Vertebrate Model Research Articles

PACS-1 variant protein is aberrantly localized in Caenorhabditis elegans model of PACS1/PACS2 syndromes.

PACS (phosphofurin acidic cluster sorting) proteins are known for their roles in sorting cargo proteins to organelles and can physically interact with WD40 repeat-containing protein WDR37. PACS1, PACS2, and WDR37 variants are associated with multisystemic syndromes and neurodevelopmental disorders characterized by intellectual disability, seizures, developmental delays, craniofacial abnormalities, and autism spectrum disorder. However, the functional effects of syndromic variants at the cellular level remain unknown. Here, we report the expression pattern of Caenorhabditis elegans orthologs of PACS and WDR37 and their interaction. We show that cePACS-1 and ceWDR-37 colocalize to somatic cytoplasm of many types of cells and are mutually required for expression, supporting a conclusion that the intermolecular dependence of PACS1/PACS2/PACS-1 and WDR37/WDR-37 is evolutionarily conserved. We further show that editing in PACS1 and PACS2 variants in cePACS-1 changes protein localization in multiple cell types, including neurons. Moreover, expression of human PACS1 can functionally complement C. elegans PACS-1 in neurons, demonstrating conserved functions of the PACS-WDR37 axis in an invertebrate model system. Our findings reveal effects of human variants and suggest potential strategies to identify regulatory network components that may contribute to understanding molecular underpinnings of PACS/WDR37 syndromes.

The brittle star genome illuminates the genetic basis of animal appendage regeneration

Species within nearly all extant animal lineages are capable of regenerating body parts. However, it remains unclear whether the gene expression programme controlling regeneration is evolutionarily conserved. Brittle stars are a species-rich class of echinoderms with outstanding regenerative abilities, but investigations into the genetic bases of regeneration in this group have been hindered by the limited genomic resources. Here we report a chromosome-scale genome assembly for the brittle star Amphiura filiformis. We show that the brittle star genome is the most rearranged among echinoderms sequenced so far, featuring a reorganized Hox cluster reminiscent of the rearrangements observed in sea urchins. In addition, we performed an extensive profiling of gene expression during brittle star adult arm regeneration and identified sequential waves of gene expression governing wound healing, proliferation and differentiation. We conducted comparative transcriptomic analyses with other invertebrate and vertebrate models for appendage regeneration and uncovered hundreds of genes with conserved expression dynamics, particularly during the proliferative phase of regeneration. Our findings emphasize the crucial importance of echinoderms to detect long-range expression conservation between vertebrates and classical invertebrate regeneration model systems.

Conserved Signaling Pathways in the Ciona robusta Gut.

The urochordate Ciona robusta exhibits numerous functional and morphogenetic traits that are shared with vertebrate models. While prior investigations have identified several analogies between the gastrointestinal tract (i.e., gut) of Ciona and mice, the molecular mechanisms responsible for these similarities remain poorly understood. This study seeks to address this knowledge gap by investigating the transcriptional landscape of the adult stage gut. Through comparative genomics analyses, we identified several evolutionarily conserved components of signaling pathways of pivotal importance for gut development (such as WNT, Notch, and TGFβ-BMP) and further evaluated their expression in three distinct sections of the gastrointestinal tract by RNA-seq. Despite the presence of lineage-specific gene gains, losses, and often unclear orthology relationships, the investigated pathways were characterized by well-conserved molecular machinery, with most components being expressed at significant levels throughout the entire intestinal tract of C. robusta. We also showed significant differences in the transcriptional landscape of the stomach and intestinal tract, which were much less pronounced between the proximal and distal portions of the intestine. This study confirms that C. robusta is a reliable model system for comparative studies, supporting the use of ascidians as a model to study gut physiology.

Multisite Assembly of Gateway Induced Clones (MAGIC): a flexible cloning toolbox with diverse applications in vertebrate model systems.

