Efficacy and safety of same-day preparation with sodium picosulfate plus magnesium citrate on the day of colonoscopy for bowel preparation: Multicenter, single-arm, open-label study.

BACKGROUND AND AIM: Sodium picosulfate plus magnesium citrate (SP + MC) is a well-tolerated bowel preparation agent. However, Japan currently approves only two methods of taking SP + MC: the day-before and split-dose preparation, without approval of same-day preparations. This study aimed to evaluate the efficacy and safety of same-day SP + MC preparations. METHODS: This was a multicenter, single-arm, nonrandomized, open-label study. We enrolled 145 Japanese patients between June and December 2023. The patients received two sachets of SP + MC dissolved in 300 ml of water and 1200 mL or more of clear liquid on the day of colonoscopy. Bowel cleansing efficacy, adverse events (AEs), and patient satisfaction were evaluated. RESULTS: Of the enrolled patients, 137 underwent colonoscopy according to our protocol. Bowel preparation was adequate in 133 patients (97.1%). The mean total Boston Bowel Preparation Score was 8.3 ± 1.2. Five patients experienced AEs (3.6%): two (1.5%), abdominal pain; one (0.73%), ischemic enteritis; one (0.73%), vomiting or nausea; and one (0.73%), headache. All AEs were treated conservatively. None of the patients exhibited abnormal blood test results or clinical symptoms after receiving SP + MC. Regarding patient satisfaction, all patients were able to take SP + MC as directed; 136 (99.2%) expressed a preference for this bowel preparation for future colonoscopies. CONCLUSION: The same-day SP + MC preparation showed high bowel-cleansing efficacy and satisfaction in Japanese patients without serious AEs.

Zinc transport via ZNT5-6 and ZNT7 is critical for cell surface glycosylphosphatidylinositol-anchored protein expression.

Glycosylphosphatidylinositol (GPI)-anchored proteins play crucial roles in various enzyme activities, cell signaling and adhesion, and immune responses. While the molecular mechanism underlying GPI-anchored protein biosynthesis has been well studied, the role of zinc transport in this process has not yet been elucidated. Zn transporter (ZNT) proteins mobilize cytosolic zinc to the extracellular space and to intracellular compartments. Here, we report that the early secretory pathway ZNTs (ZNT5-ZNT6 heterodimers [ZNT5-6] and ZNT7-ZNT7 homodimers [ZNT7]), which supply zinc to the lumen of the early secretory pathway compartments are essential for GPI-anchored protein expression on the cell surface. We show, using overexpression and gene disruption/re-expression strategies in cultured human cells, that loss of ZNT5-6 and ZNT7 zinc transport functions results in significant reduction in GPI-anchored protein levels similar to that in mutant cells lacking phosphatidylinositol glycan anchor biosynthesis (PIG) genes. Furthermore, medaka fish with disrupted Znt5 and Znt7 genes show touch-insensitive phenotypes similar to zebrafish Pig mutants. These findings provide a previously unappreciated insight into the regulation of GPI-anchored protein expression and protein quality control in the early secretory pathway.

Wnt4a Is Indispensable for Genital Duct Elongation but Not for Gonadal Sex Differentiation in the Medaka, Oryzias latipes.

