Investigate Ddx3x functions during murine placentation and embryogenesis

• Main Authors: Chia-Yu Chen, 陳佳郁
• Other Authors: Yan-Hwa Wu Lee
• Format: Others
• Language: zh-TW
• Published: 2014
• Online Access: http://ndltd.ncl.edu.tw/handle/49435142456821406867

碩士 === 國立陽明大學 === 生化暨分子生物研究所 === 102 === DDX3X gene is an X-lined member of DEAD-box RNA helicase family. Previous evidences have revealed that DDX3 plays important roles in various cellular processes and functions which involving in mRNA transport, transcriptional regulation and translation initiation. However, the biological function of Ddx3x during development remains largely unknown. To study the physiological role of Ddx3x during development, we generated Ddx3x conditional knockout and null alleles. Our data revealed that loss of Ddx3x leads to early embryonic lethality of Ddx3x-/Y male mutant. In addition, the preferential paternal X-inactivation in extra-embryonic tissues of F2 Ddx3x heterozygous (Ddx3x+/-) females with a maternally inherited null allele led to the placenta abnormalities and embryonic lethality during development. Gross appearance showed that F2 Ddx3x+/- embryos and placentas were smaller than their littermate controls. PAS staining and RNA in situ hybridization confirmed that the thickness of the spongiotrophoblast layer was dramatically reduced in the placenta from F2 Ddx3x+/- embryo. The expression levels of trophoblast specific markers, Hand1, Mash2, Tpbpa, Tfeb and Cdx2, were significantly decreased in the placentas from F2 E9.5 Ddx3x+/- embryos. To investigate the molecular mechanisms by which DDX3X regulates trophoblast development, we applied the siRNA methodology to knockdown the expression of DDX3X in JAR and BeWo human choriocarcinoma cells. Consistent with the in vivo result, we confirmed that loss of DDX3X affects the expression of HAND1 in JAR and BeWo cells. These findings reveal that Ddx3x regulates Hand1 gene expression, which is required for proper differentiation of trophoblast giant cells during mouse placental development. Moreover, we used epiblast-specific Sox2-cre to ablate Ddx3x gene at E6.5 to explore the biological function of Ddx3x in embryo proper. Sox2-cre/+; Ddx3xflox/Y mutants exhibited growth retardation around E8.5 and died around E11.5 with various defects, including headfold malformation, failure of body turning, pericardial effusion and abnormal caudal structure. A preliminary investigation revealed that Sox2-cre/+; Ddx3xflox/Y embryos display normal mitotic indices but show excessive apoptosis and DNA damage. These results implied that Ddx3x may involve in DNA damage induced apoptotic signaling pathways. How Ddx3x modulates cell proliferation, differentiation, and death during murine placentation and embryogenesis remains to be elucidated.