Cao et al. and (2020) 11:859 https://doi.org/10.1038/s41419-020-03078-5 Cell Death & Disease

ARTICLE Open Access Restoring BMP4 expression in vascular endothelial progenitors ameliorates maternal diabetes-induced and neural tube defects Songying Cao1,E.AlbertReece1,2, Wei-Bin Shen1 and Peixin Yang1,2

Abstract During mouse embryonic development, vasculogenesis initially occurs in the yolk sac, preceding neurulation. Our previous studies have demonstrated that maternal diabetes induces embryonic vasculopathy at early embryonic developmental stage by suppressing the expression of vascular growth factors including BMP4 (bone morphogenetic protein 4). This study aimed to determine whether restoring diabetes-inhibited BMP4 expression in Flk-1+ progenitors effectively prevented maternal diabetes-induced embryonic vasculopathy and NTDs. Transgenic (Tg) BMP4 expression in the vascular endothelial receptor 2 (Flk-1)-positive (Flk-1+) progenitors was achieved by crossing a Floxed BMP4 Tg mouse line with the Flk-1-Cre mouse line. Non-BMP4 Tg and BMP4 Tg embryos were harvested at E8.5 to assess the expression of BMP4, markers of endoplasmic reticulum , and expression of the Id genes, direct targets of BMP4; and the presence of cleaved 3 and 8, apoptosis, and Smad signaling. BMP4 Tg overexpression neutralized its down-regulation by maternal diabetes in E8.5 embryos. Maternal diabetes-induced Flk-1+ progenitor apoptosis, impairment of blood island formation, and reduction of Flk-1+ progenitor number and blood vessel density, which were reversed by BMP4 Tg expression. BMP4 Tg expression in Flk-1+ progenitors blocked maternal diabetes- induced vasculopathy in early stage embryos (E7.5-E8.5) and consequently led to amelioration of maternal diabetes- 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; induced neural tube defects (NTDs) at E10.5. BMP4 Tg expression inhibited maternal diabetes-induced endoplasmic reticulum stress and caspase cascade activation in the developing neuroepithelium, and reduced neuroepithelial cell apoptosis. BMP4 Tg expression re-activated Smad1/5/8 phosphorylation and reversed maternal diabetes-suppressed Smad4 expression. BMP4 Tg expression restored Id1 and Smad6 expression inhibited by maternal diabetes. In vitro, recombinant BMP4 protein blocked high glucose-induced Flk-1+ progenitor apoptosis and NTDs. These data demonstrate that BMP4 down-regulation in Flk-1+ progenitors are responsible for diabetes-induced yolk sac vasculopathy, and that restoring BMP4 expression prevents vasculopathy and rescues neuroepithelial cells from cellular stress, leading to NTD reduction.

Introduction defects1,2, collectively called as diabetic embryopathy. The Pregestational maternal diabetes mellitus contributes to neural tube and the yolk sac of the early stage conceptuses the increased incidence of congenital malformations, during organogenesis are the most vulnerable structures including neural tube defects (NTDs) and cardiovascular to the deleterious effect of maternal diabetes3. The embryonic vasculature derived from vasculogenesis is the first organ system to be developed during embryonic Correspondence: Peixin Yang ([email protected]) development. 1Department of Obstetrics, Gynecology & Reproductive Science, University of In mammals, embryonic vasculogenesis occurs first in Maryland School of Medicine, Baltimore, MD 21201, USA the yolk sac and then in the developing embryo proper4. 2Department of and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD 21201, USA Arrested development of the yolk sac vasculature at early Edited by B. Zhivotovsky

