Cao et al. Cell Death and Disease (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 apoptosis 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 growth factor 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 stress, and expression of the Id genes, direct targets of BMP4; and the presence of cleaved caspase 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 organelle 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 Biochemistry 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 © The Author(s) 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a linktotheCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Official journal of the Cell Death Differentiation Association Cao et al. Cell Death and Disease (2020) 11:859 Page 2 of 14 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 Animals 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 animal 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