J . of Evans Blue ( EB ) of in - vivo brain permeability at P90 ( top ) representative pictures ( bottom ) of perfused brains from Ctrl ( left ; 0 . 70 ± 0 . 47 ) Poly I : C ( right ; 0 . 10 ± 0 . 10 ) offspring . In accompanying pseudocolor - blue pixels are represented as a gradient of - to - blue depending on of color in original photo while absence of blue tones is represented as green . Data information : Ctrl = bars with purple border . Poly I : C = purple bars with purple border . Numbers in bars indicate number of ( N ) ( n ) . Bars represent mean ± .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O

J. Quantification of Evans Blue (EB) analysis of in-vivo brain permeability at P90 (top) and representative pictures (bottom) of perfused brains from Female Ctrl (left; 0.70 ± 0.47) and Poly I:C (right; 0.10 ± 0.10) offspring. In accompanying pseudocolor images - blue pixels are represented as a gradient of light-to-dark blue depending on the intensity of the color in the original photo while the absence of blue tones is represented as green. Data information: Ctrl females = white bars with purple border. Poly I:C females = purple bars with purple border. Numbers in bars indicate the number of animals (N) and images (n). Bars represent mean ± SEM.


K . Quantitative of optical density of Claudin - 5 ( CLDN5 ) immunoreactive bands normalized by CANX in cortices from P90 Ctrl ( 1 . 00 ± 0 . 16 ) Poly I : C ( 1 . 39 ± 0 . 13 ) offspring . Data information : Ctrl = bars with purple border . Poly I : C = purple bars with purple border . Numbers in bars indicate number of ( N ) ( n ) . Bars represent mean ± .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O

K. Quantitative analysis of the optical density of Claudin-5 (CLDN5) immunoreactive bands normalized by CANX in cortices from P90 female Ctrl (1.00 ± 0.16) and Poly I:C (1.39 ± 0.13) offspring. Data information: Ctrl females = white bars with purple border. Poly I:C females = purple bars with purple border. Numbers in bars indicate the number of animals (N) and images (n). Bars represent mean ± SEM.


L . Quantitative of optical density of ZO - 1 immunoreactive bands normalized by αTUB in cortices from P90 Ctrl ( 1 . 00 ± 0 . 11 ) Poly I : C ( 1 . 19 ± 0 . 19 ) offspring . Data information : Ctrl = bars with purple border . Poly I : C = purple bars with purple border . Numbers in bars indicate number of ( N ) ( n ) . Bars represent mean ± .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O

L. Quantitative analysis of the optical density of ZO-1 immunoreactive bands normalized by αTUB in cortices from P90 female Ctrl (1.00 ± 0.11) and Poly I:C (1.19 ± 0.19) offspring. Data information: Ctrl females = white bars with purple border. Poly I:C females = purple bars with purple border. Numbers in bars indicate the number of animals (N) and images (n). Bars represent mean ± SEM.


M . Representative of P90 Ctrl Poly I : C offspring cortices stained for TJs . Claudin - 5 ( top - green ) ZO - 1 ( bottom - red ) - Scale bar = 10 µm . N . Quantitative of mean of CLDN5 in cortices from P90 Ctrl ( 75 . 14 ± 1 . 08 ) Poly I : C ( 67 . 39 ± 2 . 00 ) offspring . Student ' s t , * p < . 05 . O . Quantitative of CLDN5 signal volume in cortices from P90 Ctrl ( 5 . 54 ± 0 . 59 ) Poly I : C ( 3 . 70 ± 0 . 48 ) offspring . Student ' s t , * p < . 05 . P . Quantitative of mean of ZO - 1 in cortices from P90 Ctrl ( 73 . 16 ± 0 . 59 ) Poly I : C ( 73 . 86 ± 1 . 94 ) offspring . Q . Quantitative of ZO - 1 signal volume in cortices from P90 Ctrl ( 23 . 29 ± 1 . 42 ) Poly I : C ( 16 . 78 ± 2 . 18 ) offspring . Student ' s t , * p < . 05 . Data information : Ctrl = bars with blu border . Poly I : C = blu bars with blu border . Numbers in bars indicate number of ( N ) ( n ) . Bars represent mean ± .

O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O

M. Representative images of P90 Ctrl and Poly I:C male offspring cortices stained for TJs proteins. Claudin-5 (top-green) and ZO-1 (bottom-red) - Scale bar = 10 µm. N. Quantitative analysis of mean intensity of CLDN5 in cortices from P90 male Ctrl (75.14 ± 1.08) and Poly I:C (67.39 ± 2.00) offspring. Student's t test, *p < .05. O. Quantitative analysis of the CLDN5 signal volume in cortices from P90 male Ctrl (5.54 ± 0.59) and Poly I:C (3.70 ± 0.48) offspring. Student's t test, *p < .05. P. Quantitative analysis of mean intensity of ZO-1 in cortices from P90 male Ctrl (73.16 ± 0.59) and Poly I:C (73.86 ± 1.94) offspring. Q. Quantitative analysis of the ZO-1 signal volume in cortices from P90 male Ctrl (23.29 ± 1.42) and Poly I:C (16.78 ± 2.18) offspring. Student's t test, *p < .05. Data information: Ctrl males = white bars with blu border. Poly I:C males = blu bars with blu border. Numbers in bars indicate the number of animals (N) and images (n). Bars represent mean ± SEM.


T . [ Left ] of in - vivo cerebrovascular permeability to EB of P90 Ctrl ( 0 . 16 ± 0 . 16 ) Poly I : C - treated ( 0 . 89 ± 0 . 89 ) injected at P20 . [ Right ] Representative pictures of saline perfused brains 24h after EB injection accompanied by pseudocolor - blue pixels are represented as a gradient of - to - blue depending on of color in original photo while absence of blue tones is represented as green - from Ctrl ( top ) Poly I : C ( bottom ) mice . Data information : Ctrl = bars with blu border . Poly I : C = blu bars with blu border . Numbers in bars indicate number of ( N ) ( n ) . Bars represent mean ± .

O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O

T. [Left] Quantification of in-vivo cerebrovascular permeability to EB of P90 Ctrl (0.16 ± 0.16) and Poly I:C-treated (0.89 ± 0.89) males injected at P20. [Right] Representative pictures of saline perfused brains 24h after the EB injection accompanied by pseudocolor images - blue pixels are represented as a gradient of light-to-dark blue depending on the intensity of the color in the original photo while the absence of blue tones is represented as green - from Ctrl (top) and Poly I:C (bottom) male mice. Data information: Ctrl males = white bars with blu border. Poly I:C males = blu bars with blu border. Numbers in bars indicate the number of animals (N) and images (n). Bars represent mean ± SEM.


U . Quantitative of optical density of CLDN5 immunoreactive bands normalized to CANX in cortices from P90 Ctrl ( 0 . 99 ± 0 . 12 ) Poly I : C ( 0 . 98 ± 0 . 10 ) treated at P20 . Data information : Ctrl = bars with blu border . Poly I : C = blu bars with blu border . Numbers in bars indicate number of ( N ) ( n ) . Bars represent mean ± .

O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O

U. Quantitative analysis of the optical density of CLDN5 immunoreactive bands normalized to CANX in cortices from P90 Ctrl (0.99 ± 0.12) and Poly I:C (0.98 ± 0.10) males treated at P20. Data information: Ctrl males = white bars with blu border. Poly I:C males = blu bars with blu border. Numbers in bars indicate the number of animals (N) and images (n). Bars represent mean ± SEM.


V . Quantitative of optical density of ZO - 1 immunoreactive bands normalized to CANX in cortices from P90 Ctrl ( 1 . 00 ± 0 . 11 ) Poly I : C ( 0 . 73 ± 0 . 05 ) treated at P20 . Data information : Ctrl = bars with blu border . Poly I : C = blu bars with blu border . Numbers in bars indicate number of ( N ) ( n ) . Bars represent mean ± .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O

V. Quantitative analysis of the optical density of ZO-1 immunoreactive bands normalized to CANX in cortices from P90 Ctrl (1.00 ± 0.11) and Poly I:C (0.73 ± 0.05) males treated at P20. Data information: Ctrl males = white bars with blu border. Poly I:C males = blu bars with blu border. Numbers in bars indicate the number of animals (N) and images (n). Bars represent mean ± SEM.