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embryonic stages can result in embryonic lethality or reduced in the embryonic heart by maternal diabetes6,26. congenital malformations in major organs. Previous stu- Thus, we hypothesize that reduced BMP4 contributes to + dies have shown that hyperglycemia induces vasculopathy maternal diabetes-decreased number of Flk-1 progeni- in the yolk sac correlating with structural anomalies in the tors and blood islands leading to vasculopathy and later embryo5,6. Embryos exposed to maternal hyperglycemia NTD formation, and that restoring BMP4 expression + manifest decreased conceptus size and gross malforma- prevents Flk-1 progenitor loss, vasculopathy and NTD. tions7. Additionally, the visceral yolk sac capillaries and The present study utilized a transgenic mouse model vitelline vessels of conceptuses are morphologically dis- which expressed BMP4 and demonstrated that restoring + rupted by maternal diabetes7. These data strongly impli- diabetes-inhibited BMP4 expression in Flk-1 progenitors cate that vasculopathy in early development contributes effectively prevented maternal diabetes-induced embryo- to the induction of structural birth defects later in nic vasculopathy. Blockade of vasculopathy by BMP4 embryogenesis. Thus, correcting the disruption of vas- transgenic expression led to suppression of ER stress and cular signaling induced by maternal diabetes may prevent neuroepithelial cell apoptosis, which ultimately resulted in diabetic embryopathy. Some of the critical molecular amelioration of NTDs in diabetic pregnancy. Thus, cor- events in the developing neuroepithelium including ER recting vascular growth factor signaling in early embryo- stress and excessive apoptosis has been revealed as the nic development significantly alleviates structural birth cause of NTDs in diabetic pregnancy8. In the mouse, defects in relatively late stage of development comparing neurulation occurs during E8.0 to E9.5 and any insults to to that of vasculogenesis. vasculogenesis that occur before this period may be a causative event for NTD formation. Materials and methods Yolk sac vasculopathy is associated with increased and reagents malformation rates in diabetic pregnancy3,5,9,10, support- Wild-Type (WT) C57BL/6J mice (12-week to 14-week) ing the yolk sac vasculopathy hypothesis in maternal were purchased from The Jackson Laboratory (Bar Har- diabetes-induced NTDs. In mice, vasculogenesis starts bor, ME). We generated the pCALL2-BMP4-GFP con- with the emergence of Flk-1 (VEGFR2) progenitors in the struct (Fig. 1a). Pronuclei microinjection in the C57BL/6J + yolk sac11,12. Flk-1 progenitors form blood islands, which background was carried out in the Genome Modification subsequently fuse to form blood vessels13. A critical Facility of Harvard University (Cambridge, MA). Strep- + number of Flk-1 progenitors and blood islands are cru- tozotocin (STZ; Sigma, St. Louis, MO) was dissolved in cial for normal vasculogenesis. Vasculogenesis dynami- sterile 0.1 mol/L citrate buffer (pH4.5). The procedures – cally modulated by growth factors and morphogens14 16. for use were approved by the University of Bone morphogenetic proteins (BMPs) are secreted growth Maryland School of Medicine Institutional Animal Care factors that belong to the transforming growth factor beta and Use Committee. (TGFβ) superfamily17. BMPs bind to BMP type I and type II receptors, which leads to phosphorylation of receptor BMP4 transgenic (Tg) mice Smads (R-Smads), including Smad1, Smad5, and Smad8. We created the BMP4 Tg mice that contained the CAG The phosphorylated R-Smads then form complexes with promoter-driven floxed stop codon followed by the Bmp4 the common mediator Smad4, translocate into the transgene, using an approach previously described27. Cre- – nucleus and induce gene expression18 20. The inhibitor of Flox recombination was used to specifically overexpress + DNA binding family of proteins (Id proteins) is major BMP4 in Flk-1 progenitors using the Flk-1-Cre mice28. downstream mediators of BMP-Smad signaling21. Genetic Nondiabetic male Flk1-Cre mice bred with nondiabetic or deletion or misexpression of different components of the diabetic female BMP4 Tg mice to produce embryos with BMP-Smad signaling leads to embryonic death due to or without BMP4 Tg expression. severe cardiovascular malformations22. Proper BMP- Smad signaling is required for appropriate vasculogen- Mouse models of diabetic embryopathy esis and angiogenesis23. Our mouse model of diabetic embryopathy has been – BMP4 is an important vascular growth factor that is described previously8,29 32. Briefly, female mice in the required for the initiation of embryonic vasculogenesis4. diabetic group (DM) were intravenously injected daily Gene deletion studies have demonstrated that BMP4 is with 65 mg/kg STZ over 2 days to induce diabetes. Dia- + essential for the induction of Flk-1 progenitors from betes was defined as a 12-h fasting blood glucose level of ≥ mesoderm24. The critical role of BMP4 in embryonic 250 mg/dl. Female mice in the nondiabetic group (ND) vasculogenesis is also supported by gene deletion studies were injected with vehicle. Females were randomized into of its downstream Smad protein. Targeted inactivation of experimental groups. Pregnancy was identified by the Smad5 induces vascular defects in the yolk sac and the presence of the vaginal plug the next morning. On E8.5 or embryo proper25. Additionally, BMP4 expression is E10.5 mice were euthanized and conceptuses were

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A loxp loxp CAG 3 x Lacz Bmp4 IRES GFP poly A promoter poly A

B non-BMP4-Tg BMP4-Tg D ND DM non-BMP4-Tg BMP4-Tg non-BMP4-Tg BMP4-Tg BMP4 (47 kD) β-actin (42 kD)

C

BMP4/b-actin * * BMP4/b-actin

non-BMP4-Tg BMP4-Tg non-BMP4-Tg BMP4-Tg non-BMP4-Tg BMP4-Tg non-BMP4-Tg BMP4-Tg

ND DM ND DM E ND DM 2 1.5 non-BMP4-Tg BMP4-Tg non-BMP4-Tg BMP4-Tg 1

density 0.5 *

BMP4 relative 0 non-TgBMP4-Tgnon-Tg BMP4-Tg 2 ND DM 1.5 1

density 0.5 *

BMP4 relative 0 non-Tg non-Tg BMP4-Tg BMP4-Tg ND DM

Fig. 1 BMP4 Tg expression in Flk-1+ progenitors abrogate maternal diabetes-suppressed BMP4 expression. a Diagram of the Bmp4 transgene construct. b Nuclear GFP positive cells (green) in the Flk-1+ progenitors (red) of E8.5 BMP4 Tg embryos. Scale bar = 20 μm. c Levels of Bmp4 mRNA in E8.5 non-BMP4 Tg embryos and BMP4 Tg embryos from nondiabetic (ND) or diabetes mellitus (DM) dams. d BMP4 protein levels in E8.5 non-BMP4 Tg and BMP4 Tg embryos from ND or DM dams. Quantification of relative expression levels of BMP4 proteins was shown in the graphs. e. BMP4 staining (green) in E8.5 non-BMP4 Tg and BMP4 Tg embryos from nondiabetic (ND) or diabetes mellitus (DM) dams. Cell nuclei were stained with DAPI (blue), Scale bar = 30μm. Double immunostaining BMP4 (green) and Flk-1 (red) in E8.5 non-BMP4 Tg and BMP4 Tg yolk sac from nondiabetic (ND) or diabetes mellitus (DM) dams. Cell nuclei were stained with DAPI (blue), Scale bar = 20μm. Quantification of relative expression levels of BMP4 protein expression (normalized by DAPI in the V-shape neuroepithelium and by in the yolk sac) was shown in the graphs. Experiments were repeated three times with embryos from three different dams in each group (n = 3). * indicates significant difference compared to the other groups (P < 0.05) in one-way ANOVA followed by Tukey tests. ND: Nondiabetic; DM: Diabetes Mellitus.

