49 Trascription factor TEAD4 is not required for bovine blastocyst formation

The first lineage differentiation in mammals gives rise to the inner cell mass (ICM) and the trophectoderm (TE), the first extraembryonic lineage. This differentiation event is governed by transcription factors that have been thoroughly studied by means of knockout (KO) murine models. In mice, the nuclear translocation of the transcription factor TEAD4 in the outer blastomeres triggers TE differentiation by repressing the ICM-specific gene OCT4 and enhancing the expression of TE-specific genes such as CDX2 and GATA3. The predominant role of TEAD4 on TE specification in mice is evidenced by Tead4 KO embryos, which are unable to form blastocoel. The objective of this study was to determine whether TEAD4 is required for initial TE specification in bovine by the evaluation of preimplantation development of TEAD4 KO bovine embryos. Bovine IVM oocytes were microinjected in three independent replicates with Cas9-encoding mRNA and a single guide (sg)RNA against TEAD4 (167 oocytes, C+G group, containing KO embryos) or with Cas9-encoding mRNA alone (75 oocytes, C group, formed by wild-type (WT) embryos) as microinjection control. sgRNA was produced using Guide-it sgRNA Kit (Takara). Microinjected oocytes were fertilised in vitro and allowed to develop up to Day 8, when they were fixed and subjected to immunohistochemistry (IHC) to detect TEAD4 (ab151274; AbCam) and the TE-specific marker CDX2 (MU392A-UC; Biogenex). Following IHC, embryos from the C+G group were genotyped by clonal sequencing to identify those harbouring only KO alleles. Embryo development to blastocyst was similar for both groups (19.2 ± 5.3 vs. 22.7 ± 3.6%, mean ± s.e.m., for C+G and C groups, respectively; t-test P > 0.05), and seven out of 13 blastocysts genotyped in C+G were identified as KO for TEAD4. The TEAD4 IHC signal was not detected in KO blastocysts, which showed normal morphology and expressed the TE marker CDX2 in outer cells. Total and CDX2+ cell number were similar in WT and KO embryos (Total: 97.4 ± 16.1 vs. 71.7 ± 8.4; CDX2+ : 52.5 ± 15.6 vs. 22.3 ± 11.4, mean ± s.e.m. for WT and KO, respectively, seven embryos analysed/group; t-test P > 0.05). In conclusion, the role of TEAD4 is not conserved in mammals, being dispensable for TE specification in ungulates, in striking contrast to its essential role in mouse development.