O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O

A. qPCR analyses of GD9+6h male embryos for: Pecam1 (Ctrl = 1.00 ± 0.17 N=8, Poly I:C = 0.89 ± 0.23 N=13), CD248 (Ctrl = 1.00 ± 0.20 N=6, Poly I:C = 1.35 ± 0.31 N=11), Vegfa (Ctrl = 1.00 ± 0.19 N=8, Poly I:C = 0.15 ± 0.04 N=8, Student's t test, ***p < .001), Pdfgb (Ctrl = 1.00 ± 0.23 N=5, Poly I:C = 1.10 ± 0.18 N=13), Tgfb1 (Ctrl = 1.00 ± 0.21 N=7, Poly I:C = 1.76 ± 0.22 N=10, Mann-Whitney U test, *p < .05), Tgfb2 (Ctrl = 1.00 ± 0.28 N=6, Poly I:C = 1.37 ± 0.28 N=13), Tgfb3 (Ctrl = 1.00 ± 0.51 N=6, Poly I:C = 0.73 ± 0.19 N=10), Foxf2 (Ctrl = 1.00 ± 0.45 N=8, Poly I:C = 0.46 ± 0.18 N=13) mRNAs. B. qPCR analyses of E17 male cortices for: Pecam1 (Ctrl = 1.00 ± 0.63 N=10, Poly I:C = 3.07 ± 1.58 N=8; Mann-Whitney U test, *p < .05), CD248 (Ctrl = 1.00 ± 0.23 N=10, Poly I:C = 3.33 ± 0.79 N=9; Mann-Whitney U test, **p < .01), Vegfa (Ctrl = 1.00 ± 0.10 N=8, Poly I:C = 1.70 ± 0.29 N=5; Mann-Whitney U test, *p < .05), Pdfgb (Ctrl = 1.00 ± 0.75 N=9, Poly I:C = 2.05 ± 0.96 N=9; Mann-Whitney U test, *p < .05), Tgfb1 (Ctrl = 1.00 ± 0.16 N=7, Poly I:C = 15.27 ± 5.75 N=7; Mann-Whitney U test, ***p < .001), Tgfb2 (Ctrl = 1.00 ± 0.16 N=8, Poly I:C = 3.82 ± 1.05 N=6; Mann-Whitney U test, *p < .05), Tgfb3 (Ctrl = 1.00 ± 0.10 N=9, Poly I:C = 10.58 ± 6.73 N=8; Mann-Whitney U test, ***p < .001); Foxf2 (Ctrl = 1.00 ± 0.06 N=5, Poly I:C = 2.60 ± 0.57 N=8; Mann-Whitney U test, *p < .05) mRNAs. C. qPCR analyses of P90 male cortices for: Pecam1 (Ctrl = 1.00 ± 0.18 N=8, Poly I:C = 0.85 ± 0.08 N=8), CD248 (Ctrl = 1.00 ± 0.20 N=9, Poly I:C = 0.93 ± 0.11 N=8), Vegfa (Ctrl = 1.00 ± 0.07 N=8, Poly I:C = 0.75 ± 0.06 N=8; Mann-Whitney U test, *p < .05); Pdfgb (Ctrl = 1.00 ± 0.20 N=8, Poly I:C = 1.11 ± 0.17 N=4), Tgfb1 (Ctrl = 1.00 ± 0.29 N=5, Poly I:C = 2.37 ± 0.47 N=7; Mann-Whitney U test, *p < .05); Tgfb2 (Ctrl = 1.00 ± 0.29 N=4, Poly I:C = 1.19 ± 0.26 N=7), Tgfb3 (Ctrl = 1.00 ± 0.18 N=10, Poly I:C = 1.78 ± 0.50 N=11) mRNAs. D. qPCR analyses of GD9+6h female embryos for: Pecam1 (Ctrl = 1.00 ± 0.27 N=10, Poly I:C = 2.22 ± 0.50 N=6), CD248 (Ctrl = 1.00 ± 0.27 N=10, Poly I:C = 2.32 ± 0.57 N=6; Mann-Whitney U test, *p < .05), Vegfa (Ctrl = 1.00 ± 0.38 N=8, Poly I:C = 0.29 ± 0.03 N=5; Mann-Whitney U test, *p < .05), Pdfgb (Ctrl = 1.00 ± 0.36 N=10, Poly I:C = 1.31 ± 0.47 N=6), Tgfb1 (Ctrl = 1.00 ± 0.16 N=8, Poly I:C = 1.63 ± 0.42 N=7), Tgfb2 (Ctrl = 1.00 ± 0.33 N=9, Poly I:C = 1.75 ± 0.29 N=6; Mann-Whitney U test, *p < .05), Tgfb3 (Ctrl = 1.00 ± 0.29 N=9, Poly I:C = 1.33 ± 0.51 N=6), Foxf2 (Ctrl = 1.00 ± 0.28 N=9, Poly I:C = 1.71 ± 0.52 N=6) mRNAs. E. qPCR analyses of E17 female cortices for: Pecam1 (Ctrl = 1.00 ± 0.34 N=6, Poly I:C = 0.27 ± 0.08 N=8), CD248 (Ctrl = 1.00 ± 0.27 N=8, Poly I:C = 1.04 ± 0.28 N=9), Vegfa (Ctrl = 1.00 ± 0.70 N=5, Poly I:C = 0.32 ± 0.13 N=5), Pdfgb (Ctrl = 1.00 ± 0.38 N=6, Poly I:C = 0.19 ± 0.07 N=9), Tgfb1 (Ctrl = 1.00 ± 0.33 N=5, Poly I:C = 0.09 ± 0.03 N=6), Tgfb2 (Ctrl = 1.00 ± 0.32 N=7, Poly I:C = 0.45 ± 0.11 N=8), Tgfb3 (Ctrl = 1.00 ± 0.38 N=7, Poly I:C = 0.40 ± 0.20 N=9), Foxf2 (Ctrl = 1.00 ± 0.18 N=5, Poly I:C = 1.04 ± 0.28 N=8) mRNAs. F. qPCR analyses of P90 female cortices for: Pecam1 (Ctrl = 1.00 ± 0.15 N=5, Poly I:C = 0.69 ± 0.15 N=5), CD248 (Ctrl = 1.00 ± 0.13 N=5, Poly I:C = 0.58 ± 0.09 N=5), Vegfa (Ctrl = 1.00 ± 0.38 N=6, Poly I:C = 1.53 ± 0.38 N=8), Pdfgb (Ctrl = 1.00 ± 0.16 N=9, Poly I:C = 1.77 ± 0.35 N=17), Tgfb1 (Ctrl = 1.00 ± 0.26 N=13, Poly I:C = 1.30 ± 0.23 N=14), Tgfb2 (Ctrl = 1.00 ± 0.27 N=11, Poly I:C = 0.59 ± 0.06 N=4), Tgfb3 (Ctrl = 1.00 ± 0.45 N=11, Poly I:C = 0.51 ± 0.06 N=4) mRNAs. G. qPCR analyses of GD9+6h male embryos for Il1α (Ctrl = 1.00 ± 0.29 N=7, Poly I:C = 2.10 ± 0.57 N=17), Il1β (Ctrl = 1.00 ± 0.39 N=6, Poly I:C = 10.84 ± 4.70 N=6; Mann-Whitney U test, **p < .01), Il1rn (Ctrl = 1.00 ± 0.27 N=7, Poly I:C = 3.60 ± 1.58 N=11), Il6 (Ctrl = 1.00 ± 0.17 N=6, Poly I:C = 0.82 ± 0.12 N=7) mRNAs. H. qPCR analyses of E17 male cortices for: Il1α (Ctrl = 1.00 ± 0.27 N=9, Poly I:C = 1.77 ± 0.53 N=11), Il1β (Ctrl = 1.00 ± 0.21 N=7, Poly I:C = 1.33 ± 0.33 N=8), Il1rn (Ctrl = 1.00 ± 0.25 N=10, Poly I:C = 0.82 ± 0.40 N=18), Il6 (Ctrl = 1.00 ± 0.24 N=7, Poly I:C = 2.17 ± 0.43 N=8) mRNAs. I. qPCR analyses of P90 male cortices for: Il1α (Ctrl = 1.00 ± 0.24 N=8, Poly I:C = 1.51 ± 0.26 N=10), Il1β (Ctrl = 1.00 ± 0.27 N=8, Poly I:C = 1.19 ± 0.34 N=8), Il1rn (Ctrl = 1.00 ± 0.31 N=18, Poly I:C = 1.30 ± 0.50 N=14), Il6 (Ctrl = 1.00 ± 0.27 N=7, Poly I:C = 2.40 ± 0.70 N=8) mRNAs. J. qPCR analyses of GD9+6h female embryos for: Il1α (Ctrl = 1.00 ± 0.93 N=6, Poly I:C = 0.03 ± 0.00 N=4), Il1β (Ctrl = 1.00 ± 0.61 N=5, Poly I:C = 0.17 ± 0.04 N=7), Il1rn (Ctrl = 1.00 ± 0.25 N=9, Poly I:C = 0.33 ± 0.11 N=7), Il6 (Ctrl = 1.00 ± 0.48 N=7, Poly I:C = 0.59 ± 0.24 N=5) mRNAs. K. qPCR analyses of E17 female cortices for: Il1α (Ctrl = 1.00 ± 0.31 N=5, Poly I:C = 1.38 ± 0.20 N=12), Il1β (Ctrl = 1.00 ± 0.30 N=5, Poly I:C = 1.05 ± 0.35 N=7), Il1rn (Ctrl = 1.00 ± 0.55 N=6, Poly I:C = 0.45 ± 0.22 N=6), Il6 (Ctrl = 1.00 ± 0.26 N=7, Poly I:C = 0.94 ± 0.14 N=8) mRNAs. L. qPCR analyses of P90 female cortices for Il1α (Ctrl = 1.00 ± 0.08 N=5, Poly I:C = 1.32 ± 0.11 N=9), Il1β (Ctrl = 1.00 ± 0.13 N=5, Poly I:C = 0.65 ± 0.09 N=8), Il1rn (Ctrl = 1.00 ± 0.61 N=4, Poly I:C = 0.85 ± 0.29 N=7), Il6 (Ctrl = 1.00 ± 0.11 N=9, Poly I:C = 1.28 ± 0.20 N=20) mRNAs. Values are normalized over control (dashed line). Data information: Ctrl males = white bars with blu border. Poly I:C males = blu bars with blu border. Ctrl females = white bars with purple border. Poly I:C females = purple bars with purple border. Numbers in bars indicate the number of animals (N). Bars represent mean ± SEM.


M . ELISA of total TGF - β1 in E17 brains as pg / mL ( Ctrl = 132 . 6 ± 18 . 14 , Poly I : C = 129 . 6 ± 8 . 62 ) . N . ELISA of active free TGF - β1 in E17 brains as pg / mL ( Ctrl = undetectable , Poly I : C = 0 . 92 ± 0 . 25 ) . O . ELISA of total TGF - β1 in E17 brains as pg / mL ( Ctrl = 177 . 6 ± 10 . 88 , Poly I : C = 134 . 4 ± 13 . 59 ) . P . ELISA of active free TGF - β1 in E17 brains as pg / mL ( undetectable ) . Data information : Ctrl = bars with blu border . Poly I : C = blu bars with blu border . Ctrl = bars with purple border . Poly I : C = purple bars with purple border . Numbers in bars indicate number of ( N ) . Bars represent mean ± .

O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O

M. ELISA quantification of total TGF-β1 in E17 male embryos brains as pg/mL (Ctrl = 132.6 ± 18.14, Poly I:C = 129.6 ± 8.62). N. ELISA quantification of active free TGF-β1 in E17 male embryos brains as pg/mL (Ctrl = undetectable, Poly I:C = 0.92 ± 0.25). O. ELISA quantification of total TGF-β1 in E17 female embryos brains as pg/mL (Ctrl = 177.6 ± 10.88, Poly I:C = 134.4 ± 13.59). P. ELISA quantification of active free TGF-β1 in E17 female embryos brains as pg/mL (undetectable). Data information: Ctrl males = white bars with blu border. Poly I:C males = blu bars with blu border. Ctrl females = white bars with purple border. Poly I:C females = purple bars with purple border. Numbers in bars indicate the number of animals (N). Bars represent mean ± SEM.


