Reimagining Preclinical Research: Zebrafish Models Driving Advances in Pharmacology and Toxicology

2.1 Zebrafish growth cycle

A common model organism in scientific studies, especially in developmental biology and genetics, is the little freshwater zebrafish. Figure 2 illustrates how the zebrafish development cycle may be broken down into numerous important phases.¹⁷ Understanding the growth cycle of zebrafish is crucial for both laboratory studies and ecological assessments.¹⁸

Figure 2. The diagram illustrates the progression of zebrafish embryogenesis, beginning with cleavage and advancing through blastula and gastrula stages, followed by larval development.

The blue side represents embryonic stages within the first 24 hours, while the other side shows subsequent larval and juvenile growth leading to the adult fish.

Zebrafish exhibit external fertilization, where eggs are laid and fertilized in the water. The fertilization process occurs when a male fish releases sperm over the eggs laid by a female. After fertilization, the eggs begin to develop rapidly.¹⁹ Within approximately 36 hours, precursors to all major organs appear, and the embryo becomes transparent, allowing for easy observation of developmental processes.²⁰ Hatching occurs around 48 hours post-fertilization (hpf ), at which point the embryos transition into free-swimming larvae. This stage is characterized by the absorption of the yolk. After hatching, zebrafish enter the larval stage, which lasts until about 14 days post-hatching. They experience major morphological changes at this time, including the development of adult fin structures and the disappearance of the larval fin fold. The larvae are extremely sensitive to their surroundings, and elements like temperature and water quality can have an impact on their growth.¹⁸ From about 14 to 60 days, zebrafish are considered juveniles. They continue to grow and develop more distinct adult features. This stage is critical for establishing social hierarchies and behaviours, which can affect growth rates and health.²¹ Zebrafish mature and reproduce within 3-4 months, enabling rapid multigenerational studies. Males are typically more brightly colored with distinct markings.²² Adult zebrafish can live for up to 5 years under optimal conditions. They are capable of spawning frequently, with females laying hundreds of eggs every few days. This prolific spawning behaviour is advantageous for research purposes, allowing for the collection of large numbers of embryos for experimental use.²³

2.2 Differences in male and female zebrafish

Zebrafish are widely used in genetics, developmental, and behavioral research. Males and females differ in appearance, behavior, and physiology, which can affect outcomes. Table 1 and Figure 3 summarize these key differences.

Figure 3. Comparison of males and females showing distinctions in coloration, body shape, activity, metabolite profiles, and brain gene expression.

2.3 Embryogenesis and organogenesis

Zebrafish are ideal for studying embryogenesis and organogenesis due to their quick development and transparent embryos. External fertilization enables real-time visualization of cleavage, gastrulation, and organ formation (heart, kidney, endocrine tissues). Embryogenesis in zebrafish begins with external fertilization, leading to cleavage and the formation of a blastula and gastrula. Fluorescence microscopy reveals cell migration and differentiation, while key pathways like Wnt, Notch, and Hedgehog regulate these processes.²⁹

In zebrafish, several organs develop sequentially. The heart begins to form around 24-hpf and is fully functional by 48-hpf. It develops from mesodermal precursors through a series of morphogenetic movements.³⁰ Zebrafish develop pronephros, which is the first kidney structure, followed by the mesonephros. The pronephros consists of a simple structure with two nephrons, while the mesonephros develops more complex nephron structures during larval stages, supporting ongoing nephrogenesis.³¹ The inter-renal gland, the teleostean equivalent of the adrenal gland, forms alongside the embryonic kidney. Recent studies have identified genes involved in the development and steroidogenesis of this organ, revealing similarities between zebrafish and mammalian organogenesis ( Figure 4).³² Zebrafish also provide insights into the effects of genetic mutations and environmental factors on organ development. The use of transgenic lines and gene knockdown techniques has enabled researchers to dissect the molecular mechanisms underlying organogenesis, revealing conserved pathways that are critical for understanding vertebrate development.

Figure 4. Representative histological images showing the anatomical localization and microscopic structure of key zebrafish organs, including the heart, brain, kidney, gills, liver, pancreas, testes, and musculoskeletal tissue.

