Real‐time imaging of actin filaments in the zebrafish oocyte and embryo

Abstract

Dynamic changes of cytoplasmic and cortical actin filaments drive various cellular and developmental processes. Although real-time imaging of actin filaments in living cells has been developed, imaging of actin filaments in specific cells of living organisms remains limited, particularly for the analysis of gamete formation and early embryonic development. Here, we report the production of transgenic zebrafish expressing the C-terminus of Moesin, an actin filament-binding protein, fused with green fluorescent protein or red fluorescent protein (GFP/RFP-MoeC), under the control of a cyclin B1 promoter. GFP/RFP-MoeC was expressed maternally, which labels the cortical actin cytoskeleton of blastula-stage cells. High levels of GFP/RFP fluorescence were detected in the adult ovary and testis. In the ovaries, GFP/RFP-MoeC was expressed in oocytes but not in follicle cells, which allows us to clearly visualize the organization of actin filaments in different stages of the oocyte. Using full-grown oocytes, we revealed the dynamic changes of actin columns assembled in the cortical cytoplasm during oocyte maturation. The number of columns slightly decreased in the early period before germinal vesicle breakdown (GVBD) and then significantly decreased at GVBD, followed by recovery after GVBD. Our transgenic fish are useful for analyzing the dynamics of actin filaments in oogenesis and early embryogenesis.

Zebrafish as a Model for the Study of Live in vivo Processive Transport in Neurons

“…Zebrafish embryos are amenable to pharmacological treatment by bath application, allowing for treatment of intact, live embryos with compounds known for the modulation of cytoskeletal dynamics, for instance, targeting microtubules: Colchicine (Roche et al, 1994), vinblastine (Keiter et al, 2016; Yao et al, 2017), vincristine (Mizgirev and Revskoy, 2010; Khan et al, 2012; Holloway et al, 2016), nocodazole (Plucińska et al, 2012; Jayachandran et al, 2016) and paclitaxel (Jayachandran et al, 2016). For the actin cytoskeleton: Cytochalasin D (Nukada et al, 2015; Artelt et al, 2018) and latrunculin A (Artelt et al, 2018), jasplakinolide (Artelt et al, 2018), phalloidin oleate (Dutta and Kumar Sinha, 2015), and the inhibitor of actin–myosin interaction BDM (Norden et al, 2009) have been used with success.…”

High-Sensitivity and High-Resolution In Situ Hybridization of Coding and Long Non-coding RNAs in Vertebrate Ovaries and Testes

“…Large numbers of cyclin B1 RNA granules were localized at the animal polar cytoplasm in stage III (Fig. 4d ) and fully grown, stage IV oocytes [ 31 , 32 , 50 ]. The localization and granular structure of dazl mRNA were maintained in stage III and IV oocytes, but the granules were broadly distributed in the vegetal polar cytoplasm (Fig.…”

“…A previous study showed that zebrafish cyclin B1 mRNA is initially distributed throughout the cytoplasm of oocytes (stages I and II) and becomes localized at the animal polar cytoplasm after stage III, when DIG-labeled RNA probes were recognized by an AP-coupled anti-DIG antibody, followed by detection of signals by precipitation of AP substrates [ 28 ]. Fluorescence in situ hybridization with the TSA system has revealed the localization of cyclin B1 mRNA at the animal polar cytoplasm as RNA granules [ 31 , 32 , 50 ], and this study revealed changes in cyclin B1 mRNA distribution and localization during oocyte development; i.e., cyclin B1 mRNA was initially distributed throughout the cytoplasm of oocytes in stage I and subsequently moved to the animal pole in stage II (Fig. 4a - c ), and a granular structure was first found in stage Ib oocytes and persisted in later stages (Fig.…”

Tools of the trade: studying actin in zebrafish

“…The C-terminus of the actin-binding protein, Moesin, has been used in zebrafish to image actin in vivo, through a transgenic line that allows the visualisation of actin filaments and actin dynamics during oogenesis and early development (Nukada et al 2015 ).…”

“…The yolk granules are kept at the vegetal pole by means of another actin-based mechanism which is the formation of actin comet-like structures (Shamipour et al 2019 ). Actin dynamics has been studied in oocytes and early embryos using a Moesin-GFP fusion (Nukada et al 2015 ). Germ plasm segregation and localisation of many maternal RNAs in the early embryo have been found to be actin-dependent (Eno and Pelegri 2018 ; Theusch et al 2006 ; Eno et al 2019 ; Nair et al 2013 ).…”