2023)

rut and rad expression in b1 and b3 motor neurons, I looked for cells express 6 genes (rut, rad, FoxP, VGlut, AD TF, DBD TF) at the same time above certain threshold:

Retro-tango confocal images and Torquemeter practice results

Retro-tango:

(2023) retro-Tango enables versatile retrograde circuit tracing in Drosophila eLife 12:e85041. https://doi.org/10.7554/eLife.85041

Retro-tango genotype: y[1] w[*] P{y[+t7.7] w[+mC]=QUAS-mtdTomato-3xHA.S}su(Hw)attP8; P{y[+t7.7] w[+mC]=retro-Tango(panneuronal)}attP40/SM6b; P{y[+t7.7] w[+mC]=10xUAS-retro-Tango(ligand)-P2A-EGFP-F}attP2

Crossed with IS69306 (BDSC 601295 and 75552) split-Gal4 driver to express in b1 motor neurons. CNSs stained with anti-GFP Rabbit, (2nd ab: goat anti-rabbit 488), anti-HA Rat (2nd ab: goat anti-rat 555), nc82 (2nd ab: goat anti-mouse 647)

Channel 1 (Green: Laser Line ( 496 nm) Intensity: 23.99%) Spectral Positions/Gain/Offset: (501nm – 556nm) / 865.7 / -0.03

Channel 2 (Blue: Laser Line ( 561 nm) Intensity: 13.10%) Spectral Positions/Gain/Offset: (566nm – 639nm) / 537.7 / 0.03

Channel 3 (Red: Laser Line ( 633 nm) Intensity: 23.99%) Spectral Positions/Gain/Offset: (644nm – 776nm) / 802.9 / -0.01

Second line retro-tango crossed with SS98650 (split-Gal4 driver line from Janelia targeting b3 motor neurons)

Settings are same as above

Torquemeter Practice with WTB Flies

N=10 out of 24 glued flies

Optomotor at start:

Optomotor end:

Performance index:

Performance subtracted:

rut and rad expression in ventral nerve cord:

cells that express all 4 genes more than 2 fold, 889 cells in total

Top genes expressed in all of these cells: