Animals and reagents
The procedures for animal use were approved by the University of Maryland School of Medicine Institutional Animal Care and Use Committee. Wild-type (WT) C57BL/6 J mice were purchased from the Jackson Laboratory (Bar Harbor, ME). The DNA construct for the FGF2 transgenic mice (FGF2-Tg) was derived from the pTTR1ExV3 vector provided by Dr. Terry A. Van Dyke (Department of Biological Sciences, University of Pittsburgh, PA). The vector contains 3 kb transthyretin (TTR) promoter, its exon 1, intron 1, and exon2 fused with the SV40 polyA. We cloned the FGF2 open reading frame (ORF) followed by an IRES site and a nuclear specific expressing GFP (Clontech, Mountain View, CA94043) in exon 1. After oocyte injection, 6 founders carried the FGF2 transgene, 3 of them showed GFP expression and one line (#134) was selected for experiment. The DNA construct for the Survivin transgenic mice was driven by the Flk-1 promoter, which was cloned based on a previous report60. The mouse Flk-1 promoter fragment (−640 bp ~ +299 bp, the Transcriptional Start Site was defined as +1) was amplified by PCR, and then was inserted into 5′ of the Survivin ORF (Cat#: MR223428, Origene, Rockville, MD 20850). An IRES site and a nuclear specific expressing GFP were added to the 3′ of the Survivin ORF. An intronic fragment with the 3′ region of the first intron (+1677 bp ~ +3947 bp) of the Flk-1 gene was PCR cloned and ligated to the 3′ of GFP. There were 15 founders that carried the Survivin transgene, and one was selected for our experiment.
All Tg animal lines were generated in the Genome Modification Facility at Harvard University using the C57BL/6J background as previously described11. Streptozotocin (STZ; Sigma (St. Louis, MO) was dissolved in sterile 0.1 M citrate buffer (pH 4.5).
Mouse models of diabetic embryopathy
Our mouse model of diabetic embryopathy has been described previously8,10,36,61,62. Briefly, 10-week-old female mice were intravenously injected daily with 75 mg/kg STZ over two days to induce diabetes. Diabetes was defined as 12 h fasting blood glucose level of ≥250 mg/dL. Male and female mice were paired at 3:00 P.M, and day 0.5 (E0.5) of pregnancy was established by the presence of the vaginal plug at 8:00 A.M the next morning. Female mice injected with vehicle served as the nondiabetic controls. On E7.5, E8.5, E9.5, or E10.5 mice were euthanized and conceptuses were dissected out of the uteri for biochemical and molecular analysis. The exosome inhibitor GW4869 (Sigma, St, Louis, MO) was injected daily to nondiabetic pregnant dams from E7.0 to E8.0. NTDs were examined at E10.5.
Cell culture and transfection
The C17.2 mouse neural stem cell line the European Collection of Cell Culture (Salisbury, UK), and the yolk sac endoderm PYS2 cell line (ATCC® CRL2745™) were maintained in DMEM (5 mM glucose) supplemented with 10% FBS, 100 U/ml penicillin, and 100 mg/mL streptomycin at 37 °C in a humidified atmosphere of 5% CO2. Mouse recombinant FGF2 (#3139-FB; R&D Systems, Minneapolis, MN) was used at a final concentration of 10 ng/ml. Lipofectamine 2000 (Invitrogen, Carlsbad, CA) was used according to the manufacturer’s protocol for transfection of siFGF2 and control siRNA (Invitrogen, Carlsbad, CA) into the cells. After seeding for 12 h, cells were transfected with recombinant FGF2 and cultured in 1% FBS for 8 h, after which cells were cultured in 10% FBS and collected after 48 h for analysis.