3.1 Acute toxicity tests

Acute toxicity testing in zebrafish has gained prominence as an efficient and relevant alternative to traditional methods involving adult fish, particularly in assessing chemical safety. The zebrafish model, specifically its embryos and larvae, is utilized in the Zebrafish Embryo Toxicity Test (FET) and the Zebrafish Larvae Acute Toxicity Test (FLT), which evaluate developmental endpoints and acute toxicity, respectively.³⁵ These tests offer advantages such as rapid development, transparency, cost-effectiveness, and high throughput, making them valuable tools in chemical risk assessment, although they may have limitations in detecting neurotoxicants.³⁶ The FLT test exposes larvae to toxicants at a later stage, whereas the FET test concentrates on early embryo development. These two assays provide partially overlapping toxicity profiles and unique information, indicating that their combination can enhance the use of zebrafish embryos and larvae as standard toxicity testing models.³⁷ The use of zebrafish embryos to evaluate acute toxicity is described in the OECD TG-236 guideline. Coagulation, lack of somite development, absence of heartbeat, and non-detachment of the tail are the four main endpoints that the test focuses on. In the extended FET Test, beyond the standard endpoints, additional morphological observations can be included to identify different classes of toxicants, although specificity is higher at sublethal concentrations.^38,39 FLT uses zebrafish larvae for four days post-fertilization and is exposed for 48 hours in 6-well plates. It has shown high correlation with traditional acute fish toxicity tests and is simpler to implement than the FET test.⁴⁰ A modified version of the OECD 236 test uses 4-128 cell stage embryos in a 96-well microtiter plate, improving testing efficiency and throughput. This method has shown good correlation with the standard OECD 236 method and is less time-consuming.⁴¹

3.2 Chronic toxicity tests

Chronic toxicity testing in zebrafish is increasingly recognized for its effectiveness in evaluating the long-term effects of chemical exposure, leveraging the species' rapid development and genetic similarities to humans.⁴² Zebrafish can be subjected to various chronic exposure protocols, often involving prolonged exposure to test substances from early life stages through adulthood. This approach allows researchers to assess a range of toxicological endpoints, including developmental, reproductive, and behavioural impacts.⁴³ The transparency of the zebrafish model throughout early development makes it very useful for observing physiological changes and any harmful consequences in real time. Additionally, research has demonstrated that long-term exposure can yield important insights into toxicity processes, such as neurotoxicity and endocrine disruption. It offers a more thorough comprehension of chemical safety in aquatic ecosystems and its consequences for human health.¹⁵

3.2.1 Developmental toxicity

Developmental toxicity testing in zebrafish has emerged as a critical approach for evaluating the effects of chemicals on embryonic development, offering a high-throughput and ethically sound alternative to traditional mammalian models. Recent studies have validated the zebrafish developmental toxicity assay by demonstrating its ability to predict teratogenic effects with high sensitivity and specificity; for instance, a study found that 74.2% of known teratogenic compounds were accurately identified using this model, highlighting its reliability in screening potential developmental toxicants.⁴⁴

3.2.2 Neurotoxicity

Neurotoxicity testing in zebrafish has become essential in pharmaceutical research for evaluating the neurotoxic effects of drugs and environmental chemicals. Various behavioural assays can be employed to assess neurotoxicity, including locomotor activity tests and assessments of anxiety-like behaviours, which have been shown to correlate well with mammalian responses. Studies indicate that exposure to neurotoxicants, such as heavy metals and certain pharmaceuticals, can lead to significant behavioural changes and alterations in neural morphology in zebrafish.⁴⁵

3.2.3 Cardiotoxicity

Zebrafish cardiotoxicity testing is useful for evaluating the possible cardiac effects of pharmacological medications and phytomolecules. Using zebrafish models, various endpoints such as heart rate, contractility, and structural abnormalities can be evaluated, providing insights into the mechanisms of drug-induced cardiotoxicity.⁴⁶ Studies have demonstrated that zebrafish models can effectively detect the cardiotoxic effects of chemotherapeutic agents like doxorubicin, recreational drugs, and environmental pollutants.⁴⁷ Furthermore, zebrafish have been used to screen for cardioprotective compounds, such as angiotensin receptor blockers and beta-blockers, which can ameliorate drug-induced heart dysfunction.⁴⁸

3.2.4 Hepatotoxicity

Zebrafish hepatotoxicity testing has emerged as a significant technique for assessing the liver toxicity of medications and phytomolecules, providing a high-throughput and morally sound substitute for conventional animal models. Various studies have demonstrated that exposure to hepatotoxic compounds, such as acetaminophen,⁴⁹ and certain herbal extracts, like Polygonii multiflori, can induce significant liver damage in zebrafish, characterized by histopathological changes, increased levels of liver enzymes, and alterations in liver morphology.⁵⁰ For instance, fraxinellone, a compound derived from Cortex Dictamni, has been shown to cause hepatotoxicity through oxidative stress mechanisms, leading to apoptosis and liver degeneration in zebrafish larvae.⁵¹

3.2.5 Nephrotoxicity

Due to their transparent bodies and rapid development, zebrafish larvae allow for real-time observation of kidney function and morphology, making them ideal for assessing nephrotoxic effects. Studies have shown that exposure to nephrotoxic agents such as gentamicin and aristolochic acid leads to significant renal damage, characterized by tubular injury, necrosis, and impaired kidney function.⁵² For example, studies showed that exposure to gentamicin caused renal tubules to widen and elevated the expression of biomarkers for kidney damage, including kidney injury molecule-1 (kim-1) and heme oxygenase 1 (hmox1). Additionally, zebrafish models have been used to screen for different compounds' protective properties, which has helped identify prospective therapeutic agents against drug-induced kidney damage.⁵³

