E . Representative images of Δypt7 , Δopi3Δypt7 cells expressing GFP - Atg8 ( projection ) . Cells were grown to log phase in SD - URA medium and starved in SD - N , with supplementation of choline ( 1 mM ) to SD and SD - N as indicated . Images were taken during growth and starvation by confocal microscopy ( left panel ) . Scale bar 1 µm . Right panel - quantification of cell with more than two GFP - Atg8 puncta , in Δypt7 and Δopi3Δypt7 at growth ( SD ) or starvation ( SD - N ) in the presence ( + choline : SD , SD - N ) or absence ( - choline ) of choline ( 1 mM ) . Incidence were normalized to Δypt7 ( SD - N ) control . Statistical analysis was done by Anova multiple comparisons test - Sidak ' s ( * * * , p ≤ 0 . 001 ) . Error bars represent SEM of least 3 independent experiments . Number of cells analyzed for each strain and condition , SD : Δypt7 ( n = 209 ) , Δopi3Δypt7 ( n = 111 ) , SD - N : Δypt7 ( n = 118 ) , Δopi3Δypt7 ( n = 103 ) , SD + Choline : Δypt7 ( n = 107 ) , Δopi3Δypt7 ( n = 120 ) , Δypt7 ( n = 84 ) , Δopi3Δypt7 ( n = 74 ) .

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E. Representative images of Δypt7, Δopi3Δypt7 cells expressing GFP-Atg8 (projection). Cells were grown to log phase in SD-URA medium and starved in SD-N, with supplementation of choline (1 mM) to SD and SD-N as indicated. Images were taken during growth and starvation by confocal microscopy (left panel). Scale bar 1 µm. Right panel- quantification of cell with more than two GFP-Atg8 puncta, in Δypt7 and Δopi3Δypt7 at growth (SD) or starvation (SD-N) in the presence (+choline: SD, SD-N) or absence (-choline) of choline (1 mM). Incidence were normalized to Δypt7 (SD-N) control. Statistical analysis was done by Anova multiple comparisons test- Sidak's (***, p≤0.001). Error bars represent SEM of least 3 independent experiments. Number of cells analyzed for each strain and condition, SD: Δypt7 (n=209), Δopi3Δypt7 (n=111), SD-N: Δypt7 (n=118), Δopi3Δypt7 (n=103), SD+Choline: Δypt7 (n=107), Δopi3Δypt7 (n=120), Δypt7 (n=84), Δopi3Δypt7 (n=74).


( B ) Phase - contrast images of wild - type or PABPC4KO HeLa cells depleted of PABPC1 by siRNA - mediated knockdown . Scale bar , 100 μm . ( C ) Total numbers of viable cells in ( B ) were quantified with acridine orange and propidium iodide staining and graphed as a percentage of wild - type HeLa cells . Error bars represent the SEM of three biological replicates .

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(B) Phase-contrast images of wild-type or PABPC4KO HeLa cells depleted of PABPC1 by siRNA-mediated knockdown. Scale bar, 100 μm. (C) Total numbers of viable cells in (B) were quantified with acridine orange and propidium iodide staining and graphed as a percentage of wild-type HeLa cells. Error bars represent the SEM of three biological replicates.


( C ) Phase - contrast images of PABPDHFR cells , or PABPDHFR cells transfected with a plasmid coding for wild - type PABPC1 , 48 hours post - TMP removal . Scale bar , 100 μm .

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(C) Phase-contrast images of PABPDHFR cells, or PABPDHFR cells transfected with a plasmid coding for wild-type PABPC1, 48 hours post-TMP removal. Scale bar, 100 μm.


( D ) Western blot analysis of lysates derived from cells imaged in ( C ) .

(D) Western blot analysis of lysates derived from cells imaged in (C).


( B ) SUnSET assay of PABPDHFR cells grown in the presence or absence of TMP for 12 hours to maintain or deplete PABP . Cells were subsequently pulsed with either puromycin or puromycin and cycloheximide ( control ) , lysed and equal protein amounts were resolved by SDS - PAGE . Western blot analysis was performed using a monoclonal antibody against puromycin or actin ( loading control ) .

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(B) SUnSET assay of PABPDHFR cells grown in the presence or absence of TMP for 12 hours to maintain or deplete PABP. Cells were subsequently pulsed with either puromycin or puromycin and cycloheximide (control), lysed and equal protein amounts were resolved by SDS-PAGE. Western blot analysis was performed using a monoclonal antibody against puromycin or actin (loading control).


( D ) Scatter plot comparing log2 fold - changes [ ( - ) PABP vs ( + ) PABP ] in polysome - associated mRNA ( y - axis ) to corresponding changes in total cytoplasmic mRNA . Transcripts identified as regulated via altered translation efficiency ( orange and red ) or abundance ( light and dark green ) according to anota2seq are visualized together with non - regulated transcripts ( grey ) .

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(D) Scatter plot comparing log2 fold-changes [(-) PABP vs (+) PABP] in polysome-associated mRNA (y-axis) to corresponding changes in total cytoplasmic mRNA. Transcripts identified as regulated via altered translation efficiency (orange and red) or abundance (light and dark green) according to anota2seq are visualized together with non-regulated transcripts (grey).