Here we present the Multisite Assembly of Gateway Induced Clones (MAGIC) system, which harnesses site-specific recombination-based cloning via Gateway technology for rapid, modular assembly of between 1 and 3 "Entry" vector components, all into a fourth, standard high copy "Destination" plasmid backbone. The MAGIC toolkit spans a range of in vitro and in vivo uses, from directing tunable gene expression, to driving simultaneous expression of microRNAs and fluorescent reporters, to enabling site-specific recombinase-dependent gene expression. All MAGIC system components are directly compatible with existing multisite gateway Tol2 systems currently used in zebrafish, as well as existing eukaryotic cell culture expression Destination plasmids, and available mammalian lentiviral and adenoviral Destination vectors, allowing rapid cross-species experimentation. Moreover, herein we describe novel vectors with flanking piggyBac transposon elements for stable genomic integration in vitro or in vivo when used with piggyBac transposase. Collectively, the MAGIC system facilitates transgenesis in cultured mammalian cells, electroporated mouse and chick embryos, as well as in injected zebrafish embryos, enabling the rapid generation of innovative DNA constructs for biological research due to a shared, common plasmid platform.

Exploring vertebral bone density changes in a trunk with adolescent idiopathic scoliosis: a mechanobiological modeling investigation of intact and unilaterally paralyzed muscles

This study aimed to elucidate the vertebral bone density variations associated with adolescent idiopathic scoliosis (AIS), specifically examining the impact of unilateral muscle paralysis using an integrated approach combining Frost’s Mechanostat theory, a three-dimensional subject-specific finite element model and a musculoskeletal model of the L2 vertebra. The findings revealed a spectrum of bone density values ranging from 0.29 to 0.31 g/cm3, along with vertebral micro-strain levels spanning from 300 to 2200, consistent with existing literature. Furthermore, the ratio of maximum von Mises stress between the concave and convex side in the AIS model with intact muscles was approximately 1.08, which decreased by 4% due following unilateral paralysis of longissimus thoracis pars thoracic muscle. Overall, this investigation contributes to a deeper understanding of AIS biomechanics and lays the groundwork for future research endeavors aimed at optimizing clinical management approaches for individuals with this condition.

Combined Pituitary Hormone Deficiency in lhx4-Knockout Zebrafish.

LIM homeobox 4 (LHX4) is a transcription factor crucial for anterior pituitary (AP) development. Patients with LHX4 mutation suffer from combined pituitary hormone deficiency (CPHD), short statures, reproductive and metabolic disorders and lethality in some cases. Lhx4-knockout (KO) mice fail to develop a normal AP and die shortly after birth. Here, we characterize a zebrafish lhx4-KO model to further investigate the importance of LHX4 in pituitary gland development and regulation. At the embryonic and larval stages, these fish express lower levels of tshb mRNA compared with their wildtype siblings. In adult lhx4-KO fish, the expressions of pituitary hormone-encoding transcripts, including growth hormone (gh), thyroid stimulating hormone (tshb), proopiomelanocortin (pomca) and follicle stimulating hormone (fshb), are reduced, the pomca promoter-driven expression in corticotrophs is dampened and luteinizing hormone (lhb)-producing gonadotrophs are severely depleted. In contrast to Lhx4-KO mice, Lhx4-deficient fish survive to adulthood, but with a reduced body size. Importantly, lhx4-KO males reach sexual maturity and are reproductively competent, whereas the females remain infertile with undeveloped ovaries. These phenotypes, which are reminiscent of those observed in CPHD patients, along with the advantages of the zebrafish for developmental genetics research, make this lhx4-KO fish an ideal vertebrate model to study the outcomes of LHX4 mutation.

Review of Pseudoloma neurophilia (Microsporidia): A common neural parasite of laboratory zebrafish (Danio rerio).

Zebrafish (Danio rerio) is now the second most used animal model in biomedical research. As with other vertebrate models, underlying diseases and infections often impact research. Beyond mortality and morbidity, these conditions can compromise research end points by producing nonprotocol induced variation within experiments. Pseudoloma neurophilia, a microsporidium that targets the central nervous system, is the most frequently diagnosed pathogen in zebrafish facilities. The parasite undergoes direct, horizontal transmission within populations, and is also maternally transmitted with spores in ovarian fluid and occasionally within eggs. This transmission explains the wide distribution among research laboratories as new lines are generally introduced as embryos. The infection is chronic, and fish apparently never recover following the initial infection. However, most fish do not exhibit outward clinical signs. Histologically, the parasite occurs as aggregates of spores throughout the midbrain and spinal cord and extends to nerve roots. It often elicits meninxitis, myositis, and myodegeneration when it infects the muscle. There are currently no described therapies for the parasite, thus the infection is best avoided by screening with PCR-based tests and removal of infected fish from a facility. Examples of research impacts include reduced fecundity, behavioral changes, transcriptome alterations, and autofluorescent lesions.

