1 supporting information10.1038...119 were collected from n = 1 biological replicate. * t = 0min t =...

Supporting Information 1

Modeling Environmentally-Induced Motor neuron Degeneration in Zebrafish 2

Jessica R Morrice1, Cheryl Y Gregory-Evans1-3 and Christopher A Shaw1-3* 3

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1Department of Experimental Medicine 6

2Graduate Program in Neuroscience 7

3Department of Ophthalmology and Visual Sciences 8

*[email protected] 9

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Supplementary data 24

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Supplementary Figure S1. Neurotoxic effect of time-dependent exposure to BPA. 27

Wild type embryos were exposed to control, vehicle control or BPA at 6 hpf and fixed at 28

a) 24 hpf (N= 2 – 5 biological replicates) and b) 48 hpf (P = 0.0001; N = 7-10 biological 29

replicates). Error bars represent ± s.d., P values were determined by Mann-Whitney U 30

test. 31

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Supplementary Table S1. Lethality of delayed exposure at 12 hpf. Delayed exposure 33

to BPA at 12 hpf did not increase embryonic lethality at 24 hpf or 48 hpf (% morality of 34

total embryos). N = 11 biological replicates. 35

24 hpf 48 hpf

Vehicle Control* 0% 0%

50 μM BPA 0% 0%

Embryos were subject to chronic non-static exposure starting at 12 hpf in vehicle control 36

and treatment groups. 37

*1% DMSO dissolved in E3 media 38

Abbreviations: BPA – Bisphenol A; hpf – hours post fertilization 39

BisphenolA(BPA)-exposedembryosat48hpf

ControlVehicle1 5 15 50Control BPA(μM)

P=0.0001NS



Aver

age A

xon

Len

gth

(µm

)

Aver

age A

xon

Len

gth

(µm

)

160

140

120

100

80

60

40

20

0

NS

A

AverageAx

onLen

gth(μm)

b



P=0.0001NS



Aver

age A

xon

Len

gth

(µm

)

Aver

age A

xon

Len

gth

(µm

)

160

140

120

100

80

60

40

20

0

NS

B

AverageAx

onLen

gth(μm)

a a b

Supplementary Table S2. Motor function of delayed exposure at 12 hpf. Embryos 40

subject to delayed BPA exposure at 12 hpf reduced motor behaviour at 48 hpf using 41

TEER (% failed motor response). N = 11 biological replicates; N = 1 technical replicate. 42

24 hpf 48 hpf

Vehicle Control*^ 0% 0%

50 μM BPA^ 0% 100%


and treatment groups 44


Abbreviations: BPA – Bisphenol A; hpf – hours post fertilization 46

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Supplementary Figure S2. The effect of neurodevelopmental exposure of BPA on 49

motor axon length. Motor axon length was reduced in Tg:mnx1-GFP embryos subject to 50

delayed exposure at 12 hpf in groups exposed to 50 μM BPA as compared to vehicle 51

control groups (P = 0.04, N = 8 biological replicates, N = 5 - 6 technical replicates; 52

Mann-Whitney U test). 53

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Table 3.1 Embryonic mortality

Table 3.2 Embryos with a failed motor

Supplementary Table S3. Motor Function of delayed exposure at 3 dpf. Embryos 55

subject to delayed exposure to BPA starting at 3 dpf. Values indicate percent of Tg:mnx1-56

GFP embryos with failed motor response at 5 dpf. N = 15 biological replicates in 3 dpf, 57

N = 8 biological replicates in 5 dpf group; N = 1 technical replicate. 58

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Embryos were subject to chronic non-static exposure of treatment starting at 3 dpf in all 63

groups. 64

Ψ Motor function of randomly selected embryos grown normally in embryo media was 65

analyzed prior to either treatment exposure 66


Abbreviations: BPA – Bisphenol A; dpf – days post fertilization 68

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Supplementary Table S4. Lethality of delayed exposure at 3 dpf. Delayed exposure to 70

BPA at 3 dpf increased embryonic lethality at 5 dpf (% morality of total embryos). N = 71

120 biological replicates. Embryos subject to delayed exposure to BPA starting at 3 dpf 72

instead of 6 hpf. 73

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Embryos were subject to chronic non-static exposure of treatment starting at 3 dpf in all 78

groups. 79


3 dpf ψ Exposure at 3 dpf 5 dpf

6.7% Vehicle Control* 12.5%

50 μM BPA 100%

4 dpf 5 dpf

Vehicle Control* 0% 0%

50 μM BPA 3.3% 93.7%

Abbreviations: BPA – Bisphenol A; dpf – days post fertilization 81

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Supplementary Figure S3. Exposure to BPA at 3 dpf on motor axon length at 5 dpf. 85

Tg:mnx1-GFP embryos subject to 42 hours of treatment exposure at 3 dpf instead of 6 86

hpf showed reduced motor axon length at 5 dpf as compared to vehicle controls. (P = 87