dissected out of the uteri for analyses. NTD rates were Fisher Scientific, Waltham, MA). Yolk sacs were digested examined in a blinded manner. with 0.1% collagenase/Trypsin (Sigma, St. Louis, MO) in PBS for 30 minutes at 37 °C. Cells were washed twice with Flk-1+ progenitor culture growth medium containing 10% FBS. Cell clumps were Embryonic yolk sacs were carefully dissected from E8.5 removed by pouring cells through a 70-μm nylon mesh embryos in phosphate-buffered saline (PBS) (Thermo (BD Biosciences, Discovery Labware, Bedford, MA), and

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+ then cells were centrifuged, washed, and Flk-1 progeni- conceptuses and mounted on positively charged slides. tors were pulled down using the CD309 (Flk-1) CD31-positive structures (vessel area) in the yolk sac and MicroBead Kit (Miltenyi Biotec, San Diego, CA). The embryo were determined by capturing images and ana- + Flk-1 progenitors were cultured under normal glucose lyzing the CD31-stained areas with NIH Image Analysis (5 mM, NG) or high glucose (25 mM, HG) conditions software (Version 1.62; National Institutes of Health, supplemented with 10% FBS, 100 U/ml penicillin, and Bethesda, MD) by setting a consistent threshold for all 100 mg/mL streptomycin at 37 °C in a humidified atmo- slides so that only CD31-stained cells were apparent. The sphere of 5% CO2. Cells were treated with different con- CD31-positive area was expressed as pixels-squared per centrations of recombinant BMP4 protein (R&D Systems, high-power field. The CD31-positive area was measured Minneapolis, MN) for 48 hours. for each yolk sac and embryo of the four groups. Infor- mation of antibodies are in Table S3. Whole embryo cultures WT female and male mice were mated overnight. The Immunofluorescent staining day on which a vaginal plug was observed was designated Tissue sections were fixed in 4% paraformaldehyde E0.5. At E7.5, mouse embryos with an intact visceral yolk (pH7.4) in PBS for 30 min at room temperature, followed sac were dissected from the uteri into PBS (Invitrogen, La by permeabilization with 0.25% Triton-X100 (Sigma, Jolla, CA). Then, embryos were cultured in 4 ml of culture St. Louis, MO) in PBS for 10 min at room temperature. medium containing 2.66 ml of rat serum, 1.34 ml of Samples were blocked for 30min in 10% donkey serum Tyrode’s salt solution (Cat#: T2397, Sigma, St. Louis, in PBS and incubated with anti-Flk1 antibody (1:200; MO), 100 units/mL of penicillin and 100 µg/mL of Santa Cruz Biotechnology, Dallas, TX) overnight at 4 °C. streptomycin with NG, HG, and NG + BMP4 at 37 °C in a After washing with PBS, samples incubated with roller bottle system. The embryos were cultured for 3 days 594 secondary antibody (1:1000, Invitrogen, Carlsbad, under the following conditions: 5% O2–5% CO2–90% N2 CA) for 2 h at room temperature, in the dark, followed by for the first two days and 10% O2–5% CO2–85% N2 for DAPI cell nuclear counterstaining for 10 min at room the last day. temperature, mounted using aqueous mounting med- ium. For confocal fluorescent images, the cells were H&E staining recorded by a laser scanning microscope (LSM 510 + Nondiabetic-non-BMP4 Tg, nondiabetic BMP4 Tg, META;ZEISS,Germany).AllFlk-1 progenitors (red) in diabetic-non-BMP4 Tg, and diabetic BMP4 Tg embryos the yolk sac were counted under a microscope. Experi- were collected and fixed with 4% paraformaldehyde in ments were repeated three times using three embryos phosphate-buffered saline (PBS). Embryos were dehy- (n = 3) from three different dams and three images were drated in alcohol, embedded in paraffin, and 5-µm sec- obtained from each embryo. Information of antibodies tions were stained with hematoxylin and eosin (Sigma, are in Table S3. St. Louis, MO) and observed under a light microscope. TUNEL assay Blood island quantification The TUNEL assay was performed using the ApopTag E8.5 conceptuses were collected and processed as Fluorescein in Situ Apoptosis Detection kit (Millipore, described above. Five micrometer cross-sections (vertical) Billerica, MA). 5-µm embryonic sections were fixed with were stained with H&E. Yolk sac blood islands were 4% paraformaldehyde in PBS and incubated with TUNEL counted. Ten sections from each conceptus were used and reaction agents. Three embryos from three different dams the data were averaged. Five conceptuses from different (n = 3) in each group were used, and two sections per pregnant mice in each group were used to determine the embryo were examined. TUNEL-positive cells in an area numbers of blood islands. of neuroepithelium and yolk sac (about 200 cells) were counted. Blood vessel density measurement The conceptuses were fixed with 4% paraformaldehyde Real-time PCR (RT-PCR) in PBS overnight at 4 °C. For immunostaining analyses, Total mRNA was isolated from E8.5 embryos using controls were processed by omitting the primary anti- the RNeasy Mini kit (Qiagen, Hilden, Germany) and body. We use CD31 antibody (Abcam, Cambridge, MA) mRNA was reverse transcribed to cDNA using the to stain the whole conceptus. Samples were incubated in Quanti Tect Reverse Transcription Kit (Qiagen, Hilden, ABC solution (elite ABC kit, Vector Laboratories, Bur- Germany). Real time-PCR for Bmp4, Id1, Id2, Id3, Id4, lingame, CA) for 30 min, and then with stable diamino- Smad6, and β-actin were performed using the Maxima benzidine substrate solution (Vector Laboratories, SYBR Green/ROX qPCR Master Mix assay (Thermo Burlingame, CA). Yolk sacs were removed from the Fisher Scientific; Waltham, MA) in the Step One Plus