In most vertebrates, the oviducts and sperm ducts are derived from the Müllerian ducts and Wolffian ducts, respectively. However, in teleosts, the genital ducts are formed by the posterior extension of gonads in both sexes. Whether the genital ducts of teleosts are newly evolved organs or variants of Müllerian ducts is an important question for understanding evolutionary mechanisms of morphogenesis. One of the genes essential for Müllerian duct formation in mice is Wnt4, which is expressed in the mesenchyme and induces invagination of the coelomic epithelium and its posterior elongation. Here, we addressed the above question by examining genital duct development in mutants of two Wnt4 genes in the medaka (wnt4a is orthologous to mouse Wnt4, and wnt4b is paralogous). The wnt4b mutants had a short body but were fertile with normal genital ducts. In contrast, both male and female wnt4a mutants had their posterior elongation of the gonads stopped within or just outside the coelom. The mutants retained the posterior parts of ovarian cavities or sperm duct primordia, which are potential target tissues of Wnt4a. The gonads of female scl mutants (unable to synthesize sex steroids) lacked these tissues and did not develop genital ducts. Medaka wnt4a was expressed in the mesenchyme ventral to the genital ducts in both sexes. Taken together, the data strongly suggest that the mouse Müllerian ducts and the medaka genital ducts share homologous developmental processes. Additionally, the wnt4a or wnt4b single mutants and the double mutants did not show sex-reversal, implying that both genes are dispensable for gonadal sex differentiation in the medaka.

Establishment of transgenic epithelium-specific Cre-recombinase driving medaka (Oryzias latipes) by homology repair mediated knock-in.

Deletion of gene expression in the target tissues and cells is an effective strategy for elucidating the physiological functions of the protein of interest. For tissue-specific and/or inducible gene deletion, the Cre-loxP system has been widely used in various model organisms including medaka (Oryzias latipes). The epithelium is the key tissue, locating at the outermost area and playing a role in barrier to external stimuli. Despite a large genetic toolbox developed in medaka, there is no available Cre-driver line that works in an epithelium-specific manner. Here, we established epithelium-specific Cre-driver lines in medaka using a homology-directed repair mediated knock-in approach with CRISPR/Cas9, targeting each of periplakin and keratin genes. We show that Cre-recombinase is expressed exclusively in the epithelium in the knock-in lines and that it efficiently and specifically induces recombination in the tissues. These Cre-driver lines are useful for studying the functions of proteins expressed in the epithelium.

Sexually dimorphic role of oxytocin in medaka mate choice.

Oxytocin is a central neuromodulator required for facilitating mate preferences for familiar individuals in a monogamous rodent (prairie vole), irrespective of sex. While the role of oxytocin in mate choice is only understood in a few monogamous species, its function in nonmonogamous species, comprising the vast majority of vertebrate species, remains unclear. To address this issue, we evaluated the involvement of an oxytocin homolog (isotocin, referred herein as oxt) in mate choice in medaka fish (Oryzias latipes). Female medaka prefer to choose familiar mates, whereas male medaka court indiscriminately, irrespective of familiarity. We generated mutants of the oxt ligand (oxt) and receptor genes (oxtr1 and oxtr2) and revealed that the oxt-oxtr1 signaling pathway was essential for eliciting female mate preference for familiar males. This pathway was also required for unrestricted and indiscriminate mating strategy in males. That is, either oxt or oxtr1 mutation in males decreased the number of courtship displays toward novel females, but not toward familiar females. Further, males with these mutations exhibited enhanced mate-guarding behaviors toward familiar females, but not toward novel females. In addition, RNA-sequencing (seq) analysis revealed that the transcription of genes involved in gamma-amino butyric acid metabolism as well as those encoding ion-transport ATPase are up-regulated in both oxt and oxtr1 mutants only in female medaka, potentially explaining the sex difference of the mutant phenotype. Our findings provide genetic evidence that oxt-oxtr1 signaling plays a role in the mate choice for familiar individuals in a sex-specific manner in medaka fish.

The medaka fish Tol2 transposable element is in an early stage of decay: identification of a nonautonomous copy.

The majority of DNA-based transposable elements comprise autonomous and nonautonomous copies, or only nonautonomous copies, where the autonomous copy contains an intact gene for a transposase protein and the nonautonomous copy does not. Even if autonomous copies coexist, they are generally less frequent. The Tol2 element of medaka fish is one of the few elements for which a nonautonomous copy has not yet been found. Here, we report the presence of a nonautonomous Tol2 copy that was identified by surveying the medaka genome sequence database. This copy contained three local sequence alterations that affected the deduced amino acid sequence of the transposase: a deletion of 15 nucleotides resulting in a deletion of 5 amino acids, a base substitution causing a single amino acid change, and another base substitution giving rise to a stop codon. Transposition assays using cultured human cells revealed that transposase activity was reduced by the 15-nucleotide deletion and abolished by the nonsense mutation. This is the first example of a nonautonomous Tol2 copy. Thus, Tol2 is in an early stage of decay in the medaka genome, and is therefore a unique element to observe an almost complete decay process that progresses in natural populations.

