Cell division requires proper sister chromatid cohesion, a process mediated by the cohesin complex. ESCO2 plays a critical role in establishing sister chromatid cohesion by acetylating SMC3; stabilizing cohesin on chromatin. However, despite the necessity of ESCO2 in chromosome cohesion and cell division, individuals with ESCO2 mutations are born with the Roberts Syndrome, a devastating developmental disorder characterized by limb and craniofacial abnormalities. These pregnancies can be viable nonetheless, challenging the prevailing model of cell cycle regulation of chromosome dynamics in the developing embryo.

This study aimed to characterize the phenotypic impact of depleting a key cohesin complex regulatory protein, ESCO2, on X. laevis embryo cell division to further elucidate how chromosome cohesion is regulated during the earliest stages of life.

To assess the impact of post-translation ESCO2 depletion on chromosome segregation during early embryonic cell divisions, an exogenous E3 ubiquitin ligase (TRIM21) was conjugated with an anti-ESCO2 antibody. The TRIM21 complex, as well as synthetic mRNA encoding fluorescent cell structure marker proteins, was injected into the fertilized egg prior to the first cell division to induce targeted protein degradation. Confocal imaging at early gastrulation enabled real-time visualization of chromosome dynamics.

TRIM21/anti-ESCO2 treated embryos exhibited a significant reduction in ESCO2 quantity as early as stage 4 compared to untreated embryos (p<.001), with little detectable signal through gastrulation on immunoblot analysis. TRIM21/anti-ESCO2 treated embryos and mock depleted controls demonstrated increased RR for cell division error phenotypes (RR 12.64 (95% CI 3.41-47.96); RR 13.20 (95% CI 3.49-50.92) and binucleate cell frequency (RR 28.70 (95% CI 15.63-53.18); RR 24.56 (95% CI 13.14-46.10) relative to non-TRIM21 treated embryos.

ESCO2 depletion may increase errors in chromosome segregation. However, interpretation of the findings here is limited; observations of the experimental cohort did not significantly differ from the mock-depleted controls, suggesting that the phenotypic differences observed may not have been reflective of the consequence of ESCO2 depletion, but rather the result of experimental or procedural artifact.

Nevertheless, this study demonstrated that ESCO2 is normally expressed during the earliest stages of X. laevis embryogenesis, and can be effectively and persistently depleted though gastrula stage via microinjection of the TRIM21 E3 ubiquitin ligase degradation system in Stage 1 embryos demonstrating the effectiveness of a novel application of the TRIM21-mediated protein degradation system to achieve targeted ESCO2 depletion in the intact embryo. This methodological contribution can provide a foundation for future studies in reproductive biology.