Expression of the novel maternal centrosome assembly factor Wdr8 is required for vertebrate embryonic mitoses

Abstract

The assembly of the first centrosome occurs upon fertilisation when male centrioles recruit pericentriolar material (PCM) from the egg cytoplasm. The mechanisms underlying the proper assembly of centrosomes during early embryogenesis remain obscure. We identify Wdr8 as a novel maternally essential protein that is required for centrosome assembly during embryonic mitoses of medaka (Oryzias latipes). By CRISPR–Cas9-mediated knockout, maternal/zygotic Wdr8-null (m/zWdr8−/−) blastomeres exhibit severe defects in centrosome structure that lead to asymmetric division, multipolar mitotic spindles and chromosome alignment errors. Via its WD40 domains, Wdr8 interacts with the centriolar satellite protein SSX2IP. Combining targeted gene knockout and in vivo reconstitution of the maternally essential Wdr8–SSX2IP complex reveals an essential link between maternal centrosome proteins and the stability of the zygotic genome for accurate vertebrate embryogenesis. Our approach provides a way of distinguishing maternal from paternal effects in early embryos and should contribute to understanding molecular defects in human infertility.

Loss and Rebirth of the Animal Microtubule Organizing Center: How Maternal Expression of Centrosomal Proteins Cooperates with the Sperm Centriole in Zygotic Centrosome Reformation

“…When entering the 2nd mitotic division, however, the lack of maternal Wdr8 stopped further centrosome formation leading to progressive spindle formation defects. Consistent with that, mRNA injection, mimicking maternal Wdr8, into the eggs of homozygous ko mothers could fully restore chromosome segregation in fish, which survived to fertile adults suggesting that only maternal Wdr8 is essential for development . In contrast, mutant variants, in which two amino acids defining one of the WD40 repeats in Wdr8 were replaced, failed to display rescue activity of Wdr8 knockout zygotes despite efficient protein expression.…”

“…In contrast to somatic cells, wdr8 ko blastomeres displayed structural defects in centrosome formation, the integrity of the PCM and centriolar satellites, and, finally, chromosome segregation errors. Of note, the first mitotic spindle assembly still seemed to work as judged by spindle morphology and chromosome alignment . The sperm's contribution of the centriole pair may have sufficed to build up the first mitotic spindle independently of the presence of the history of the sperm − expressing or not expressing paternal Wdr8.…”

“…Pulldown experiments in amphibian egg lysates, done in parallel, revealed that both wt and mutants interacted with the γ–TuRC but differed in Ssx2IP binding. While the wt readily associated with Ssx2ip, the mutant variants did not . Such a straighforward in vivo rescue assay allowed analysing the structure‐function relationship of the Wdr8‐Ssx2ip module in zygotic centrosome reformation in a vertebrate model system on single amino acid level.…”

The centriolar satellite protein Cfap53/Ccdc11 facilitates the formation of the first zygotic microtubule organizing center in the zebrafish embryo

“…Forward genetic screens in zebrafish is an unbiased approach to identify parental factors that have an important biological role in early development (Pelegri and Mullins, 2016). Several zebrafish and medaka mutants have been generated via this approach and proven to be very informative for the identification and characterization of some of these maternal and paternal factors (Abrams et al, 2020;Dekens et al, 2003;Inoue et al, 2017;Yabe et al, 2007). The zebrafish gene cellular atoll (cea) encodes for the centriole duplication factor Sas-6 (Yabe et al, 2007).…”

“…In mouse embryos, nuclear congression appears different as it is facilitated by two bipolar spindles formed by two clusters of MTOCs around each pronucleus (Reichmann et al, 2018). The medaka (Oryzias latipes) maternal WD40 repeatcontaining protein, Wdr8, (the orthologue of human WRAP73), is important during MTOC assembly (Inoue et al, 2017). Maternal zygotic wdr8 mutant embryos form multipolar mitotic spindles resulting in chromosome alignment errors.…”