Cellular Energy Sensor Sirt1 Augments Mapk Signaling to Promote Hypoxia/Reoxygenation-Induced Catch-up Growth in Zebrafish Embryo.

Animal growth is blunted in adverse environments where catabolic metabolism dominates; however, when the adversity disappears, stunted animals rapidly catch up to age-equivalent body size. This phenomenon is called catch-up growth, which we observe in various animals. Since growth retardation and catch-up growth are sequential processes, catabolism or stress response molecules may remain active, especially immediately after growth resumes. Sirtuins (Sirt1-7) deacetylate target proteins in a nicotinamide adenine dinucleotide-dependent manner, and these enzymes govern diverse alleys of cellular functions. Here, we investigated the roles of Sirt1 and its close paralog Sirt2 in the hypoxia/reoxygenation-induced catch-up growth model using zebrafish embryos. Temporal blockade of Sirt1/2 significantly reduced the growth rate of the embryos in reoxygenation, but it was not evident in constant normoxia. Subsequent gene knockdown and chemical inhibition experiments demonstrated that Sirt1, but not Sirt2, was required for the catchup growth. Inhibition of Sirt1 significantly reduced the activity of mitogen-activated kinase (Mapk) of embryos in the reoxygenation condition. In addition, co-inhibition of Sirt1- and Igf-signaling did not further reduce the body growth or Mapk activation compared to those of the Igf-signaling-alone-inhibited embryos. Furthermore, in the reoxygenation condition, Sirt1- or Igf-signaling inhibition similarly blunted Mapk activity, especially in anterior tissues and trunk muscle, where the sirt1 expression was evident in the catching-up embryos. These results suggest that the catch-up growth requires Sirt1 action to activate the somatotropic Mapk pathway, likely by modifying the Igf-signaling.

The involvement of Nile tilapia (Oreochromis niloticus) Neu4 sialidase in neural differentiation during early ontogenesis.

Neurogenesis is an important process for the formation of the central nervous system during ontogenesis. Mammalian sialidases are involved in neurogenesis through desialylation of sialo-glycoconjugates. However, the significance of fish sialidases, unlike that of mammals, in neurogenesis has not been investigated. The present study focuses on Nile tilapia (Oreochromis niloticus) because of its unique profiles of sialidases related to enzymatic properties, subcellular localization, and tissue-specific gene expression. First, the fish were cultured under aphotic condition, which is known to cause the delayed development of the retina and brain in various fish. Next, we investigate the effect of aphotic condition on the levels of tilapia sialidases. Our results revealed that the tilapia showed a decrease in the number of ganglion cell in the retina. The expression level of neu4 mRNA is up-regulated in the eyes from tilapia reared in Dark accompanied by the increase of retinal differentiation markers. These results indicated that tilapia Neu4 is involved in retinal development in Nile tilapia. Furthermore, we tried to clarify the function of tilapia Neu4 in the neuronal cells using two neuroblast cell lines (SH-SY5Y and Neuro2a cell lines). Tilapia Neu4 decreased sialic acid level of both nuclear glycoproteins as well as glycolipids. Moreover, tilapia Neu4 accelerated neurite formation in both two neural cell lines and, increased the acetylcholinesterase activity, but it did not affect cell proliferation. Collectively, these results suggest that Neu4 accelerates neurite differentiation during ontogenesis in tilapia.

Threshold and spectral sensitivity of vision in medaka Oryzias latipes determined by a novel template wave matching method.

We propose a new analytical method for determining the response threshold in electroretinogram (ERG) in which the wave shows a biphasic slow dc-potential shift. This method uses the recorded wave to the highest intensity stimuli in each wavelength tested as a template wave f(t), and it was compared with other recorded waves obtained under lower intensities g(t). Our test recordings in medaka Oryzias latipes were analogous between the template and the compared waveforms, although there were differences in amplitude and time lag (τ, peak time difference) which occurred as a result of the difference in stimulus intensity. Cross-correlation analysis was applied. Based on the obtained cross-correlation function Cfg(τ) in each comparison, τ was determined as the time lag at which the cross-correlation coefficient Rfg(τ) showed the maximum value. Determined thresholds that were based on both the experimenter's visual inspection and this new method agreed well when the adoption condition was set to satisfy R(τ) ≥ 0.7 and τ ≤ 150 ms in scotopic or τ ≤ 120 ms in photopic conditions. We concluded that this "template wave matching method" is a quick and reliable objective assessment that can be used to determine the threshold. This study analyzed ERG recordings in response to 6 kinds of wavelength light stimuli (380 nm to 620 nm) at different photon flux densities. We report the threshold levels and relative spectral sensitivities in scotopic and photopic vision of medaka.

Establishment and characterization of Neu1-knockout zebrafish and its abnormal clinical phenotypes.

