Sonic Hedgehog Pathway Regulates Murine Placental Development

Survival and growth of the fetus are critically dependent on the placenta. It forms the interface between the maternal and fetal circulation, facilitating metabolic and gas exchange as well as fetal waste disposal. The development of a functional mouse placenta is a dynamic phenomenon involving a number of different processes: trophoblast cell type specification, branching of the chorion and fetal vascular morphogenesis, as well as formation of a maternal blood circulation.Sonic hedgehog (Shh) is a member of the hedgehog family of signaling molecules identified by homology to the Drosophila hedgehog (HH). It acts as a morphogen in normal development of various vertebrate tissues and organs. Shh signaling is transduced by the receptor Patched (Ptch) and co-receptor Smoothened (Smo). Shh binding to Ptch relieves Ptch-mediated repression of Smo leading to activation of downstream Gli transcription factors in vertebrates.Hedgehog-dependent transcripitional activation is mediated by three vertebrate homologs to the Drosphila cubitus interruptus (Ci) transcription factor:Gli-1,Gli-2and Gli-3. They have similarly amino acid motifs of highly conserved five tandem zinc fingers and a consensus cysteine-histidine link. Their structures suggest that Gli-1and Gli-2are mainly activators, whereas Gli-3is predominantly a repressor. The transcriptional outcome of HH signaling is thought to depend on the balance between the activator and repressor forms. Downstream target genes include Ptch, whose upregulation serves as an indicator of a hedgehog response, as well as bone morphogenic proteins (BMPs).Whether this signaling pathway is involved in the development of mouse placenta remains unknown. In the present study, we first examined the locolization of the key molecules related to hedgehog signaling pathway by immunohistochemistry in mouse placenta from E8.0to E11.5. Then, we use a combination of transgenic mice and a lentiviral vector-mediated placenta-specific expression system to study the function of Shh in murine placental development.First we investigated the expression pattern of the key molecules related to Shh signaling in blastocyst and developing mouse placenta from E8.0to E11.5. We observed that Shh and Ptchl were experessed in the blastocyst trophectoderm (Tr) at E3.5. At E8.0we could detect expression of Shh in the uterus and first trophoblat giant cells. As to receptor localization, both deciduas and first trophoblat giant cells expressed Ptchl and Smo. Smo expression was also in the uterus. Gli-2and Gli-3transcription factors staining were detected in the uterus, deciduas, trophoblat giant cells, the extraembryonic ectoderm and the ectoplacental cone. After the chorioallantoic attachment, at E9.5we found that Shh, Ptchl,and Smo expressed strong in the deciduas, trophoblat giant cells, spongiotrophoblast and labyrinth. These expression was also strong at E11.5. At E9.5both Gli-2and Gli-3transcription factors staining were detected broadly in the deciduas, trophoblat giant cells, spongiotrophoblast and labyrinth. These results suggest that the Shh signaling play a role in the blastocyst and early junctional zone after the chorioallantoic attachment.Next we use Shh knockout mice to study the function in placenta. We found that Shh+/-mice were viable, no homozygous knockout pups obtained from heterozygous intercrosses, indicating that the Shh disruption causes embryonic lethality. To determine the developmental stages when the lethality occurred, embryos were collected and analyzed at different stages. As a result, Shh-/-embryos up to E12.5were observed with expected Mendelian frequency. By E13.5, all Shh-/-embryos were dead. These finding show that Shh is essential for normal embryonic development. Noteworthy, the living Shh-/-embryos at E12.0were paler and displayed decreased vascularization of their extraembryonic annexes. Blood vessels branching at the surface of the yolk sac and on the fetal side of the placenta of mutant embryos were barely visible. Meanwhile, measurement of transplacental passage by using the fluorescent dye rhodamine123injected into the mother revealed a reduction in the fluorescence recovered in the living E12.0Shh-/-embryos compared with their heterozygous littermates, consistent with reduced placental transport capacity. The Shh-/-STGCs layer is much thinner The Shh-/-ST-II layer contained less lipid inclusions indicating that this layer developed abnormally. There is also many abnormal cavities in the Shh-/-ST-I layer.As Gli-3is predominantly a repressor, we used a lentiviruse carrying shRNA for Gli-3to activate Shh signaling. First, the efficiency and specificity of the LV-Gli-3-shRNA was validated by western blotting and IHC. Then, all those phenotypes of Shh-/-can be partial rescued by LV-Gli-3-shRNA. Although the rescued Shh-/-embryos still died, the morphological defects were ameliorated at E12.0. The labyrinth architecture in rescued Shh-/-placenta become more compact. Besides, the electron microscopy showed that rescued Shh-/-STGCs become thicker and ST-II layer contained more lipid inclusions. However, Gli2knockdown in Shh-/-placentas led to a partial phenocopy of the labyrinthine defects of Shh-/-placentas.The results of this study have revealed an important role of Shh signaling in murine placental development. The Shh-/-placental phenotype is similar to preeclampsia (PE) which is a serious complication that affects7%of first-time pregnancies. Our results suggest that the Shh signaling may be involve in the pathological process of PE and a candidate target for PE treatment.