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− system (Applied Biosystems, Grand Island, NY). The expression (embryo genotype: Floxed BMP4; Flk-1-Cre ) primers for RT-PCR are listed in Table S2. under nondiabetic and diabetic conditions. The mRNA and protein levels of BMP4 were slightly increased but not Immunoblotting significantly different in embryos with BMP4 Tg than in A protease inhibitor cocktail (Sigma, St. Louis, MO) embryos without BMP4 Tg from nondiabetic dams dissolved in lysis buffer (Cell Signaling Technology, (Fig. 1c, d). The mRNA and protein levels of BMP4 were Danvers, MA) was used to extract protein. Equal amounts significantly lower in embryos without BMP4 Tg from of protein and the Precision Plus Protein Standards (Bio- diabetic dams than in embryos without BMP4 Tg from Rad Laboratories, Hercules, CA) were resolved by SDS- nondiabetic dams (Fig. 1c, d). BMP4 Tg expression PAGE and transferred onto Immobilon-P membranes reversed the decrease of BMP4 expression in embryos (Millipore, Billerica, MA). Membranes were blocked in 5% from diabetic dams (Fig. 1c, d). BMP4 immunofluorescent nonfat milk for 2 h at room temperature and then were staining confirmed that under nondiabetic conditions, incubated with the following primary antibodies: BMP4, BMP4 expression was not significantly different in the p-PERK, p-IRE1α, p-eIF2α, CHOP, p-Smad1/5/8, and neuroepithelia of embryos with BMP4 Tg than those Smad4 at 1:1000 (all from Cell Signaling Technologies, without BMP4 Tg (Fig. 1e). BMP4 Tg expression reversed Danvers, MA); caspase 3 at 1:500 (Millipore, Billerica, the decrease of BMP4 expression in the neuroepithelia MA) and caspase 8at 1:1000 (Enzo Life Sciences, Farm- of embryos from diabetic dams (Fig. 1e). Strong + ingdale, NY) overnight at 4 oC. The membranes were BMP4 staining signal was present in the Flk-1 progeni- + washed with TBST. And then exposed to related sec- tors in the yolk sac (Fig. 1e). BMP4 expression in Flk-1 ondary antibodies. To ensure that equivalent amounts of progenitors was significantly lower in the yolk sacs of protein samples were loaded, membranes were incubated embryos without BMP4 Tg from diabetic dams compared with a mouse antibody against β-actin (1:10,000; Abcam, to those in the yolk sacs of embryos from nondiabetic Cambridge, MA). Signals were detected using the Super dams (Fig. 1e). BMP4 Tg expression restored BMP4 + Signal West Femto Maximum Sensitivity Substrate kit expression in Flk1 progenitors in yolk sacs of embryos (Thermo Fisher Scientific, Waltham, MA). All experi- from diabetic dams (Fig. 1e). These findings collectively ments were repeated in triplicate with the use of inde- support that BMP4 Tg expression reverses maternal pendently prepared tissue lysates. Information of diabetes-suppressed BMP4. antibodies are in Table S3. BMP4Tg expression re-activates Smad signaling Statistical analysis Smad1/5/8 and Smad4 are downstream effectors of Data are presented as means ± SD. Embryonic samples BMP4 and their phosphorylation transmits the down- – from each replicate were from different dams. One-way stream signals of the BMP4-Smad pathway33 35.We ANOVA was performed using Sigma Stat 3.5 software examined the levels of p-Smad1/5/8, total Smad1/5/8, and (Systat Software Inc., San Jose, CA). In ANOVA analysis, Smad4. Protein levels of p-Smad1/5/8 and Smad4 in Turkey test was used to estimate the significance of the embryos without BMP4 Tg expression from diabetic dams results. To determine significant difference between were significantly decreased, compared with those in groups for NTDs incidence, the Fisher Exact Tests were conceptuses from nondiabetic dams (Fig. 2a, b). BMP4 Tg used. Statistical significance was accepted at P < 0.05. expression restored Smad1/5/8 phosphorylation and Smad4 expression in conceptuses exposed to maternal Results diabetes (Fig. 2a, b). The protein levels of p-Smad1/5/8 BMP4 Tg expression reverses maternal diabetes- and Smad4 were not significantly different in embryos suppressed BMP4 with BMP4 Tg than in embryos without BMP4 Tg from We generated a BMP4 transgenic (Tg) mouse line in nondiabetic dams (Fig. 2a, b). Protein levels of Smad1/5/8 which contained the CAG promoter-driven floxed stop in the conceptuses from diabetic dams showed no dif- codon followed and a separate nuclear GFP (Fig. 1a). ference when compared with those in conceptuses from Nuclear GFP positive cells were detected specifically in nondiabetic dams (Fig. 2a). The experiments showed that + Flk-1 progenitors in the yolk sac of BMP4 transgenic BMP4 Tg expression restored BMP4 expression and its (Tg) embryos (Fig. 1b), supporting the BMP4 transgene downstream effectors of Smad 1/5/8, Smad 4, Smad 6. + expression specifically in the Flk-1 progenitors. To + determine whether BMP4 Tg expression in Flk-1 pro- BMP4 Tg rescues BMP4 target gene expression – genitors restores BMP4 expression suppressed by mater- Id genes and Smad 6 are BMP4 target genes34 36. Thus, nal diabetes, we assessed mRNA and protein levels of we examined the expression of Id1, Id2, Id3, Id4, and BMP4 in embryos with BMP4 Tg expression (embryo Smad6. At the mRNA level, maternal diabetes suppressed + genotype: Floxed BMP4; Flk-1-Cre ) or without BMP4 Tg the expression of Smad6, Id1, Id2, and Id3 (Fig. 3a–d).