Q - S . Western Blotting of SMAD in E17 cortices for ( Q ) pSMAD1 - 5 ( Ctrl = 0 . 99 ± 0 . 21 , Poly I : C = 2 . 67 ± 0 . 46 ) ; Mann - Whitney U , * p < . 05 , ( R ) pSMAD2 ( Ctrl = 1 . 00 ± 0 . 04 , Poly I : C = 0 . 88 ± 0 . 24 ) , ( S ) SMAD4 ( Ctrl = 1 . 00 ± 0 . 12 , Poly I : C = 0 . 96 ± 0 . 21 ) . were normalized to GAPDH . T - V . Western Blotting of SMAD in E17 cortices for ( T ) pSMAD1 - 5 ( Ctrl = 1 . 00 ± 0 . 02 , Poly I : C = 0 . 70 ± 0 . 18 ) , ( U ) pSMAD2 ( Ctrl = 1 . 00 ± 0 . 01 , Poly I : C = 1 . 09 ± 0 . 12 ) , ( V ) SMAD4 ( Ctrl = 1 . 00 ± 0 . 16 , Poly I : C = 1 . 46 ± 0 . 57 . were normalized to GAPDH . Data information : Ctrl = bars with blu border . Poly I : C = blu bars with blu border . Ctrl = bars with purple border . Poly I : C = purple bars with purple border . Numbers in bars indicate number of ( N ) . Bars represent mean ± .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O

Q-S. Western Blotting quantification of SMAD proteins in E17 male cortices for (Q) pSMAD1-5 (Ctrl = 0.99 ± 0.21, Poly I:C = 2.67 ± 0.46); Mann-Whitney U test, *p < .05, (R) pSMAD2 (Ctrl = 1.00 ± 0.04, Poly I:C = 0.88 ± 0.24), (S) SMAD4 (Ctrl = 1.00 ± 0.12, Poly I:C = 0.96 ± 0.21). Protein levels were normalized to GAPDH. T-V. Western Blotting quantification of SMAD proteins in E17 female cortices for (T) pSMAD1-5 (Ctrl = 1.00 ± 0.02, Poly I:C = 0.70 ± 0.18), (U) pSMAD2 (Ctrl = 1.00 ± 0.01, Poly I:C = 1.09 ± 0.12), (V) SMAD4(Ctrl = 1.00 ± 0.16, Poly I:C = 1.46 ± 0.57. Protein levels were normalized to GAPDH. Data information: Ctrl males = white bars with blu border. Poly I:C males = blu bars with blu border. Ctrl females = white bars with purple border. Poly I:C females = purple bars with purple border. Numbers in bars indicate the number of animals (N). Bars represent mean ± SEM.


A . TEER of in vitro BBB models composed by a monolayer of ECs sorted from E17 Ctrl ( 1 . 00 ± 0 . 02 ) , Poly I : C ( 0 . 92 ± 0 . 04 ) , Ctrl ( 1 . 15 ± 0 . 04 ) , Poly I : C ( 1 . 00 ± 0 . 07 ) offspring . Data are normalized to Ctrl . Data information : prenatally exposed to vehicle ( Ctrl ) or Poly I : C on GD9 were dissected , cortex derived vascular were used to build in - vitro barrier models . derived from Ctrl = bars with blu border . derived from Poly I : C = blu bars with blu border . derived from Ctrl = bars with purple border . derived from Poly I : C = purple bars with purple border . Numbers in bars indicate number of ( N ) or independent cultures / pictures . Bars represent mean ± .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. TEER of the in vitro BBB models composed by a monolayer of ECs sorted from E17 Male Ctrl (1.00 ± 0.02), Male Poly I:C (0.92 ± 0.04), Female Ctrl (1.15 ± 0.04), Female Poly I:C (1.00 ± 0.07) offspring. Data are normalized to male Ctrl. Data information: Embryos prenatally exposed to vehicle (Ctrl) or Poly I:C on GD9 were dissected, and cortex derived vascular cells were used to build in-vitro barrier models. Cells derived from Ctrl males = white bars with blu border. Cells derived from Poly I:C males = blu bars with blu border. Cells derived from Ctrl females = white bars with purple border. Cells derived from Poly I:C females = purple bars with purple border. Numbers in bars indicate the number of animals (N) or independent cultures/pictures. Bars represent mean ± SEM.


B . Permeability to 10 kDa Dextran of in vitro BBB models composed by a monolayer of sorted ECs from Ctrl ( 1 . 00 ± 0 . 06 ) , Poly I : C ( 1 . 13 ± 0 . 02 ) , Ctrl ( 0 . 97 ± 0 . 05 ) , Poly I : C ( 0 . 95 ± 0 . 01 ) offspring . Mann - Whitney U , * p < . 05 . Data are normalized to Ctrl . Data information : prenatally exposed to vehicle ( Ctrl ) or Poly I : C on GD9 were dissected , cortex derived vascular were used to build in - vitro barrier models . derived from Ctrl = bars with blu border . derived from Poly I : C = blu bars with blu border . derived from Ctrl = bars with purple border . derived from Poly I : C = purple bars with purple border . Numbers in bars indicate number of ( N ) or independent cultures / pictures . Bars represent mean ± .

O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O

B. Permeability to 10 kDa Dextran of the in vitro BBB models composed by a monolayer of sorted ECs from Male Ctrl (1.00 ± 0.06), Male Poly I:C (1.13 ± 0.02), Female Ctrl (0.97 ± 0.05), Female Poly I:C (0.95 ± 0.01) offspring. Mann-Whitney U test, *p < .05. Data are normalized to male Ctrl. Data information: Embryos prenatally exposed to vehicle (Ctrl) or Poly I:C on GD9 were dissected, and cortex derived vascular cells were used to build in-vitro barrier models. Cells derived from Ctrl males = white bars with blu border. Cells derived from Poly I:C males = blu bars with blu border. Cells derived from Ctrl females = white bars with purple border. Cells derived from Poly I:C females = purple bars with purple border. Numbers in bars indicate the number of animals (N) or independent cultures/pictures. Bars represent mean ± SEM.


C . TEER of barriers composed of bEND . 3 alone ( 1 . 00 ± 0 . 00 ) or co - cultured with sorted PCs from Ctrl ( 1 . 17 ± 0 . 07 ) , Poly I : C ( 0 . 95 ± 0 . 05 ) , Ctrl ( 1 . 15 ± 0 . 08 ) , Poly I : C ( 1 . 15 ± 0 . 08 ) offspring . Mann - Whitney U , * p < . 05 . Data are normalized to bEnd . 3 . Data information : prenatally exposed to vehicle ( Ctrl ) or Poly I : C on GD9 were dissected , cortex derived vascular were used to build in - vitro barrier models . derived from Ctrl = bars with blu border . derived from Poly I : C = blu bars with blu border . derived from Ctrl = bars with purple border . derived from Poly I : C = purple bars with purple border . Numbers in bars indicate number of ( N ) or independent cultures / pictures . Bars represent mean ± .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. TEER of barriers composed of bEND.3 alone (1.00 ± 0.00) or co-cultured with sorted PCs from Male Ctrl (1.17 ± 0.07), Male Poly I:C (0.95 ± 0.05), Female Ctrl (1.15 ± 0.08), Female Poly I:C (1.15 ± 0.08) offspring. Mann-Whitney U test, *p < .05. Data are normalized to bEnd.3. Data information: Embryos prenatally exposed to vehicle (Ctrl) or Poly I:C on GD9 were dissected, and cortex derived vascular cells were used to build in-vitro barrier models. Cells derived from Ctrl males = white bars with blu border. Cells derived from Poly I:C males = blu bars with blu border. Cells derived from Ctrl females = white bars with purple border. Cells derived from Poly I:C females = purple bars with purple border. Numbers in bars indicate the number of animals (N) or independent cultures/pictures. Bars represent mean ± SEM.


D . Permeability to 10 kDa Dextran of barriers composed by a monolayer of bEND . 3 alone ( 1 . 00 ± 0 . 06 ) or co - cultured with sorted PCs from Ctrl ( 0 . 47 ± 0 . 09 ) , Poly I : C ( 0 . 46 ± 0 . 07 ) , Ctrl ( 0 . 46 ± 0 . 11 ) , Poly I : C ( 0 . 65 ± 0 . 05 ) offspring . One sample t , * * p < . 01 . Data are normalized to bEnd . 3 . Data information : prenatally exposed to vehicle ( Ctrl ) or Poly I : C on GD9 were dissected , cortex derived vascular were used to build in - vitro barrier models . derived from Ctrl = bars with blu border . derived from Poly I : C = blu bars with blu border . derived from Ctrl = bars with purple border . derived from Poly I : C = purple bars with purple border . Numbers in bars indicate number of ( N ) or independent cultures / pictures . Bars represent mean ± .

O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. Permeability to 10 kDa Dextran of barriers composed by a monolayer of bEND.3 alone (1.00 ± 0.06) or co-cultured with sorted PCs from Male Ctrl (0.47 ± 0.09), Male Poly I:C (0.46 ± 0.07), Female Ctrl (0.46 ± 0.11), Female Poly I:C (0.65 ± 0.05) offspring. One sample t test, **p < .01. Data are normalized to bEnd.3. Data information: Embryos prenatally exposed to vehicle (Ctrl) or Poly I:C on GD9 were dissected, and cortex derived vascular cells were used to build in-vitro barrier models. Cells derived from Ctrl males = white bars with blu border. Cells derived from Poly I:C males = blu bars with blu border. Cells derived from Ctrl females = white bars with purple border. Cells derived from Poly I:C females = purple bars with purple border. Numbers in bars indicate the number of animals (N) or independent cultures/pictures. Bars represent mean ± SEM.