Boxplots of 5 ´ UTR ( A ) , 3 ´ UTR ( B ) lengths for mRNAs whose abundance decreased ( ' DOWN ' , dark green ) or increased ( ' UP ' , light green ) in PABP - depleted cells as compared to cells expressing PABP . Solid horizonal middle lines in box plots denote the median ; lower and upper box limits correspond to first and third quartiles ; whiskers extend from box limits to the most extreme values . Wilcoxon - Mann - Whitney tests ( two - sided ) were used to determine differences between upregulated mRNAs and downregulated mRNAs relative to each other and to the background ( mRNAs whose abundance did not change in the absence of PABP relative to PABP - expressing cells ) .

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Boxplots of 5´UTR (A), 3´UTR (B) lengths for mRNAs whose abundance decreased ('DOWN', dark green) or increased ('UP', light green) in PABP-depleted cells as compared to cells expressing PABP. Solid horizonal middle lines in box plots denote the median; lower and upper box limits correspond to first and third quartiles; whiskers extend from box limits to the most extreme values. Wilcoxon-Mann-Whitney tests (two-sided) were used to determine differences between upregulated mRNAs and downregulated mRNAs relative to each other and to the background (mRNAs whose abundance did not change in the absence of PABP relative to PABP-expressing cells).


( A ) Boxplots of mRNA half - lives ( hrs ) assessed by metabolic labeling of normal human umbilical vein endothelial cells for mRNAs whose abundance decreased ( ' DOWN ' , dark green ) or increased ( ' UP ' , light green ) in PABP - depleted cells as compared to cells expressing PABP comparing ( Tiana et al . , 2020 ) . Solid horizonal middle lines in box plots denote the median ; lower and upper box limits correspond to first and third quartiles ; whiskers extend from box limits to the most extreme values but not further than 1 . 5 × the inter - quartile range ( IQR ) . ( B - H ) PABPDHFR cells were cultured in the presence or absence of TMP for 12 hours . The stabilities of select mRNAs was assessed by using actinomycin D ( 5 μg / ml ) for the indicated amounts of time . Total RNA was isolated , reverse transcribed with random hexamer oligonucleotide primers and quantified by qPCR . mRNA decay rates were normalized to an in vitro synthesized spike - in RNA with the zero time point set at 1 . Error bars represent the SEM of three independent experiments . First order exponential decay trend lines were generated by non - linear regression analysis .

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(A) Boxplots of mRNA half-lives (hrs) assessed by metabolic labeling of normal human umbilical vein endothelial cells for mRNAs whose abundance decreased ('DOWN', dark green) or increased ('UP', light green) in PABP-depleted cells as compared to cells expressing PABP comparing (Tiana et al., 2020). Solid horizonal middle lines in box plots denote the median; lower and upper box limits correspond to first and third quartiles; whiskers extend from box limits to the most extreme values but not further than 1.5× the inter-quartile range (IQR). (B- H) PABPDHFR cells were cultured in the presence or absence of TMP for 12 hours. The stabilities of select mRNAs was assessed by using actinomycin D (5 μg/ml) for the indicated amounts of time. Total RNA was isolated, reverse transcribed with random hexamer oligonucleotide primers and quantified by qPCR. mRNA decay rates were normalized to an in vitro synthesized spike-in RNA with the zero time point set at 1. Error bars represent the SEM of three independent experiments. First order exponential decay trend lines were generated by non-linear regression analysis.


( B ) PABPDHFR cells were transfected with plasmids encoding RL - 6xB or RL - 6xBMUT . 24 hours after transfections , the cells were split and cultured for an additional 12 hours in the presence or absence of TMP . The stabilities of reporter mRNAs was assessed by using actinomycin D ( 5 μg / ml ) for the indicated amounts of time . Total RNA was isolated , reverse transcribed with random hexamer oligonucleotide primers and quantified by qPCR . mRNA decay rates were normalized to an in vitro synthesized spike - in RNA with the zero time point set at 1 . Error bars represent the SEM of three independent experiments . First order exponential decay trend lines were generated by non - linear regression analysis .

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(B) PABPDHFR cells were transfected with plasmids encoding RL-6xB or RL-6xBMUT. 24 hours after transfections, the cells were split and cultured for an additional 12 hours in the presence or absence of TMP. The stabilities of reporter mRNAs was assessed by using actinomycin D (5 μg/ml) for the indicated amounts of time. Total RNA was isolated, reverse transcribed with random hexamer oligonucleotide primers and quantified by qPCR. mRNA decay rates were normalized to an in vitro synthesized spike-in RNA with the zero time point set at 1. Error bars represent the SEM of three independent experiments. First order exponential decay trend lines were generated by non-linear regression analysis.


( A ) Western blot analysis PABPDHFR cells depleted of LSM1 , DCP2 or XRN1 by siRNA - mediated knockdown . siGFP represents a negative control .

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(A) Western blot analysis PABPDHFR cells depleted of LSM1, DCP2 or XRN1 by siRNA-mediated knockdown. siGFP represents a negative control.


( B ) Phase - contrast images of PABPDHFR cells previously transfected with siRNAs targeting GFP ( control ) , LSM1 , DCP2 or XRN1 and cultured in the presence or absence of TMP for 48 hours . Total numbers of viable cells , denoted in the upper right hand corner of images , were quantified with acridine orange and propidium iodide and calculated as a percentage of PABPDHFR cells grown in the presence of TMP , along with the SEM . Scale bar , 100 μm .