Synthesis and Structure of Novel Phenothiazine Derivatives, and Compound Prioritization via In Silico Target Search and Screening for Cytotoxic and Cholinesterase Modulatory Activities in Liver Cancer Cells and In Vivo in Zebrafish.

Phenothiazines (PTZ) are antipsychotics known to modulate a variety of neurotransmitter activities that include dopaminergic and cholinergic signaling and have been identified as potential anticancer agents in vitro. However, it is important to also test whether a highly cytotoxic, repurposed, or novel PTZ has low toxicity and neuromodulatory activity in vivo using vertebrate model organisms, such as zebrafish. In this study, we synthesized novel phenothiazines and screened them in vitro in liver cancer and in vivo in zebrafish embryos/larvae. The syntheses of several intermediate PTZ 10-yl acyl chlorides were followed by elemental analysis and determination of 1H NMR and 13C NMR mass (ESI+) spectra of a large number of novel PTZ 10-carboxamides. Cytotoxicities of 28 PTZ derivatives (1-28) screened against Hep3B and SkHep1 liver cancer cell lines revealed five intermediate and five novel leads along with trifluoperazine (TFP), prochlorperazine (PCP), and perphenazine, which are relatively more cytotoxic than the basic PTZ core. Overall, the derivatives were more cytotoxic to Hep3B than SkHep1 cells. Moreover, in silico target screening identified cholinesterases as some of the commonest targets of the screened phenothiazines. Interestingly, molecular docking studies with acetylcholinesterase (AChE) and butyrylcholinesterase proteins showed that the most cytotoxic compounds 1, 3, PCP, and TFP behaved similar to Huprin W in their amino acid interactions with the AChE protein. The highly cytotoxic intermediate PTZ derivative 1 exhibited a relatively lower toxicity profile than those of 2 and 3 during the zebrafish development. It also modulated in vivo the cholinesterase activity in a dose-dependent manner while significantly increasing the total cholinesterase activity and/or ACHE mRNA levels, independent of the liver cancer cell type. Our screen also identified novel phenothiazines, i.e., 8 and 10, with significant cytotoxic and cholinesterase modulatory effects in liver cancer cells; yet both compounds had low levels of toxicity in zebrafish. Moreover, they modulated the cholinesterase activity or expression of ACHE in a cancer cell line-specific manner, and compound 10 significantly inhibited the cholinesterase activity in zebrafish. Accordingly, using a successful combination of in silico, in vitro, and in vivo approaches, we identified several lead anticancer and cholinesterase modulatory PTZ derivatives for future research.

Myo5B plays a significant role in the hyphal growth and virulence of the human pathogenic fungus Mucor lusitanicus.

Mucormycosis is an emerging and deadly invasive fungal infection caused by fungi belonging to the Mucorales order. We investigated the myosin superfamily, which encompasses diverse actin-based motor proteins with various cellular functions. Specifically, the role of the Myo5B (ID 179665) protein from the myosin class V family in Mucor lusitanicus was explored by generating silencing phenotypes and null mutants corresponding to the myo5B gene. Silencing fungal transformants exhibited a markedly reduced growth rate and a nearly complete absence of sporulation compared to the wild-type strain. The myo5BΔ null mutant strain displayed atypical characteristics, including abnormally short septa and inflated hyphae. Notably, there were a majority of small yeast-like cells instead of filamentous hyphae in the mutant. These yeast-like cells cannot germinate normally, resulting in a loss of polarity. In vivo virulence assays conducted in the Galleria mellonella invertebrate model revealed that the myo5BΔ mutant strain was avirulent. These findings shed light on the crucial contributions of the Myo5B protein to the dimorphism and pathogenicity of M. lusitanicus. Therefore, the myosin V family is a potential target for future therapeutic interventions aimed at treating mucormycosis.

