Tube, Neural

For testing the electroporation parameters, embryos from different test conditions were injected with the same volume using the same capillary. Electroporation using the standard setting (20 V/50 ms/1 s/8 x) was always performed at the end of the test series as a control.
12 h after electroporation, the success rate was estimated on live anesthetized embryos under a fluorescence stereoscopic microscope (MZFLII, Leica). Each embryo was scored according to the fluorescence intensity and spread of the signal (0 = no signal, 0.25 = dim, 0.5 = high but restricted, 1 = high and widespread). As absolute efficiency varies with DNA preparations and embryo batches, embryos electroporated with the standard setting were scored first to set the index. Results from different experiments were normalized to the standard settings (100%). The pictures presented and archived were taken under the same conditions (same magnification, time after electroporation and exposure). Embryos exhibiting any apparent damage such as smaller eye, local head depression, defect in eye pigmentation or persistent skin peeling were scored as damaged.
The fraction of transfected cells was quantified on serial frontal sections of embryos 6 h, 12 h, 24 h or 48 h after electroporation with nls-GFP. Sections were screened at low power (5×) to identify the rostral most and caudal most positive sections. All inclusive sections were then photographed at 20× (Eclipse 80i, Nikon) using fixed acquisition parameters separately set for the 24 h (for analysis of electroporation kinetics) and 12 h (for analysis of stage and pulse parameters) time points (Orca, Hamamatsu, Open lab, Improvision). Regions of interest (ROIs) corresponding to the hemi-neural tube and superficial regions of the brain were outlined based on DAPI counter-staining and used for subsequent quantification. Thresholds were set for DAPI and GFP fluorescence intensity and the total area in ROIs above threshold was calculated. Thresholds for DAPI and GFP were calibrated so that the ratio of GFP area to DAPI area matched the manual percentage count at 24 h (kinetics) or 12 h (stages and pulse parameters). The centers of mass of DAPI and GFP signals were also calculated. All the quantifications were done in ImageJ (NIH).

Embryonated eggs were incubated at 38.5°C in a humidified incubator until HH14 stage. Fresh or frozen patient samples were dissociated in Hank's Balanced Salt Solution (HBSS) with 156 units/ml of type IV collagenase, 200 mM CaCl2 and 50 units/ml DNase I for 20 min at 37°C and then incubated with 5 mg/ml trypsin for 2 min at 37°C under gentle mixing. Non‐dissociated tissue was removed by filtration trough 0.4 μm nylon cell strainer (BD falcon). Non‐fluorescent cell lines or patient samples were labeled with an 8 μM CFSE solution (Life Technologies). Stage HH13 chick embryos were grafted with fluorescent cells at the top of the dorsal neural tube within the migration staging area, with a glass capillary connected to a pneumatic PicoPump (PV820, World Precision Instruments) under a fluorescence stereomicroscope. For cell lines, approximately 2,500 living cells were grafted in each embryo, 200–300 for patient samples. For patient samples, the full cellular content obtained after dissociation was engrafted possibly including stromal and/or immune cells. Sample size (number of grafted embryos) for patient samples was not estimated as the total and limited amount of material available was used. For engraftment of cell lines, sample size was not estimated using statistical methods but was rather based on previous studies using avian embryos (Delloye‐Bourgeois et al, 2017 (link); Jarrosson et al, 2021 (link); Ben Amar et al, 2022 (link)).

The BaP-induced NTD mouse model was performed as previously described [10 (link)]. Briefly, ICR mice of 8–9 weeks old weighing 28 ± 2 g were used in the experiment. Female mice were mated with males overnight and vaginal plugs were examined the following morning. Noon on the day of finding a vaginal plug was considered 0.5 days of embryonic development (E0.5). Pregnant mice were randomly divided into 2 groups. In BaP-treatment groups, mice were given BaP (CAS-No. 192-97-2, purity 98%, Sigma-Aldrich, St. Louis, MO, USA) intraperitoneally, dissolved in corn oil (CAS-No. 8001-30-7, aladdin, Shanghai, China) (25 mg mL⁻¹), from E6.5 for three consecutive days at a dose of 250 mg kg⁻¹. Mice in the control group were treated with corn oil (10 mL kg⁻¹). On E9.5, pregnant mice were sacrificed by cervical dislocation and the embryos were removed by caesarean section. Embryos were carefully inspected for visible external malformations under a dissecting microscope. NTD-affected embryos were classified as showing distinct evidence of failed closure of the cephalic neural tube. Finally, 16.4% of the embryos were observed with open neural tube (Table S1). The somite numbers were carefully counted. E9.5 embryos with 22–27 somites were used for further analysis [19 (link)].