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Mouse SAS-6 is required for centriole formation in embryos and integrity in embryonic stem cells.

ABSTRACT: SAS-6 (SASS6) is essential for centriole formation in human cells and other organisms but its functions in the mouse are unclear. Here, we report that Sass6-mutant mouse embryos lack centrioles, activate the mitotic surveillance cell death pathway, and arrest at mid-gestation. In contrast, SAS-6 is not required for centriole formation in mouse embryonic stem cells (mESCs), but is essential to maintain centriole architecture. Of note, centrioles appeared after just one day of culture of Sass6-mutant blastocysts, from which mESCs are derived. Conversely, the number of cells with centrosomes is drastically decreased upon the exit from a mESC pluripotent state. At the mechanistic level, the activity of the master kinase in centriole formation, PLK4, associated with increased centriolar and centrosomal protein levels, endow mESCs with the robustness in using a SAS-6-independent centriole-biogenesis pathway. Collectively, our data suggest a differential requirement for mouse SAS-6 in centriole formation or integrity depending on PLK4 activity and centrosome composition.

SUBMITTER: Grzonka M

PROVIDER: S-EPMC10917421 | biostudies-literature | 2024 Feb

REPOSITORIES: biostudies-literature

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Mouse SAS-6 is required for centriole formation in embryos and integrity in embryonic stem cells.

Grzonka Marta M Bazzi Hisham H

eLife 20240226

SAS-6 (SASS6) is essential for centriole formation in human cells and other organisms but its functions in the mouse are unclear. Here, we report that <i>Sass6</i>-mutant mouse embryos lack centrioles, activate the mitotic surveillance cell death pathway, and arrest at mid-gestation. In contrast, SAS-6 is not required for centriole formation in mouse embryonic stem cells (mESCs), but is essential to maintain centriole architecture. Of note, centrioles appeared after just one day of culture of <i ...[more]

PMID: 38407237

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Project description:Mutations in the transcription factor gene grainyhead-like 2 (GRHL2) are associated with progressive non-syndromic sensorineural deafness autosomal dominant type 28 (DFNA28) in humans. Since complete loss of Grhl2 is lethal in mouse embryos, we studied its role during inner ear pathology and hearing loss in vitro. To this end, we generated different homozygous deletions to knockout Grhl2 in mouse embryonic stem cells (Grhl2-KO ESCs), including some mimicking naturally occurring truncations in the dimerisation domain related to human DFNA28. Under naïve culture conditions, Grhl2-KO cells in suspension were more heterogenous in size and larger than wild-type controls. Adherent Grhl2-KO cells were also larger, with a less uniform shape, flattened, less circular morphology, forming loose monolayer colonies with poorly defined edges. These changes correlated with lower expression of epithelial cadherin Cdh1 but no changes in tight junction markers (Ocln, Tjp2) or other Grhl isoforms (Grhl1, Grhl3). Clonogenicity from single cells, proliferation rates of cell populations and proliferation markers were reduced in Grhl2-KO ESCs. We next induced stepwise directed differentiation of Grhl2-KO ESCs along an otic pathway, giving rise to three-dimensional inner ear-like organoids (IELOs). Quantitative morphometry revealed that Grhl2-KO cells initially formed larger IELOs with a less compacted structure, more eccentric shape and increased surface area. These morphological changes persisted for up to one week. They were partially rescued by forced cell aggregation and fully restored by stably overexpressing exogenous Grhl2 in Grhl2-KO ESCs, indicating that Grhl2 alters cell-cell interactions. On day 8, aggregates were transferred into minimal maturation medium to allow self-guided organogenesis for another two weeks. During this period, Grhl2-KO cells and wild-type controls developed similarly, expressing neural, neuronal and sensory hair cell markers, while maintaining their initial differences in size and shape. In summary, Grhl2 is required for morphological maintenance of ESCs and orderly formation of IELOs, consistent with an essential role in organising epithelial integrity during inner ear development. Our findings validate quantitative morphometry as a useful, non-invasive screening method for molecular phenotyping of candidate mutations during organoid development.

Project description:Embryonic stem (ES) cells and germ cells have the potential to give rise to an entire organism. A common requirement is that both must have very robust mechanisms to preserve the integrity of their genomes. This is particularly true since somatic cells have very high mutation frequencies approaching 10-4 in vivo that would lead to unacceptable levels of fetal lethality and congenital defects. Notably, between 70% and 80% of mutational events monitored at a heterozygous endogenous selectable marker were loss of heterozygosity due to mitotic recombination, a mechanism that affects multiple heterozygous loci between the reporter gene and the site of crossing over. This chapter examines three mechanisms by which mouse embryonic stem cells preserve their genomic integrity. The first entails suppression of mutation and recombination between chromosome homologues by two orders of magnitude when compared with isogenic mouse embryo fibroblasts which had a mutation frequency similar to that seen in adult somatic cells. The second renders mouse ES cells hypersensitive to environmental challenge and eliminates damaged cells from the self-renewing population. Mouse ES cells lack a G1 checkpoint so that cells damaged by exogenous insult such as ionizing radiation do not arrest at the G1/S phase checkpoint but progress into the S phase where the damaged DNA is replicated, the damage exacerbated and the cells driven to apoptosis. The third mechanism examines how mouse ES cells repair double strand DNA breaks. Somatic cells predominantly utilize error prone nonhomologous end joining which, from a teleological perspective, would be disadvantageous for ES cells since it would promote accumulation of mutations. When ES cells were tested for the preferred pathway of double strand DNA break repair, they predominantly utilized the high fidelity homology-mediated repair pathway, thereby minimizing the incurrence of mutations during the repair process. When mouse ES cells are induced to differentiate, the predominant repair pathway switches from homology-mediated repair to nonhomologous end joining that is characteristic of somatic cells.

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Mouse SAS-6 is required for centriole formation in embryos and integrity in embryonic stem cells.

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Mouse SAS-6 is required for centriole formation in embryos and integrity in embryonic stem cells.

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