Isolation of Flk-1+ endothelial progenitor cells
Flk-1+ progenitor cells were isolated from the E8 yolk sac. Yolk sacs were minced in phosphate-buffered saline PBS (Thermo Fisher Scientific, Waltham, MA), digested with 1 mg/ml of collagenase type I (Sigma, St Louis, MO) and incubated on an orbital shaker at 37 °C for 30–45 min. Following the collagenase treatment, cells were washed twice with growth medium [EBM-2, fetal bovine serum (10%), hFGF-basic (0.5 ml), hydrocortisone (0.2 ml), VEGF (0.5 ml), IGF-1 (0.5 ml), ascorbic acid (0.5 ml), hEGF (0.5 ml), GA-100 (0.5 ml)] before being plated. Cells were trypsinized (Lonza, Walkersville, MD) and sorted for Flk-1 with MACS microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) as per the kit instructions. The Flk-1+ progenitor cells were collected and cultured in growth medium as mentioned above.
Tissue sections were fixed with 4% paraformaldehyde (pH7.4) for 30 min at room temperature, followed by permeabilization with 0.25% Triton-X100 (Sigma, St. Louis, MO) for 10 min. Samples were blocked for 30 min in PBS with 10% donkey serum and incubated with CD63 (Santa Cruz Biotechnology, Dallas, TX), Nestin (1:1000, Invitrogen, Carlsbad, CA), Flk-1 (1:200, Santa Cruz Biotechnology, Dallas, TX) and Myc-tag antibodies (1:200, Cell Signaling Technology, Danvers, MA) overnight at 4 °C. After washing three times with PBS, samples were incubated with Alexa Fluor 488 or 594-conjugated secondary antibody (1:1000, Invitrogen, Carlsbad, CA) for 2 h, followed by DAPI (Invitrogen, Carlsbad, CA) cell nuclear counterstaining for 10 min, then mounted with aqueous mounting medium (Sigma, St Louis, MO). Confocal immunofluorescent images were recorded by a laser scanning microscope (LSM 510 META; Zeiss, Germany).
Exosome extraction and labeling
Cells and debris were removed from cell culture media via centrifugation at 2000 × g for 30 min at 4 °C. Supernatants containing the cell-free culture media were transferred to a new tube without disturbing the pellet. Total Exosome Isolation (#4478359, Invitrogen, Carlsbad, CA) at 0.5 volume of the collected supernatants was added to each tube. The cell culture media/reagent mixture was mixed well via vortex and pipetting up and down until it became a homogenous solution, and then was incubated at 4 °C overnight. After incubation, the samples were centrifuged at 10,000 × g for 1 h at 4 °C and the supernatants were discarded. Exosomes were contained in the pellet at the bottom of the tube (not visible in most cases). The pellet was re-suspended in 1 ml of 1 X PBS and NanoSight (#NS300, Malvern Panalytical) was used to measure the exosomes. The presence of exosomes was confirmed by Western blot using the exosomal marker CD63 (Santa Cruz Biotechnology, Dallas, TX). Exosomes were suspended in 1 ml Diluent C (Sigma, St Louis, MO) and labeled by incubating with PKH67 fluorescent dye (Sigma, St Louis, MO) for 5 min in the dark at room temperature. An equal volume of 1% BSA in PBS was added to each tube to stop the staining, with an additional incubation period of 1 min to allow binding of excess dye. The labeled exosomes were separated from free dye using the Total Exosome Isolation kit (#4478359, Invitrogen, Carlsbad, CA).
In utero ultrasound-guided microinjection
E8.0 pregnant dams were anesthetized by 3% isoflurane (Vetone, Boise, ID) in 100% oxygen initially, followed by 2% isoflurane during the rest of the procedure. The dams were placed supine on a mouse handling table where the integrated warmer of the heating platform set to 37 °C and a rectal thermometer was put in place to monitor body temperature. The dams’ eyes were covered with a lubricant to prevent drying of the sclera. Abdomen hairs of the dams were removed. A 2-cm ventral midline incision was made approximately 1 cm above the vagina to open the abdomen and peritoneum. The number of embryos was counted to ensure adequate record keeping of which embryos were injected. To stabilize the uterus, a modified Petri dish with a 3-cm hole cut into the middle covered by a thin silicone membrane with a small slit cut in the center was placed over the dams’ abdomens. The uterine horns were gently pulled up through the silicon membrane of the dish, which contained 0.9% saline to prevent dehydration. Under ultrasound (Vevo 770, VisualSonics, Canada) guidance, the uterus was penetrated by a microinjection needle (#C060609, Coopersurgical) to reach the amniotic cavity. A total of 5 × 109 exosomes suspended in 280 nl of PBS were injected into the amniotic cavity. Following one injection, the handling table was repositioned to image and inject the next embryo. After injecting the desired number of embryos, the uterine horns were placed back into the abdomen. The maternal abdomen was sutured closed.