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Fig. 2 BMP4 Tg expression re-activates Smads. a Protein levels of phosphorylated Smad1/5/8 and Smad1/5/8 in E8.5 non-BMP4 Tg and BMP4 Tg embryos from nondiabetic (ND) or diabetes mellitus (DM) dams. Quantification of relative expression levels of phosphorylation of Smad1/5/8 versus total Samd1/5/8 was shown in the graphs. b Protein levels of Smad4 in E8.5 non-BMP4 Tg and BMP4 Tg embryos from nondiabetic (ND) or diabetes mellitus (DM) dams. Quantification of relative expression levels of Smad4 was shown in the graphs. Experiments were performed using three embryos from three different dams per group (n = 3); * indicates significant difference compared to the other groups (P < 0.05) in one-way ANOVA followed by Tukey tests. ND: nondiabetic; DM: diabetes mellitus.

Fig. 3 BMP4 overexpression rescues BMP4 target gene expression. a–e mRNA levels of Smad6and Id genes in E8.5 non-BMP4 Tg and BMP4 Tg embryos from nondiabetic (ND) or diabetes mellitus (DM) dams. Quantification of relative expression levels of Smad6, Id1, Id2, Id3, and Id4 was shown in the graphs. Experiments were performed using three embryos from three different dams per group (n = 3); * indicates significant difference compared to the other groups (P < 0.05) in one-way ANOVA followed by Tukey tests. ND: nondiabetic; DM: diabetes mellitus.

BMP4 Tg expression significantly restored Smad6 and BMP4 Tg expression mitigates maternal diabetes-induced Id1expression (Fig. 3a, b). BMP4 Tg expression also vasculopathy + restored Id2 and Id3 expression, but they were not sta- We assessed whether BMP4 Tg expression in Flk-1 tistically significant (Fig. 3c, d). The expression of Id4 progenitors ameliorate maternal diabetes-induced vasculo- + showed no difference when compared with nondiabetic pathy. We observed that maternal diabetes-induced Flk-1 dams and diabetic dams (Fig. 3e). progenitor apoptosis in the yolk sac (Fig. 4a).Invitro,we

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Fig. 4 BMP4 overexpression block Flk-1+ progenitor apoptosis in vivo and in vitro. a Representative TUNEL staining images showing apoptotic cells (red dots) in Flk-1+ progenitors (green) in yolk sac. Cell nuclei were stained with DAPI (blue), Scale bar = 100 μm. Quantification of TUNEL positive in Flk-1+ progenitors was shown in the graph. B: Representative TUNEL staining images showing apoptotic cells (red dots) in Flk-1+ progenitors (green) treated with different concentrations of recombinant BMP4 protein. Cell nuclei were stained with DAPI (blue), Scale bar = 50 μm. Quantification of TUNEL positive in Flk-1+ progenitors was shown in the graph. Experiments were performed using three embryos from three different dams per group (n = 3); * indicates significant difference compared to the other groups (P < 0.05) in one-way ANOVA followed by Tukey tests. ND: nondiabetic; DM: diabetes mellitus. NG: normal glucose; HG: high glucose.