E . TEER ( Ω . cm2 ) was monitored over 72h treatment with TGF - β1 normalized to baseline in BBB models composed by a monolayer of sorted ECs from ( 72h TGF - β1 = 82 . 85 ± 1 . 56 ) or ( 72h TGF - β1 = 85 . 92 ± 2 . 03 ) . insert shows values at 72h ( bars ) normalized to 0h values ( dashed ) . One sample t , * * * p < . 001 , * * * * p < . 0001 . Data information : prenatally exposed to vehicle ( Ctrl ) or Poly I : C on GD9 were dissected , cortex derived vascular were used to build in - vitro barrier models . In Fig . 3 E blue purple dotted lines represent derived from brain respectively .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. TEER (Ω.cm2) was monitored over 72h treatment with TGF-β1 and normalized to baseline in BBB models composed by a monolayer of sorted ECs from Male (72h TGF-β1 = 82.85 ± 1.56) or Female (72h TGF-β1 = 85.92 ± 2.03) embryos. The insert shows the values at 72h (bars) normalized to the 0h values (dashed line). One sample t test, ***p < .001, ****p < .0001. Data information: Embryos prenatally exposed to vehicle (Ctrl) or Poly I:C on GD9 were dissected, and cortex derived vascular cells were used to build in-vitro barrier models. In Fig.3 E blue and purple dotted lines represent cells derived from male and female brain respectively.


F . TEER ( Ω . cm2 ) was monitored over 72h treatment with TGF - β1 normalized to baseline in barrier models composed by a monolayer of bEND . 3 alone ( 72h TGF - β1 = 89 . 85 ± 2 . 05 ) or co - cultured with sorted PCs from ( 72h TGF - β1 = 88 . 83 ± 1 . 93 ) or ( 72h TGF - β1 = 89 . 02 ± 2 . 22 ) . insert shows values at 72h ( bars ) normalized to 0h values ( dashed ) . Wilcoxon signed - rank , * p < . 05 . Data information : prenatally exposed to vehicle ( Ctrl ) or Poly I : C on GD9 were dissected , cortex derived vascular were used to build in - vitro barrier models . In Fig . 3 F blue purple dotted lines represent derived from brain respectively .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F. TEER (Ω.cm2) was monitored over 72h treatment with TGF-β1 and normalized to baseline in barrier models composed by a monolayer of bEND.3 alone (72h TGF-β1 = 89.85 ± 2.05) or co-cultured with sorted PCs from Male (72h TGF-β1 = 88.83 ± 1.93) or Female (72h TGF-β1 = 89.02 ± 2.22) embryos. The insert shows the values at 72h (bars) normalized to the 0h values (dashed line). Wilcoxon signed-rank test, *p < .05. Data information: Embryos prenatally exposed to vehicle (Ctrl) or Poly I:C on GD9 were dissected, and cortex derived vascular cells were used to build in-vitro barrier models. In Fig.3 F blue and purple dotted lines represent cells derived from male and female brain respectively.


G . 10 kDa Dextran permeability after 72h TGF - β1 treatment of in vitro BBBs prototypes composed by a monolayer made of sorted ECs from NT ( 0 . 43 ± 0 . 02 ) , TGF - β1 ( 0 . 43 ± 0 . 03 ) ; NT ( 0 . 45 ± 0 . 00 ) , TGF - β1 ( 0 . 47 ± 0 . 01 ) . Data information : prenatally exposed to vehicle ( Ctrl ) or Poly I : C on GD9 were dissected , cortex derived vascular were used to build in - vitro barrier models . derived from Ctrl = bars with blu border . derived from Ctrl = bars with purple border . Numbers in bars indicate number of ( N ) or independent cultures / pictures . Bars represent mean ± .

O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G. 10 kDa Dextran permeability after 72h TGF-β1 treatment of the in vitro BBBs prototypes composed by a monolayer made of sorted ECs from Male NT (0.43 ± 0.02), Male TGF-β1 (0.43 ± 0.03); Female NT (0.45 ± 0.00), Female TGF-β1 (0.47 ± 0.01). Data information: Embryos prenatally exposed to vehicle (Ctrl) or Poly I:C on GD9 were dissected, and cortex derived vascular cells were used to build in-vitro barrier models. Cells derived from Ctrl males = white bars with blu border. Cells derived from Ctrl females = white bars with purple border. Numbers in bars indicate the number of animals (N) or independent cultures/pictures. Bars represent mean ± SEM.


H . 10 kDa Dextran permeability 72h after TGF - β1 treatment of NVU prototypes composed by a monolayer made of bEND . 3 alone ( NT , solid black bar = 0 . 45 ± 0 . 01 , TGF - β1 , dotted black bar = 0 . 49 ± 0 . 00 ) co - cultured with sorted PCs from ( NT = 0 . 45 ± 0 . 04 , TGF - β1 = 0 . 48 ± 0 . 00 ) , or ( NT = 0 . 49 ± 0 . 03 , TGF - β1 = 0 . 53 ± 0 . 02 ) . Student ' s t , * p < . 05 . Data information : prenatally exposed to vehicle ( Ctrl ) or Poly I : C on GD9 were dissected , cortex derived vascular were used to build in - vitro barrier models . bEend . 3 = bars with black border . derived from Ctrl = bars with blu border . derived from Ctrl = bars with purple border . Numbers in bars indicate number of ( N ) or independent cultures / pictures . Bars represent mean ± .

O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H. 10 kDa Dextran permeability 72h after TGF-β1 treatment of NVU prototypes composed by a monolayer made of bEND.3 alone (NT, solid black bar = 0.45 ± 0.01, TGF-β1, dotted black bar = 0.49 ± 0.00) and co-cultured with sorted PCs from Male (NT = 0.45 ± 0.04, TGF-β1 = 0.48 ± 0.00), or Female (NT = 0.49 ± 0.03, TGF-β1 = 0.53 ± 0.02) embryos. Student's t test, *p < .05. Data information: Embryos prenatally exposed to vehicle (Ctrl) or Poly I:C on GD9 were dissected, and cortex derived vascular cells were used to build in-vitro barrier models. bEend.3 cells = bars with black border. Cells derived from Ctrl males = white bars with blu border. Cells derived from Ctrl females = white bars with purple border. Numbers in bars indicate the number of animals (N) or independent cultures/pictures. Bars represent mean ± SEM.


I . FACS quantitative data of normalized MFI of CD146 ( NT = 100 . 00 ± 1 . 14 , TGF - β1 = 54 . 08 ± 9 . 42 ; Student ' s t , * p < . 05 ) representative histogram of untreated ( solid gray ) TGF - β1 treated ( hollow black bold ) . J . FACS quantitative data of normalized MFI of Claudin - 5 ( CLDN5 ; NT = 100 . 00 ± 8 . 94 , TGF - β1 = 65 . 22 ± 5 . 29 ; Student ' s t , * * p < . 01 ) representative histogram of untreated ( solid gray ) TGF - β1 treated ( hollow black bold ) . Data information : bEend . 3 = bars with black border . black dotted represent bEnd . 3 cultures . Numbers in bars indicate number of ( N ) or independent cultures / pictures . Bars represent mean ± .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

I. FACS quantitative data of the normalized MFI of CD146 (NT = 100.00 ± 1.14, TGF-β1 = 54.08 ± 9.42; Student's t test, *p < .05) and representative histogram of untreated (solid gray) and TGF-β1 treated cells (hollow black bold line). J. FACS quantitative data of the normalized MFI of Claudin-5 (CLDN5; NT = 100.00 ± 8.94, TGF-β1 = 65.22 ± 5.29; Student's t test, **p < .01) and representative histogram of untreated (solid gray) and TGF-β1 treated cells (hollow black bold line). Data information: bEend.3 cells = bars with black border. The black dotted line represent bEnd.3 cultures. Numbers in bars indicate the number of animals (N) or independent cultures/pictures. Bars represent mean ± SEM.


K . Representative of monolayers of endothelial isolated from brains stained for CD146 ( red ) CLDN5 ( green ) under control conditions ( NT ) or upon treatment with different cytokines . Scale bar = 50 µm . L . Quantitative of CD146 immunofluorescence mean measured in brain EC monolayers under control conditions or after 72h treatments with different cytokines : NT = 100 . 00 ± 6 . 55 , TGF - β1 = 94 . 03 ± 2 . 01 , IL - 1β + TGF - β1 = 84 . 87 ± 4 . 59 , IL - 1β = 92 . 34 ± 2 . 65 ; Kruskal - Wallis , * p < . 05 . M . Quantitative of Claudin - 5 immunofluorescence mean in brain EC monolayers under control conditions or after 72h treatments with different cytokines : NT = 99 . 76 ± 5 . 19 , TGF - β1 = 86 . 74 ± 6 . 76 , IL - 1β + TGF - β1 = 51 . 64 ± 3 . 31 , IL - 1β = 107 . 3 ± 7 . 37 ; Ordinary one - way ANOVA , * * p < . 01 , * * * * p < . 0001 . N . Quantitative of Claudin - 5 immunofluorescence ( µm2 ) in brain EC monolayers under control conditions or after 72h treatments with different cytokines : NT = 39781 ± 2342 , TGF - β1 = 31707 ± 5939 , IL - 1β + TGF - β1 = 5415 ± 1037 , IL - 1β = 46303 ± 2986 ; Ordinary one - way ANOVA , * p < . 05 , * * p < . 01 , * * * * p < . 0001 . Data information : Cortex - derived - EC isolated from mice at 2 mouths were used to build in - vitro barrier models analyzed . derived from Ctrl = bars with blu border . Numbers in bars indicate number of ( N ) or independent cultures / pictures . Bars represent mean ± .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

K. Representative images of monolayers of primary endothelial cells isolated from male brains and stained for CD146 (red) and CLDN5 (green) under control conditions (NT) or upon treatment with different cytokines. Scale bar = 50 µm. L. Quantitative analysis of CD146 immunofluorescence mean intensity measured in primary male brain EC monolayers under control conditions or after 72h treatments with different cytokines: NT = 100.00 ± 6.55, TGF-β1 = 94.03 ± 2.01, IL-1β + TGF-β1 = 84.87 ± 4.59, IL-1β = 92.34 ± 2.65; Kruskal-Wallis, *p < .05. M. Quantitative analysis of Claudin-5 immunofluorescence mean intensity in primary male brain EC monolayers under control conditions or after 72h treatments with different cytokines: NT = 99.76 ± 5.19, TGF-β1 = 86.74 ± 6.76, IL-1β + TGF-β1 = 51.64 ± 3.31, IL-1β = 107.3 ± 7.37; Ordinary one-way ANOVA, **p < .01, ****p < .0001. N. Quantitative analysis of Claudin-5 immunofluorescence area (µm2) in primary male brain EC monolayers under control conditions or after 72h treatments with different cytokines: NT = 39781 ± 2342, TGF-β1 = 31707 ± 5939, IL-1β + TGF-β1 = 5415 ± 1037, IL-1β = 46303 ± 2986; Ordinary one-way ANOVA, *p < .05, **p < .01, ****p < .0001. Data information: Cortex-derived-EC isolated from male mice at 2 mouths were used to build in-vitro barrier models and analyzed. Cells derived from Ctrl males = white bars with blu border. Numbers in bars indicate the number of animals (N) or independent cultures/pictures. Bars represent mean ± SEM.