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(B) Phase-contrast images of PABPDHFR cells previously transfected with siRNAs targeting GFP (control), LSM1, DCP2 or XRN1 and cultured in the presence or absence of TMP for 48 hours. Total numbers of viable cells, denoted in the upper right hand corner of images, were quantified with acridine orange and propidium iodide and calculated as a percentage of PABPDHFR cells grown in the presence of TMP, along with the SEM. Scale bar, 100 μm.


( A ) Western blot analysis PABPDHFR cells depleted of LSM1 by siRNA - mediated knockdown . Transfected cells were subsequently cultured in the presence or absence of TMP to maintain or deplete endogenous PABP .

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(A) Western blot analysis PABPDHFR cells depleted of LSM1 by siRNA-mediated knockdown. Transfected cells were subsequently cultured in the presence or absence of TMP to maintain or deplete endogenous PABP.


( C and D ) Poly ( A ) tail analysis of GAPDH and ACTB mRNAs from total RNA isolated from PABPDHFR cells described in ( B ) was determined by extension poly ( A ) tail ( ePAT ) . PCR products were run on high - resolution agarose gels . ' A12 ' represents the size of ePAT RT - PCR amplicons derived from a mRNA with a fixed ( A12 ) - tail .

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(C and D) Poly(A) tail analysis of GAPDH and ACTB mRNAs from total RNA isolated from PABPDHFR cells described in (B) was determined by extension poly(A) tail (ePAT). PCR products were run on high-resolution agarose gels. 'A12' represents the size of ePAT RT-PCR amplicons derived from a mRNA with a fixed (A12)-tail.


C Similar to CSR - 1 , the majority of small RNAs interacting with CSR - 2 in vivo have 5 ′ PPP ends . The RNAs in the immunopurified Argonaute complexes were treated with or without Vaccinia virus capping enzyme followed by an alkaline phosphatase , and finally labeled with [ γ - 32P ] ATP and a polynucleotide kinase . RNA products were resolved by 15 % sequencing gels .

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C Similar to CSR-1, the majority of small RNAs interacting with CSR-2 in vivo have 5′PPP ends. The RNAs in the immunopurified Argonaute complexes were treated with or without Vaccinia virus capping enzyme followed by an alkaline phosphatase, and finally labeled with [γ-32P]ATP and a polynucleotide kinase. RNA products were resolved by 15% sequencing gels.


G Three csr - 1 hypomorphic mutants showed a diminished RNAi response to exo - dsRNA . The WT , the csr - 1 ( fj150 ) mutant , and several strains were injected with dsRNA ( 1 . 25 µg / µL ) targeting the maternal gene pos - 1 , essential for embryogenesis . The incidence of lethal embryos in the F1 progeny was examined . The number of F1 progeny counted is indicated by n . Error bars represent 95 % confidence intervals ( CIs ) .

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G Three csr-1 hypomorphic mutants showed a diminished RNAi response to exo-dsRNA. The WT, the csr-1(fj150) mutant, and several strains were injected with dsRNA (1.25 µg/µL) targeting the maternal gene pos-1, essential for embryogenesis. The incidence of lethal embryos in the F1 progeny was examined. The number of F1 progeny counted is indicated by n. Error bars represent 95% confidence intervals (CIs).


A Nuclei of the WT strain were stained by the immuno - FISH method using an anti - histone H3K9me2 antibody and a probe against the right region of the X chromosome . B Shapes of H3K9me2 - positive chromosomes in male testes of the WT and the csr - 2 mutant . The relative frequency of extended chromosomes in H3K9me2 - positive chromosomes was examined . H3K9me2 - positive chromosomes ≥ 2 . 5 µm were interpreted to be in an extended state . The number of nuclei counted is indicated by n . Error bars represent 95 % CIs .

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A Nuclei of the WT strain were stained by the immuno-FISH method using an anti-histone H3K9me2 antibody and a probe against the right region of the X chromosome. B Shapes of H3K9me2-positive chromosomes in male testes of the WT and the csr-2 mutant. The relative frequency of extended chromosomes in H3K9me2-positive chromosomes was examined. H3K9me2-positive chromosomes ≥ 2.5 µm were interpreted to be in an extended state. The number of nuclei counted is indicated by n. Error bars represent 95% CIs.


E Immunofluorescence images of H3K9me2 and SYP - 1 in male nuclei of the WT and the Argonaute mutants . High - resolution fluorescence images , 3D reconstruction images ( 3D CG ) , and maximum intensity projection ( MIP ) images of segmented H3K9me2 signals are presented . In the male nuclei of the csr - 1 mutant and the csr - 2 ; csr - 1 double mutant , some H3K9me2 segments showed abnormally large volumes ( narrow arrow ) and / or overlapped partially with SYP - 1 signals ( arrowhead ) . In the double mutant , DAPI signals were often detected in an aggregated state ( dashed line ) .

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E Immunofluorescence images of H3K9me2 and SYP-1 in male nuclei of the WT and the Argonaute mutants. High-resolution fluorescence images, 3D reconstruction images (3D CG), and maximum intensity projection (MIP) images of segmented H3K9me2 signals are presented. In the male nuclei of the csr-1 mutant and the csr-2; csr-1 double mutant, some H3K9me2 segments showed abnormally large volumes (narrow arrow) and/or overlapped partially with SYP-1 signals (arrowhead). In the double mutant, DAPI signals were often detected in an aggregated state (dashed line).