Involvement of Rev1 in alkylating agent-induced loss of heterozygosity in Oryzias latipes.

Translesion synthesis (TLS) polymerases mediate DNA damage bypass during replication. The TLS polymerase Rev1 has two important functions in the TLS pathway, including dCMP transferase activity and acting as a scaffolding protein for other TLS polymerases at the C-terminus. Because of the former activity, Rev1 bypasses apurinic/apyrimidinic sites by incorporating dCMP, whereas the latter activity mediates assembly of multipolymerase complexes at the DNA lesions. We generated rev1 mutants lacking each of these two activities in Oryzias latipes (medaka) fish and analyzed cytotoxicity and mutagenicity in response to the alkylating agent diethylnitrosamine (DENA). Mutant lacking the C-terminus was highly sensitive to DENA cytotoxicity, whereas mutant with reduced dCMP transferase activity was slightly sensitive to DENA cytotoxicity, but exhibited a higher tumorigenic rate than wild-type fish. There was no significant difference in the frequency of DENA-induced mutations between mutant with reduced dCMP transferase activity and wild-type cultured cell. However, loss of heterozygosity (LOH) occurred frequently in cells with reduced dCMP transferase activity. LOH is a common genetic event in many cancer types and plays an important role on carcinogenesis. To our knowledge, this is the first report to identify the involvement of the catalytic activity of Rev1 in suppression of LOH.

Establishment of culture and microinjection methods for false clownfish embryos without parental care.

Anemonefish, including the false clownfish Amphiprion ocellaris, are attractive model organisms because of their unique features, such as sex change and brilliant color patterns in mutants. However, anemonefish are not widely used to study gene function using reverse genetic approaches owing to microinjection difficulties and subsequent rearing and hatching of embryos without parental care. A. ocellaris embryos are spawned on a hard substrate and cared for by their parents until hatching. However, the eggs need to be detached from the substrate and raised without their parents to perform successful microinjection. We established a method to culture and hatch A. ocellaris embryos without spawning substrates or parental care. We found that changing water and generating water flow are critical for culturing the embryos, and that water flow (as physical stimulation) and complete darkness in the dark period are necessary for successful hatching. We further investigated the effectiveness of microinjection into the yolk sac of fertilized eggs rather than into the cytoplasm, which makes microinjection easier. A reporter RNA injected into the yolk sac was transferred to the cytoplasm and translated, indicating that yolk sac microinjection is an efficient alternative as has been used for zebrafish. These findings highlight the potential of A. ocellaris as an experimental model organism for reverse genetics, and our methods could be applied to other anemonefish species.

Pre-validation of choriogenin H transgenic medaka eleutheroembryos as a quantitative estrogenic activity test method.

The choriogenin H - EGFP transgenic medaka (Oryzias melastigma) has been used to test estrogenic substances and quantify estrogenic activity into 17ß-estradiol (E2) equivalency (EEQ). The method uses 8 eleutheroembryos in 2 ml solution per well and 3 wells per treatment in 24-well plates at 26 ± 1 °C for 24 ± 2 h, with subsequent measurements of induced GFP signal intensity. EEQ measurements are calculated using a E2 probit regression model with a coefficient of determination (R2) > 0.90. The selectivity was confirmed evaluating 27 known estrogenic and 5 known non-estrogenic compounds. Limit of quantitation (LOQ), recovery rate and bias were calculated to be 1 ng/ml EEQ, 104% and 4% respectively. Robustness analysis revealed exposure temperature is a sensitive parameter that should be kept at 26 ± 1 °C. The repeatability of intra- and inter-laboratories achieved CV < 30% for most tested food and cosmetics samples. The lot-lot stability was confirmed by the stable EEQ qualitative control (QC, 1 ng/mL E2) and calibration curve results. The stability of standard reagents, samples and sample extracts was also investigated. These data demonstrated this method to be an accurate indicator of estrogenic activity for both chemicals and extracts.