+ + treated Flk-1 progenitor isolated from yolk sacs at E8.5 restored the number of Flk-1 progenitors, blood islands, with different concentrations of recombinant BMP4 protein and blood vessel density (Fig. 5b–d), leading to amelioration under normal glucose (NG, 5 mM) and high glucose (HG, of maternal diabetes-induced vasculopathy (Fig. 5a). + 25 mM). After 48 h, using TUNEL staining to detect Flk-1 progenitor apoptosis. We found that high glucose triggered BMP4 Tg expression blocks caspase activation and + Flk-1 progenitor apoptosis and that different concentra- neuroepithelial cell apoptosis tions of recombinant BMP4 protein inhibited high glucose- Apoptosis is a hallmark of diabetic embryopathy and + induced Flk-1 progenitor apoptosis (Fig. 4b). BMP4 Tg aberrant cell death in key embryonic tissues leads to + expression specifically in the Flk-1 progenitors alleviated major structural malformations in the fetus and even maternal diabetes-induced yolk sac vasculopathy (Fig. 5a). death37,38. To test whether BMP4 Tg expression inhibits + Flk-1 progenitor reduction and subsequent impaired maternal diabetes-induced neuroepithelial cell apoptosis, blood island formation resulted in vasculopathy, manifested we performed a TUNEL assay in E8.5 embryos. The by reduced blood vessel density in conceptuses exposed to number of apoptotic cells in the neuroepithelia of maternal diabetes (Fig. 5b). The key process during embryos from diabetic dams was significantly greater than embryonic vasculogenesis is the formation of blood islands that in embryos from nondiabetic dams (Fig. 6a). BMP4 + from Flk-1 progenitors. Blood island formation at E8.5 was Tg expression significantly reduced maternal diabetes- significantly impaired in the yolk sacs of conceptuses induced neuroepithelial cell apoptosis to a level compar- exposed to maternal diabetes (Fig. 5c). The total number of able to that of the nondiabetic group (Fig. 6a). Maternal + Flk-1 progenitors was reduced by maternal diabetes (Fig. diabetes triggered caspase 3 and caspase 8 cleavage in 5d). Reversal of BMP4 reduction via BMP4 Tg expression embryos, whereas BMP4 Tg expression abrogated

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Fig. 5 BMP4 Tg expression ameliorates maternal diabetes-induced vasculopathy. a Blood vessel density in yolk sac and embryos. Scale bars = 1 mm. b The vascular endothelial cell marker CD31 staining in the yolk sac (Scale bar = 100 μm) and the embryo (Scale bar = 250 μm). The red arrow indicates NTDs. Quantification of blood vessel density in yolk sac was shown in the graphs. c HE staining shows blood islands. The red arrow indicates blood island. Scale bar = 100 μm. Quantification of blood island number per section was shown in the graph. d Immunofluorescent staining shows Flk-1+ progenitors (red dots) in yolk sac. Cell nuclei were stained with DAPI (blue). Scale bar = 100 μm. Quantification of Flk-1+ progenitors cell number per section was shown in the graph. Experiments were performed using three embryos from three different dams per group (n = 3); * indicates significant difference compared to the other groups (P < 0.05) in one-way ANOVA followed by Tukey tests. ND: nondiabetic; DM: diabetes mellitus.

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Fig. 6 BMP4 Tg expression reduces maternal diabetes-induced apoptosis in the developing neuroepithelium. a Representative TUNEL staining images showing apoptotic cells (red dots) in the E8.5 neuroepithelium. Cell nuclei were stained with DAPI (blue), Scale bar = 30μm. Quantification of TUNEL positive cells per section was shown in the graph. b Protein levels of cleaved caspase 8 and caspase 3 in E8.5 embryos. Experiments were performed using three embryos from three different dams per group (n = 3). * indicates significant difference compared to the other groups (P < 0.05) in one-way ANOVA followed by Tukey tests. ND: nondiabetic; DM: diabetes mellitus. maternal diabetes-induced caspase 3 and caspase 8 clea- BMP4 Tg expression ameliorates maternal diabetes- vage (Fig. 6b). induced NTDs whereas recombinant BMP4 alleviates high glucose-induced NTDs + BMP4 Tg expression abrogates maternal diabetes To determine whether BMP4 Tg expression in Flk-1 -induced-ER stress progenitors ameliorates maternal diabetes-induced NTDs, ER stress is a primary molecular mechanism under- NTDs were examined morphologically in E10.5 and the lying diabetic embryopathy32. To determine whether open neural tube in NTD embryos was confirmed by BMP4 Tg expression affects maternal diabetes-induced- histological sectioning (Fig. 7e). In E10.5, growth retar- ER stress, we measured the expression levels of the ER dation and embryonic lethality were not yet manifested, stress markers IRE1α,PERK,eIF2α, and total CHOP. and NTD embryos could be clearly identified based on the Protein levels of p-IRE1α,p-PERK,p-eIF2α, and total existence of an open neural tube (Fig. 7e). Embryos with CHOP were significantly up-regulated in the con- BMP4 Tg were morphologically indistinguishable from ceptuses from diabetic dams when compared with those embryos without BMP4 Tg at nondiabetic conditions in in conceptuses from nondiabetic dams (Fig. 7a–d). E10.5 (Fig. 7e). As shown in Table 1 and Fig. 7e, 21 out of BMP4 Tg expression inhibited the expression of these 87 embryos without BMP4 Tg expression (non-BMP4 Tg) ER stress markers induced by maternal diabetes from diabetic dams had NTDs (NTD rate was 24.14%), (Fig. 7a–d). Protein levels of IRE1α, PERK, and eIF2α in whereas embryos with or non-BMP4 Tg from nondiabetic the conceptuses from diabetic dams showed no differ- dams did not exhibit any NTDs. Thus, BMP4 Tg ence when compared with those in conceptuses from expression didn’t affect neurulation under nondiabetic nondiabetic dams (Fig. 7a–d). conditions (Table 1). Under diabetic conditions, 2 of 31

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Fig. 7 (See legend on next page.)

