(34) FGF8 is not Required for Bovine Epiblast Formation and Early Development

Inés Flores-Borobia¹, Aitana Salvo-Jiménez¹, Leopoldo González-Brusi¹, Priscila Ramos-Ibeas¹, Pablo Bermejo-Álvarez¹

¹Animal Reproduction Department, INIA, CSIC, Spain

Abstract Text:

Early embryo development entails a series of cellular differentiation, proliferation and migration events that are mediated by specific transcription, growth and signaling factors. The formation of the epiblast, the lineage that evolve into the fetus, requires the accomplishment of a first lineage differentiation event that generates trophectoderm and inner cell mass cells and a second differentiation event that gives rise to epiblast and hypoblast cells from the inner cell mass. The molecular regulation of such events has been thoroughly studied in the mouse model, but the roles of key regulators of first lineage differentiation in mice such as TEAD4 is not conserved in other mammals such as cattle or rabbits. FGF8 is a secreted factor required for mouse gastrulation that is also expressed by the bovine embryonic disc. The aim of this study has been to determine if FGF8 is required for epiblast formation and early proliferation in cattle by assessing the developmental ability of FGF8 KO bovine embryos generated by introducing a stop codon by base editing technology.

In vitro matured bovine oocytes (n=424) were divided in two groups: one was microinjected with cytosine base editor (CBE) encoding mRNA and a sgRNA against FGF8 (n=286, BE+G group, containing KO embryos), and the other was microinjected with CBE encoding mRNA alone, serving as microinjection control (n=138, BE group, formed by wild-type WT embryos). To assess if FGF8 embryos were able to complete first and second lineage differentiation events, microinjected oocytes were fertilized in vitro and developed to Day (D) 12 by conventional culture in SOF medium to D7 followed by culture in N2B27 medium. D12 embryos were fixed and subjected to immunohistochemistry to detect trophectoderm (CDX2+) and the two lineages derived from the ICM in a second differentiation event: epiblast (SOX2+) and hypoblast (SOX17+). Following image acquisition, embryos from BE+G group were genotyped by Sanger to identify KO, Hz and WT embryos.

No significant differences were observed in developmental rates between both microinjection groups (Cleavage rate: 81.5±3.7 vs. 80.2±6.1 %; blastocyst rate: 25.0±4.4 vs. 21.0±5.8 %; D7 to D12 survival rate: 73.0±1.0 vs. 72.7±7.8 %; for BE vs. BE+G, mean±s.e.m., t-test p >0.05). 35 out of 50 D12 embryos genotyped in C+G group (70 %) were edited, and 32 were KO (64 %). FGF8 KO embryos were able to develop up to D12 normally. D12 embryo diameter was similar between WT and KO embryos (0.66±0.05 vs. 0.75±0.08 mm, for WT vs. KO, respectively, mean±s.e.m, ANOVA, p >0.05). Epiblast survival was also unaffected by FGF8 ablation (24/42 vs. 16/32, for WT vs. KO, respectively, Chi-Square, p >0.05), and the number of epiblast (SOX2+) cells in the embryonic disc was similar between the two genotypes (26±5 vs. 15±4, for WT vs. KO, respectively, mean±s.e.m, ANOVA, p >0.05). No significant differences were observed either in the rate of complete hypoblast migration between WT and KO embryos (14/42 for WT vs. 13/32 for KO, Chi-Square, p >0.05).

In conclusion, FGF8 KO bovine embryos form epiblast that proliferate normally at early stages of development.

Work supported by PID2020-117501RB-I00.