Adult wild-type zebrafish (Danio rerio) were maintained at 28°C on a 14 h:10 h (light:dark) cycle, and fed twice daily. Embryos were generated from natural crosses. Fertilized eggs were raised in embryo medium at 28.5°C and staged according to Kimmel et al. (23). This study was carried out in accordance with the recommendations of the Animal Research and Ethics Committee of Ocean University of China. The protocol was approved by the Animal Research and Ethics Committee of Ocean University of China.

All chemicals and reagents were purchased from Fisher Scientific (Pittsburgh, PA, USA), unless otherwise noted. Restriction endonucleases were purchased from New England BioLabs (Beverly, MA, USA). The anti-Akt and anti-Phospho-Akt antibodies (Ser473) and the anti-p70s6 kinase and anti-Phospho-p70s6 kinase (Ser411) antibodies were purchased from Cell Signaling (Danvers, MA, USA).

Capped mRNA synthesis was carried out using a commercial kit using mouse myr-Akt1 and GFP-linearized plasmid DNA (linearized with NotI digests) as template (Megascript kit; Ambion Inc., Austin, TX, USA). mRNA were injected into 1–2 cell embryos as previously reported (24). As a control, we injected mRNA encoding GFP at a concentration of 200 pg per embryo. After injection, embryos were placed in embryo rearing medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl₂, and 0.33 mM MgSO₄) and kept at 28.5°C.

Whole mount in situ hybridization using digoxigenin-labeled RNA riboprobes was carried out as reported previously (25).

Embryos were injected with 200 pg of either GFP mRNA (control) or mouse myr-Akt1 mRNA. At 20 hpf, 50 embryos in each treatment group were subjected to either heatshock (10 min incubation in a dry-air 37°C incubator in 50 ml of embryo rearing solution) or UV light treatment (60 s in a UV cross linker set at 100 mJ/cm² in 5 ml of embryo rearing solution). Embryos were then transferred to non-treated embryo rearing solution and allowed to incubate for 8 h, at which they were fixed in 4% paraformaldehyde for later analysis.

Embryo embedding, freezing, and sectioning protocols were performed according to Brunet et al. (26). Sections (10 µm) were collected and air dried at room temperature for 2 h before staining or storage at −20°C. For TUNEL assays, sections were stained using the In Situ Cell Death Detection Kit, TMR Red, according to the manufacturer’s instructions (Roche, Nutlet, NJ, USA). Nuclei were counterstained with 50 nM Sytox (Molecular Probes, Carlsbad, CA, USA). Images of TUNEL and Sytox staining were captured separately and then merged using Adobe Photoshop. Quantification of TUNEL-positive cells and total cell number (Sytox) was done using the particle analysis function of ImageJ. All thresholds were set and used for the analysis of every image. A total of six images for TUNEL and Sytox staining were quantified per treatment group.

The PI3K inhibitor, LY294002, was dissolved in dimethyl sulfoxide (Amresco, Solon, OH, USA) as a 50 mM stock solution and then added to embryo rearing medium; the final concentration of LY294002 was 8, 16, or 32 µM according to different experiments. For treatment of PI3K inhibitor, embryos at 10 hpf were transferred into embryo rearing medium containing different concentrations of LY294002, and then washed out and dechorionated at 24 hpf for inspection.

Dechorionate embryos and place in 5 µg/ml acridine orange (Sigma, USA) in embryo rearing medium. After 30 min of staining, wash the embryos twice with embryo rearing medium and view using fluorescence microscope with green filter. Quantification of apoptosis cells was done using the particle analysis function of ImageJ 1.41 (NIH, USA). All thresholds were set and used for the analysis of every image.

Twenty-five embryos from each treatment group were dechorionated, deyolked, and homogenized in 25 µl of RIPA buffer (50 mM Tris–HCl, 150 mM NaCl, 2 mM EGTA, 0.1% Triton X-100, pH 7.5) containing 10 µg/ml aprotinin, 10 μg/ml leupeptin, 10 µg/ml pepstatin, 100 mM PMSF, and 0.1 M sodium orthovanadate. The homogenates were briefly centrifuged to pellet cellular debris and the supernatant was retained. Each sample was subjected to SDS-PAGE (12.0%) and transferred to Immoblin-P membrane (Millipore Corp., Billerica, MA, USA). The total Akt antibody and the phospho-Akt antibody (Ser473) were used at a 1:1,000 and 1:2,000 dilutions, respectively.

Brain sizes were determined at 24 hpf by measuring the linear distance from the forebrain to the mid–hindbrain boundary, bisecting the lens (called the horizontal brain length, H), and by measuring the linear distance from the yolk sac to the top of the brain, bisecting the lens (called the vertical brain height, V). Each value was then placed over embryo length, which is the curvilinear distance from the forebrain and midbrain boundary to the tail (L) unless otherwise noted. This calculation is called relative brain height (H/L) and relative brain length (V/L) and was determined with the ImageJ 1.41 (NIH, USA) for each embryo from pictures. Each line was then measured using a scale taken at the same magnification.

Data are presented as means ± SE. Differences among groups were analyzed by One-Way ANOVA followed by Fisher Post hoc Test or t-test (SPSS Inc., Chicago, IL, USA). Significance was accepted at P < 0.05.

