Centrosomes in the zebrafish (Danio rerio): a review including the related basal body

Abstract

Ever since Edouard Van Beneden and Theodor Boveri first formally described the centrosome in the late 1800s, it has captivated cell biologists. The name clearly indicated its central importance to cell functioning, even to these early investigators. We now know of its role as a major microtubule-organizing center (MTOC) and of its dynamic roles in cell division, vesicle trafficking and for its relative, the basal body, ciliogenesis. While centrosomes are found in most animal cells, notably it is absent in most oocytes and higher plant cells. Nevertheless, it appears that critical components of the centrosome act as MTOCs in these cells as well. The zebrafish has emerged as an exciting and promising new model organism, primarily due to the pioneering efforts of George Streisinger to use zebrafish in genetic studies and due to Christiane Nusslein-Volhard, Wolfgang Driever and their teams of collaborators, who applied forward genetics to elicit a large number of mutant lines. The transparency and rapid external development of the embryo allow for experiments not easily done in other vertebrates. The ease of producing transgenic lines, often with the use of fluorescent reporters, and gene knockdowns with antisense morpholinos further contributes to the appeal of the model as an experimental system. The added advantage of high-throughput screening of small-molecule libraries, as well as the ease of mass rearing together with low cost, makes the zebrafish a true frontrunner as a model vertebrate organism. The zebrafish has a body plan shared by all vertebrates, including humans. This conservation of body plan provides added significance to the existing lines of zebrafish as human disease models and adds an impetus to the ongoing efforts to develop new models. In this review, the current state of knowledge about the centrosome in the zebrafish model is explored. Also, studies on the related basal body in zebrafish and their relationship to ciliogenesis are reviewed.

Figure 1

Western blot of zebrafish oocyte or embryo extracts at the indicated developmental stages probed by anti-γ-tubulin antibodies (GTU-88 or TU-30). Molecular weight standards (in kilodaltons) are shown at left. Each sample lane was loaded with 0.8 mg of protein (wet weight equivalent). (A) Original 40,000 × g supernatant probed by TU-30. (B) Original 40,000 × g supernatant probed by GTU-88. Arrowhead indicates γ-tubulin breakdown product. (C) Original 40,000 × g pellet probed by TU-30. (D) Original 40,000 × g pellet probed by GTU-88. Lane 1: Fully grown oocyte. Lane 2: one to four cells. Lane 3: eight to sixty-four cells. Lane 4: 64 to 128 cells. Lane 5: Midblastula (> 1,000 cells). Lane 6: Gastrula. Lane 7: Pharyngula. Reprinted with permission [53].

Figure 2

Centrosomes at the spindle poles of the blastomere of an eight- or thirty-two-cell paraformalin-fixed zebrafish embryo depicted by γ-tubulin antibody GTU-88 or TU-30, respectively, and Alexa Fluor 488 dye-conjugated second antibody (green) as well as chromosomes stained with 4',6-diamidino-2-phenylindole (blue). Z-axis projections of approximately 60 optical sections photographed with an Olympus spinning disk confocal microscope using a 60 × oil immersion lens objective. Note the size differences, with the γ-tubulin array being smaller in centrosomes of 32-cell compared to 8-cell embryos.

Figure 3

Models of nucleation complex attachment and activation. (a) In the absence of c-tubulin ring complex (c-TuRC)-specific components, as in Saccharomyces, Spc110 or its equivalent directly attaches c-tubulin small complex (c-TuSC) to microtubule-organizing centers (MTOCs), promoting ring assembly. (b) In organisms with complete c-TuRCs, active complexes attach to organizing centers directly through c-TuSCs or potentially through unique sites in the c-TuRC-specific components. Localization of c-TuRCs at non-MTOC locations, such as within the mitotic spindle, is mediated through the c-TuRC-specific proteins. In both models, c-TuSC interactions define the geometry of the nucleating template. Reprinted with permission [59].

Figure 4

In situ hybridization (probed with fluoroscein isothiocyanate-oligo-γ-tubulin probe) of different stages of zebrafish oocytes and embryos. (A) and (G) Fully grown, immature oocytes. (B) and (H) Mature oocytes or eggs) and embryos. (C) and (I) 32-cell embryos. (D) and (J) 256-cell embryos. (E) and (K) Approximately 30% epiboly gastrula. (F) and (L) pharyngula). The top panels in (A) through (F) show the "with probe" treatments, and the lower panels in (G) through (L) show the corresponding "without probe" treatments. The primordial blastodisc (b) that forms at the animal pole of the egg during oocyte meiotic maturation labeled intensely with the probe (B) Inset: Immature oocyte probed after hemisection showing cortical label. Anti-FITC secondary antibody conjugated to alkaline phosphatase and the substrate 3,3'-diaminobenzidine were used to develop color. The specimens were dehydrated in 100% MeOH and placed in clearing media containing benzyl benzoate and benzyl alcohol (2:1 dilution) prior to being mounted on slides. Scale bar = 250 μm. Reprinted with permission [64].