Mammalian sialidase Neu1 is involved in various physiological functions, including cell adhesion, differentiation, cancer metastasis, and diabetes through lysosomal catabolism and desialylation of glycoproteins at the plasma membrane. Various animal models have been established to further explore the functions of vertebrate Neu1. The present study focused on zebrafish (Danio rerio) belonging to Cypriniformes as an experimental animal model with neu1 gene deficiency. The results revealed that the zebrafish Neu1 desialyzed both α2-3 and α2-6 sialic acid linkages from oligosaccharides and glycoproteins at pH 4.5, and it is highly conserved with other fish species and mammalian Neu1. Furthermore, Neu1-knockout zebrafish (Neu1-KO) was established through CRISPR/Cas9 genome editing. Neu1-KO fish exhibited slight abnormal embryogenesis with the accumulation of pleural effusion; however, no embryonic lethality was observed. Although Neu1-KO fish were able to be maintained as homozygous, they showed smaller body length and weight than the wild-type (WT) fish, and muscle atrophy and curvature of the vertebra were observed in adult Neu1-KO fish (8 months). The expression patterns of myod and myog transcription factors regulating muscle differentiation varied between Neu1-KO and WT fish embryo. Expression of lysosomal-related genes, including ctsa, lamp1a, and tfeb were up-regulated in adult Neu1-KO muscle as compared with WT. Furthermore, the expression pattern of genes involved in bone remodeling (runx2a, runx2b, and mmp9) was decreased in Neu1-KO fish. These phenotypes were quite similar to those of Neu1-KO mice and human sialidosis patients, indicating the effectiveness of the established Neu1-KO zebrafish for the study of vertebrate Neu1 sialidase.

Neuropeptide Y deficiency induces anxiety-like behaviours in zebrafish (Danio rerio).

Neuropeptide Y (NPY) controls energy homeostasis including orexigenic actions in mammalians and non-mammalians. Recently, NPY has attracted attention as a mediator of emotional behaviour and psychosomatic diseases. However, its functions are not fully understood. We established npy gene-deficient (NPY-KO) zebrafish (Danio rerio) to assess the relationship between NPY and emotional behaviours. The NPY-KO zebrafish exhibited similar growth, but pomc and avp mRNA levels in the brain were higher as compared to wild-type fish. NPY-KO zebrafish exhibited several anxiety-like behaviours, such as a decrease in social interaction in mirror test and decreased locomotion in black-white test. The acute cold stress-treated NPY-KO zebrafish exhibited anxiety-like behaviours such as remaining stationary and swimming along the side of the tank in the mirror test. Moreover, expression levels of anxiety-associated genes (orx and cck) and catecholamine production (gr, mr, th1 and th2) were significantly higher in NPY-KO zebrafish than in wild-type fish. We demonstrated that NPY-KO zebrafish have an anxiety phenotype and a stress-vulnerability like NPY-KO mice, whereby orx and/or catecholamine signalling may be involved in the mechanism actions.

Novel Nile tilapia Neu1 sialidases: Molecular cloning and biochemical characterization of the sialidases Neu1a and Neu1b.

Lysosomal desialylation is the initial step in the degradation of sialo-glycopeptides that is essential for regenerating sialo-glycoconjugates. Neu1 sialidase is the enzyme responsible for the removal of sialic acid in the mammalian lysosome. Although Neu1 sialidases are conserved in fish similar to mammals, their physiological functions remain to be fully understood. Nile tilapia (Oreochromis niloticus) is known to possess two putative Neu1 sialidases (Neu1a and Neu1b) in the genome that may have arisen by gene duplication (specifically in cichlidae family members). This suggests that understanding the Neu1 sialidase in fish, particularly cichlids, could provide insights into the (novel) physiological functions of these genes. Moreover, characterization of the tilapia Neu1 sialidase is paramount to ensure clarity of the desialylation reaction performed by the fish sialidases (like the characterized tilapia sialidases Neu3 and Neu4). Therefore, this study focused on the characterization of the tilapia Neu1 sialidases. Neu1b exhibited narrow substrate specificity when compared with Neu1a, whereas the properties of these two Neu1 sialidases, such as cathepsin A-induced activation, optimal pH, and lysosomal localization, were conserved. Neu1a mRNA levels were detected in various tissues of tilapia as compared to the mRNA levels of Neu1b. Although the cloned construct of Neu1b contained an extra exon unlike tilapia Neu1a, the exon did not affect the enzymatic properties of Neu1b. This study suggests that tilapia Neu1a profiles were highly conserved with other vertebrate Neu1 isoforms, while Neu1b probably evolved independently in other members of the cichlidae family. Moreover, the expression of sialidase genes (neu1a, neu1b, neu3a, and neu4) were determined in various stages of tilapia embryogenesis using real-time PCR; sialidase gene expression is reported to be drastically and individually altered during embryogenesis in Japanese medaka (Oryzias latipes). The mRNA levels of neu1a drastically increased between 72 and 84 hpf and mildly decreased from 84 to 144 hpf. In contrast, the transcript levels of neu1b did not change between 84 and 144 hpf and the expression of neu3a gradually increased between 84 and 120 hpf and drastically decreased at 144 hpf. The highest level of the neu4 transcripts was detected at 84 hpf. These expression patterns were different from those in Japanese medaka, possibly due to the different developmental program found in the tilapia embryo accompanied with the unique profiles of the tilapia sialidases.

Green light irradiation during sex differentiation induces female-to-male sex reversal in the medaka Oryzias latipes.

This study investigated whether irradiation of a specific light wavelength could affect the sex differentiation of fish. We first found that the photoreceptor genes responsible for receiving red, green, and ultraviolet light were expressed in the eyes of medaka during the sex differentiation period. Second, we revealed that testes developed in 15.9% of genotypic females reared under green light irradiation. These female-to-male sex-reversed fish (i.e. neo-males) showed male-specific secondary sexual characteristics and produced motile sperm. Finally, progeny tests using the sperm of neo-males (XX) and eggs of normal females (XX) revealed that all F1 offspring were female, indicating for the first time in animals that irradiation with light of a specific wavelength can trigger sex reversal.