Remote Magneto–Thermal Modulation of Reactive Oxygen Species Balance Enhances Tissue Regeneration In Vivo

AbstractOne of the hallmarks of tissue repair is the production of reactive oxygen species (ROS), which modulate processes such as cell proliferation. Although several attempts have been made to manipulate ROS levels to increase tissue repair, the lack of techniques able to remotely manipulate the redox homeostasis with spatio–temporal fashion has hindered its progress. Herein, a new approach for tuning the ROS levels using magnetic nanoparticles (MNPs) that act as nanoheaters when exposed to an alternating magnetic field is presented. Two manganese–iron oxide (MnxFe3−xO4) MNPs (with a low and a high Mn2+ content) are designed and probed for the possibility of modulating the ROS balance by magneto–thermal stimulation in the invertebrate model organism Hydra vulgaris, able to fully regenerate. By evaluating the expression of selected genes involved in the maintenance of ROS homeostasis and proliferation pathways, a biphasic modulation of the ROS levels played by the MNPs is found. While MNPs with a lower Mn2+ content are able to positively modulate the regeneration potential under magnetostimulation, MNPs with a higher Mn2+ content cause a different redox imbalance, negatively affecting the regeneration dynamic. This innovative approach reveals a novel way of manipulating redox homeostasis that can advance in the field of tissue engineering.

Canine inherited retinal degeneration as model to study disease mechanisms and therapy for ciliopathies

Humans have a highly developed retina and obtain approximately 80% of their external information from vision. Photoreceptor cells, which are located in the outermost layer of the neuroretina and recognize light signals, are highly specialized sensory cilia that share structural and functional features with primary cilia. Genetic disorders of the retina or photoreceptor cells are termed inherited retinal diseases (IRDs) and are caused by variants in one of more than 280 genes identified to date. Among the genes responsible for IRDs, many are shared with those responsible for ciliopathies. In studies of inherited diseases, mouse models are commonly used due to their advantages in breeding, handling, and relative feasibility in creating pathological models. On the other hand, structural, functional, and genetic differences in the retina between mice and humans can be a barrier in IRD research. To overcome the limitations of mouse models, larger vertebrate models of IRDs can be a useful research subject. In particular, canines have retinas that are structurally and functionally similar and eyes that are anatomically comparable to those of humans. In addition, due to their unique veterinary clinical surveillance and genetic background, naturally occurring canine IRDs are more likely to be identified than in other large animals. To date, pathogenic mutations related to canine IRDs have been identified in more than 30 genes, contributing to the understanding of pathogeneses and to the development of new therapies. This review provides an overview of the roles of the canine IRD models in ciliopathy research.

Finite element analysis of a three-dimensional cervical spine model with muscles based on CT scan data

Background The incidence of cervical spondylosis is increasing, gradually affecting people’s normal lives. Establishing a finite element model of the cervical spine is one of the methods for studying cervical spondylosis. MRI (Magnetic Resonance Imaging) still has certain difficulties in transitioning from human imaging to establishing muscle models suitable for finite element analysis. Medical software provides specific morphologies and can generate muscle finite element models. Additionally, there is little research on the static analysis of cervical spine finite element models with solid muscle. Purpose A new method is proposed for establishing a finite element model of the cervical spine based on CT (Computed Tomography) data and medical software, and the model’s effectiveness is validated. Human movement characteristics based on the force distribution in various parts are analyzed and predicted. Methods The muscle model is reconstructed in medical software and a three-dimensional finite element model of the entire cervical spine (C0–C7) is established by combining muscle models with CT vertebral data models. 1.5 Nm of load is applied to the finite element model to simulate the cervical spine movement. Results The finite element model was successfully established, and effectiveness was verified. Stress variations in various parts under six movements were obtained. The effectiveness of the model was basically verified. Conclusion The finite element model of the cervical spine for mechanical analysis can be successfully established by using medical software and CT data. In daily life, the C2–3, C3–4, C4–C5 intervertebral discs, rectus capitis posterior major, longus colli, and obliquus capitis inferior are more prone to injury.

Cell cycle length governs heterochromatin reprogramming during early development in non-mammalian vertebrates

Heterochromatin marks such as H3K9me3 undergo global erasure and re-establishment after fertilization, and the proper reprogramming of H3K9me3 is essential for early development. Despite the widely conserved dynamics of heterochromatin reprogramming in invertebrates and non-mammalian vertebrates, previous studies have shown that the underlying mechanisms may differ between species. Here, we investigate the molecular mechanism of H3K9me3 dynamics in medaka (Japanese killifish, Oryzias latipes) as a non-mammalian vertebrate model, and show that rapid cell cycle during cleavage stages causes DNA replication-dependent passive erasure of H3K9me3. We also find that cell cycle slowing, toward the mid-blastula transition, permits increasing nuclear accumulation of H3K9me3 histone methyltransferase Setdb1, leading to the onset of H3K9me3 re-accumulation. We further demonstrate that cell cycle length in early development also governs H3K9me3 reprogramming in zebrafish and Xenopus laevis. Together with the previous studies in invertebrates, we propose that a cell cycle length-dependent mechanism for both global erasure and re-accumulation of H3K9me3 is conserved among rapid-cleavage species of non-mammalian vertebrates and invertebrates such as Drosophila, C. elegans, Xenopus and teleost fish.