Immunofluorescence experiments were performed on the pachytene nuclei of X0 males . A The affinities of the recombinant chromodomain peptides of CEC - 5 and CEC - 8 for the N - terminus of histone H3 were assayed by in vitro pull - down experiments . CEC - 4 served as a control .

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Immunofluorescence experiments were performed on the pachytene nuclei of X0 males. A The affinities of the recombinant chromodomain peptides of CEC-5 and CEC-8 for the N-terminus of histone H3 were assayed by in vitro pull-down experiments. CEC-4 served as a control.


Immunofluorescence experiments were performed on the pachytene nuclei of X0 males . C H3K9me2 signals at the pachytene region in the males of cec mutants and cohesin mutants . Worms were cultured at 20 ° C , except for the smc - 1 ( e870 ) mutant that was cultured at 23 ° C . The intensity of the H3K9me2 signal in the him - 8 mutant male was similar to that in the WT male .

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Immunofluorescence experiments were performed on the pachytene nuclei of X0 males. C H3K9me2 signals at the pachytene region in the males of cec mutants and cohesin mutants. Worms were cultured at 20°C, except for the smc-1(e870) mutant that was cultured at 23°C. The intensity of the H3K9me2 signal in the him-8 mutant male was similar to that in the WT male.


Immunofluorescence experiments were performed on the pachytene nuclei of X0 males . D The nuclear intensities of H3K9me2 signals were measured from wide - field ( WF ) images . The image quantification was performed E The nuclear intensities of H3K9me2 and H3K9me3 signals were measured from deconvolved ( DV ) images . The H3K9me2 signals in the major strains were double - checked using deconvolution microscopy .

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Immunofluorescence experiments were performed on the pachytene nuclei of X0 males. D The nuclear intensities of H3K9me2 signals were measured from wide-field (WF) images. The image quantification was performed E The nuclear intensities of H3K9me2 and H3K9me3 signals were measured from deconvolved (DV) images. The H3K9me2 signals in the major strains were double-checked using deconvolution microscopy.


A The meiosis - specific kleisin ( s ) COH - 3 / 4 was detected on paired chromosomes and the heterochromatinized unpaired X chromosome . The testes of WT males were immunostained with anti - COH - 3 / 4 antibodies and an anti - H3K9me2 antibody .

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A The meiosis-specific kleisin(s) COH-3/4 was detected on paired chromosomes and the heterochromatinized unpaired X chromosome. The testes of WT males were immunostained with anti-COH-3/4 antibodies and an anti-H3K9me2 antibody.


B Immunofluorescence images of COH - 3 / 4 and SYP - 1 on paired chromosomes in the gonads of WT hermaphrodites . COH - 3 / 4 kleisin ( s ) was detected laterally to the string - like signal of SYP - 1 , which corresponds to the central region of the SC .

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B Immunofluorescence images of COH-3/4 and SYP-1 on paired chromosomes in the gonads of WT hermaphrodites. COH-3/4 kleisin(s) was detected laterally to the string-like signal of SYP-1, which corresponds to the central region of the SC.


C SC formation in the WT and multiple mutants were analyzed by immunostaining against SYP - 1 and COH - 3 / 4 . The SC formed string - like structures in the csr - 2 ; csr - 1 double mutant and the ego - 1 mutant , but the superstructure of the SC was often abnormally branched .

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C SC formation in the WT and multiple mutants were analyzed by immunostaining against SYP-1 and COH-3/4. The SC formed string-like structures in the csr-2; csr-1 double mutant and the ego-1 mutant, but the superstructure of the SC was often abnormally branched.


A Multicolor FISH for the 5S rRNA region ( rDNA , green ) of autosome V and the right region of the X chromosome ( LGX - R , magenta ) in nuclei of the WT strain , coh - 3 / 4 double mutant , csr - 1 mutants , csr - 2 ; csr - 1 double mutant , and ego - 1 mutant . Arrows indicate occasional colocalizations of the 5S rDNA and X - R loci detected in the mutant strains .

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

A Multicolor FISH for the 5S rRNA region (rDNA, green) of autosome V and the right region of the X chromosome (LGX-R, magenta) in nuclei of the WT strain, coh-3/4 double mutant, csr-1 mutants, csr-2; csr-1 double mutant, and ego-1 mutant. Arrows indicate occasional colocalizations of the 5S rDNA and X-R loci detected in the mutant strains.


D Simultaneous staining of the 5S rRNA region ( rDNA , green ) by FISH , and SYP - 1 ( magenta ) by immunofluorescence .

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

D Simultaneous staining of the 5S rRNA region (rDNA, green) by FISH, and SYP-1 (magenta) by immunofluorescence.


E csr - 1 ( fj67 ) and csr - 1 ( fj126 ) in - frame mutations reduced the abundance of CSR - 1 at the chromosomal regions in pachytene nuclei . The WT strain and the csr - 1 mutants were stained by anti - CSR - 1 antibodies .