CRISPR/Cas9 nickase-mediated efficient and seamless knock-in of lethal genes in the medaka fish Oryzias latipes.

The CRISPR/Cas system offers new opportunities for targeted gene modifications in a wide range of organisms. In medaka (Oryzias latipes), a vertebrate model organism, a wild-type Cas9-based approach is commonly used to establish desired strains, however, its use in lethal genes is still challenging due to excess gene disruptions triggered by DNA double strand breaks (DSBs). To overcome this problem, we aimed to develop a new knock-in system using Cas9 nickase (Cas9n) that can reduce DNA DSBs. We revealed that Cas9n allowed reduction of the DSB-induced unwanted mutagenesis via non-homologous end-joining at both on- and off- target sites. Further, with a new donor plasmid (p2BaitD) that provides a linear template through Cas9n-mediated nicks, we successfully integrated reporter cassettes via homology-directed repair (HDR) into all three loci tested, including a lethal gene. In the experiment targeting the lethal gene, the combination of p2BaitD and Cas9n achieved higher survival rates than the Cas9-based approach, which enabled the desired knock-in founders. Additionally, through a technical blend of our knock-in system with a recently developed One-step mating protocol, we successfully established a homozygous knock-in strain in one generation period. This study presents evidence of an effective method to generate an HDR-mediated gene knock-in in medaka and other organisms, which is useful for establishing screening platforms for genes or drugs toxicity or other applications.

Peroral endoscopic myotomy alone is effective for esophageal motility disorders and esophageal epiphrenic diverticulum: a retrospective single-center study.

BACKGROUND: Esophageal epiphrenic diverticulum (ED) is associated with esophageal motility disorder (EMD). If a diverticulum associated with EMD is enlarging with worsening symptoms, surgical intervention, including laparoscopic epiphrenic diverticulectomy with myotomy and fundoplication, is indicated. However, some studies suggest that myotomy alone, with less adverse events, is sufficient to improve symptoms. Additionally, peroral endoscopic myotomy (POEM) is considered effective and safe for EMD. Since theoretically, POEM is endoscopic Heller myotomy, POEM without diverticulectomy is considered a less invasive, promising treatment option for EMD and ED. We aimed to determine the efficacy and safety of POEM alone for ED with EMD. METHODS: This single-center study was retrospective. A total of 298 patients underwent POEM in Kobe University Hospital from April 2015 to October 2018. Of them, 14 patients had ED. Procedure-related outcomes and treatment outcomes 3 months post POEM were evaluated in these patients. RESULTS: The median maximum ED diameter was 29 (range 9-90) mm; and the median POEM procedure time, 77.5 (range 41-123) min. Pneumoperitoneum, which required needle decompression, occurred in one patient, but no fatal adverse events occurred. The median Eckardt score significantly decreased from 5 [range 2-11] pre POEM to 0 [range 0-2] post POEM (P < 0.0001). The median integrated relaxation pressure significantly decreased from 22.5 [13.9-34.3] mmHg pre POEM to 10.2 [0.7-23.9] mmHg post POEM (P < 0.0001). Of 14 patients, only one patient complained of gastroesophageal reflux disease symptoms, which could be controlled with a potassium-competitive acid blocker. CONCLUSIONS: POEM alone seemed effective and safe for patients with EMD and ED.

The sp7 gene is required for maturation of osteoblast-lineage cells in medaka (Oryzias latipes) vertebral column development.