Magnesium malate-modified calcium phosphate bone cement promotes the repair of vertebral bone defects in minipigs via regulating CGRP

Active artificial bone substitutes are crucial in bone repair and reconstruction. Calcium phosphate bone cement (CPC) is known for its biocompatibility, degradability, and ability to fill various shaped bone defects. However, its low osteoinductive capacity limits bone regeneration applications. Effectively integrating osteoinductive magnesium ions with CPC remains a challenge. Herein, we developed magnesium malate-modified CPC (MCPC). Incorporating 5% magnesium malate significantly enhances the compressive strength of CPC to (6.18 ± 0.49) MPa, reduces setting time and improves disintegration resistance. In vitro, MCPC steadily releases magnesium ions, promoting the proliferation of MC3T3-E1 cells without causing significant apoptosis, proving its biocompatibility. Molecularly, magnesium malate prompts macrophages to release prostaglandin E2 (PGE2) and synergistically stimulates dorsal root ganglion (DRG) neurons to synthesize and release calcitonin gene-related peptide (CGRP). The CGRP released by DRG neurons enhances the expression of the key osteogenic transcription factor Runt-related transcription factor-2 (RUNX2) in MC3T3-E1 cells, promoting osteogenesis. In vivo experiments using minipig vertebral bone defect model showed MCPC significantly increases the bone volume fraction, bone density, new bone formation, and proportion of mature bone in the defect area compared to CPC. Additionally, MCPC group exhibited significantly higher levels of osteogenesis and angiogenesis markers compared to CPC group, with no inflammation or necrosis observed in the hearts, livers, or kidneys, indicating its good biocompatibility. In conclusion, MCPC participates in the repair of bone defects in the complex post-fracture microenvironment through interactions among macrophages, DRG neurons, and osteoblasts. This demonstrates its significant potential for clinical application in bone defect repair.

Different stressors uniquely affect the expression of endocannabinoid-metabolizing enzymes in the central ring ganglia of Lymnaea stagnalis.

The endocannabinoid system (ECS) plays an important role in neuroprotection, neuroplasticity, energy balance, modulation of stress, and inflammatory responses, acting as a critical link between the brain and the body's peripheral regions, while also offering promising potential for novel therapeutic strategies. Unfortunately, in humans, pharmacological inhibitors of different ECS enzymes have led to mixed results in both preclinical and clinical studies. As the ECS has been highly conserved throughout the eukaryotic lineage, the use of invertebrate model organisms like the pond snail Lymnaea stagnalis may provide a flexible tool to unravel unexplored functions of the ECS at the cellular, synaptic, and behavioral levels. In this study, starting from the available genome and transcriptome of L. stagnalis, we first identified putative transcripts of all ECS enzymes containing an open reading frame. Each predicted protein possessed a high degree of sequence conservation to known orthologues of other invertebrate and vertebrate organisms. Sequences were confirmed by qualitative PCR and sequencing. Then, we investigated the transcriptional effects induced by different stress conditions (i.e., bacterial LPS injection, predator scent, food deprivation, and acute heat shock) on the expression levels of the enzymes of the ECS in Lymnaea's central ring ganglia. Our results suggest that in Lymnaea as in rodents, the ECS is involved in mediating inflammatory and anxiety-like responses, promoting energy balance, and responding to acute stressors. To our knowledge, this study offers the most comprehensive analysis so far of the ECS in an invertebrate model organism.

Synaptic Mechanisms of Ethanol Tolerance and Neuroplasticity: Insights from Invertebrate Models.

Alcohol tolerance is a neuroadaptive response that leads to a reduction in the effects of alcohol caused by previous exposure. Tolerance plays a critical role in the development of alcohol use disorder (AUD) because it leads to the escalation of drinking and dependence. Understanding the molecular mechanisms underlying alcohol tolerance is therefore important for the development of effective therapeutics and for understanding addiction in general. This review explores the molecular basis of alcohol tolerance in invertebrate models, Drosophila and C. elegans, focusing on synaptic transmission. Both organisms exhibit biphasic responses to ethanol and develop tolerance similar to that of mammals. Furthermore, the availability of several genetic tools makes them a great candidate to study the molecular basis of ethanol response. Studies in invertebrate models show that tolerance involves conserved changes in the neurotransmitter systems, ion channels, and synaptic proteins. These neuroadaptive changes lead to a change in neuronal excitability, most likely to compensate for the enhanced inhibition by ethanol.