Akt phosphorylation levels at 24 hpf were obviously attenuated in zebrafish embryos treated with LY294002, while total Akt levels remained unchanged (Figure 1A). Meanwhile, embryos with 8 or 16 µM LY294002 treatment at 24 hpf displayed an overall reduction of body length and an uncharacteristic cloudiness or opacity in the head region (Figure 1B). However, the trunk region appeared to be relatively normal. We also observed other phenotypes commonly associated with PI3K-treated embryos, such as hypoplasia of the yolk sac extension, and delayed pigmentation. On close-up examination of those embryos, we determined significant reduction of brain thickness (linear distance form yolk sac to top of head, bisecting the lens) as that found in IGF1R knockdown research (Figure 1C); and the relative brain thickness (expressing brain thickness over whole embryo length, which is the linear distance from inner forehead to tail) also displayed an obvious trend of decrease (Figure 1D). It elucidated that the brain exhibited more severe size reduction over the whole retardation of growth.

An additional myristoylation sequence to Akt has been previously shown to be able to localize Akt to the plasma membrane where it can be more readily phosphorylated and rendered constitutively active (27–29). In order to test the effects of Akt overexpressing on zebrafish embryogenesis, considering the high degree of conservation, mRNA encoding mouse myr-Akt1 was transcribed in vitro and injected into embryos at the 1–2 cell stage. Injection of myr-Akt at concentrations above 200 pg mRNA per embryo caused severe and inconsistent morphological deformities (data not shown). As shown in Figure 2, overexpression of myr-Akt induced a significant increase of the levels of Akt and phosphorylation of its downstream molecule p70S6, comparing to controls and to embryos with reduced IGF1R-mediated signaling (IGF1R MOs and dnIGF1R: GFP). Therefore, these results provide in vivo biochemical verification that injection of myr-Akt caused increases in Akt and p70S6 kinase phosphorylation.

Akt is activated via receptor tyrosine kinases in a PI3K-dependent manner and phosphorylated on Thr308 and Ser473 by upstream kinases. We used western immunoblot to analyze the phosphorylation levels of Akt in embryos. The results illustrated that treating with a series concentration of LY294002 markedly reduced phosphorylation of Akt in embryos with 200 pg myr-Akt mRNA injection (Figure 3A) via blocking the PI3K function. As shown in Figures 3B,C, Akt overexpressing could partially rescue phenotypes of PI3K inhibition. Morphological inspection shows their brain size were obviously larger than those with LY294002 treatment. Furthermore, they appeared to be relatively normal comparing to wild type and GFP mRNA-injected embryos.

At a concentration of 200 pg per embryo, injection of myr-Akt mRNA caused an increase in brain size in zebrafish embryos (Figure 4A), evident in ~77% of injected embryos at 24 hpf (N = 151 total embryos from three independent microinjection experiments). We observed a significant increase in relative brain height and length in embryos injected with myr-Akt mRNA compared to control mRNA-injected embryos (Figure 4B).

Since the embryos overexpressing Akt displayed abnormity mainly in the brain region during early developmental stage (24 hpf), the whole mount in situ hybridization was performed to analyze the expression pattern of several marker genes, egr2b (third and fifth rhombomeres of the hindbrain), emx1 (forebrain), pax2a (optic stalk, mid-hindbrain boundary), and rx1 (retina), involved in neural patterning of the brain (Figure 5A, a–d). In all cases, there is an increase in the expression domain of these genes, consistent with a larger brain size. In contrast, the expression domain of these genes decreased in embryos treated with LY294002 (Figure 5B). However, the localization of those neural markers in the embryos overexpressing Akt or treated with PI3K inhibitor was the same as that of wild-type embryos, suggesting that cell fate was unaffected. Thus, it indicates that overexpression of a constitutively active Akt in zebrafish embryos increases brain size but does not significantly alter brain patterning.

Next, we performed acridine orange staining on PI3K inhibitor-treated embryos of 24 hpf control and myr-Akt mRNA-injected embryos. As shown in Figures 5C,D embryos with myr-Akt mRNA injection had obviously lower numbers of AO-stained cells throughout the brain region. It revealed that PI3K inhibition-associated brain-size reduction might be realized via increased cell apoptosis.

Activation of Akt has been shown to be important for protecting neurons from apoptosis (18, 30). To test the ability of constitutively active Akt at protecting neuronal cell from apoptosis in vivo during zebrafish embryogenesis, we subjected control and myr-Akt mRNA-injected embryos to heatshock and UV light treatment. After administration of heatshock or UV light exposure at 20 hpf, embryos were allowed to continue to develop until 28 hpf and then fixed and stained for apoptotic cells using TUNEL. Compared to untreated control and myr-Akt mRNA-injected embryos, heatshock and UV light treatment showed significantly higher numbers of TUNEL-positive cells in the brains of control mRNA-injected embryos (Figures 6A–D,G). However, injection of myr-Akt mRNA significantly attenuated the heatshock and UV light-induced apoptosis (*P < 0.05; ^#P < 0.001, respectively; Figures 6E–G). These results indicate that Akt overexpression in zebrafish embryos could inhibit neuronal apoptosis in vivo, and suggest that apoptotic inhibition may be part of the underlying causes of the increased brain size in untreated myr-Akt mRNA-injected embryos.