Targets of Elf5 in Mouse Trophoblast Stem Cells

Abstract

The Placenta is an essential organ for all mammalian embryonic development as it provides the nutritional link between maternal and foetal blood streams. The cells which go on to proliferate and contribute to all the major cell types of the embryo derived placenta have been located to the trophoblast (TE) cells overlying the Inner Cell Mass (ICM) of the embryo. Immortal cell lines have been subsequentially derived from this tissue and called Trophoblast Stem (TS) cells. In parallel with their in vivo counterparts they are also reliant on Fibroblast growth factor 4 (FgF4) (Tanaka et al., 1998) and Activin/Nodal signalling (Guzman-Ayala et al., 2004).

The Ets family transcription factor, Elf5, has been shown to be specifically expressed in the early placental trophoblast and subsequent derived tissues. Mice deficient in Elf5 failed to form a placenta post implantation. Furthermore TS cells were unable to be derived from Elf5 knockout embryos (Donnison et al., 2005). This work suggested that Elf5 plays an essential role in TS cells and their differentiation. The aim of this study was to determine the downstream target genes of Elf5 in mouse TS cells. The target genes of Fgf4 and Activin/Nodal signalling in TS cells were also investigated. This work is hoped to contribute to an overall greater understanding of the molecular networks underlying TS cell maintenance and to contribute to our knowledge of early placental development.

Small interfering RNA (siRNA) targeted reduction of Elf5 mRNA expression in mTS cells was achieved using two independent siRNAs; with Elf5 reduction exceeding 80%. The resulting changes in gene expression were measured in order to determine the downstream targets of Elf5. Selected genes known to be important for trophoblast differentiation and maintenance were measured using real-time PCR in a candidate gene approach .Global changes in gene expression as a consequence of Elf5 silencing were measured using an Affymetrix microarray. Global changes in gene expression due to growth factor (Fgf4 and/or Activin) removal were also measured. Expression of 22 genes was changed using either of the Elf5 siRNA oligonucleotides. Of these, 9 were also significantly changed by growth factor removal. Included in this set were Synopl, Hst3st3b1, Cyr61 and Sox2. In the overall analysis, many genes whose expression changed upon loss of Elf5 are known to play important roles in trophectoderm cell specification. Real-time PCR validation agreed closely with the up or down regulation measured using the microarray. This work has thus led to the discovery of sets of Elf5 target genes potentially involved in trophoblast stem cell function and has provided the foundation for future work exploring the molecular pathways of trophoblast development.