Variations in sister chromatid cohesion dysfunction in esco2 mutant zebrafish reflects the phenotypic diversity of Roberts Syndrome

Abstract

Mutations in ESCO2, one of two establishment of cohesion factors necessary for proper sister chromatid cohesion (SCC), cause a spectrum of developmental defects in the autosomal-recessive disorder Roberts syndrome (RBS), warranting in vivo analysis of the consequence of cohesion dysfunction. Through a genetic screen in zebrafish targeting embryonic-lethal mutants that have increased genomic instability, we have identified an esco2 mutant zebrafish. Utilizing the natural transparency of zebrafish embryos, we have developed a novel technique to observe chromosome dynamics within a single cell during mitosis in a live vertebrate embryo. Within esco2 mutant embryos, we observed premature chromatid separation, a unique chromosome scattering, prolonged mitotic delay, and genomic instability in the form of anaphase bridges and micronuclei formation. Cytogenetic studies indicated complete chromatid separation and high levels of aneuploidy within mutant embryos. Amongst aneuploid spreads, we predominantly observed decreases in chromosome number, suggesting that either cells with micronuclei or micronuclei themselves are eliminated. We also demonstrated that the genomic instability leads to p53-dependent neural tube apoptosis. Surprisingly, although many cells required Esco2 to establish cohesion, 10-20% of cells had only weakened cohesion in the absence of Esco2, suggesting that compensatory cohesion mechanisms exist in these cells that undergo a normal mitotic division. These studies provide a unique in vivo vertebrate view of the mitotic defects and consequences of cohesion establishment loss, and they provide a compensation-based model to explain the RBS phenotypes.

How many roads lead to cohesinopathies?

“…These esco2 morphants exhibited craniofacial cartilage defects, underdeveloped jaws, and shorter pectoral fins. Similar findings are observed in esco2 mutants (Morita et al, ; Percival et al, ). In Zebrafish esco2 hi2865 mutants, the apoptotic response is p53‐dependent, consistent with mitotic failure.…”

“…In Zebrafish esco2 hi2865 mutants, the apoptotic response is p53‐dependent, consistent with mitotic failure. However, a subset of mutant cells exhibit normal mitoses, suggesting the potential for multiple mechanisms underlying RBS phenotypes (Percival et al, ). Moreover, an apoptotic‐independent mechanism underlying bone and tissue growth defects was reported in the regenerating‐fin Zebrafish model (Banerji et al, ).…”

Observing Mitotic Division and Dynamics in a Live Zebrafish Embryo

“…Multiple possibilities for visualizing mitotic components ranging from microtubule-kinetochore attachments to centriole dynamics can be applied. Combining the capability of imaging mitosis in vivo with the recent advances in genome editing in zebrafish will make it easy to generate mutants in various aspects of mitosis to model human disease in a vertebrate organism 25,26,52-56 .…”

“…It acetylates cohesin on the SMC3 portion of the ring thus stabilizing cohesin to ensure proper chromosome segregation at the metaphase-anaphase transition 23 . Loss of Esco2 in zebrafish leads to chromosome missegregation, premature sister chromatid separation, genomic instability, and p53-dependent and independent apoptosis 24,25 . Due to the availability, auroraB hi1045 , and esco2 hi2865 mutant zebrafish (hereafter referred to as aurB m/m and esco2 m/m , respectively) will be used to illustrate this technique 25-27 .…”

“…Coupling confocal microscopy with fluorescent-tagged cell machinery has enabled researchers to visualize chromatin and cell membrane dynamics during mitosis 25,28,29 . Fluorescent-tagged histones have historically been used to visualize chromatin.…”