Immunoblotting was performed as described by Yang et al.8. Lysis buffer (Cell Signaling Technology, Danvers, MA) containing a protease inhibitor cocktail (Sigma, St Louis, MO) was used to extract protein. Equal amounts of protein and Precision Plus Protein Standards (Bio-Rad, Hercules, CA) were resolved by SDS-PAGE and transferred onto Immunobilon-P membranes (Millipore, Billerica, MA). Membranes were incubated in 5% nonfat milk for 45 min at room temperature, and then were incubated for 18 h at 4 °C with the following primary antibodies at dilutions of 1:1000 in 5% nonfat milk: FGF2, phosphorylated (p-)FGFR, p-AKT, BMP4, mVEGFR1, p-VEGFR2, p-PERK, p-IRE1α, p-eIF2α, CHOP, caspase 8 and caspase 3. Following primary antibody incubation, membranes were washed with PBS and then exposed to HRP-conjugated related secondary antibodies at dilution of 1:10000. Signals were detected using the SuperSignal West Femto Maximum Sensitivity Substrate kit (Thermo Fisher Scientific, Waltham, MA). To ensure that equivalent amounts of protein were loaded on the SDS-PAGE gel, membranes were stripped and incubated with a mouse antibody against β-actin (Abcam, Cambridge, MA). All experiments were repeated in triplicate with the use of independently prepared tissue lysates. The detailed antibody information is provided in Supplementary Table 5, and whole Western blots are shown in Supplementary Fig. 9.
Real-time PCR (RT-PCR)
mRNA was isolated from embryos using the Rneasy Mini kit (Qiagen, Hilden, Germany), and then reversely transcribed using the high-capacity cDNA archive kit (Applied Biosystem, Grand Island, NY). Real time-PCR for Fgf2, Fgfr, Bmp4, Vegfr2, mVegfr1, Calnexin, GRP94, PDIA, BiP, IRE1α, CHOP, Survivin, and β-actin were performed using the Maxima SYBR Green/ROX qPCR Master Mix assay (Thermo Fisher Scientific, Waltham, MA) in the Step One Plus system (Applied Biosystem, Grand Island, NY). All primer sequences were listed in Supplementary Table 6.
Tissue section staining
Nondiabetic wild-type (WT), nondiabetic Tg, diabetic WT and diabetic Tg embryos were collected for a morphological examination at E10.5 and fixed in methacarn (methanol, 60%; chloroform, 30%; and glacial acetic acid, 10%). Embryos were dehydrated in alcohol and embedded in paraffin. And cut into 5-μm sections. After deparaffinization and rehydration, and then stained with hematoxylin and eosin (H&E) and imaged under a Nikon Ni-U microscope (Nikon, Tokyo, Japan).
Blood island quantification
Five micrometer cross (vertical) sections of E8.5 conceptuses were stained by H&E. Yolk sac blood islands were counted and expressed against the yolk sac circumference. The yolk sac in an E8.5 conceptus cross-section is near-circular, so it was assumed to be a circle for calculating circumference. Ten sections of each conceptus were used and the data were averaged. Five conceptuses from different pregnant dams in each group were used to determine the numbers of blood islands in yolk sacs.
