Maternal-zygotic zebrafish γ-tubulin expression and subcellular localization during early development. A. RT-PCR analysis revealed by electrophoresis in agarose gel showed that γ-tubulin mRNA is detected during the first 8 h of the zebrafish embryogenesis. B. qPCR analysis indicated that γ-tubulin mRNA expression levels were invariant during the first 8 h of zebrafish embryogenesis. β-actin and eef1a were used as reference genes. C. Immunoblot analysis showed that γ-tubulin protein is present during the cleavage (1–3 hpf) and post-MBT stages (3–8 hpf) of zebrafish development. Actin was used as a loading control. D–E. Fluorescence microscopy images of zebrafish embryos (2 hpf) double-stained with anti-γ-tubulin antibody (green) and DAPI (red). Metaphase (E), anaphase, and telophase (F) stages of the cell cycle are shown.

γ-tubulin silencing by morpholino-based knockdown causes developmental arrest during gastrulation. A. Bright field images of the representative phenotypes obtained in a tubg1-Mo (20 nM) injection experiment and complete rescue phenotype. Control injected embryos did not show a phenotype. Images were taken at 8 and 24 hourspost-fertilization (hpf). The frequency of each phenotypic class (severe, moderate and wild type) is indicated as a percentage. The standard deviations correspond to three independent biological replicas. B. Phenotypes distribution of 24 hpf embryos injected with tubg1-Mo (20 and 10 nM), control Mo (20 nM) and tubg1-Mo (20 mM) + γ-tubulin polypeptide.

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Nuclear and microtubule organization defects in γ-tubulin morphant embryos. A–D. Fluorescence microscopy images of control and morphant embryos at 2 (A and C) and 4 (B and D) hpf, showing centrosomal (γ-tubulin, green) and chromosomal (DAPI, red) distribution. E–H. anti-α-tubulin antibodies (green) and DAPI (red) double-stained embryos at 2 (E and G) and 4 (F and H) hpf. In the morphant embryos, γ-tubulin and microtubule organizations were severely affected and γ-TuSC formation impairment generated multinucleated blastomeres (arrowheads in D and H). I, J. Confocal projection images of α-tubulin-immunofluorescence staining of blastoderm in 8 hpf control and morphant embryos. Microtubule-stained spindle structures (arrowheads in I) and bundle organizations are shown. N, nucleus. K, L. DAPI-stained nuclei of control and tubg1-Mo embryos show a severe karyokinesis defect after γ-tubulin silencing.

Cell proliferation arrest and apoptosis triggered in the absence of γ-TuSC formation. A. Zebrafish control and morphant embryos treated through the BrdU incorporation procedure. Embryos (8 hpf) were exposed to a BrdU pulse for 1 h, then fixed and treated for DNA labeling with DAPI (red) and immunolabeled for the incorporated BrdU (green). tubg1-Mo embryos did not incorporate BrdU in their DNA, indicating proliferation arrest. B. Apoptotic response revealed by TUNEL assay in embryos (8 hpf). The tubg1-morphant showed many clusters of cells positive for TUNEL stain (arrowheads) distributed throughout the blastoderm.