0.03, N = 8 biological replicates, N = 3 - 4 technical replicates). Error bars represent ± 88

s.d., P value was determined by a student’s t-test using Welch’s correction. 89

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Vehicl

e Contro

l

50 µ

M BPA

0

50

100

150

200

250

Delayed BPA exposure at 3 dpf

Ave

rage

Axo

n Le

ngth

(µ

m)

P = 0.03

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Supplementary Figure S4. Environmental toxin exposure is suggestive of retrograde 93

motor neuron degeneration. Reduced motor axon length at 48 hpf is associated with 94

increased motor cell death at 72 hpf in embryos exposed to 50 μMBPA. Symbol 95

represents the group mean, error bars represent s.d.; N = 9 – 10 biological replicates. 96

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Supplementary Table S5. Localization of activated microglia. Proportion of amoeboid 98

microglia spatially associated with degenerating motor axons as compared to the motor 99

cell soma in the spinal cord at 48 hpf in Tg:mnx1-GFP/pU1-RFP embryos (% of total 100

microglial cells). N = 8-10 biological replicates; N = 1-15 technical replicates. 101

Microglia associated

with motor axons* (%)

Microglia associated

with soma* (%)

Vehicle Controlψ 97 3

50 µM BPA 100 0


and treatment groups. 103

*Data are based on number of activated microglial cells spatially associated with motor 104

axons or located in the spinal cord in 1 hemisegment within the 6 – 9 somite region. 105

Ψ 1% DMSO dissolved in E3 media 106

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Supplementary Figure S5. Microglial engulfment of apoptotic neurons at 72 hpf. 109

Representative static images from live imaging of a microglial cell engulfing an apoptotic 110

neuron in the spinal cord at 72 hpf following BPA exposure in a double transgenic 111

Tg:pU1:Gal4-UAS-RFP/NBT:DLexPR-secA5-TagBFP embryo, which have pU1+ 112

microglia cells labeled in red and apoptotic neurons labeled in blue. Asterisks denotes 113

pU1+ cell (in red) in the process of engulfing a neuron undergoing apoptosis (in blue) in 114

the spinal cord and its subsequent migration away from this site. White arrow denotes an 115

apoptotic neuron being removed from the spinal cord over a time course of 20 minutes. 116

Note that specific microglia were not followed from 48 hpf to 72 hpf, and cells imaged in 117

this figure illustrate the general microglial response in the spinal cord at 72 hpf. Images 118

were collected from N = 1 biological replicate. 119

*

t=0min t=14min t=20minOverla

yNBT

-Ann

exinV

pU1

*

*

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Supplementary Figure S6. Quantification of muscle birefringence (a-b) Birefringence 123

of skeletal muscle at 48 hpf in wild type embryos exposed to vehicle control or BPA at 6 124

hpf. c) Wild type embryos exposed to vehicle control or 50 μM BPA had similar skeletal 125

muscle birefringence at 48 hpf (P=0.27; N=5 biological replicates, N=1 technical 126

replicate). Birefringence values represent the mean intensity of the total trunk normalized 127

to the area of trunk analyzed. Scale bar = 500 µm. Data was analyzed by students t-test 128

using Welch’s correction. 129

Vehicl

e Contro

l

50 µ

M BPA

0.00

0.01

0.02

0.03

0.04

Bir

efri

ngen

ce

c

b

Vehicle

Control

50μMBPA

b

a

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Supplementary Figure S7. Cell death caused by BPA is not specific to motor 131

neurons. Representative images of non-specific cell death following BPA exposure. PI 132

staining in spinal cord and musculature of embryos exposed to 50 µM BPA or vehicle 133

control. Images represent one hemisegment within the 6 – 9 somite region of Tg:mnx1-134

GFP embryos at 3dpf. Inset images show that the neurotoxic effect of BPA is not motor 135

neuron-specific in the spinal cord. Spinal cord (SC) is indicated by the white capped bar. 136

Scale bar = 20 µm. 137

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mnx1PI

VehicleCo

ntrol

50μMBPA

SC SC

SC SC

SCSCSC

α-Bungarotoxin An1-synaptotagmin2 overlay

Control

SCSCSC

SCSCSC

VehicleControl

50μMBPA

α-Bungarotoxin An/-synaptotagmin2 overlay

Control

VehicleControl

50μMBPA

overlay

Supplementary Video S1. Activated microglia spatially interact with degenerating 141

motor neurons. Representative 3D reconstruction of microglia interacting with motor 142

axons in a double transgenic Tg:mnx1-GFP/pU1-RFP BPA-exposed embryo, which have 143

pU1+ microglia cells shown here in magenta and motor neurons labeled in green, 144

extending from the spinal cord. Image collected at 30 hpf by confocal microscopy and 145

reconstructed in ImageJ. X, Y and Z axes are labeled in green. Z-stack images were 146

collected and reconstructed from N=1 biological replicate. 147

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1 supporting information10.1038...119 were collected from n = 1 biological replicate. * t = 0min t =...

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