O . Graph showing TEER measured by volt - ohmmeter at 72h normalized in NT ( 100 . 00 ± 7 . 46 ) IL - 1β + TGF - β1 ( 49 . 93 ± 10 . 96 ) treated brain EC cultures . Student ' s t , * * p < . 01 . Data are normalized to t0 . Data information : Cortex - derived - EC isolated from mice at 2 mouths were used to build in - vitro barrier models analyzed . derived from Ctrl = bars with blu border . Numbers in bars indicate number of ( N ) or independent cultures / pictures . Bars represent mean ± .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

O. Graph showing TEER measured by volt-ohmmeter at 72h normalized in NT (100.00 ± 7.46) and IL-1β + TGF-β1 (49.93 ± 10.96) treated primary male brain EC cultures. Student's t test, **p < .01. Data are normalized to t0. Data information: Cortex-derived-EC isolated from male mice at 2 mouths were used to build in-vitro barrier models and analyzed. Cells derived from Ctrl males = white bars with blu border. Numbers in bars indicate the number of animals (N) or independent cultures/pictures. Bars represent mean ± SEM.


P . Permeability to 3kDa dextran quantified by spectrophotometry in NT ( 1 . 00 ± 0 . 10 ) IL - 1β + TGF - β1 ( 1 . 92 ± 0 . 33 ) treated brain EC cultures . Student ' s t , * p < . 05 . Q . Permeability to 10kDa dextran quantified by spectrophotometry in NT ( 1 . 00 ± 0 . 11 ) IL - 1β + TGF - β1 ( 1 . 80 ± 0 . 26 ) treated brain EC cultures . Student ' s t , * p < . 05 . Data information : Cortex - derived - EC isolated from mice at 2 mouths were used to build in - vitro barrier models analyzed . derived from Ctrl = bars with blu border . Numbers in bars indicate number of ( N ) or independent cultures / pictures . Bars represent mean ± .

O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

P. Permeability to 3kDa dextran quantified by fluorescence spectrophotometry in NT (1.00 ± 0.10) and IL-1β + TGF-β1 (1.92 ± 0.33) treated primary male brain EC cultures. Student's t test, *p < .05. Q. Permeability to 10kDa dextran quantified by fluorescence spectrophotometry in NT (1.00 ± 0.11) and IL-1β + TGF-β1 (1.80 ± 0.26) treated primary male brain EC cultures. Student's t test, *p < .05. Data information: Cortex-derived-EC isolated from male mice at 2 mouths were used to build in-vitro barrier models and analyzed. Cells derived from Ctrl males = white bars with blu border. Numbers in bars indicate the number of animals (N) or independent cultures/pictures. Bars represent mean ± SEM.


A . [ left ] Representative dot plots showing gating strategy for E17 samples to distinguish NVU . were pre - gated on single live ( Zombie Aqua negative ) . Live were pre - gated on CD45 ( CD45 - ) . [ right ] Brain Pericytes ( PC ) were defined as CD146 + CD31 - brain Endothelial ( EC ) as CD31 + . B . [ left ] of between PC number to EC numbers for ( blue ) ( purple ) E17 offspring cortex samples . Ctrl = 1 . 10 ± 0 . 08 , Poly I : C = 1 . 34 ± 0 . 09 , Ctrl = 1 . 02 ± 0 . 08 , Poly I : C = 1 . 12 ± 0 . 09 . Mann - Whitney U , * p < . 05 . Bars represent average of ± , numbers in bars indicate number of . [ right up ] Percentages of ECs in Ctrl ( 0 . 79 ± 0 . 05 ) , Poly I : C ( 0 . 90 ± 0 . 09 ) , Ctrl ( 0 . 65 ± 0 . 07 ) , Poly I : C ( 0 . 80 ± 0 . 07 ) samples [ right down ] PCs in Ctrl ( 0 . 60 ± 0 . 05 ) , Poly I : C ( 0 . 57 ± 0 . 06 ) , Ctrl ( 0 . 53 ± 0 . 07 ) , Poly I : C ( 0 . 62 ± 0 . 06 ) samples . C . [ left ] Gating strategy used to analyze P30 samples : single , live ( zombie Aqua negative ) , CD45 - . [ right ] PCs are identified as CD146 + CD13 + CD31 - ; ECs are CD13 - CD31 + D . [ left ] of EC / PC number for P30 offspring cortex samples , obtained by manual gating . Ctrl = 1 . 78 ± 0 . 29 , Poly I : C = 1 . 74 ± 0 . 32 , Ctrl = 1 . 49 ± 0 . 11 , Poly I : C = 1 . 60 ± 0 . 19 . [ right up ] Percentages of ECs in Ctrl ( 17 . 07 ± 1 . 88 ) , Poly I : C ( 17 . 52 ± 1 . 90 ) , Ctrl ( 14 . 58 ± 0 . 66 ) , Poly I : C ( 15 . 73 ± 1 . 07 ) samples , [ right down ] of PCs in Ctrl ( 10 . 55 ± 1 . 89 ) , Poly I : C ( 10 . 67 ± 1 . 04 ) , Ctrl ( 10 . 07 ± 0 . 85 ) , Poly I : C ( 10 . 20 ± 0 . 79 Data information : prenatally exposed to vehicle ( Ctrl ) or Poly I : C on GD9 were dissected , cortex single - suspension was analyzed by multi - color flow cytometry . Ctrl = bars with blu border . Poly I : C = blu bars with blu border . Ctrl = bars with purple border . Poly I : C = purple bars with purple border . Bars represent mean ± , numbers in bars indicate number of .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O

A. [left] Representative dot plots showing the gating strategy for E17 samples to distinguish NVU cells. The cells were pre-gated on single cells and live cells (Zombie Aqua negative). Live cells were pre-gated on CD45 (CD45-). [right] Brain Pericytes (PC) were defined as CD146+CD31- cells and brain Endothelial Cells (EC) as CD31+cells. B. [left] Quantification of the ratio between PC number to EC numbers for male (blue) and female (purple) E17 offspring cortex samples. Male Ctrl = 1.10 ± 0.08, Male Poly I:C = 1.34 ± 0.09, Female Ctrl = 1.02 ± 0.08, Female Poly I:C = 1.12 ± 0.09. Mann-Whitney U test, *p < .05. Bars represent average ratio of individual embryos ± SEM, numbers in bars indicate the number of embryos. [right up] Percentages of ECs in Male Ctrl (0.79 ± 0.05), Male Poly I:C (0.90 ± 0.09), Female Ctrl (0.65 ± 0.07), Female Poly I:C (0.80 ± 0.07) samples and [right down] PCs in Male Ctrl (0.60 ± 0.05), Male Poly I:C (0.57 ± 0.06), Female Ctrl (0.53 ± 0.07), Female Poly I:C (0.62 ± 0.06) samples. C. [left] Gating strategy used to analyze P30 samples: single cells, live (zombie Aqua negative), CD45- cells. [right] PCs are identified as CD146+CD13+CD31-; ECs are CD13-CD31+ D. [left] Quantification of EC/PC number ratio for male and female P30 offspring cortex samples, obtained by manual gating. Male Ctrl = 1.78 ± 0.29, Male Poly I:C = 1.74 ± 0.32, Female Ctrl = 1.49 ± 0.11, Female Poly I:C = 1.60 ± 0.19. [right up] Percentages of ECs in Male Ctrl (17.07 ± 1.88), Male Poly I:C (17.52 ± 1.90), Female Ctrl (14.58 ± 0.66), Female Poly I:C (15.73 ± 1.07) samples, and [right down] percentage of PCs in Male Ctrl (10.55 ± 1.89), Male Poly I:C (10.67 ± 1.04), Female Ctrl (10.07 ± 0.85), Female Poly I:C (10.20 ± 0.79 Data information: Embryos prenatally exposed to vehicle (Ctrl) or Poly I:C on GD9 were dissected, and cortex single-cell suspension was analyzed by multi-color flow cytometry. Ctrl males = white bars with blu border. Poly I:C males = blu bars with blu border. Ctrl females = white bars with purple border. Poly I:C females = purple bars with purple border. Bars represent mean ± SEM, numbers in bars indicate the number of animals.


E . Normalized of live ( CD45 - ) EdU - positive at E17 in Ctrl ( 1 . 00 ± 0 . 05 ) , Poly I : C ( 0 . 62 ± 0 . 11 ) , Ctrl ( 0 . 61 ± 0 . 04 ) , Poly I : C ( 0 . 59 ± 0 . 11 ) samples . Mann - Whitney U Kruskal - Wallis , * p < . 05 , * * p < . 001 . F . of PCs EdU - positive at E17 in Ctrl ( 7 . 88 ± 1 . 57 ) , Poly I : C ( 4 . 55 ± 0 . 70 ) , Ctrl ( 7 . 03 ± 1 . 04 ) , Poly I : C ( 4 . 93 ± 0 . 83 ) samples . Student ' s t , * p < . 05 . G . of ECs EdU - positive at E17 in Ctrl ( 4 . 30 ± 0 . 71 ) , Poly I : C ( 2 . 80 ± 0 . 53 ) , Ctrl ( 2 . 84 ± 0 . 43 ) , Poly I : C ( 2 . 29 ± 0 . 77 ) samples . H . Normalized of live ( CD45 - ) EdU - positive at P30 in Ctrl ( 1 . 16 ± 0 . 14 ) , Poly I : C ( 0 . 66 ± 0 . 16 ) , Ctrl ( 1 . 00 ± 0 . 08 ) , Poly I : C ( 0 . 91 ± 0 . 13 ) samples . Mann - Whitney U , * p < . 05 . I . of PCs EdU - positive at P30 in Ctrl ( 0 . 80 ± 0 . 24 ) , Poly I : C ( 0 . 24 ± 0 . 06 ) , Ctrl ( 0 . 26 ± 0 . 08 ) , Poly I : C ( 0 . 38 ± 0 . 13 ) samples . Mann - Whitney U , * p < . 05 . J . of ECs EdU - positive at P30 in Ctrl ( 0 . 58 ± 0 . 10 ) , Poly I : C ( 0 . 44 ± 0 . 19 ) , Ctrl ( 0 . 38 ± 0 . 22 ) , Poly I : C ( 0 . 25 ± 0 . 06 ) samples . Data information : Ctrl = bars with blu border . Poly I : C = blu bars with blu border . Ctrl = bars with purple border . Poly I : C = purple bars with purple border . Bars represent mean ± , numbers in bars indicate number of .