Figure 5

Spindle organization and centrosome duplication defects in maternally mutant cellular atoll (cea) zebrafish embryos. (A) to (D') Embryos labeled with anti-α-tubulin antibody. (A) and (B') Fixed wild-type embryos. (C) and (D') Mutant embryos. Spindle organization is normal in the mutant embryos immediately prior to the first cell division (35 minutes postfertilization (pf) (C); compare with (A)) but is defective in a fraction of blastomeres in the next cleavage cycle (50 minutes pf (D) and (D'); compare with (B) and (B')). (E) to (H') Embryos labeled with anti-γ-tubulin antibody. (E) and (F') Fixed wild-type. (G) and (H') Mutant. Centrosome duplication appears normal immediately prior to the first cleavage division (35 minutes pf (G); compare with (E)) but is defective in a fraction of the blastomeres in the following cleavage cycle (50 minutes pf (H) and (H'); compare with (F) and (F')). Animal views: (B'), (D'), (F') and (H') are enlargements of the area indicated by the squares in (B), (D), (F) and (H), respectively. Reprinted with permission [71].

Figure 6

Cea/Sas-6 localizes to zebrafish centrosomes. Expressed fusions of mCherry and Sas-6 splice forms colocalize in cytoplasmic structures containing γ-tubulin (arrowheads), markers for centrosomes. Fields of cells in embryos are fixed at 50% epiboly. Reprinted with permission [71].

Figure 7

Model of SAS-6 ring assembly in zebrafish. (A) Ribbon presentation of a modeled SAS-6 tetramer based on the observed coiled-coil and head-to-head dimers. The distance between the base regions of the two coiled-coil domains is indicated. (B) Cryo-electron microscopic image of a face-on view of a thin crystal of N-SAS-61-217. The pixel size is 3.74 Å/pixel. Scale bar = 60 Å. The image was Fourier-filtered and symmetry-averaged. Overlay shows the SAS-6 tetramer presented in (A). The overlaid structure is based on N-SAS-61-179, which has a shorter coiled-coil domain than N-SAS-61-217. (C) Models of SAS-6 rings with different symmetries. The approximate diameters of these rings (the double-distance from head domain center to ring center) are indicated above the modeled rings. The diameter of cartwheel hubs observed in procentrioles by cryo-electron tomography is shown as a dotted circle. To model these rings, a change in the orientation of the head domains relative to the coiled-coil domain was allowed for. To compare the required changes, the root-mean-square deviation between the N-SAS-61-179 structure and two equivalent head domains in the modeled ring was calculated. These values are indicated under the modeled rings. Reprinted with permission [74].

Figure 8

Centrosome duplication is not affected in futile cycle (fue) mutants. Centrosomes at the eight-cell stage during interphase visualized with an antibody directed against γ-tubulin (green) and nuclei labeled with 4',6-diamidino-2-phenylindole (blue) from (A) a wild-type embryo, (B) a fue embryo and (C) an embryo treated with the DNA replication inhibitor aphidicolin. (D) through (F) Enlargements of single-centrosome pairs shown in (A) through (C), respectively. Centrosomes are present and able to duplicate in all fue cells as well as the (anucleate) cells of aphidicolin-treated embryos, demonstrating that the centrosomes can duplicate independently of nuclei. Scale bars = 20 mm. Reprinted with permission [54].

Figure 9

Dual use of the centrioles during cell cycle and primary cilium formation. In most cells, primary cilium formation first occurs during the G₁ phase following centrosomal docking to the membrane. Intraflagellar transport (IFT) and accessory proteins build the ciliary axoneme, which extends directly from the mother centriole's triplet microtubules. During this stage of the cell cycle, as well as during the G₀ phase, the cilium functions as a cellular antenna, interpreting extracellular signals such as Hedgehog and platelet-derived growth factor (PDGF). Upon entry into the S phase, the cell's centrioles and the DNA begin to replicate. The centrioles reach maturity during the late G₂ phase, at which point the cilium is disassembled so that the engaged centrioles can be liberated for mitotic spindle formation. Once cell division is complete, the centrioles can proceed to ciliary reassembly in G₁. Reprinted with permission [18].

Figure 10

Summary of centrosome and basal body research in zebrafish. (A) Cellular and molecular findings include, from left, (1) Western blot with anti-γ-tubulin demonstrating γ-tubulin in 40,000 × g supernatants and pellets of ovarian oocytes and different embryonic stages [53], (2) in situ hybridization of γ-tubulin probe to mRNA in the cortices of ovarian oocytes and in embryonic cells [64] and (3) ninefold homodimer of coiled-coil sas-6 protein reveals structural basis for centriole assembly in zebrafish [74]. (B) The results of studies of mutants include, from left, (1) failure to replicate centrioles due to sas-6 mutation in cellular atoll (cea) [71], (2) failure to organize a furrow microtubule array due to aurora B kinase mutation in cellular island (cei) [77], (3) failure to organize DNA and spindle, though centrosome duplication and placement are normal in futile cycle mutant (fue) [54] and (4) the maternal-zygotic ovl (MZovl) mutant lacks cilia due to IFT88 mutation, though basal bodies appear normal [108]. (C) The results of transgenic reporters and morphants in zebrafish, from left, (1) centrin 2-GFP marks apical polarity in retinal neuroepithelium development, as does γ-tubulin-YFP [91], and (2) Cep290 morpholino injection produces morphants with curved body phenotype and reduced Kupffer vesicle size characteristic of mutations in ciliopathy-associated genes; analogous gene mutations cause a number of human ciliopathy syndromes [116].