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

E csr-1(fj67) and csr-1(fj126) in-frame mutations reduced the abundance of CSR-1 at the chromosomal regions in pachytene nuclei. The WT strain and the csr-1 mutants were stained by anti-CSR-1 antibodies.


A , B ChIP - seq data for CSR - 1 , SMC - 1 , COH - 3 / 4 , and histone H3K9me2 were compared with sub - populations in CSR - 1 - interacting small RNAs . Adult worms were used for experiments . ChIP signals on autosome III ( A ) and the X chromosome ( B ) from the WT hermaphrodites were visualized by aggregation plots with a bin size of 20 kbp . H3K9me2 ChIP signals derived from the him - 8 mutant population rich in X0 males are shown in the lower part . The common ChIP regions computed from the technical duplicates were used for panels A , B , In the adult body , about half of the nuclei correspond to germ cells . Most H3K9me2 ChIP signals from the WT hermaphrodites were likely to originate in somatic cells because staining signals of H3K9me2 in adult hermaphrodites were detected abundantly in somatic cells and very weakly in the germline . Most meiosis - specific COH - 3 / 4 ChIP signals were thought to originate in the germline . For comparison , aggregation plots were also constructed from the following sub - populations of CSR - 1 - interacting small RNAs : small RNAs mapped in sense orientation to coding genes ( exons and introns ) , and repeat - derived small RNAs . The small RNA data merged from the technical duplicates were used for panels A and B . Densities of protein - coding exons , all non - simple repeats , and five major repeats on the reference genome are attached . The CeRep55 sequences were partially degenerate , and no clear ChIP signal for CSR - 1 was detected in CeRep55 cluster # 4 on the X chromosome in the ce10 reference genome .

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

A, B ChIP-seq data for CSR-1, SMC-1, COH-3/4, and histone H3K9me2 were compared with sub-populations in CSR-1-interacting small RNAs. Adult worms were used for experiments. ChIP signals on autosome III (A) and the X chromosome (B) from the WT hermaphrodites were visualized by aggregation plots with a bin size of 20 kbp. H3K9me2 ChIP signals derived from the him-8 mutant population rich in X0 males are shown in the lower part. The common ChIP regions computed from the technical duplicates were used for panels A, B, In the adult body, about half of the nuclei correspond to germ cells. Most H3K9me2 ChIP signals from the WT hermaphrodites were likely to originate in somatic cells because staining signals of H3K9me2 in adult hermaphrodites were detected abundantly in somatic cells and very weakly in the germline. Most meiosis-specific COH-3/4 ChIP signals were thought to originate in the germline. For comparison, aggregation plots were also constructed from the following sub-populations of CSR-1-interacting small RNAs: small RNAs mapped in sense orientation to coding genes (exons and introns), and repeat-derived small RNAs. The small RNA data merged from the technical duplicates were used for panels A and B. Densities of protein-coding exons, all non-simple repeats, and five major repeats on the reference genome are attached. The CeRep55 sequences were partially degenerate, and no clear ChIP signal for CSR-1 was detected in CeRep55 cluster #4 on the X chromosome in the ce10 reference genome.


G Frequencies of small RNAs expressed from the major non - simple repeats were counted in the CSR - 1 and CSR - 2 complexes and the input cell lysates . The small RNA reads obtained by the experiments in were analyzed . The data from technical duplicates ( # 1 and # 2 ) are individually presented . The y - axis indicates reads per million ( rpm ) . Error bars represent 95 % CIs . The red border indicates the expected value of non - specific recovery , which was predicted with the recovery rate of miRNAs .

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

G Frequencies of small RNAs expressed from the major non-simple repeats were counted in the CSR-1 and CSR-2 complexes and the input cell lysates. The small RNA reads obtained by the experiments in were analyzed. The data from technical duplicates (#1 and #2) are individually presented. The y-axis indicates reads per million (rpm). Error bars represent 95% CIs. The red border indicates the expected value of non-specific recovery, which was predicted with the recovery rate of miRNAs.


F The CeRep55 lncRNA signals partially colocalized with those of COH - 3 / 4 in hermaphrodites . Pachytene nuclei in the WT strain and the csr - 2 ; csr - 1 double mutant were stained .

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

F The CeRep55 lncRNA signals partially colocalized with those of COH-3/4 in hermaphrodites. Pachytene nuclei in the WT strain and the csr-2; csr-1 double mutant were stained.


H The CeRep55 lncRNA signal was also detected at a region of the unpaired X chromosome where the CEC - 5 and COH - 3 / 4 signals overlapped . The pachytene chromosome showing the strongest CEC - 5 enrichment was considered to be the unpaired X chromosome in males . The RNA - directed in situ hybridizations were performed with stringent conditions that detected only long RNAs .

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

H The CeRep55 lncRNA signal was also detected at a region of the unpaired X chromosome where the CEC-5 and COH-3/4 signals overlapped. The pachytene chromosome showing the strongest CEC-5 enrichment was considered to be the unpaired X chromosome in males. The RNA-directed in situ hybridizations were performed with stringent conditions that detected only long RNAs.


Pachytene nuclei were analyzed . B Multicolor FISH using the 5S rDNA and LGX - R probes in the csr - 1 ( fj150 ) mutant and the csr - 1 ( fj150 ) ; CeRep55 _ X ( Q ) mutant .