Sp7 is a zinc finger transcription factor that is essential for osteoblast differentiation in mammals. To verify the characteristic features of osteoblast-lineage cells in teleosts, we established medaka sp7 mutants using a transcription activator-like effector nuclease (TALEN) genome editing system. These mutants showed severe defects in the formation of skeletal structures. In particular, the neural and the hemal arches were not formed, although the chordal centra were formed. Analysis of the transgenic medaka revealed that sp7 mutant had normal distribution of type X collagen a1 a (col10a1a)-positive osteoblast-like cells around the centrum and at the proximal region of the vertebral arch. The sp7 mutant phenotype could be rescued by exogenous sp7 expression in col10a1a-positive cells, as well as in sp7-positive osteoblast cells. Furthermore, runx2-positive osteoblast progenitors were observed on the vertebral arches, but not on the centrum, during vertebral column development. In addition, these osteoblast progenitors differentiated into the col10a1a-positive cells. In sp7 mutant, the runx2-positive cells were normally distributed at the region of unformed vertebral arch but failed to differentiate into col10a1a-positive cells. These results indicate that osteoblast-lineage cells undergo two distinct differentiation processes during development of the vertebral arch and the centrum. Nevertheless, our results verified that sp7 gene expression in osteoblast-lineage cells is required for differentiation into mature osteoblasts to form the vertebral column and other skeletal structures.

Loss of zinc finger MYND-type containing 10 (zmynd10) affects cilia integrity and axonemal localization of dynein arms, resulting in ciliary dysmotility, polycystic kidney and scoliosis in medaka (Oryzias latipes).

Cilia and flagella are hair-like organelles that project from the cell surface and play important roles in motility and sensory perception. Motility defects in cilia and flagella lead to primary ciliary dyskinesia (PCD), a rare human disease. Recently zinc finger MYND-type containing 10 (ZMYND10) was identified in humans as a PCD-associated gene. In this study, we use medaka fish as a model to characterize the precise functions of zmynd10. In medaka, zmynd10 is exclusively expressed in cells with motile cilia. Embryos with zmynd10 Morpholino knockdown exhibited a left-right (LR) defect associated with loss of motility in Kupffer's vesicle (KV) cilia. This immotility was caused by loss of the outer dynein arms, which is a characteristic ultrastructural phenotype in PCD. In addition, KV cilia in zmynd10 knockdown embryos had a swollen and wavy morphology. Together, these results suggest that zmynd10 is a multi-functional protein that has independent roles in axonemal localization of dynein arms and in formation and/or maintenance of cilia. The C-terminal region of zmynd10 has a MYND-type zinc finger domain (zf-MYND) that is important for its function. Our rescue experiment showed that the zmynd10-ΔC truncated protein, which lacks zf-MYND, was still partially functional, suggesting that zmynd10 has another functional domain besides zf-MYND. To analyze the later stages of development, we generated a zmynd10 knockout mutant using transcription activator-like effector nuclease (TALEN) technology. Adult mutants exhibited sperm dysmotility, scoliosis and progressive polycystic kidney.

Viable neuronopathic Gaucher disease model in Medaka (Oryzias latipes) displays axonal accumulation of alpha-synuclein.

Homozygous mutations in the glucocerebrosidase (GBA) gene result in Gaucher disease (GD), the most common lysosomal storage disease. Recent genetic studies have revealed that GBA mutations confer a strong risk for sporadic Parkinson's disease (PD). To investigate how GBA mutations cause PD, we generated GBA nonsense mutant (GBA-/-) medaka that are completely deficient in glucocerebrosidase (GCase) activity. In contrast to the perinatal death in humans and mice lacking GCase activity, GBA-/- medaka survived for months, enabling analysis of the pathological progression. GBA-/- medaka displayed the pathological phenotypes resembling human neuronopathic GD including infiltration of Gaucher cell-like cells into the brains, progressive neuronal loss, and microgliosis. Detailed pathological findings represented lysosomal abnormalities in neurons and alpha-synuclein (α-syn) accumulation in axonal swellings containing autophagosomes. Unexpectedly, disruption of α-syn did not improve the life span, formation of axonal swellings, neuronal loss, or neuroinflammation in GBA-/- medaka. Taken together, the present study revealed GBA-/- medaka as a novel neuronopathic GD model, the pahological mechanisms of α-syn accumulation caused by GCase deficiency, and the minimal contribution of α-syn to the pathogenesis of neuronopathic GD.