O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O

E. Normalized percentage of live(CD45-) EdU-positive cells at E17 in Male Ctrl (1.00 ± 0.05), Male Poly I:C (0.62 ± 0.11), Female Ctrl (0.61 ± 0.04), Female Poly I:C (0.59 ± 0.11) samples. Mann-Whitney U test and Kruskal-Wallis test, *p < .05, **p < .001. F. Percentage of PCs EdU-positive cells at E17 in Male Ctrl (7.88 ± 1.57), Male Poly I:C (4.55 ± 0.70), Female Ctrl (7.03 ± 1.04), Female Poly I:C (4.93 ± 0.83) samples. Student's t test, *p < .05. G. Percentage of ECs EdU-positive cells at E17 in Male Ctrl (4.30 ± 0.71), Male Poly I:C (2.80 ± 0.53), Female Ctrl (2.84 ± 0.43), Female Poly I:C (2.29 ± 0.77) samples. H. Normalized percentage of live(CD45-) EdU-positive cells at P30 in Male Ctrl (1.16 ± 0.14), Male Poly I:C (0.66 ± 0.16), Female Ctrl (1.00 ± 0.08), Female Poly I:C (0.91 ± 0.13) samples. Mann-Whitney U test, *p < .05. I. Percentage of PCs EdU-positive cells at P30 in Male Ctrl (0.80 ± 0.24), Male Poly I:C (0.24 ± 0.06), Female Ctrl (0.26 ± 0.08), Female Poly I:C (0.38 ± 0.13) samples. Mann-Whitney U test, *p < .05. J. Percentage of ECs EdU-positive cells at P30 in Male Ctrl (0.58 ± 0.10), Male Poly I:C (0.44 ± 0.19), Female Ctrl (0.38 ± 0.22), Female Poly I:C (0.25 ± 0.06) samples. Data information: Ctrl males = white bars with blu border. Poly I:C males = blu bars with blu border. Ctrl females = white bars with purple border. Poly I:C females = purple bars with purple border. Bars represent mean ± SEM, numbers in bars indicate the number of animals.


K . [ left ] CD146 normalized MFI of EC at P30 in Ctrl ( 100 . 00 ± 8 . 27 ) , Poly I : C ( 65 . 73 ± 3 . 63 ) , Ctrl ( 57 . 08 ± 14 . 10 ) , Poly I : C ( 43 . 07 ± 8 . 34 ) . Mann - Whitney U Ordinary one - way ANOVA , * * p < . 01 . [ right ] Representative histograms CD146 expression on ECs in Ctrl ( blue hollow ) , Poly I : C ( solid blue ) , Ctrl ( purple hollow ) , Poly I : C ( solid purple ) samples . Data information : prenatally exposed to vehicle ( Ctrl ) or Poly I : C on GD9 were dissected , cortex single - suspension was analyzed by multi - color flow cytometry . Ctrl = bars with blu border . Poly I : C = blu bars with blu border . Ctrl = bars with purple border . Poly I : C = purple bars with purple border . Bars represent mean ± , numbers in bars indicate number of .

O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O

K. [left] CD146 normalized MFI of the EC cells at P30 in Male Ctrl (100.00 ± 8.27), Male Poly I:C (65.73 ± 3.63), Female Ctrl (57.08 ± 14.10), Female Poly I:C (43.07 ± 8.34). Mann-Whitney U test and Ordinary one-way ANOVA, **p < .01. [right] Representative histograms CD146 expression on ECs in Male Ctrl (blue hollow), Male Poly I:C (solid light blue), Female Ctrl (purple hollow), Female Poly I:C (solid purple) samples. Data information: Embryos prenatally exposed to vehicle (Ctrl) or Poly I:C on GD9 were dissected, and cortex single-cell suspension was analyzed by multi-color flow cytometry. Ctrl males = white bars with blu border. Poly I:C males = blu bars with blu border. Ctrl females = white bars with purple border. Poly I:C females = purple bars with purple border. Bars represent mean ± SEM, numbers in bars indicate the number of animals.


L . Percentages of PC CD146 + + , CD146 + CD146 - respectively at P30 in Ctrl ( 26 . 32 / 36 . 75 / 23 . 48 ± 2 . 84 / 3 . 31 / 4 . 62 ) , Poly I : C ( 27 . 53 / 42 . 40 / 20 . 60 ± 2 . 25 / 0 . 58 / 1 . 94 ) , Ctrl ( 29 . 05 / 40 . 98 / 2 . 19 ± 1 . 99 / 1 . 32 / 2 . 19 ) , Poly I : C ( 27 . 17 / 46 . 60 / 17 . 10 ± 3 . 09 / 3 . 86 / 1 . 96 ) samples . Data information : CD13 positive pericytes were represented ad CD146 - ( greed bars ) , CD146 + ( green bars ) CD46 + + ( green bars ) . Bars represent mean ± , numbers in bars indicate number of .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

L. Percentages of PC CD146++, CD146+ and CD146- cells respectively at P30 in Male Ctrl (26.32/36.75/23.48 ± 2.84/3.31/4.62), Male Poly I:C (27.53/42.40/20.60 ± 2.25/0.58/1.94), Female Ctrl (29.05/40.98/2.19 ± 1.99/1.32/2.19), Female Poly I:C (27.17/46.60/17.10 ± 3.09/3.86/1.96) samples. Data information: CD13 positive pericytes were represented ad CD146- (dark greed bars), CD146+ (green bars) and CD46++ (light green bars). Bars represent mean ± SEM, numbers in bars indicate the number of animals.


M . [ left ] CD105 normalized MFI of EC at P30 in Ctrl ( 100 . 00 ± 7 . 39 ) , Poly I : C ( 99 . 58 ± 6 . 23 ) , Ctrl ( 100 . 30 ± 1 . 61 ) , Poly I : C ( 96 . 67 ± 3 . 72 ) . [ right ] Representative histograms indicating mean ( MFI ) of CD105 on EC in Ctrl ( blue hollow ) , Poly I : C ( solid blue ) , Ctrl ( purple hollow ) , Poly I : C ( solid purple ) samples . N . [ left ] Quantitative data of normalized MFI of CD105 in PCs at P30 in Ctrl ( 100 . 00 ± 3 . 43 ) , Poly I : C ( 98 . 01 ± 5 . 14 ) , Ctrl ( 98 . 57 ± 4 . 16 ) , Poly I : C ( 87 . 99 ± 4 . 21 ) . Mann - Whitney U , * p < . 05 . [ right ] Representative histograms for CD105 expression on PCs in Ctrl ( blue hollow ) , Poly I : C ( solid blue ) , Ctrl ( purple hollow ) , Poly I : C ( solid purple ) samples . Data information : prenatally exposed to vehicle ( Ctrl ) or Poly I : C on GD9 were dissected , cortex single - suspension was analyzed by multi - color flow cytometry . Ctrl = bars with blu border . Poly I : C = blu bars with blu border . Ctrl = bars with purple border . Poly I : C = purple bars with purple border . Bars represent mean ± , numbers in bars indicate number of .

O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O

M. [left] CD105 normalized MFI of the EC cells at P30 in Male Ctrl (100.00 ± 7.39), Male Poly I:C (99.58 ± 6.23), Female Ctrl (100.30 ± 1.61), Female Poly I:C (96.67 ± 3.72). [right] Representative histograms indicating mean fluorescence intensity (MFI) of CD105 on EC in Male Ctrl (blue hollow), Male Poly I:C (solid light blue), Female Ctrl (purple hollow), Female Poly I:C (solid purple) samples. N. [left] Quantitative data of the normalized MFI of CD105 in PCs at P30 in Male Ctrl (100.00 ± 3.43), Male Poly I:C (98.01 ± 5.14), Female Ctrl (98.57 ± 4.16), Female Poly I:C (87.99 ± 4.21). Mann-Whitney U test, *p < .05. [right] Representative histograms for CD105 expression on PCs in Male Ctrl (blue hollow), Male Poly I:C (solid light blue), Female Ctrl (purple hollow), Female Poly I:C (solid purple) samples. Data information: Embryos prenatally exposed to vehicle (Ctrl) or Poly I:C on GD9 were dissected, and cortex single-cell suspension was analyzed by multi-color flow cytometry. Ctrl males = white bars with blu border. Poly I:C males = blu bars with blu border. Ctrl females = white bars with purple border. Poly I:C females = purple bars with purple border. Bars represent mean ± SEM, numbers in bars indicate the number of animals.


O . [ top ] Representative of CD13 ( red ) positive pericytes covering Lectin - positive vessels ( green ) in sections of P90 untreated brains . [ bottom ] 3D rendering , Scale bar = 20 µm , enlargement scale bar = 10 µm . P . of CD13 - positive pericyte coverage at P90 in Ctrl ( 53 . 42 ± 9 . 22 ) Poly I : C ( 27 . 73 ± 5 . 29 ) . Student ' s t , * p < . 05 . Data information : Ctrl = bars with blu border . Poly I : C = blu bars with blu border . Bars represent mean ± , numbers in bars indicate number of .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O

O. [top] Representative images of CD13 (red) positive pericytes covering Lectin-positive vessels (green) in sections of P90 untreated male brains. [bottom] 3D rendering, Scale bar = 20 µm, enlargement scale bar = 10 µm. P. Percentage of CD13-positive pericyte coverage at P90 in Male Ctrl (53.42 ± 9.22) and Poly I:C (27.73 ± 5.29). Student's t test, *p < .05. Data information: Ctrl males = white bars with blu border. Poly I:C males = blu bars with blu border. Bars represent mean ± SEM, numbers in bars indicate the number of animals.


