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.
Highlights
The assembly of the first centrosome occurs upon fertilisation when male centrioles recruit pericentriolar material (PCM) from the egg cytoplasm
Alongside the centrosome assembly factor SSX2IP, our screen revealed an upregulation of Wdr[8], a previously uncharacterised WD40 repeat-containing protein, which is well conserved among species (Supplementary Fig. 1a,b)
To analyse Wdr8’s functions in early vertebrate development, we took advantage of the transparency of medaka embryos to carry out a cell biological analysis combined with a CRISPR–Cas[9] genome editing approach to inactivate medaka Wdr[8] (O. latipes Wdr[8], OlWdr[8], hereafter referred to as Wdr8)
Summary
The assembly of the first centrosome occurs upon fertilisation when male centrioles recruit pericentriolar material (PCM) from the egg cytoplasm. The Drosophila PCM protein Spd-2, and the centriolar protein Sas-4, for instance, do not appear to be strictly required for somatic mitosis in cultured Drosophila cells and late development (after midblastula transition), but are essential for early embryonic mitoses via their centrosome assembly functions[12,13,15]. We applied CRISPR–Cas9-mediated targeted gene inactivation, and elicited specific centrosome assembly defects in the absence of maternal and zygotic Wdr[8]. We used mRNA injection, which mimicked maternal gene expression, and observed a remarkable reconstitution of centrosome assembly, proper cell divisions and gross development until adulthood This in vivo reconstitution strategy of maternal Wdr[8] functions allowed us to perform a straightforward screening of mutant variants that revealed domains/modules of the Wdr[8] protein essential for centrosome assembly in living vertebrate embryos. Our system delivered molecular insights into Wdr8’s essential function in embryonic centrosome assembly
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