An essential role of the arginine vasotocin system in mate-guarding behaviors in triadic relationships of medaka fish (Oryzias latipes).

To increase individual male fitness, males of various species remain near a (potential) mating partner and repel their rivals (mate-guarding). Mate-guarding is assumed to be mediated by two different types of motivation: sexual motivation toward the opposite sex and competitive motivation toward the same sex. The genetic/molecular mechanisms underlying how mate presence affects male competitive motivation in a triadic relationship has remained largely unknown. Here we showed that male medaka fish prominently exhibit mate-guarding behavior. The presence of a female robustly triggers male-male competition for the female in a triadic relationship (2 males and 1 female). The male-male competition resulted in one male occupying a dominant position near the female while interfering with the other male's approach of the female. Paternity testing revealed that the dominant male had a significantly higher mating success rate than the other male in a triadic relationship. We next generated medaka mutants of arginine-vasotocin (avt) and its receptors (V1a1, V1a2) and revealed that two genes, avt and V1a2, are required for normal mate-guarding behavior. In addition, behavioral analysis of courtship behaviors in a dyadic relationship and aggressive behaviors within a male group revealed that avt mutant males displayed decreased sexual motivation but showed normal aggression. In contrast, heterozygote V1a2 mutant males displayed decreased aggression, but normal mate-guarding and courtship behavior. Thus, impaired mate-guarding in avt and V1a2 homozygote mutants may be due to the loss of sexual motivation toward the opposite sex, and not to the loss of competitive motivation toward rival males. The different behavioral phenotypes between avt, V1a2 heterozygote, and V1a2 homozygote mutants suggest that there are redundant systems to activate V1a2 and that endogenous ligands activating the receptor may differ according to the social context.

Leucophores are similar to xanthophores in their specification and differentiation processes in medaka.

Animal body color is generated primarily by neural crest-derived pigment cells in the skin. Mammals and birds have only melanocytes on the surface of their bodies; however, fish have a variety of pigment cell types or chromatophores, including melanophores, xanthophores, and iridophores. The medaka has a unique chromatophore type called the leucophore. The genetic basis of chromatophore diversity remains poorly understood. Here, we report that three loci in medaka, namely, leucophore free (lf), lf-2, and white leucophore (wl), which affect leucophore and xanthophore differentiation, encode solute carrier family 2, member 15b (slc2a15b), paired box gene 7a (pax7a), and solute carrier family 2 facilitated glucose transporter, member 11b (slc2a11b), respectively. Because lf-2, a loss-of-function mutant for pax7a, causes defects in the formation of xanthophore and leucophore precursor cells, pax7a is critical for the development of the chromatophores. This genetic evidence implies that leucophores are similar to xanthophores, although it was previously thought that leucophores were related to iridophores, as these chromatophores have purine-dependent light reflection. Our identification of slc2a15b and slc2a11b as genes critical for the differentiation of leucophores and xanthophores in medaka led to a further finding that the existence of these two genes in the genome coincides with the presence of xanthophores in nonmammalian vertebrates: birds have yellow-pigmented irises with xanthophore-like intracellular organelles. Our findings provide clues for revealing diverse evolutionary mechanisms of pigment cell formation in animals.

Modular development of the teleost trunk along the dorsoventral axis and zic1/zic4 as selector genes in the dorsal module.