3.2.6 Endocrine toxicity

Endocrine toxicity testing in zebrafish has become a crucial approach for evaluating the effects of pharmaceuticals and phytomolecules on the endocrine system, particularly in the context of endocrine-disrupting compounds (EDCs). Research has shown that exposure to EDCs, such as bisphenol A and certain pesticides, can disrupt sexual differentiation, reproductive functions, and hormone signaling pathways in zebrafish.⁵⁴ For example, studies have demonstrated that exposure to 17α-ethinylestradiol leads to feminization of male zebrafish and altered reproductive behaviors.⁵⁵

3.2.7 Environmental and industrial toxicity studies

An important technique for determining how chemicals and contaminants affect aquatic ecosystems and human health is environmental and industrial toxicity testing. To assess the impact of several environmental pollutants, including heavy metals, pesticides, and nanomaterials, on embryonic development and behaviour, the zebrafish embryo toxicity test (ZET) has been used extensively.⁵⁶ For instance, studies have shown that exposure to polychlorinated biphenyls (PCBs) and heavy metals can lead to significant morphological abnormalities and behavioural changes in zebrafish larvae.⁵⁷

4.1 Use of zebrafish embryos and larvae for pharmacological screening

Zebrafish screens have identified several compounds in early clinical trials for various conditions. Zebrafish embryos are suitable for high-throughput screening in 96- or 48-well plates, allowing for cheaper and efficient screening of compounds. Treatments like EPX-100 (clemizole) for Dravet⁵⁸ and Trifluoperazine for Diamond-Blackfan⁵⁹ are also under investigation. Additionally, DB-041, aimed at treating hearing loss after aminoglycoside antibiotic use, is in Phase-I trials. Zebrafish models are also being used to create treatments for lymphatic diseases, melanoma, and graft-versus-host disease (GvHD). Pathogens are delivered into zebrafish embryos either by immersion or microinjection, making them useful as infection models. Five weakly water-soluble nitronaphthofuran compounds were tested for in vivo toxicity and effectiveness using the zebrafish embryo TB model.⁶⁰

The zebrafish model allows researchers to investigate the pharmacokinetics and pharmacodynamics of drugs at a cellular level. Blood sampling from zebrafish larvae allowed for the measurement of paracetamol and its metabolites. The PK parameters, including clearance and distribution volume, correlated well with those in higher vertebrates. One challenge in using zebrafish, especially larvae, is the difficulty in measuring internal drug exposure. Accurate quantification of drug and metabolite levels is essential for reliable PK and PD studies.⁶¹ A case study demonstrated the use of zebrafish larvae to assess the ADME profile of naloxone, an opioid antagonist. The research confirmed that zebrafish metabolize naloxone into the same major metabolites observed in humans. Furthermore, the spatial distribution of naloxone within the larvae mirrored known human pharmacological data, underscoring the model's translational relevance.⁶² In the context of Traditional Chinese medicine (TCM), researchers have developed an integrated high-throughput strategy using zebrafish to study the ADME characteristics of complex herbal formulations. Utilising techniques like ultra-performance liquid chromatography-high-resolution mass spectrometry (UPLC-HRMS) and desorption electrospray ionisation-mass spectrometry (DESI-MS) imaging, the study successfully identified and characterised multiple metabolites, demonstrating the model's efficacy in complex compound analysis.⁶³ Research comparing immersion and intrayolk microinjection methods in zebrafish embryos revealed insights into compound absorption and distribution. Evidence suggests that not all small molecules are equally absorbed, even if immersion of ZFE is the primary exposure method utilized. This might lead to false-negative readouts and inaccurate findings. Fluorescent dyes administered through these methods allowed for real-time imaging of pharmacokinetics, highlighting the model's utility in studying compound behaviour over time.⁶⁴

4.2 Inflammation, pain, and its modulation in zebrafish models

Zebrafish possess a range of nociceptors that respond to noxious stimuli, similar to mammals. Research has identified various molecular pathways involved in pain perception, including the opioid system, transient receptor potential (TRP) channels, and acid-sensing ion channels (ASIC).^65–67 Zebrafish models have been used to test various analgesics, including opioids, NSAIDs, and local anaesthetics, demonstrating their effectiveness in reducing pain-induced behaviours. Recent studies have shown that various analgesics acting on COX-1 or COX-2 can effectively alleviate pain-related behaviours in zebrafish following induced injuries like fin amputation.⁶⁷ Studies have shown that female zebrafish may exhibit more pronounced pain-related behaviours compared to males, indicating the importance of considering sex as a variable in pain research.⁶⁸ Essential oils from the Piper genus have shown potential antinociceptive and anti-inflammatory effects in zebrafish models.⁶⁹ Similar to adult zebrafish and other vertebrates, larval zebrafish react to unpleasant stressors, and analgesics can lessen the impact of nociception on activity.^70–72