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

Pachytene nuclei were analyzed. B Multicolor FISH using the 5S rDNA and LGX-R probes in the csr-1(fj150) mutant and the csr-1(fj150); CeRep55_X(Q) mutant.


Pachytene nuclei were analyzed . D Nuclei of male testes carrying an unpaired X chromosome were immunostained with an antibody against histone H3K9me2 . H3K9me2 - positive chromosomes showing an extended state ( arrow ) were observed in the males of the CeRep55 _ X ( Q ) deletion mutant more frequently than in WT males .

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

Pachytene nuclei were analyzed. D Nuclei of male testes carrying an unpaired X chromosome were immunostained with an antibody against histone H3K9me2. H3K9me2-positive chromosomes showing an extended state (arrow) were observed in the males of the CeRep55_X(Q) deletion mutant more frequently than in WT males.


Pachytene nuclei were analyzed . E In male pachytene nuclei , the relative frequency of extended chromosomes in H3K9me2 - positive chromosomes was examined . H3K9me2 - positive chromosomes ≥ 2 . 5 µm were interpreted to be in an extended state . The number of nuclei counted is indicated by n . Error bars represent 95 % CIs . P - value ( two - sided ) was determined by two - proportion z - test .

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 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

Pachytene nuclei were analyzed. E In male pachytene nuclei, the relative frequency of extended chromosomes in H3K9me2-positive chromosomes was examined. H3K9me2-positive chromosomes ≥ 2.5 µm were interpreted to be in an extended state. The number of nuclei counted is indicated by n. Error bars represent 95% CIs. P-value (two-sided) was determined by two-proportion z-test.


( B ) Disintegration reaction ( 1 hour ) performed by RAG1 aa 216 - 1008 R848M / E649V , RAG2 aa 1 - 361 and full length human HMGB1 with Mg2 + or Mn2 + at the indicated temperatures . Denaturing gel displays the fluorophore - labeled DNA strand from the RAG STC before ( 16 nt band , lane 2 ) and after ( 37 nt ) the disintegration reaction . Lane 1 , fluorophore - labeled 37 nt DNA marker . Representative of 2 independent experiments .

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

(B) Disintegration reaction (1 hour) performed by RAG1 aa 216-1008 R848M/E649V, RAG2 aa 1-361 and full length human HMGB1 with Mg2+ or Mn2+ at the indicated temperatures. Denaturing gel displays the fluorophore-labeled DNA strand from the RAG STC before (16 nt band, lane 2) and after (37 nt) the disintegration reaction. Lane 1, fluorophore-labeled 37 nt DNA marker. Representative of 2 independent experiments.


( D ) Results of in vivo plasmid - to - plasmid transposition assay using WT or R848M RAG1 and the indicated forms of RAG2 containing or lacking ( ∆ L ) the four amino acids at the tip of the 333 - 342 loop , performed as described in ( Zhang et al . , 2019 ) ; ( * * * * , p < 0 . 0001 ) . Three biological replicates for each condition except in the last two columns where data from 11 biological replicates are presented .

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

(D) Results of in vivo plasmid-to-plasmid transposition assay using WT or R848M RAG1 and the indicated forms of RAG2 containing or lacking (∆L) the four amino acids at the tip of the 333-342 loop, performed as described in (Zhang et al., 2019); (****, p<0.0001). Three biological replicates for each condition except in the last two columns where data from 11 biological replicates are presented.


( E ) Results of in vivo plasmid - to - genome transposition assay using WT or R848M RAG1 and different forms of RAG2 as indicated . As previously demonstrated ( Zhang et al . , 2019 ) , R848 ( present in WT RAG1 ) and RAG2 acidic hinge residues 362 - 383 each potently suppress transposition . Substantial transposition is observed with the combination of RAG1 R848M and RAG2 1 - 361 , which is further increased by ∆ L ; ( * * * , p = 0 . 001 ) . Four biological replicates for each condition .

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

(E) Results of in vivo plasmid-to-genome transposition assay using WT or R848M RAG1 and different forms of RAG2 as indicated. As previously demonstrated (Zhang et al., 2019), R848 (present in WT RAG1) and RAG2 acidic hinge residues 362-383 each potently suppress transposition. Substantial transposition is observed with the combination of RAG1 R848M and RAG2 1-361, which is further increased by ∆L; (***, p=0.001). Four biological replicates for each condition.


( F ) In vivo recombination activity in HEK293T cells of mouse wild - type RAG1 or RAG1 R848M with RAG2 1 - 361 or 1 - 361 ∆ L . ( p = 0 . 42 for wild - type RAG1 group , p = 0 . 41 for RAG1 R848M group ) . Three biological replicates for each condition .

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

(F) In vivo recombination activity in HEK293T cells of mouse wild-type RAG1 or RAG1 R848M with RAG2 1-361 or 1-361 ∆L. (p=0.42 for wild-type RAG1 group, p=0.41 for RAG1 R848M group). Three biological replicates for each condition.