Caenorhabditis elegans as a Convenient Animal Model for Microbiome Studies.

Microbes constitute the most prevalent life form on Earth, yet their remarkable diversity remains mostly unrecognized. Microbial diversity in vertebrate models presents a significant challenge for investigating host-microbiome interactions. The model organism Caenorhabditis elegans has many advantages for delineating the effects of host genetics on microbial composition. In the wild, the C. elegans gut contains various microbial species, while in the laboratory it is usually a host for a single bacterial species. There is a potential host-microbe interaction between microbial metabolites, drugs, and C. elegans phenotypes. This mini-review aims to summarize the current understanding regarding the microbiome in C. elegans. Examples using C. elegans to study host-microbe-metabolite interactions are discussed.

Assessing metabolic rates in zebrafish using a 3D-printed intermittent-flow respirometer and swim tunnel system.

Zebrafish have become a widely used vertebrate model in physiology and reliable measures of their metabolic rate are needed. We have developed a 3D-printed respirometer and swim tunnel system and used it for obtaining accurate measurement of standard metabolic rate (SMR) and maximal, aerobic metabolic rate (MMR) in zebrafish under rest and maximal exercise, respectively. We compared a slow (stepwise) protocol to a fast (continuous) protocol for determining MMR. The fast protocol yielded slightly (but not significantly) higher oxygen consumption rates than the slow protocol and the data, in contrast to the slow protocol, followed a normal distribution. These findings point to the fast protocol as a fast and reliable method for obtaining accurate values of MMR in zebrafish. We make the 3D drawings for printing the system available to researchers, to help streamline the field of metabolic research in zebrafish and other smaller fish species.

Sex-dependent regulation of vertebrate somatic growth and aging by germ cells.

The function of germ cells in somatic growth and aging has been demonstrated in invertebrate models but remains unclear in vertebrates. We demonstrated sex-dependent somatic regulation by germ cells in the short-lived vertebrate model Nothobranchius furzeri. In females, germ cell removal shortened life span, decreased estrogen, and increased insulin-like growth factor 1 (IGF-1) signaling. In contrast, germ cell removal in males improved their health with increased vitamin D signaling. Body size increased in both sexes but was caused by different signaling pathways, i.e., IGF-1 and vitamin D in females and males, respectively. Thus, vertebrate germ cells regulate somatic growth and aging through different pathways of the endocrine system, depending on the sex, which may underlie the sexual difference in reproductive strategies.

Functional Test of a Naturally Occurred Tumor Modifier Gene Provides Insights to Melanoma Development.

Occurrence of degenerative interactions is thought to serve as a mechanism underlying hybrid unfitness. However, the molecular mechanisms underpinning the genetic interaction and how they contribute to overall hybrid incompatibilities are limited to only a handful of examples. A vertebrate model organism, Xiphophorus , is used to study hybrid dysfunction and it has been shown from this model that diseases, such as melanoma, can occur in certain interspecies hybrids. Melanoma development is due to hybrid inheritance of an oncogene, xmrk , and loss of a co-evolved tumor modifier. It was recently found that adgre5 , a G protein-coupled receptor involved in cell adhesion, is a tumor regulator gene in naturally hybridizing Xiphophorus species X. birchmanni and X. malinche . We hypothesized that one of the two parental alleles of adgre5 is involved in regulation of cell proliferation, migration and melanomagenesis. Accordingly, we assessed the function of adgre5 alleles from each parental species of the melanoma-bearing hybrids using in vitro cell proliferation and migration assays. In addition, we expressed each adgre5 allele with the xmrk oncogene in transgenic medaka. We found that cells transfected with the X. birchmanni adgre5 exhibited decreased proliferation and migration compared to those with the X. malinche allele. Moreover, X. birchmanni allele of adgre5 completely inhibited melanoma development in xmrk transgenic medaka, while X. malinche adgre5 expression did not exhibit melanoma suppressive activity in medaka. These findings showed that adgre5 is a natural melanoma suppressor and provide new insight in melanoma etiology.