4.3 Zebrafish models to screen drugs for neurological disorders

4.3.1 Zebrafish models to screen drugs for neurodevelopmental disorders

Autism: Deficits in social interaction, hyperactivity, anxiety, communication problems, repetitive behaviors, and impaired sensory processing are characteristics of autism. Zebrafish, with their genetic tractability, clear development, preserved brain architecture, and aptitude for high-throughput screening, have lately become a potent supplementary system to rodent models, which have long been at the forefront of ASD research.⁷³ The Simons Foundation Autism Research Initiative (SFARI) has validated 12 ASD risk genes with established zebrafish mutant lines, enabling cross-species comparisons with human and mouse models to identify conserved neurodevelopmental mechanisms.⁷³ For example, oxytocin treatment during the larval stage, particularly at 50μM for 48 hours, significantly ameliorated autism-like behaviors in the zebrafish model. This behavioural improvement was accompanied by the upregulation of key autism-related genes such as Shank3a, Shank3b, and the oxytocin receptor.⁷⁴ Similarly, tanc2 knockout zebrafish exhibited increased brain size, selective expansion of glutamatergic neurons, and impaired social preference phenotypes consistent with TANC2-associated ASD. These changes reflect an underlying excitatory/inhibitory (E/I) imbalance, a common feature in many neurodevelopmental disorders.⁷⁵ Other gene models further underscore the utility of zebrafish in elucidating ASD mechanisms. shank2b-/- zebrafish displayed core ASD-like features, including reduced social and kin preference, stereotypic behaviors, and sensory hypersensitivity, which were linked to GABA receptor downregulation and increased seizure susceptibility, implicating GABAergic dysfunction as a major contributor to ASD symptoms.⁷⁶ The adult brain's downregulation of synaptic structure and function genes was also linked to poor shoaling, decreased social interaction, and motor coordination problems caused by haploinsufficiency of setd5.⁷⁷ In ctnnd2b heterozygous zebrafish, ectopic Isl1-positive neurons and reduced GABAergic populations disrupted neuronal differentiation in the forebrain, leading to hyperactivity that mirrors CTNND2-related neurodevelopmental disorders.⁷⁸ Loss of GluN2B, encoded by grin2B, resulted in selective social deficits without broader neurodevelopmental impairments. While grin2B-/- larvae retained normal learning and locomotor function, they exhibited reduced inhibitory neuron marker expression in the subpallium, a region implicated in ASD. This highlights the potential of zebrafish for modeling NMDA receptor-related autism in ways that are not possible in lethal rodent knockouts.⁷⁹ In a valproic acid (VPA)-induced ASD model, acute treatment with duloxetine (DLX) from 4.5 to 6 dpf reversed hyperactivity, anxiety, and social deficits. DLX also normalized “acetylcholine esterase (AChE)” activity and “Akt-mTOR signaling”, yielding persistent improvements into adulthood.⁸⁰ Another study developed a comprehensive VPA-induced zebrafish model that integrates behavioural despair assays, molecular alterations, and predictive endpoints into a unified system. Using 7-day-old larvae for early screening and 21-day-old juveniles for assessing social behaviour, this paradigm supports scalable and rapid identification of lead compounds.⁸¹

ADHD: Recent zebrafish studies have illuminated novel mechanisms and therapeutic leads for attention deficit hyperactivity disorder (ADHD). CFTR-deficient zebrafish exhibit reduced dopaminergic neuron populations and hyperactivity, both of which are ameliorated by CFTR modulators such as lumacaftor and ivacaftor. Separately, amlodipine, a calcium channel blocker, reduced hyperactivity and impulsivity in both zebrafish and rat ADHD models by targeting L-type calcium channel genes, which have been implicated in ADHD via human GWAS studies.⁸² Environmental factors also play a significant role: embryonic exposure to bisphenol A (BPA) and perfluorooctanesulfonic acid (PFOS) induced dopamine dysfunction, reduced GABAergic neuron density, and ADHD-like behaviors in zebrafish, highlighting the neurodevelopmental risks of endocrine disruptors during pregnancy.⁸³ CRISPR-generated chmp7+/- zebrafish modeled an ADHD-associated risk variant and displayed significant hyperactivity at 6 dpf, along with reduced brain volume. These early-stage hyperactivity phenotypes correlated with reduced chmp7 transcript levels, mimicking the human risk allele profile. Importantly, methylphenidate treatment successfully reversed the hyperactivity, supporting the model’s pharmacological validity.⁸⁴ Moderate embryonic exposure to estrogen and the environmental pollutant bisphenol A (BPA) in zebrafish induced distinct neurodevelopmental phenotypes that parallel features of ADHD and ASD, respectively. Estrogen exposure led to hyperactivity and impulsivity, linked to increased Cyp19a1b and reduced tyrosine hydroxylase expression, while BPA exposure caused reduced GABAergic neuron populations, particularly in the midbrain, along with social deficits, repetitive behaviors, and heightened escape responses.⁸⁵ Using adgrl3.1-/- zebrafish larvae as a genetic model of ADHD, researchers identified robust hyperactivity that was responsive to standard non-stimulant treatments, albeit with sleep-related side effects.⁸⁶

