Cell Death Dis 6:e1653 (2015) 

Sonic hedgehog through Gli2 and Gli3 is required for the proper development of placental labyrinth



Sonic hedgehog (Shh) functions as a conserved morphogen in the development of various organs in metazoans ranging from Drosophila to humans. Here, we have investigated the potential roles and underlying mechanisms of Shh signaling in murine placentation. Immunostaining revealed the abundant expression of the main components of Shh pathway in both the trophectoderm of blastocysts and developing placentas. Disruption of Shh led to impaired vascularogenesis of yolk sac, less branching and malformation of placental labyrinth, thereby leading to a robust decrease in capacity of transplacental passages. Moreover, placenta-specific gene incorporation by lentiviral transduction of mouse blastocysts and blastocyst transplantation robustly knocked down the expression of Gli3 and Gli2 in placenta but not in embryos. Finally, Gli3 knockdown in Shh−/− placentas partially rescued the defects of both yolk sac and placental labyrinth, and robustly restored the capacity of transplacental passages. Gli2 knockdown in Shh+/− placentas affected neither the capacity of tranplacental passages nor the vascularogenesis of yolk sac, however, it partially phenocopied the labyrinthine defects of Shh−/− placentas. Taken together, these results uncover that both Shh/Gli2 and Shh/Gli3 signals are required for proper development of murine placentas and are possibly essential for pregnant maintenance.To examine the localization of the main components of Shh pathway, we performed immunostaining for the blastocysts at E3.5 and placentas from E8.0 to E11.5. In blastocysts, Shh and Ptc1 were relatively restricted in the trophectoderms, caudal-related homeobox 2 (Cdx2), a trophoblast stem cell marker, was evenly expressed in both the trophectoderms and inner cell masses (ICMs), while octamer-binding transcription factor 3/4 (OCT3/4), an embryonic stem cell marker, was more highly expressed in ICMs than in the trophectoderms (Supplementary Figures 1a–d”). Shh-derived immunohistochemistry signal was detectable in the primary TGC and chorion at E8.0, and was diffusely localized in decidium, junctional (TGC and SP layers) and labyrinthine zones at E9.5, whereas it was relatively restricted in the placenta but not in the decidium at E11.5 (Figures 1a–b''). Ptc1 and Smo were barely detectable at E8.0, whereas they were abundantly and region-specifically localized at placentas of E9.5 and E11.5; Ptc1 was more highly expressed in junctional zone than in labyrinthine zone, whereas Smo was almost evenly expressed in both of them (Figures 1c–d”). Gli2 was broadly but not cell lineage specifically localized in the uterus and placenta at E8.0, and was diffusely distributed in all three layers of murine placentas at E9.5 and E11.5 (Figures 1e–e”). Gli3 was diffusely expressed in uterus and placenta, especially in ectoplacental cone (EPC) at E8.0, however, it was robustly localized in both the junctional zone and labyrinthine zone at E9.5 and E11.5 (Figures 1f–f”). To gain quantitative information about the expression levels, we performed RT-PCR assays in developing placentas from E8.5 to E11.5. Dhh and Ihh were expressed at relatively higher levels than Shh at E8.5 and E9.5, but Shh was expressed at a higher level than either Dhh or Ihh at E11.5; Ptc1, Gli2, and Gli3 were expressed at relatively higher levels than Smo (Supplementary Figures 1e and f). Overall, the main components of Hh pathway are abundantly expressed in murine developing placentas.Shh+/– mice on a C57BL/6J genetic background were generated as previously described,20, 21 and were obtained from Cyagen Biosciences (Santa Clara, CA, USA). Adult wild-type and Shh+/– mice were housed at Zhejiang University Animal Care Facility according to the institutional guidelines for laboratory animals. The animal protocol was approved by the Zhejiang University Institutional Animal Care and Use Committee. Founder mice and their progenies were genotyped by PCR using tail genomic DNA. Females were mated with fertile males of heterozygous mice to induce pregnancy; the day that the vaginal plug was first observed was considered day 1 of pregnancy. Pre-implantation embryos at blastocyst stages were harvested by flushing the uterus as appropriate, and the littermates were harvested from E8.0 to E12.0 for analyses of phenotypes of placenta and embryo as well.We thank Drs Hong-Mei Wang and Qi zhou from Institute of Zoology, Chinese Academy, and Dr Beibei Wang from Facility Core of Electron Microscopy of Zhejiang University for their excellent technical support. This work was supported by 973 Program (No. 2011CB944403), National Natural Science Foundation of China (No. 30973580, No. 31071292, No. 81370713, and No. 81072658), and Natural Science Foundation of Zhejiang Province, China (No. R2110269) to XW.Supplementary Information accompanies this paper on Cell Death and Disease website (http://www.nature.com/cddis)Edited by A Stephanou
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