Science - USA (2022-02-18)

EDC exposure
For EDC chemical exposure of fetal progeni-
tors, cells were seeded in six-well plates. When
70 to 80% confluency was reached, 0.1% DMSO
or the other compounds diluted in DMSO,
namely the five different concentrations of
MIX N (from 0.1X to 1000X), 0.04 nM BPA,
and 5 nM T3 were added to the culture med-
ium and used to culture cells for 48 hours or
for 15 days (in the chronic setting), adding the
chemical every time media change was per-
formed (every other day). After the exposure
cells were collected for RNA extraction.
For EDC chemical exposure of organoids,
0.1% DMSO, or the other compounds diluted
in DMSO, MIX N in the two different concen-
trations used (1X and 1000X), 0.04 nM BPA, or
5 nM T3 was added to the culture media and
used to culture cells from days 0 to 50. The
concentration we used for T3 is higher than
the physiological dose of T3 in the develop-
ing human brain ( 102 , 103 ) in order to have a
reference transcriptional impact on TH tar-
gets. After exposure, organoids were collected
for both RNA extraction and fixed for immu-
nofluorescence analysis.

Immunohistochemistry for neuronal systems

Protocols and antibodies used for immuno-
histochemistry are described in the supple-
mentary materials.

RNA sequencing

Total RNA was isolated with the RNeasy
Micro Kit (Qiagen) according to the manufac-
turer’s instructions. RNA was quantified with
Nanodrop, and then the integrity was eval-
uated with Agilent 2100 Bioanalyzer (only
if the quality ratios were not optimal after
Nanodrop analysis). TruSeq Stranded Total
RNA LT Sample Prep Kit (Illumina) was used
to run the library for each sample. Sequencing
was performed with the Illumina HiSeq 2000
platform, sequencing on average 10 million
50 – base pair paired-end reads per sample.

RNA-seq data analysis

All the data and the bioinformatic analyses to
reproduce the results presented have been
organized in a repository publicly available at
Zenodo ( 104 ). This includes both Rmd files
for bioinformaticians to reproduce the analysis
and html interactive reports for noncompu-
tational scientists to explore, in detail, all the
analytical steps. A description of the bioin-
formatic analyses performed for alignment,
filtering, normalization, batch correction, dif-
ferential expressions analysis, GO, and GSEA
and the master regulator analysis are included
in the supplementary materials.

Gene network analyses

For connection analysis, Genomatix soft-
ware was used. Cytoscape was used to visual-

ize the network (https://cytoscape.org/). More

detailed information on the gene network analy-

sis is available in the supplementary materials.

X. laevisrearing and strains

Xenopus laevisstrains were maintained in ac-

cordance with institutional and European guide-

lines (2010/63/UE Directive 2010). Heterozygous

X. laevistadpoles used for the XETA were

obtained by crossing adult homozygous

Tg(thibz:eGFP) adults with wild-type animals

( 105 ). Wild-type tadpoles were obtained by

crossing wild-type adults. More detailed in-

formation on theX. laevishusbandry and

breeding routine is available in the sup-

plementary materials.

Chemical exposure protocol for X. laevis

T3 (Sigma-Aldrich) was prepared in 70%

milliQWater,30%NaOH,at0.01M,aliquoted

in volumes of 100ml in 1.5 ml low-binding

Eppendorf (100% polypropylene) tubes, and

stored at−20°C until use. For XETA, mobility,

and qPCR analyses, 15 tadpoles per well (stage

Nieuwkoop and Faber 45) were incubated in a

six-well plate (TPP). Each well contained 8 ml

of exposure solution prepared in water (Evian)

or T3 5 nM (prepared in Evian) and DMSO

(either containing or not containing mixtures

or BPA at different concentrations). All expo-

sure solutions were extemporaneously prepared

in Greiner polypropylene tubes (50 ml tubes)

and transferred in the wells of the six-well plate

with a final 0.01% DMSO concentration in all

groups. MIX N and BPA screenings were

done in the presence of T3 5 nM for XETA.

BPA was tested at the actual concentration as

described in the mixture. Furthermore, a total

mixture concentration 0.1mM was tested as

well as 1mMforBPA.Theexposuretimewas

72 hours in the dark at 23°C with 24-hour

renewal. After 72 hours of exposure, tadpoles

were rinsed and tested for mobility or anes-

thetized with 0.01% tricaine methanesulfonate

(MS222, Sigma-Aldrich) either for fluorescent

screening (XETA) or euthanasia in 0.1% MS-

222 for brain gene expression analysis.

XETA

After 72 hours of exposure, tadpoles were

anesthetized with 0.01% MS-222 and placed

dorsally, one per well, in a black, conic-based

96-well plate (Greiner). All images were ac-

quired as described in ( 54 ). Quantifications

were carried out on whole pictures, and data

were expressed in relative units of fluores-

cence. GraphPad Prism 7 software was used

for statistical analysis. All values were normal-

ized (100%) to T3 when mixtures were tested

in coexposure. Results are expressed as scatter

dot plots with mean ± SEM. A d’Agostino and

Pearson normality test was carried out to

determine the distribution of values in each

of the exposure groups. For all groups, normal

distribution was found, and a one-way analy-

sis of variance (ANOVA) and Dunnett’s post

test was applied.

RNA extraction and qPCR analysis in X. laevis

Pooled brains ofX. laevisembryos were used

for the collection of RNA using a TissueLyser

(Qiagen) for homogenization and RNAqueous

Micro kit (AMBION) for isolation of RNA.

After quantification with NanoDrop, com-

plementary DNA (cDNA) was synthesized

with a High Capacity cDNA RT kit (Applied

BioSystem), and qPCR was carried out with

Power SyBR mix (Applied BioSystem). Pri-

mers and genes tested are listed in table S6.

More detailed information about the proto-

cols used, as well as for the analysis for gene

expression inXenopusis provided in the sup-

plementary materials.

X. laevismobility

The mobility ofXenopusembryos was recorded

by the DanioVision behavior analysis system

(Noldus). Embryos were transferred to indi-

vidual wells in a 12-well plate and a protocol

of alternating light-dark cycles (30 sec–30sec)

was used to induce locomotion. Distance

traveled was recorded for a total of 10 min

and analyzed with the EthoVision software

(11.5, Noldus). Further information on the

mobility assay and analysis is available in

the supplementary materials.

D. reriohusbandry

Adult AB-strain zebrafish were maintained

in a recirculating system and bred to ob-

tain embryos. After breeding, embryos were

collected and kept in zebrafish embryo me-

dium until the exposure was initiated. The

fish were treated in accordance with Swedish

ethical guidelines with the ethical permit

(Dnr 5.2.18-4777/16) granted by the Swedish

Board of Agriculture. More detailed infor-

mation on the zebrafish husbandry and breed-

ing routine is available in the supplementary

materials.

Exposure of D. rerio

For exposure of zebrafish, randomly selected

embryos from a minimum of three different

breeding pairs were used. Exposure solutions

in zebrafish embryo medium, including five

different MIX N concentrations (0.01-100X),

DMSO, or BPA (at concentrations correspond-

ingtothosepresentinMIXNat1Xto100Xas

well as at 0.1mM and 10mM), were prepared by

serial dilution with a final DMSO concen-

tration of 0.01% in all treatments. Embryos

were exposed for 48 hours (72 to 120 hours

postfertilization) at 28°C with a 14:10 hour

light-dark cycle. Three replicates with 20 to

25 embryos per concentration were used, and

each experiment was independently repeated

three times. After exposure, the embryos were

Caporaleet al.,Science 375 , eabe8244 (2022) 18 February 2022 12 of 15

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