4.3.2 Zebrafish models to screen drugs for neurodegenerative disorders

Alzheimer’s: Their neurogenic potential, driven by neuron-glia interactions mediated through IL-4, serotonin, and BDNF-NGFR signaling, supports studies of synaptic plasticity and regeneration. Gene manipulations of AD-associated targets such as ABCA7, BACE2, and TNFαIP1 have revealed enhanced synaptic resilience via BDNF and neuropeptide Y pathways.^87,88 Behavioural paradigms using agents like aluminium chloride (AlCl₃), D-galactose, scopolamine, streptozotocin (STZ), and okadaic acid (OKA) replicate key AD features, including amyloid aggregation, tau hyperphosphorylation, neuroinflammation, and sensorimotor decline. Spatial learning and memory are assessed through T-maze and Y-maze assays, with evidence of cognitive rescue from botanical interventions. For instance, Eulophia ochreata extract improved spontaneous alternation and reduced decision latency in scopolamine-induced zebrafish models, while Carica papaya seed extract reversed OKA-induced deficits and restored Purkinje cell architecture.⁸⁹ A range of natural compounds, neochlorogenic acid, apigenin, tangeretin, andrographolide, scoparone, and herbal agents like Ocimum sanctum, exhibited neuroprotection through Nrf2 activation, mitophagy enhancement, and GSK-3β or TNF-α inhibition.^90,91 Multi-target-directed ligands (MTDLs) such as apigenin-rivastigmine hybrids, donepezil analogs (e.g., DK02, compound 6a), and chromone derivatives showed efficacy through cholinesterase inhibition, Aβ aggregation blockade, and metal chelation with BBB permeability.^92–94

Transgenic zebrafish expressing human APP variants replicate hallmark Aβ deposition, cerebrovascular pathology, and behavioural phenotypes. Advanced nanocarriers, including PAMAM dendrimers, carbon dots, exosome-mimetic liposomes, and mesenchymal stem cell-derived vesicles, enable targeted CNS delivery of curcumin, memantine, and RNAi-based therapeutics with reduced systemic toxicity.⁹⁵ Platforms like LCSF-NR siRNA nanorings effectively silence BACE1 and reduce amyloidogenesis in vivo. Imaging breakthroughs using HBT-PON, PRS, and ESIPT-based fluorescent probes allow real-time tracking of oxidative stress, neuroinflammation, and trace metal accumulation in the zebrafish brain.⁹⁶ Gene-editing tools such as the PINE-CONE prime editor facilitate SNP-specific modeling of AD risk alleles, while systems-level analyses have revealed early mechanistic events such as serotonin-linked neuroinflammation, oligodendrocyte dysregulation, and ferroptosis.⁹⁷ Combined AD-comorbidity models incorporating osteoporosis or metal toxicity allow for synergistic drug screening with agents like notopterol and osthole, while gut-brain axis studies highlight microbial shifts induced by Aβ accumulation.⁹⁸

Parkinson's: Zebrafish possess a dopaminergic system analogous to humans, including tyrosine hydroxylase-positive neurons, enabling modeling of substantia nigra-like degeneration. Zebrafish also exhibit a functional blood-brain barrier, enabling translational screening of CNS-active drugs and nanoparticle delivery systems. Neurotoxin-based models using MPTP, rotenone, 6-OHDA, and manganese reliably induce mitochondrial dysfunction, oxidative stress, and dopaminergic cell loss, mirroring hallmark PD pathology.^72,99 Genetic models targeting pink1, parkin, dj-1, or lrrk2 further recapitulate mitophagy deficits and proteostatic imbalance observed in familial PD.^100,101 Transparent larvae enable live imaging of neurodegeneration, reactive oxygen species (ROS), and neuronal apoptosis using biosensors and fluorescent dyes. Behaviourally, zebrafish display PD-relevant phenotypes including bradykinesia, postural instability, and anxiety-like responses, assessable through high-throughput locomotor and reflex assays.¹⁰² Natural compounds like Naringenin and hesperidin have shown neuroprotective potential in the 6-OHDA zebrafish model by downregulating LRRK2, gsk3β.^103,104 Celastrus paniculatus extract reversed motor and memory deficits and restored DJ-1 and LRRK2 expression in rotenone-induced models.¹⁰⁵ Synthetic agents such as salicylaldehyde, benzoylhydrazone, and Granulathiazole A effectively counter ferroptosis and ROS-mediated damage via Nrf2/HO-1 pathway activation.^106,107 Nanocarrier systems, including DNA tetrahedral nanocages, have facilitated targeted dopamine delivery, enhancing neuronal recovery.¹⁰⁸