A . 3D rendering of the cerebrovascular tree in brain slices from male P90 Ctrl and Poly I : C MIA offspring stained with intravascular DiI ( red ) . Scale bar = 50 µm . B . DiI - positive blood vessels geometry analysis - quantification of blood vessel number , diameter ( mean and cumulative frequency distribution ) , length , number of branch segments and average branch length - of P90 male Ctrl and Poly I : C MIA offspring . Vessel number for Ctrl ( 20 . 93 ± 1 . 73 ) and Poly I : C ( 27 . 41 ± 1 . 99 ) MIA offspring , Mann - Whitney U test , * * p < . 01 . Mean diameter for Ctrl ( 5 . 58 ± 0 . 01 ) and Poly I : C ( 4 . 96 ± 0 . 01 ) MIA offspring , Mann - Whitney U test , * * * * p < . 0001 . Total length of vessel per image for Ctrl ( 6 . 12 ± 0 . 76 ) and Poly I : C ( 9 . 83 ± 1 . 48 ) MIA offspring , Mann - Whitney U test , * * p < . 01 . Number of branches for Ctrl ( 9 . 60 ± 0 . 54 ) and Poly I : C ( 15 . 80 ± 1 . 21 ) MIA offspring , Mann - Whitney U test , * p < . 05 . Average branch length for Ctrl ( 34 . 07 ± 1 . 35 ) and Poly I : C ( 33 . 47 ± 1 . 67 ) MIA offspring . C . 3D rendering of the cerebrovascular tree in brain slices from female P90 Ctrl and Poly I : C MIA offspring stained with intravascular DiI ( red ) . Scale bar = 50 µm . D . DiI - positive blood vessels geometry analysis - quantification of blood vessel number , diameter ( mean and cumulative frequency distribution ) , length , number of branch segments and average branch length - of P90 female Ctrl and Poly I : C MIA offspring . Vessel number for Ctrl ( 9 . 97 ± 1 . 18 ) and Poly I : C ( 8 . 61 ± 0 . 78 ) MIA offspring . Mean diameter for Ctrl ( 5 . 77 ± 0 . 03 ) and Poly I : C ( 6 . 16 ± 0 . 02 ) MIA offspring , Mann - Whitney U test , * * * * p < . 0001 . Total length of vessel per image for Ctrl ( 3 . 23 ± 0 . 50 ) and Poly I : C ( 2 . 97 ± 0 . 30 ) MIA offspring . Number of branches for Ctrl ( 10 . 50 ± 0 . 87 ) and Poly I : C ( 7 . 24 ± 0 . 44 ) MIA offspring . Average branch length for Ctrl ( 38 . 06 ± 2 . 17 ) and Poly I : C ( 46 . 93 ± 1 . 49 ) MIA offspring , Mann - Whitney U test , * * * p < . 001 . 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 . In Fig . 1 B and D black dotted lines represent the Ctrl while the blue and purple solid line represent the male Poly I : C and female Poly I : C offspring respectively . 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 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 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 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 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 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 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 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 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 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 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 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. 3D rendering of the cerebrovascular tree in brain slices from male P90 Ctrl and Poly I:C MIA offspring stained with intravascular DiI (red). Scale bar = 50 µm. B. DiI-positive blood vessels geometry analysis - quantification of blood vessel number, diameter (mean and cumulative frequency distribution), length, number of branch segments and average branch length - of P90 male Ctrl and Poly I:C MIA offspring. Vessel number for Ctrl (20.93 ± 1.73) and Poly I:C (27.41 ± 1.99) MIA offspring, Mann-Whitney U test, **p < .01. Mean diameter for Ctrl (5.58 ± 0.01) and Poly I:C (4.96 ± 0.01) MIA offspring, Mann-Whitney U test, ****p < .0001. Total length of vessel per image for Ctrl (6.12 ± 0.76) and Poly I:C (9.83 ± 1.48) MIA offspring, Mann-Whitney U test, **p < .01. Number of branches for Ctrl (9.60 ± 0.54) and Poly I:C (15.80 ± 1.21) MIA offspring, Mann-Whitney U test, *p < .05. Average branch length for Ctrl (34.07 ± 1.35) and Poly I:C (33.47 ± 1.67) MIA offspring. C. 3D rendering of the cerebrovascular tree in brain slices from female P90 Ctrl and Poly I:C MIA offspring stained with intravascular DiI (red). Scale bar = 50 µm. D. DiI-positive blood vessels geometry analysis - quantification of blood vessel number, diameter (mean and cumulative frequency distribution), length, number of branch segments and average branch length - of P90 female Ctrl and Poly I:C MIA offspring. Vessel number for Ctrl (9.97 ± 1.18) and Poly I:C (8.61 ± 0.78) MIA offspring. Mean diameter for Ctrl (5.77 ± 0.03) and Poly I:C (6.16 ± 0.02) MIA offspring, Mann-Whitney U test, ****p < .0001. Total length of vessel per image for Ctrl (3.23 ± 0.50) and Poly I:C (2.97 ± 0.30) MIA offspring. Number of branches for Ctrl (10.50 ± 0.87) and Poly I:C (7.24 ± 0.44) MIA offspring. Average branch length for Ctrl (38.06 ± 2.17) and Poly I:C (46.93 ± 1.49) MIA offspring, Mann-Whitney U test, ***p < .001. 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. In Fig.1 B and D black dotted lines represent the Ctrl while the blue and purple solid line represent the male Poly I:C and female Poly I:C offspring respectively. Numbers in bars indicate the number of animals (N) and images (n). Bars represent mean ± SEM.