Blood vessel density measurement
E9.5 conceptuses were fixed with 4% paraformaldehyde in PBS overnight at 4 °C. For immunostaining analyses, controls were processed by omitting the primary antibody. The rat anti-mouse PECAM-1 antibody at a dilution of 1:200 (Abcam, Cambridge, MA) was used to stain the whole conceptuses. Samples were incubated in ABC solution (elite ABC kit, Vector Laboratories) for 30 min and then with the stable diaminobenzidine substrate solution (Vector Laboratories, Burlingame, CA). Yolk sacs were removed from the conceptuses and mounted on positively charged slides. Embryos were examined directly. PECAM-1 positive structures (vessel area) in the yolk sac and embryo were determined by capturing images and analyzing the PECAM-1-stained areas with the NIH ImageJ software (Version 1.62, National Institutes of Health, Bethesda, MD) by setting a consistent threshold for all slides. The PECAM-1-positive area was expressed as pixels-squared per high-power field.
Dihydroethidium (DHE) staining
DHE staining was used for immunofluorescent detection of superoxide. DHE reacts with superoxide that binds to cellular components such as protein and DNA, and manifested with bright red fluorescence. E8.5 embryos were fixed in 4% Paraformaldehyde (PFA) (Thermo Fisher Scientific, Waltham, MA) for 30 min, washed for 5 min each for three times with PBS, and then embedded in optimal cutting temperature compound (OCT) (Sakura Finetek, Torrance, CA). 10-μm frozen embryonic sections were incubated with 1.5 μM DHE for 5 min at room temperature and then washed for 3 times with PBS. Sections were counterstained with DAPI and mounted with aqueous mounting medium (Sigma, St Louis, MO).
Lipid hydroperoxide (LPO) quantification
The degree of lipid peroxidation, an index of oxidative stress, was quantitatively assessed by the LPO assay by using the Calbiochem Lipid Hydroperoxide Assay Kit (Milliproe, Bedford, MA) following the manufacture’s manual. Briefly, E8.5 embryos were homogenized in HPLC-grade water (Thermo Fisher Scientific, Waltham, MA). The lipid hydroperoxides of each embryo tissue were extracted by deoxygenated chloroform, and then measured by the absorbance of 500 nm after reaction with chromogen. The results were expressed as μM lipid hydroperoxides per microgram protein. Protein concentrations were assessed by the BioRad DC protein assay kit (BioRad, Hercules, CA).
Detection of XBP1 mRNA splicing
mRNA was extracted from E8.5 embryos and reverse-transcribed to cDNA using the QuantiTect Reverse Transcription Kit (Qiagen, Hilden, Germany). The PCR primers for XBP1 were as follows: forward, 5′-GAACCAGGAGTTAAGAACACG-3′ and reverse, 5′-AGGCAACAGTGTCAGAGTCC-3′. If no XBP1 mRNA splicing occurred, a 205 bp band was produced. When XBP1 splicing occurred, a 205 bp band and a 179 bp main band were produced.
TUNEL assay was performed using the ApopTag Fluorescein in Situ Apoptosis Detection kit (# S7165, Millipore, Billerica, MA) as previously described8. Briefly, 5-μm frozen embryonic sections were fixed with 4% PFA in PBS and incubated with TUNEL reaction agents. Three embryos from three different dams (n = 3) in each group were used, and three sections per embryo were examined. TUNEL-positive cells in an area (about 200 cells) of the neuroepithelium were counted. The percentage of TUNEL-positive cells was calculated as a fraction of the total cell number, multiplied by 100 and averaged within the sections of each embryo.
Statistics and reproducibility
Data were presented as means ± standard errors (SE). In animal studies, experiments were repeated at least three times, and embryonic samples from each replicate were from different dams. Statistical differences were determined by one-way analysis of variance (ANOVA) using the SigmaPlot 12.5 (SigmaStat, San Jose, CA). In one-way ANOVA analysis, Tukey test was used to estimate the significance of the results (P < 0.05). The Chi square test and Fisher Exact test were used to estimate the significance of NTD incidence.
Further information on research design is available in the Nature Research Reporting Summary linked to this article.