Epilepsy: Zebrafish central nervous system shares conserved neurotransmitter systems with mammals, including GABAergic, glutamatergic, and dopaminergic circuits. Chemically induced seizure models using agents like pentylenetetrazol (PTZ), kainic acid, or pilocarpine reliably reproduce electrographic and behavioural seizure phenotypes, including hyperlocomotion, head twitching, and convulsive swim bursts. These are quantifiable using automated tracking systems, enabling high-throughput screening of both synthetic and natural anticonvulsants. Mutants like scn1lab, kcna1a, and pcdh19 show spontaneous seizures and hyperexcitability, mimicking syndromes such as Dravet and EA1 syndrome, while CRISPR/Cas9 tools now allow precise modeling of human epilepsy-linked mutations.^109–112 High-throughput drug development is made possible using adult fish to test new compounds and investigate pharmaco-resistant seizures. This also encourages the use of zebrafish in personalized medicine.¹¹³ Live calcium imaging and optogenetics in transparent zebrafish larvae further permit real-time visualization of seizure onset and neural hyperexcitability.¹¹⁴ Compounds like valproate, cannabidiol, baicalin, and resveratrol have demonstrated efficacy in zebrafish epilepsy models, validating the system for translational drug discovery.^{112,115–118}

Anxiety: They exhibit homologous neurotransmitter systems, including GABAergic, serotonergic, and dopaminergic pathways, quite closely paralleling those involved in human anxiety disorders.¹¹⁹ Using proven paradigms such as the “novel tank diving test (NTD)”, “open field test (OFT)”, and “light/dark box test (LDT)”, zebrafish exhibit measurable anxiety-like responses, such as thigmotaxis, bottom-dwelling, and light/dark zone preference.^120,121 These behaviors are modifiable by known anxiolytics such as SSRIs, benzodiazepines, and herbal extracts, demonstrating high predictive validity. Stress can be induced through environmental (e.g., novelty, isolation) or chemical (e.g., neurotoxicants like benzo [a] pyrene, valproic acid, bisphenol),¹²² while genetic models targeting anxiety-related genes (e.g., nr4a2) offer insight into molecular mechanisms. Agents like caffeine, a central nervous stimulant and adenosine receptor antagonist, exacerbate bottom-dwelling behaviour and startle responses in the NTD, resembling anxiety phenotypes in adult fish.¹²³ Similarly, MK-801, an NMDA receptor antagonist, elicits erratic swimming and avoidance responses indicative of heightened fear circuitry activation.¹²⁴ The alarm substance assay, using extracts from injured conspecifics, provides an ethologically relevant anxiety model.¹²⁵ It has been confirmed that exposure causes freezing and an increase in cortisol in both strains and sexes. According to research, female zebrafish were more active and nervous than males; melatonin caused anxiolytic reactions in both sexes, whereas diazepam only caused anxiolytic reactions in males.¹²⁶ Another robust inducer is corticotropin-releasing factor (CRF), which activates the hypothalamic-pituitary-inter-renal (HPI) axis and mimics endogenous stress hormone release; CRF-treated zebrafish display sustained thigmotaxis and elevated cortisol.¹²⁷

Depression: A versatile model for depression research, owing to its conserved monoaminergic systems, including serotonin, dopamine, and norepinephrine pathways that mirror key aspects of human neurobiology. Their functional HPI axis enables quantification of cortisol responses to chronic stressors, facilitating the study of neuroendocrine dysregulation commonly observed in depressive disorders.¹²⁸ Zebrafish display measurable depression-like behaviors, such as reduced exploration, social withdrawal, and increased bottom-dwelling, assessed through validated paradigms like the novel tank diving test, social preference test, open field test, and light/dark box test. Depression can be experimentally induced via pharmacological agents (e.g., reserpine, bisphenol), environmental stressors (e.g., social isolation, novelty exposure), or genetic modifications targeting mood-regulatory genes like nr4a2, akt3, and gnpda2. These models have high translational relevance, with zebrafish demonstrating robust responses to conventional antidepressants like fluoxetine (SSRIs, SNRIs, tricyclics).¹²⁹