E . Quantification of in - vivo brain permeability at P30 in male Ctrl ( 0 . 00 ± 0 . 00 ) and Poly I : C ( 3 . 68 ± 1 . 98 ) treated offspring , Mann - Whitney U test , p = 0 . 060 . 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 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

E. Quantification of in-vivo brain permeability at P30 in male Ctrl (0.00 ± 0.00) and Poly I:C (3.68 ± 1.98) treated offspring, Mann-Whitney U test, p=0.060. 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.


F . Quantification of Evans Blue ( EB ) analysis of in - vivo brain permeability at P90 ( top ) and representative pictures ( bottom ) of perfused brains from Male Ctrl ( left ; 0 . 00 ± 0 . 00 ) and Poly I : C ( right ; 20 . 32 ± 10 . 95 ) offspring . Mann - Whitney U test , * p < . 05 . 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 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 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 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

F. Quantification of Evans Blue (EB) analysis of in-vivo brain permeability at P90 (top) and representative pictures (bottom) of perfused brains from Male Ctrl (left; 0.00 ± 0.00) and Poly I:C (right; 20.32 ± 10.95) offspring. Mann-Whitney U test, *p < .05. 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 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.


G . Quantitative analysis of the optical density of Claudin - 5 ( CLDN5 ) immunoreactive bands normalized to Calnexin ( CANX ) in cortices from P90 male Ctrl ( 0 . 99 ± 0 . 09 ) and Poly I : C ( 0 . 58 ± 0 . 05 ) offspring . 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 images ( n ) . Bars represent mean ± SEM .

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

G. Quantitative analysis of the optical density of Claudin-5 (CLDN5) immunoreactive bands normalized to Calnexin (CANX) in cortices from P90 male Ctrl (0.99 ± 0.09) and Poly I:C (0.58 ± 0.05) offspring. 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 images (n). Bars represent mean ± SEM.


H . Quantitative analysis of the optical density of ZO - 1 immunoreactive bands normalized by α - Tubulin ( αTUB ) in cortices from P90 male Ctrl ( 1 . 00 ± 0 . 04 ) and Poly I : C ( 0 . 75 ± 0 . 08 ) 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 .

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

H. Quantitative analysis of the optical density of ZO-1 immunoreactive bands normalized by α-Tubulin (αTUB) in cortices from P90 male Ctrl (1.00 ± 0.04) and Poly I:C (0.75 ± 0.08) 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.


I . Quantification of in - vivo brain permeability at P30 in female Ctrl ( 0 . 58 ± 0 . 17 ) and Poly I : C ( 0 . 31 ± 0 . 14 ) treated 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 .

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

I. Quantification of in-vivo brain permeability at P30 in female Ctrl (0.58 ± 0.17) and Poly I:C (0.31 ± 0.14) treated 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.


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 .

O O O O O O O O O O O O O O O O O O O O O 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 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 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 .

O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR 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 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 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 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 .

O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_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 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

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 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 .

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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR 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 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-MEASURED_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 O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR 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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_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 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

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 ] 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 .

O O O 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 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 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 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 .

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 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 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

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 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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_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 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

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 B-MEASURED_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 B-MEASURED_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 B-MEASURED_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 O B-MEASURED_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 B-MEASURED_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 B-MEASURED_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 B-MEASURED_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 B-MEASURED_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 B-MEASURED_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 B-MEASURED_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 B-MEASURED_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 B-MEASURED_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 B-MEASURED_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 O B-MEASURED_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 B-MEASURED_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 O B-MEASURED_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 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 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 B-MEASURED_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 B-MEASURED_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 B-MEASURED_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 B-MEASURED_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 B-MEASURED_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 B-MEASURED_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 B-MEASURED_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 B-MEASURED_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 B-MEASURED_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 B-MEASURED_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 B-MEASURED_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 B-MEASURED_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 B-MEASURED_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 B-MEASURED_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 B-MEASURED_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 B-MEASURED_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 B-MEASURED_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 B-MEASURED_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 B-MEASURED_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 B-MEASURED_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 B-MEASURED_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 B-MEASURED_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 B-MEASURED_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 B-MEASURED_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 B-MEASURED_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 B-MEASURED_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 B-MEASURED_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 B-MEASURED_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 B-MEASURED_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 B-MEASURED_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 B-MEASURED_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 B-MEASURED_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 B-MEASURED_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 B-MEASURED_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 B-MEASURED_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 B-MEASURED_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 B-MEASURED_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 B-MEASURED_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 B-MEASURED_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 B-MEASURED_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 B-MEASURED_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 B-MEASURED_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 B-MEASURED_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 B-MEASURED_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 B-MEASURED_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 B-MEASURED_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 B-MEASURED_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 B-MEASURED_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 B-MEASURED_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 B-MEASURED_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 B-MEASURED_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 B-MEASURED_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 B-MEASURED_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 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 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 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 .

O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_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 B-MEASURED_VAR I-MEASURED_VAR I-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 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 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 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 .

O O O O O O O O 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 B-MEASURED_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 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 B-MEASURED_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 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 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 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 .

O O O O O O O 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 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 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 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 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 .

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 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 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 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 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 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 .

O O O O O O O O O O O O O O O O O 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 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 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 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 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 .

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 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 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 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 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 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 .

O O O O O O O O O O O O O O B-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 O O O O O O O O O O O O O B-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 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

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.