Influence of HIPPO signaling pathway on segregation between internal cell mass and trophectoderm in bovine embryos

Author(s)

Caroline Pereira da Costa

• Document type: Master's Dissertation
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina Veterinária e Zootecnia (FMVZ/SBD)
• Defense date: 2020-12-04

Examining board members

Marcelo Demarchi Goissis; Flavia Regina Oliveira de Barros; Marcella Pécora Milazzotto

• Advisor: Marcelo Demarchi Goissis

Abstract

The first cellular differentiation event in mammals consists of the separation between the internal cellular mass (ICM) and the trophectoderm (TE). In mice, the HIPPO signaling pathway acts to control the expression of genes that define this differentiation. In cattle, there are indications that the same genes do not participate in the same way, but the HIPPO pathway may be involved in this biological event. In the present study we aimed to test the hypothesis that the activity of LATS2 kinase is necessary for the differentiation of ICM in bovine embryos. In order to achieve this, we employed CRISPR/Cas9 gene editing system to inhibit the kinase function of LATS2. In vitro produced bovine zygotes were microinjected with RNAs guide aiming the LATS2 gene plus Cas9 restriction enzyme. The zygotes were sorted in 3 experimental groups: control group (not microinjected), Cas9 group (microinjected only with Cas9 protein) and gRNA group (microinjected with guide RNAs aiming the LATS2 gene and Cas9 protein). The embryos were cultured to evaluate the rate of blastocysts formation, cellular distribution, then fixed in 3.8% paraformol for subsequent immunofluorescence for YAP, followed by genotyping. The results suggested that the cleavage was not affected in the edited group, as rates were 58%, 36% and 50% for control , Cas9 and gRNA groups respectively; however, the blastocyst formation rates suggested a direct interference in the edited group, being 30%, 10% and 6% respectively, for the control groups, Cas9 and gRNA. Morphologically, it was observed that the embryos of the gRNA group appeared as morulas, suggestive of embryonic death or inhibition of differentation in this phase of development. There was a numeric reduction in total cell count in the structures of the gRNA group. The images obtained via confocal microscopy, after performing immunofluorescence for YAP demonstrate the marking of cells of the TE and MCI in the microinjected embryos only with Cas9 and control, however, this marking did not occur in the microinjected structures that did not form a blastocoel. After individual DNA extraction from the embryos tested in immunofluorescence, PCR of the LATS2 target region resulted in a band of 478pb, in addition to the original 650pb band, in one sample of the gRNA group. After Sanger sequencing of PCR products, genetic alteration derived from deletion of part of the LATS2 gene was observed in 2 embryos, demonstrating gene editing success and corroborating the findings. This suggests that HIPPO pathway has a significant role in the differentiation of MCI and TE in bovine embryos and that the LATS2 gene is linked to the formation of blastocysts in bovine species. (AU)

• FAPESP's process: 18/18924-8 - Influence of the HIPPO signaling pathway on the segregation between the inner cell mass and the trophectoderm in bovine embryos
• Grantee: Caroline Pereira da Costa
• Support Opportunities: Scholarships in Brazil - Master