Imaging the early zebrafish embryo centrosomes following injection of small-molecule inhibitors to understand spindle formation

Abstract

Summary During the earliest division stages, zebrafish embryos have large cells that divide rapidly and synchronously to create a cellular layer on top of the yolk. Here, we describe a protocol for monitoring spindle dynamics during these early embryonic divisions. We outline techniques for injecting zebrafish embryos with small-molecule inhibitors toward polo-like kinases, preparing and mounting embryos for three-dimensional imaging using confocal microscopy. These techniques are used to understand how the early zebrafish embryo’s centrosome constructs the mitotic spindle. For complete details on the use and execution of this protocol, please refer to Rathbun et al. (2020) .

Rab11 endosomes coordinate centrosome number and movement following mitotic exit

“…Cover slips were rinsed with dH2O and mounted on glass slides using either Prolong Diamond with DAPI mounting media or Prolong Gold (see key resource table ). For zebrafish embryo immunofluorescent protocols see (Aljiboury et al, 2021).…”

“…For zebrafish embryo imaging, fluorescent transgenic or mRNA injected embryos (refer to strains and mRNAs in key resource table, and for injection protocols refer to (Aljiboury et al, 2021)) were embedded in 2% agarose at 3.3-5 hours post fertilization (hpf) and imaged using the spinning disk or LSCM.…”

“…Aljiboury, A.A., Mujcic, A., Cammerino, T.,Rathbun, L.I., and Hehnly, H. (2021).Imaging the early zebrafish embryo centrosomes following injection of small-molecule inhibitors to understand spindle formation. STAR Protoc.2, 100293.…”

Pericentriolar matrix integrity relies on cenexin and Polo-Like Kinase (PLK)1

“…Coverslips were rinsed with diH2O and mounted on glass slides using ProLong Gold mounting media (Thermo Fisher Scientific; P36934). Zebrafish embryos were fixed using 4% PFA at 4C overnight, for immunostaining see [24,25]. See Key Resources Table for list of antibodies used in this study.…”

“…Anti-cenexin vivo translational morpholinos (Gene Tools; [23]) or vivo standard control morpholinos (Gene Tools) were constituted as 1mM stock in water and injected into zebrafish yolks at 1 cell stage in a final concentration of 2ng/nL. Injection protocols detailed in [25].…”