Functional evaluation of NANOG gene in segregation of cell lineages in bovine embryos

Author(s)

Letícia Escobar Carreiro

• Document type: Master's Dissertation
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina Veterinária e Zootecnia (FMVZ/SBD)
• Defense date: 2022-05-23

Examining board members

Marcelo Demarchi Goissis; Fabiana Fernandes Bressan; Fabíola Freitas de Paula Lopes

• Advisor: Marcelo Demarchi Goissis

Abstract

The early cell differentiation events mark the preimplantation embryonic development in mammals. The first cell differentiation event consists of the segregation between the trophectoderm (TE) and the inner cell mass (ICM), while the second event occurs within the ICM, with the segregation of epiblast (EPI) and primitive endoderm (PrE) lineages. The molecular mechanisms in the control of these events include time- related expression of specific transcription factors. Recent data indicate that the study of bovine embryos would be a good model for early human development. In mice, the transcription factor NANOG works opposite to another transcription factor, GATA6, and gene expression leads to cell population clusters in such a way that NANOG and GATA6 become mutually exclusive in EPI and PrE. This study sought to test the hypothesis that NANOG is necessary for EPI specification and PrE repression in bovine embryos. For this purpose, this study targeted CRISPR/Cas9-mediated NANOG deletion of in vitro produced zygotes. The experimental groups consisted of microinjected embryos with guide RNAs in two concentrations, 12.5 or 80 ng/µl, and Cas9 protein (Cas9-12.5 and Cas9-80) aiming to delete the NANOG gene, while the control group was not microinjected. Subsequently, embryos were cultured for evaluation of cleavage rates, blastocyst formation, and development rates, protein expression analysis by immunofluorescence, gene expression analysis, and genotyping. The protein expression of SOX17, a marker of PrE, was evaluated by immunofluorescence, however, none of the Cas9-12.5 embryos presented the expected gene deletion of NANOG. A significant decrease in NANOG expression (p=0.0360) was observed in the Cas9-80 experimental group when compared to the control group, suggesting a disturbance of gene expression, but with no evidence of NANOG full deletion under these conditions. The Cas9-80 treatment conditions, however, did not affect GATA6 gene expression. Therefore, it was possible to reduce the expression of NANOG using the CRISPR system in cattle embryos, without changing the blastocysts formation and the expression of PrE differentiation-related gene GATA6. (AU)

• FAPESP's process: 19/22649-5 - Functional evaluation of NANOG gene in segregation of cell lineages in bovine embryos
• Grantee: Letícia Escobar Carreiro
• Support Opportunities: Scholarships in Brazil - Master