Daigo Inoue

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.

The assembly of the first centrosome occurs upon fertilisation when the male centrioles recruit pericentriolar material (PCM) from the egg cytoplasm. While inaccuracy in the assembly of centrosomes leads to infertility and abnormal embryogenesis, the mechanism that ensures accurate assembly in vertebrate embryos remains obscure. Here we applied a CRISPR-Cas9-mediated gene knockout to show that Wdr8, a novel centrosomal protein, is maternally essential for PCM assembly during embryonic mitoses of medaka ( Oryzias latipes ). Maternal/zygotic Wdr8-null (Wdr8 ^−/− ) blastomeres exhibit severe defects in PCM assembly that cause them to divide asymmetrically and develop multipolar mitotic spindles and aneuploidy. We demonstrate that Wdr8 interacts via its WD40 domains with the centriolar satellite protein SSX2IP. Strikingly, exogenously provided Wdr8 fully rescues Wdr8 ^−/− embryos to adulthood, except in variants with mutations in the WD40 domains. This combination of targeted gene inactivation and in vivo reconstitution of the maternally essential Wdr8-SSX2IP complex reveals an essential link between maternal PCM and the stability of the zygotic genome in the early vertebrate embryo.

For the detection and sub-cellular (co)-localization of proteins in the context of the tissue or organism immunostaining in whole mount preparations or on sections is still the best approach. So far, each antibody required its own fixation and antigen retrieval protocol so that optimizing immunostaining turned out to be tedious and time consuming. Here we present a novel method to efficiently retrieve the antigen in a widely applicable standard protocol, facilitating fluorescent immunostaining of both cryosections and whole mount preparations in zebrafish (Danio rerio) and medaka (Oryzias latipes). Our method overcomes the loss of sections and damage of tissue and cell morphology, and allows parallel immunostaining in multiple colors, co-immunostaining with fluorescent proteins in transgenic fish lines and in combination with whole mount in situ hybridization.