4.4 Zebrafish models to screen anticancer drugs

Zebrafish larvae and adults serve as powerful platforms for xenotransplanting human cancer cells, enabling real-time studies of tumor progression and therapy responses. Advances with CRISPR/Cas9 and transgenic lines model key mutations (TP53, KRAS, BRAF) across cancers, while ZTX and zPDX models offer rapid, scalable, and physiologically relevant alternatives to murine models, aided by optical transparency and immunodeficient lines for high-throughput imaging. In solid tumours such as rhabdomyosarcoma (RMS), the use of Tg (flk1:GFP) zebrafish embryos injected with RMS cells enabled rapid screening of both chemotherapeutics and targeted agents, closely mirroring clinical efficacy.¹³⁰ In colon cancer, the cannabinoid anandamide (AEA) exerted anti-tumour and anti-angiogenic effects not through direct cytotoxicity on HCT116 cells, but by modulating the zebrafish tumour microenvironment, highlighting the model’s value in dissecting host-tumour interactions.¹³¹

In non-small cell lung cancer (NSCLC), a combined mouse-zebrafish PDX approach using cryopreserved tissue predicted drug responses and metastatic dissemination within 3 days, outperforming some clinical tests with 91% sensitivity for lymph node involvement.¹³² For glioblastoma (GBM), a highly invasive and treatment-resistant malignancy, intracranial zebrafish patient-derived orthotopic xenografts (zPDOX) retained both histological and molecular fidelity to original tumours and allowed individualized drug screening within 20 days. Single-cell RNA sequencing confirmed conservation of zebrafish BBB properties, validating their relevance in neuro-oncology. Paediatric cancer studies, including the INFORM trial, integrated zPDX models with organoids, spheroid cultures, and mouse PDXs to identify actionable drug sensitivities and effective therapeutic combinations in relapsed, heterogeneous tumours.¹³³ In hematologic malignancies, SCID zebrafish lines have enabled engraftment and drug screening of chronic myelogenous leukaemia (CML) and acute myeloid leukaemia (AML) cells.¹³⁴ These models demonstrated short-term survival of human hematopoietic cells and validated the in vivo efficacy of agents such as imatinib, cytarabine, azacitidine, and arsenic trioxide. Zebrafish avatars have shown strong clinical alignment in co-clinical trial settings. For instance, in pancreatic ductal adenocarcinoma (PDAC), zPDX responses to chemotherapy accurately predicted relapse and disease-free survival.¹³⁵ In rectal cancer, zebrafish xenografts subjected to radiotherapy effectively distinguished radiosensitive from radioresistant tumours based on apoptotic and DNA damage markers.¹³⁶

4.5 Zebrafish models to screen drugs for cardiovascular diseases

Advanced platforms such as automated heartbeat quantification systems (e.g., AISS) and wireless ECG telemetry have enabled real-time, anaesthetic-free cardiac assessment in both larvae and adults. CRISPR/Cas9 mutagenesis has facilitated the creation of cardiovascular disease models for functional genomics and therapeutic testing. Zebrafish have been used to evaluate the cardiotoxicity of tyrosine kinase inhibitors like ponatinib.¹³⁷ Microfluidic chip-based assays now allow precise anti-thrombotic drug testing, while deep learning models support rapid video-based analysis of cardiac rhythms.¹³⁸ Notably, the ZebraReg platform has identified cardiac regeneration regulators using transgenic lines.¹³⁹ These models have proven valuable for investigating metabolic regulators like isosteviol and senkyunolide I for lipid modulation and oxidative stress.¹⁴⁰ Heart rate (HR) and heart rate variability (HRV), which are markers of cardiac function, are utilized to evaluate drug-induced cardiotoxicity in zebrafish. To better understand disease causes and medication effects, zebrafish models have been created for a number of cardiovascular disorders, including atherosclerosis, congenital heart abnormalities, and dilated cardiomyopathy. HR and HRV in zebrafish models have been measured using zebrafish heartbeat detecting techniques, signal analysis technologies, and reputable commercial software like “Rvlpulse”, “LabVIEW”, and “ZebraLab”.¹⁴¹ Fully automated in vivo picture capture and analysis to extract pertinent cardiovascular data, such as heart rate, arrhythmia, AV blockage, ejection fraction, and blood flow, are made possible by high-throughput screening systems.¹⁴² “pyHeart4Fish” is a new Python-based, platform-independent application designed to automatically quantify cardiac chamber-specific metrics in zebrafish embryos, including heart rate, contractility, arrhythmia score, and conduction score.¹⁴³

