272 EXPRESSION OF Lef1 TRANSCRIPTION FACTOR AND ITS NOVEL ISOFORMS IN PRE-IMPLANTATION EMBRYOS

Abstract

Lymphoid enhancer-binding factor 1 (Lef1) is a key transcriptional factor in the Wnt signaling pathway. Recently, the Wnt pathway has been shown to function in human and mouse embryonic stem cells; however, its role in ES cell differentiation and self-renewal remains contradictory. Our lab has identified two novel Lef1 isoforms, Lef1^Δ2,3,6 and Lef1^Δ6, that are transcribed in mouse embryonic stem cells in addition to the full-length Lef1. Both Lef1^Δ2,3,6 and Lef1^Δ6 contain an N-terminal β-catenin binding domain and a C-terminal HMG DNA binding domain. However, Lef1^Δ2,3,6 lacks exons II, III, and VI, and Lef1^Δ6 lacks exon VI. Exons II, III, and VI are a part of the context-dependent repression domain, whereas exon VI contains a Groucho binding motif. The absence of these repression domains suggests that Lef1^Δ2,3,6 and Lef1^Δ6 may be regulated differently than the full-length Lef1. In this study, we determined the expression patterns of Lef1 mRNA and the two isoforms during murine and bovine pre-implantation development using RT-PCR. Total RNA was isolated from pools of 10–20 in vitro-produced bovine embryos at the 2-cell, 4–8 cell, morulae, and blastocyst stages and in vivo-derived mouse embryos at the 8-cell, morula, and blastocyst stages using a Stratagene Nanoprep kit. Reverse transcription was performed using the Invitrogen Supercript III first-strand synthesis system. PCR was then completed using Invitrogen Platinum Taq DNA Polymerase. As determined in preliminary work, full-length Lef1 was expressed in 8-cell-stage mouse embryos but was not detectable in morulae and was only slightly expressed in mouse blastocysts. In contrast, Lef1^Δ6 mRNA was not detectable in 8-cell mouse embryos, but became obvious at morula and blastocyst stages. Similarly, in bovine embryos, full-length Lef1 was detected at early cleavage stages (2–8 cells) with undetectable or low levels at the morula and blastocyst stages; however, unlike what was observed in the mouse embryos, Lef1^Δ6 was expressed in all stages of bovine embryos tested. Lef1^Δ2,3,6 was very low (mouse) or undetected (bovine). Because Lef1 appears to be playing a critical role in response to Wnt differentiation signals in mouse embryonic stem cells (He, unpublished), further exploration of these transcription factors and their regulation in developing mammalian embryos is critical to understanding the process of initial embryonic differentiation.