Teleost fish exhibit remarkable diversity in morphology, such as fins and coloration, particularly on the dorsal side. These structures are evolutionary adaptive because their back is highly visible to other individuals. However, owing to the late phenotypic appearance (from larva to adult) and lack of appropriate mutants, the genetic mechanisms that regulate these dorsoventrally asymmetric external patterns are largely unknown. To address this, we have analyzed the spontaneous medaka mutant Double anal fin (Da), which exhibits a mirror-image duplication of the ventral half across the lateral midline from larva to adult. Da is an enhancer mutant for zic1 and zic4 in which their expression in dorsal somites is lost. We show that the dorsoventral polarity in Da somites is lost and then demonstrate using transplantation techniques that somites and their derived tissues globally determine the multiple dorsal-specific characteristics of the body (fin morphology and pigmentation) from embryo to adult. Intriguingly, the zic1/zic4 expression in the wild type persists throughout life in the dorsal parts of somite derivatives, i.e. the myotome, dermis and vertebrae, forming a broad dorsal domain in the trunk. Comparative analysis further implies a central role for zic1/zic4 in morphological diversification of the teleost body. Taken together, we propose that the teleost trunk consists of dorsal/ventral developmental modules and that zic1/zic4 in somites function as selector genes in the dorsal module to regulate multiple dorsal morphologies.

In vivo recombination efficiency of two site-specific recombination systems, VCre/VloxP and SCre/SloxP, in medaka (Oryzias latipes).

The present study delineates the in vivo efficiency of two site-specific recombination systems, VCre/VloxP and SCre/SloxP, in medaka (Oryzias latipes). VCre, SCre, and Cre RNA was microinjected into fertilized medaka eggs belonging to three transgenic lines harboring VloxP, SloxP, and loxP cassette. VCre induced site-specific recombination specifically at VloxP sequence and SCre at SloxP sequence without any cross-reactivity. These findings provide two novel alternative recombination systems in vivo in addition to the existing Cre/loxP and Flp/FRT systems, thus enabling sophisticated gene expression in model organisms.

ADAM12-deficient zebrafish exhibit retardation in body growth at the juvenile stage without developmental defects.

ADAM (a disintegrin and metalloprotease) constitutes a family of multi-domain proteins that are involved in development, homeostasis, and disease. ADAM12 plays important roles in myogenesis and adipogenesis in mice; however, the precise physiological mechanisms are not known, and the function of this gene in other vertebrates has not been examined. In this study, we used a simple model vertebrate, the zebrafish, to investigate the functions of ADAM12 during development. Zebrafish adam12 is conserved with those of mammals in the synteny and the amino-acid sequence. We examined adam12 expression in zebrafish embryos by whole mount in situ hybridization and the promoter activity of the adam12 upstream sequence. We found that adam12 is strongly expressed in the cardiovascular system, erythroid progenitors, brain, and jaw cartilage during zebrafish development, and adam12-knockout zebrafish exhibited reduced body size in the juvenile stage without apparent morphological defects. Taken together, these results suggest that adam12 plays a significant role in the regulation of body growth during juvenile stage in zebrafish, although the precise molecular mechanisms await further study.

Generation of a transgenic medaka (Oryzias latipes) strain for visualization of nuclear dynamics in early developmental stages.

In this study, we verified nuclear transport activity of an artificial nuclear localization signal (aNLS) in medaka fish (Oryzias latipes). We generated a transgenic medaka strain expresses the aNLS tagged enhanced green fluorescent protein (EGFP) driven by a medaka beta-actin promoter. The aNLS-EGFP was accumulated in the nuclei of somatic tissues and yolk nuclei of oocytes, but undetectable in the spermatozoa. The fluorescent signal was observed from immediately after fertilization by a maternal contribution. Furthermore, male and female pronuclei were visualized in fertilized eggs, and nuclear dynamics of pronuclear fusion and subsequent cleavage were captured by time-lapse imaging. In contrast, SV40NLS exhibited no activity of nuclear transport in early embryos. In conclusion, the aNLS possesses a strong nuclear localization activity and is a useful probe for fluorescent observation of the pronuclei and nuclei in early developmental stage of medaka.