Q . of PDGFrB - positive pericyte coverage at P90 in Ctrl ( 66 . 15 ± 3 . 09 ) Poly I : C ( 55 . 08 ± 3 . 10 ) . Student ' s t , * p < . 05 . Data information : Ctrl = bars with blu border . Poly I : C = blu bars with blu border . Bars represent mean ± , numbers in bars indicate number of .

O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O

Q. Percentage of PDGFrB-positive pericyte coverage at P90 in Male Ctrl (66.15 ± 3.09) and Poly I:C (55.08 ± 3.10). Student's t test, *p < .05. Data information: Ctrl males = white bars with blu border. Poly I:C males = blu bars with blu border. Bars represent mean ± SEM, numbers in bars indicate the number of animals.


R . Representative of PDGFrB ( red ) NG2 ( ) positive pericytes covering Lectin - positive vessels ( blu ) in sections of P90 untreated brains . Scale bar = 10 µm , enlargement scale bar = 5 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

R. Representative images of PDGFrB (red) and NG2 (white) positive pericytes covering Lectin-positive vessels (blu) in sections of P90 untreated male brains. Scale bar = 10 µm, enlargement scale bar = 5 µm.


S . of CD13 - positive pericyte coverage at P90 in Ctrl ( 71 . 57 ± 8 . 37 ) Poly I : C ( 69 . 13 ± 6 . 63 ) . T . of PDGFrB - positive pericyte coverage at P90 in Ctrl ( 66 . 77 ± 11 . 76 ) Poly I : C ( 70 . 56 ± 8 . 32 ) . Data information : Ctrl = bars with purple border . Poly I : C = purple bars with purple border . Bars represent mean ± , numbers in bars indicate number of .

O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

S. Percentage of CD13-positive pericyte coverage at P90 in Female Ctrl (71.57 ± 8.37) and Poly I:C (69.13 ± 6.63). T. Percentage of PDGFrB-positive pericyte coverage at P90 in Female Ctrl (66.77 ± 11.76) and Poly I:C (70.56 ± 8.32). Data information: Ctrl females = white bars with purple border. Poly I:C females = purple bars with purple border. Bars represent mean ± SEM, numbers in bars indicate the number of animals.


A . Representative of brain acute bleedings detected through hemoglobin in P30 Ctrl Poly I : C MIA offspring . Scale bar = 500 µm , enlargement scale bar = 50 µm . B . Representative of brain acute bleedings detected through hemoglobin in P90 Ctrl Poly I : C MIA offspring . Scale bar = 500 µm , enlargement scale bar = 50 µm . C . of hemoglobin ( Hb ) positive signal in brain of P30 P90 MIA offspring , expressed as of . P30 Ctrl = 0 . 00 ± 0 . 00 , P30 Poly I : C = 0 . 01 ± 0 . 00 , P90 Ctrl = 0 . 00 ± 0 . 00 , P90 Poly I : C = 0 . 01 ± 0 . 00 . Mann - Whitney U , * * * p < . 001 . Data information : Ctrl = bars with blu border . Poly I : C = blu bars with blu border . Numbers in bars indicate number of ( N ) slices ( n ) . Bars represent mean ± .

O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O

A. Representative images of brain acute bleedings detected through hemoglobin IHC in P30 Ctrl and Poly I:C MIA male offspring. Scale bar = 500 µm, enlargement scale bar = 50 µm. B. Representative images of brain acute bleedings detected through hemoglobin IHC in P90 Ctrl and Poly I:C MIA male offspring. Scale bar = 500 µm, enlargement scale bar = 50 µm. C. Quantification of hemoglobin (Hb) positive signal in the brain of P30 and P90 male MIA offspring, expressed as percentage of the area. P30 Ctrl = 0.00 ± 0.00, P30 Poly I:C = 0.01 ± 0.00, P90 Ctrl = 0.00 ± 0.00, P90 Poly I:C = 0.01 ± 0.00. Mann-Whitney U test, ***p < .001. Data information: Ctrl males = white bars with blu border. Poly I:C males = blu bars with blu border. Numbers in bars indicate the number of animals (N) and slices (n). Bars represent mean ± SEM.


D . Representative 3D rendering of iron deposits detected by DAB - enhanced Perls in P30 Ctrl Poly I : C MIA offspring brains . Brain volume is rendered gold - transparent with positive signal for color - coded by surface dimension . Scale bar = 100 µm .

O O O O O O B-MEASURED_VAR O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. Representative 3D rendering of iron deposits detected by DAB-enhanced Perls staining in P30 Ctrl and Poly I:C MIA male offspring brains. Brain volume is rendered gold-transparent with positive signal for the staining color-coded by surface dimension. Scale bar = 100 µm.


E . of DAB - enhanced histochemical iron stains in brain of P30 Ctrl ( 0 . 00 ± 0 . 00 ) Poly I : C ( 0 . 01 ± 0 . 00 ) MIA offspring , expressed as Perls - positive of volume . Mann - Whitney U , * p < . 05 . Data information : Ctrl = bars with blu border . Poly I : C = blu bars with blu border . Numbers in bars indicate number of ( N ) slices ( n ) . Bars represent mean ± .

O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O

E. Quantification of DAB-enhanced histochemical iron stains in the brain of P30 Ctrl (0.00 ± 0.00) and Poly I:C (0.01 ± 0.00) male MIA offspring, expressed as Perls-positive percentage of the volume. Mann-Whitney U test, *p < .05. Data information: Ctrl males = white bars with blu border. Poly I:C males = blu bars with blu border. Numbers in bars indicate the number of animals (N) and slices (n). Bars represent mean ± SEM.


F . Poisson distribution R value of Perls / DAB positive of P30 MIA offspring , respectively in Coronal ( Ctrl = 1 . 19 ± 0 . 04 , Poly I : C = 0 . 94 ± 0 . 01 ; Mann - Whitney U , * * p < . 01 ) , Axial ( Ctrl = 1 . 16 ± 0 . 08 , Poly I : C = 1 . 03 ± 0 . 03 ) , Sagittal ( Ctrl = 1 . 09 ± 0 . 09 , Poly I : C = 1 . 01 ± 0 . 05 ) planes . Data information : Ctrl = bars with blu border . Poly I : C = blu bars with blu border . Numbers in bars indicate number of ( N ) slices ( n ) . Bars represent mean ± .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O

F. Poisson distribution R value of Perls/DAB positive staining of P30 male MIA offspring, respectively in the Coronal (Ctrl = 1.19 ± 0.04, Poly I:C = 0.94 ± 0.01; Mann-Whitney U test, **p < .01), Axial (Ctrl = 1.16 ± 0.08, Poly I:C = 1.03 ± 0.03), and Sagittal (Ctrl = 1.09 ± 0.09, Poly I:C = 1.01 ± 0.05) planes. Data information: Ctrl males = white bars with blu border. Poly I:C males = blu bars with blu border. Numbers in bars indicate the number of animals (N) and slices (n). Bars represent mean ± SEM.


G . Representative of iron deposits detected through Perls in P90 Ctrl Poly I : C MIA offspring brains . Scale bar = 500 µm , enlargement scale bar = 100 µm . H . of Perls - positive in brain of P90 Ctrl ( 0 . 25 ± 0 . 05 ) Poly I : C ( 0 . 59 ± 0 . 10 ) MIA offspring , expressed as of . Mann - Whitney U , * * p < . 01 . Data information : Ctrl = bars with blu border . Poly I : C = blu bars with blu border . Numbers in bars indicate number of ( N ) slices ( n ) . Bars represent mean ± .

O O O O B-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O

G. Representative images of iron deposits detected through Perls staining in P90 Ctrl and Poly I:C MIA male offspring brains. Scale bar = 500 µm, enlargement scale bar = 100 µm. H. Quantification of Perls-positive staining in the brain of P90 Ctrl (0.25 ± 0.05) and Poly I:C (0.59 ± 0.10) male MIA offspring, expressed as percentage of the area. Mann-Whitney U test, **p < .01. Data information: Ctrl males = white bars with blu border. Poly I:C males = blu bars with blu border. Numbers in bars indicate the number of animals (N) and slices (n). Bars represent mean ± SEM.


I . Representative image of marble burying at 0 30 minutes . 15 glass marbles were evenly spaced on a 5 x 3 grid . Marbles buried by two - thirds at least are indicated by a red circle or by a cross within a red circle when fully covered by sawdust . J . of buried marble ( n ° ) by P90 Ctrl ( 3 . 33 ± 1 . 67 ) , Poly I : C ( 5 . 72 ± 2 . 10 ) , Ctrl ( 3 . 83 ± 1 . 72 ) Poly I : C ( 3 . 00 ± 2 . 00 ) mice . Student ' s t , * * p < . 01 . Data information : Ctrl = bars with blu border . Poly I : C = blu bars with blu border . Ctrl = bars with purple border . Poly I : C = purple bars with purple border . Numbers in bars indicate number of ( N ) slices ( n ) . Bars represent mean ± .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O

I. Representative image of marble burying test at 0 and 30 minutes. 15 glass marbles were evenly spaced on a 5 x 3 grid. Marbles buried by two-thirds at least are indicated by a red circle or by a cross within a red circle when fully covered by the sawdust. J. Quantification of buried marble (n°) by P90 Ctrl Male (3.33 ± 1.67), Poly I:C Male (5.72 ± 2.10), Ctrl Female (3.83 ± 1.72) and Poly I:C Female (3.00 ± 2.00) mice. Student's t test, **p < .01. Data information: Ctrl males = white bars with blu border. Poly I:C males = blu bars with blu border. Ctrl females = white bars with purple border. Poly I:C females = purple bars with purple border. Numbers in bars indicate the number of animals (N) and slices (n). Bars represent mean ± SEM.