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(see figure on previous page) Fig. 7 BMP4 Tg expression relieves ER stress and reduces NTD formation. a–d Protein levels of ER stress markers in E8.5 non-BMP4 Tg and BMP4 Tg embryos from nondiabetic (ND) or diabetes mellitus (DM) dams. Quantification of relative expression levels of phosphorylated IRE1α, PERK, and eIF2α versus total IRE1α, PERK, and eIF2α and CHOP were shown in the graphs. Experiments were performed using three embryos from three different dams per group (n = 3); * indicates significant difference compared to the other groups (P < 0.05) in one-way ANOVA followed by Tukey tests. E: morphology of non-BMP4 Tg and BMP4 Tg embryos from ND or DM dams, bar = 1 mm in the upper panel and bar = 600 μm in the lower panel. Quantification of NTDs was shown in the graph and statistically tested by Fisher exact tests. ND: nondiabetic; DM: diabetes mellitus. F: morphology of WT with NG, WT with HG and WT with HG + recombinant BMP4 treatment embryos, bar = 1 mm. NG: normal glucose (5 mM glucose); HG: high glucose (25 mM glucose).

Table 1 BMP4 overexpression ameliorates maternal diabetes-induced NTD.

Experimental group Glucose level (mg/dl) Genotype Embryos NTDs Embryos (NTDs rate)

ND Flk-1-Cre male x BMP4-Tg female (16 litters) 157.5 ± 7.6 non-BMP4-Tg 81 0 163 ± 11.78 BMP4-Tg 27 0 DM Flk-1-Cre male x BMP4-Tg female (17litters) 476.5 ± 13.2 non-BMP4-Tg 87 21 (24.14%)a 432.56 ± 16.3 BMP4-Tg 31 2 (6.45%)

The ratio of BMP4-Tg mice to non-BMP4-Tg mice is about 1 to 3. NTD rates in E10.5 embryos. ND nondiabetic, DM diabetes mellitus, BMP4-Tg BMP4 transgenic (Floxed BMP4; Flk-1-Cre+), non-BMP4-Tg non-BMP4 transgenic (include Wild-Type mice, Flk-1-Cre+ mice, and BMP4-Tg without Flk-1-Cre+). a Indicates significant difference (P < 0.05) when compared to other groups analyzed by the Fisher Exact test.

BMP4 Tg embryos (6.45%) presented NTDs, which was vasculature is inversely related to embryonic malformation significantly lower than those in embryos without BMP4 rates3. In the present study, we demonstrated a causal Tg expression (Table 1). The above data support our relationship between early vasculopathy, mediated by BMP4, hypothesis that BMP4 Tg expression during vasculogen- and late NTD formation. esis ameliorates maternal diabetes-induced NTDs. Hyperglycemia causes yolk sac vasculopathy that ultimately To determine whether BMP4 has a direct effect on high leads to embryonic malformations or lethality44.Diabetes- glucose-induced NTDs, we cultured E7.5 embryos under induced vascular defects in the yolk sac have been directly normal glucose (NG, 5 mM) and high glucose (HG, linked to failed neural tube closure, highlighting the impor- 25 mM) conditions. As shown in Table S1, embryos under tance of studying diabetic yolk sac vasculopathy45. + NG did not exhibit any NTDs. 8 out of 12 embryos under Flk-1 vascular progenitors initiate vasculogenesis in the yolk HG conditions had NTDs, and only 1 out of 14 embryos sac by forming blood islands, which fuse to generate blood + under HG with recombinant BMP4 had NTDs. Thus, vessels46,47. A critical number of Flk-1 progenitors and – BMP4 treatment prevented high glucose-induced NTDs blood islands are crucial for normal vasculogenesis48 51.The (Table S1, Fig. 7f). findingsinthepresentstudysupportthecriticalroleofBMP4 + in restoring the numbers of Flk-1 progenitors and blood Discussion islands. BMP4 Tg expression ameliorated yolk sac vasculo- Maternal diabetes is a significant risk factor for NTDs and pathy by blocking diabetes-induced defects in vasculogenesis. previous studies of the etiology of diabetic embryopathy Under nondiabetic conditions, the BMP4 basal level was mainly focus on the direct effects of high concentrations of already very high in the embryos without BMP4 Tg. The – glucose on the developing neuroepithelium1,3,39 42.How- mRNA and protein levels of BMP4 were slightly increased ever, maternal diabetes induces yolk sac by dama- in BMP4 Tg from nondiabetic dams but were not sig- ging the morphology of cells and altering the dynamics of nificantly different in embryos with BMP4 Tg compared leading to yolk sac vasculopathy in early devel- with embryos without BMP4 Tg from nondiabetic dams opment7,10,43. The yolk sac vasculature is the firstsystemto because the endogenous BMP4 levels without the pre- develop during embryogenesis and is among the most sen- sence of BMP4 already reached a plateau. However, under sitive tissue to maternal hyperglycemiainsult3,5,44.Thecon- diabetic conditions, the BMP4 level was extremely low in sequences of yolk sac injuries may impact structural organ the embryos without BMP4 Tg; therefore, BMP4 Tg development during later development of the embryo. expression reversed the decrease of BMP4 expression in Although the functional relationship between yolk sac vas- embryos from diabetic dams. culopathy and NTDs has not been firmly established, a Diabetes-decreased BMP4 protein levels in the neu- recent study revealed that the quality of the yolk sac roepithelium were restored in BMP4 Tg embryos,