K . of self - grooming time ( sec ) by P90 Ctrl ( 19 . 34 ± 13 . 00 ) , Poly I : C ( 43 . 38 ± 35 . 21 ) , Ctrl ( 19 . 86 ± 18 . 06 ) Poly I : C ( 16 . 93 ± 11 . 19 ) mice . Student ' s t , * p < . 05 . Data information : Ctrl = bars with blu border . Poly I : C = blu bars with blu border . Ctrl = bars with purple border . Poly I : C = purple bars with purple border . Numbers in bars indicate number of ( N ) slices ( n ) . Bars represent mean ± .

O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O

K. Quantification of self-grooming time (sec) by P90 Ctrl Males (19.34 ± 13.00), Poly I:C Males (43.38 ± 35.21), Ctrl Females (19.86 ± 18.06) and Poly I:C Females (16.93 ± 11.19) mice. Student's t test, *p < .05. Data information: Ctrl males = white bars with blu border. Poly I:C males = blu bars with blu border. Ctrl females = white bars with purple border. Poly I:C females = purple bars with purple border. Numbers in bars indicate the number of animals (N) and slices (n). Bars represent mean ± SEM.


( D ) Western blot of whole zebrafish larvae , Smyhc1 stained with F59 antibody . smyhc1 + / + larvae express Smyhc1 at 24hpf , but not at 48hpf . smyhc1 - / - larvae do not express Smyhc1 .

(D) Western blot of whole zebrafish larvae, Smyhc1 stained with the F59 antibody. smyhc1+/+ larvae express Smyhc1 at 24hpf, but not at 48hpf. smyhc1-/- larvae do not express Smyhc1.


( E ) Smyhc1 immunohistochemistry stain of 24 hpf ( hour post fertilization ) zebrafish larvae . Filamentous actin is stained with phalloidin ( red ) . Nuclei are stained with DAPI ( blue ) . Smyhc1 is stained with F59 antibody ( green ) ( Elworthy et al , 2008 ) . smyhc1 + / + , smyhc1R673H / + , smyhc1R673H / R673H larvae display Smyhc1 in muscle at 24hpf . smyhc1 - / - larvae at 24 do not stain positively for Smyhc1 . Scale bar represents 50 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Smyhc1 immunohistochemistry stain of 24 hpf (hour post fertilization) zebrafish larvae. Filamentous actin is stained with phalloidin (red). Nuclei are stained with DAPI (blue). Smyhc1 is stained with the F59 antibody (green) (Elworthy et al, 2008). smyhc1+/+, smyhc1R673H/+, and smyhc1R673H/R673H larvae display Smyhc1 in the muscle fibers at 24hpf. smyhc1-/- larvae at 24 do not stain positively for Smyhc1. Scale bar represents 50 µm.


( A ) Morphologies of smyhc1 + / + , smyhc1 - / + smyhc1 - / - are grossly normal at 2 days post fertilization ( dpf ) , despite complete paralysis of smyhc1 - / - . In contrast , smyhc1R673H / + have notochord kinks in larval stages ( arrow head ) , while smyhc1R673H / R673H have much more severe notochord kinks that compress distort axis ( arrow heads ) . smyhc1R673H / - completely phenocopy smyhc1R673H / R673H . ( B ) of smyhc1R673H embryonic at 2 dpf .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Morphologies of smyhc1+/+, smyhc1-/+ and smyhc1-/- embryos are grossly normal at 2 days post fertilization (dpf), despite complete paralysis of smyhc1-/- embryos. In contrast, the smyhc1R673H/+ embryos have notochord kinks in larval stages (arrow head), while smyhc1R673H/R673H embryos have much more severe notochord kinks that compress and distort the body axis (arrow heads). The smyhc1R673H/- embryos completely phenocopy smyhc1R673H/R673H embryos. (B) Quantification of smyhc1R673H embryonic phenotypes at 2 dpf.


( B ) predicted mendelian ( 1 : 2 : 1 ) experimental of genotypes of progeny from smyhc1R673H / + in - crosses demonstrates survival at indicated ages . This quickly skewed toward an overrepresentation of smyhc1 + / + fish with no representation of smyhc1R673H / R673H after 5 dpf . There is also dropout of smyhc1 + / R673H fish into adulthood . Number of fish counted per group is indicated .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) The predicted mendelian (1:2:1) and experimental ratio of genotypes of progeny from smyhc1R673H/+ in-crosses demonstrates survival at the indicated ages. This ratio quickly skewed toward an overrepresentation of smyhc1+/+ fish with no representation of smyhc1R673H/R673H after 5 dpf. There is also dropout of smyhc1+/R673H fish into adulthood. Number of fish counted per group is indicated.


( A ) Gross of smyhc1 mutant adults . Most smyhc1 - / - adult fish display dorsal tail curvature , while smyhc1R673H / + adults have a shortened axis variable spinal curves .

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(A) Gross morphology of smyhc1 mutant adults. Most smyhc1-/- adult fish display dorsal tail curvature, while smyhc1R673H/+ adults have a shortened body axis and variable spinal curves.


( B ) Alizarin red of bone shows spinal curvature but no bony fusions in skeleton of smyhc1 - / - adults , in contrast to compression fusion of vertebrae seen in smyhc1R673H / + adults . Distal tail regions are highlighted enlarged .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Alizarin red staining of bone shows spinal curvature but no bony fusions in the skeleton of smyhc1-/- adults, in contrast to the compression and fusion of vertebrae seen in smyhc1R673H/+ adults. Distal tail regions are highlighted and enlarged.


( C ) of skeletal phenotype severity in adults . Scale bars represent a of 5 mm .

(C) Quantification of skeletal phenotype severity in adults. Scale bars represent a length of 5 mm.


( A ) Time course of - triggered larval movements were manually counted at indicated times between 24 48 hpf . smyhc1 - / - larvae ( n = 36 ) were paralyzed up to 48 hpf . smyhc1R673H / + mutants ( n = 15 ) twitched slightly less than their smyhc1 + / + siblings ( n = 21 ) at 34hpf . central bands of boxplots represent median , boxes of boxplots represent interquartile range ( between first third quartile ) , whiskers represent minimum maximum values , up to 1 . 5 times interquartile range . Outliers displayed are outside of this range .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Time course of light-triggered larval movements were manually counted at indicated times between 24 and 48 hpf. The smyhc1-/- larvae (n = 36) were paralyzed up to 48 hpf. smyhc1R673H/+ mutants (n = 15) twitched slightly less than their smyhc1+/+ siblings (n = 21) at 34hpf. The central bands of the boxplots represent the median, the boxes of the boxplots represent the interquartile range (between the first and third quartile), and the whiskers represent the minimum and maximum values, up to 1.5 times the interquartile range. Outliers displayed are outside of this range.


( B ) travelled during 5 minutes of spontaneous swimming at 6 dpf quantified by motion tracking software ( Noldus Ethovision ) . At 6 dpf , both smyhc1 - / - ( n = 24 ) smyhc1R673H / + ( n = 11 ) larvae travelled significantly less than smyhc1 + / + larvae ( n = 13 ) . central bands of boxplots represent median , boxes of boxplots represent interquartile range ( between first third quartile ) , whiskers represent minimum maximum values , up to 1 . 5 times interquartile range . Outliers displayed are outside of this range .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Distance travelled during 5 minutes of spontaneous swimming at 6 dpf quantified by motion tracking software (Noldus Ethovision). At 6 dpf, both smyhc1-/- (n = 24) and smyhc1R673H/+ (n = 11) larvae travelled significantly less than smyhc1+/+ larvae (n = 13). The central bands of the boxplots represent the median, the boxes of the boxplots represent the interquartile range (between the first and third quartile), and the whiskers represent the minimum and maximum values, up to 1.5 times the interquartile range. Outliers displayed are outside of this range.


( C ) Diagram of swim tunnel used to quantify adult swimming behavior . Water flow was gradually increased until fish fatigued were collected at grate .

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(C) Diagram of swim tunnel used to quantify adult swimming behavior. Water flow was gradually increased until the fish fatigued and were collected at the grate.


( D ) Time spent swimming in swim tunnel before fatigue in adults at 6 months post fertilization ( mpf ) . smyhc1 + / + ( n = 6 ) , smyhc1 - / - ( n = 5 ) , smyhc1R673H / + ( n = 5 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Time spent swimming in swim tunnel before fatigue in adults at 6 months post fertilization (mpf). smyhc1+/+ (n = 6), smyhc1-/- (n = 5), smyhc1R673H/+ (n = 5).


( A ) Confocal of slow skeletal muscle of 1 dpf larvae , stained with phalloidin - rhodamine to detect actin . of filamentous actin are indicated with arrowheads , misshapen myofibers are indicated with arrows outlined . ( B ) Confocal microscope of slow skeletal muscle of 3 dpf larvae , stained with phalloidin - rhodamine . of filamentous actin are indicated with arrowheads , distorted myofibers are indicated with arrows outlined , frayed myofibers are indicated with yellow arrowhead .

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(A) Confocal fluorescence images of slow skeletal muscle of 1 dpf larvae, stained with phalloidin-rhodamine to detect actin. Puncta of filamentous actin are indicated with white arrowheads, misshapen myofibers are indicated with white arrows and outlined. (B) Confocal microscope fluorescence images of slow skeletal muscle of 3 dpf larvae, stained with phalloidin-rhodamine. Puncta of filamentous actin are indicated with white arrowheads, distorted myofibers are indicated with arrows and outlined, and frayed myofibers are indicated with yellow arrowhead.


( C ) Confocal of slow skeletal muscle of 1dpf larvae , stained with phalloidin - rhodamine ( red ) , anti - α - actinin antibodies ( green ) , DAPI ( blue ) . ( D ) Confocal of slow skeletal muscle of 3dpf larvae , stained with phalloidin - rhodamine ( red ) , anti - α - actinin antibodies ( green ) , DAPI ( blue ) . Bundle of actin ringed with α - actinin indicated with - bordered black arrowhead .

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(C) Confocal fluorescence images of slow skeletal muscle of 1dpf larvae, stained with phalloidin-rhodamine (red), anti-α-actinin antibodies (green), and DAPI (blue). (D) Confocal fluorescence images of slow skeletal muscle of 3dpf larvae, stained with phalloidin-rhodamine (red), anti-α-actinin antibodies (green), and DAPI (blue). Bundle of actin ringed with α-actinin indicated with white-bordered black arrowhead.