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+ suggesting that BMP4 produced by Flk-1 progenitors contribution of yolk sac vasculopathy to these NTD reached the neuroepithelium. However, BMP4 proteins associated events has not been determined. The present + + produced by BMP4 Tg in Flk-1 progenitors were not study demonstrated that BMP4 Tg expression in Flk-1 labeled by recognized tags such GFP. Thus, there is no progenitors ameliorated ER stress in cells of the devel- direct evidence that the BMP4 Tg-produced BMP4 pro- oping neuroepithelium, leading to the blockage of caspase teins act on neuroepithelial cells. Nevertheless, BMP4 Tg activation and neuroepithelial cell apoptosis induced by can reactivate the downstream Smad signaling in maternal diabetes. Taken together, our findings establish a embryonic cells leading to BMP4 responsive gene causal relationship between early yolk sac vasculopathy, expression under diabetic conditions. Collectively BMP4 caused by BMP4 reduction, and late NTD formation, Tg blocks neuroepithelial cell apoptosis and NTD for- induced by ER stress and apoptosis in the developing mation. The beneficial effect of BMP4 may be twofold. neuroepithelium, in diabetic embryopathy. + BMP4 produced by Flk-1 progenitors directly act on the Our studies demonstrated that BMP4 Tg expression in + developing neuroepithelium leading to NTD prevention. Flk-1 progenitors prevented yolk sac vasculopathy and BMP4 protein in the neuroepithelium was decreased by ameliorated maternal diabetes-induced NTDs. Studies have maternal diabetes and this decrease was reversed in BMP4 shown the involvement of BMPs and vasculopathy in var- Tg embryos. Furthermore, recombinant BMP4 treatment ious adverse pregnancy outcomes. For example, in women alleviated NTD formation in embryos exposed to high with type 1 diabetes, vasculopathy is associated with + glucose. Thus, Flk-1 progenitor overexpressing intrauterine fetal growth abnormalities67. Vasculopathy and BMP4 secreted BMP4 protein, which reached the neu- BMPs are associated with preterm labor, preterm ruptured – roepithelium, relieved ER stress and inhibited maternal membranes and fetal growth restriction68 70.AlteredBMP4 diabetes-induced apoptosis in the neuroepithelium lead- expression and its associated vascular lesions are highly ing to amelioration of NTDs. However, embryonic vas- correlated with neurologic impairment and cerebral palsy in – culopathy is linearly linked to NTD formation52 54. the fetus71,72. It is still unclear how altered BMP4 expression Therefore, prevention of maternal diabetes-induced vas- is associated with adverse pregnancy outcomes in various culopathy may also contribute to the preventive effect of maternal conditions. Our study shows a beneficial effect of BMP4 Tg on NTDs. BMP4 prevented embryonic vascu- BMP4 in diabetic embryopathy. Therefore, further work is + lopathy through its effect on Flk-1 progenitors because needed to determine the potential involvement of BMP4 in these cells are the initiators for embryonic vasculogenesis. the induction of adverse pregnancy outcomes. + BMP4 Tg expression restored the number of Flk-1 BMPs, including BMP-2, -4, -6, and -7, play important progenitors, blood islands, and blood vessel density, roles in vasculogenesis73,74. Genetic deletion or mis- leading to amelioration of maternal diabetes-induced expression of different components of the BMP signaling + vasculopathy. The direct effect of BMP4 on Flk-1 pro- leads to embryonic lethality due to cardiovascular mal- genitors was further manifested by its inhibition on high formations and defects in vasculature22,73,75. Our study is + glucose-induced Flk-1 progenitor apoptosis. the first report that BMP4 reduction contributes to Under nondiabetic conditions, BMP4 Tg did not further maternal diabetes-induced yolk sac vasculopathy and activate Smad 1/5/8, Smad 4, Smad 6 because endogenous NTDs, and thus reveals a novel role of BMP4 in the relatively high BMP4 levels had already maximined the etiology of diabetic embryopathy. activation of these effectors. Restoring BMP4 expression Our results indicate that early vasculopathy accounts for + in Flk-1 progenitors rescued the BMP4 downstream failed neural tube closure later in diabetic pregnancy. effector, phosphorylation of Smad1/5/8, and the expres- BMP4 overexpression reversed maternal diabetes-induced + sion of BMP4-Smad1/5/8 target genes, Id genes, in con- the loss of Flk-1 progenitors, impaired blood island ceptuses exposed to maternal diabetes. Id genes function formation and reduced blood vessel densities. BMP4 Tg + as dominant-negative transcriptional regulators21,36,55. expression in the Flk-1 progenitor ameliorated diabetes- The expression and function of the four members of the induced NTDs. Furthermore, BMP4 Tg expression Id gene family depend on tissue type. Studies have sug- relieved ER stress and inhibited maternal diabetes- gested an important role of Id genes in vasculogenesis and induced apoptosis in the neuroepithelium. Our study maintaining vessel homeostasis56,57. The role of Id genes reveals a novel mechanism underlying maternal diabetes- in maternal diabetes-induced vasculopathy and NTD induced NTD formation. formation is an important topic for future investigation. Extensive studies have uncovered the molecular events Acknowledgements 32,58–63 in the embryo that contribute to failed neurulation . This work was supported by the NIH grants NIH R01HL131737, R01HL134368, ER stress-induced caspase activation and neuroepithelial R01HL139060, R01HD100195, and R01DK083243. We thank Dr. Julie Rosen, Assistant Professor of Obstetrics, Gynecology & Reproductive Sciences and cell apoptosis are key causative factors for NTD Executive Director for Medical Research and Scientific Publications at the 30,38,61–66 formation in diabetic pregnancy . However, the University of Maryland School of Medicine, for critical reading and editing.

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