4.6 Zebrafish models to screen drugs in metabolic and endocrine disorders

Humans and zebrafish have many genetic and physiological traits in common, especially regarding organs involved in metabolic control, such as the liver, adipose tissue, and the pancreas. Microbiome interactions, β-cell regeneration, and AI-driven analytics are now included in the zebrafish diabetes simulation, going beyond traditional glucose control. Zebrafish-microbiome co-models have illuminated how gut dysbiosis contributes to insulin resistance and inflammation, enabling screening of probiotics and symbiotic with anti-diabetic potential. Transgenic zebrafish lines expressing fluorescent reporters for insulin, Akt, and FoxO1 signaling now permit real-time visualization of pancreatic responses to therapeutic agents.¹⁴⁴ Furthermore, AI-based imaging tools and microfluidic platforms are increasingly employed to assess islet morphology, glucose uptake, and metabolic flux in high-throughput settings. Zebrafish models effectively mimic human diabetes, including type 2 diabetes and maturity-onset diabetes of the young (MODY), showing similar pancreatic structure and glucose homeostasis mechanisms.¹⁴⁵ Zebrafish models of insulin resistance and liver-specific insulin receptor knockdowns are used to study type-2 diabetes. Overnutrition in zebrafish larvae and juveniles can mimic human obesity, activating pathways like mTOR and increasing β-cell mass. Research using zebrafish has identified new therapeutic targets, such as the centromere protein X (CENPX), which, when inhibited, ameliorates hyperglycaemia and upregulates insulin levels.¹⁴⁶ They also replicate human lipid metabolism disorders, such as hyperlipidaemia and atherosclerosis, through genetic and dietary manipulations. Zebrafish have a simpler gut microbiota than humans, sharing some bacterial phyla but lacking the diversity and complexity of the human gut. They produce different digestive enzymes, with higher activity of proteases and lipases, affecting nutrient digestion differently. Zebrafish also have a higher metabolic rate due to their smaller size and ectothermic nature. While their lipid metabolism pathways are similar to humans, zebrafish store more fat in the liver and visceral areas rather than subcutaneously.¹⁴⁷

4.7 Zebrafish models to screen drugs for renal diseases

Researchers can investigate the underlying processes and find possible therapeutic molecules by modeling hereditary kidney disorders in zebrafish. Zebrafish larvae have substantial similarities to mammalian models in terms of drug metabolism and kidney injury, making them useful models for identifying drug-induced nephrotoxicity. Zebrafish have become an increasingly powerful model for renal disease research and drug screening owing to their conserved kidney structure, genetic tractability, and suitability for high-throughput assays. The zebrafish pronephros mirrors mammalian kidney function, enabling studies of glomerular filtration, tubular processing, and proteinuria, while genetic tools such as CRISPR/Cas9 and transgenic fluorescent reporters facilitate mechanistic insights and screening pipelines. Applications include modeling drug-induced nephrotoxicity (gentamicin, cisplatin), acute kidney injury via nephrotoxin injection or genetic disruption, and genetic kidney disorders such as polycystic kidney disease, with outputs validated against mammalian data.^148,149 Automated imaging and multiparametric pipelines now allow parallel assessment of renal morphology, function, and filtration in live larvae, supporting large-scale chemical library screening.¹⁵⁰

4.8 Zebrafish models to screen drugs for liver diseases

Despite structural differences, zebrafish livers share significant functional and genetic similarities with human livers, including comparable cytochrome P450 enzyme activity, which is crucial for drug metabolism.¹⁵¹ Zebrafish models are effective for assessing hepatotoxicity by exposing embryos and larvae to compounds, allowing real-time imaging of liver function. Researchers have identified drugs that cause liver damage, showcasing zebrafish's potential for evaluating drug-induced liver toxicity and discovering safer alternatives. Gender-specific variations in hepatotoxic reactions have been seen in zebrafish used to assess the hepatotoxicity of traditional Chinese medications, including Emodin-8-O-β-D-glucopyranoside (Em8G).¹⁵² By using genetic models or nutritional modification to create fatty liver phenotypes in zebrafish, non-alcoholic fatty liver disease (NAFLD) may be studied, and drugs that decrease lipid accumulation can be found. Additionally, zebrafish model alcoholic liver disease by exposing them to ethanol, resulting in inflammation and steatosis. Screening various compounds has revealed protective effects against alcohol-induced liver damage, aiding in the search for potential treatments. In parallel, zebrafish models of fatty liver disease (NAFLD/NASH) have recapitulated hallmark human features: hepatic lipid accumulation, ballooning degeneration, ER stress, ferroptosis, and gut-liver axis dysregulation.¹⁵³ Tools such as Oil Red O staining, RNA-seq, and fluorescent probes (e.g., ONOO^-/viscosity dual-channel sensors) have enabled mechanistic tracking and therapeutic evaluation.¹⁵⁴

4.9 Zebrafish models in regenerative medicine

Zebrafish have been identified as highly regenerative, capable of regrowing amputated fins, lesioned brain, retina, spinal cord, and heart. Studies have shown that these cardiomyocytes can dedifferentiate, re-enter the cell cycle, and proliferate to replace lost tissue. This process allows zebrafish to regenerate up to 20% of their ventricular mass within approximately two months post-injury, without scarring or arrhythmias.¹⁵⁵ Effective cardiomyocyte repopulation and scar prevention depend on early responses to cardiac injury, such as coronary revascularization aided by VEGFC signaling. Gene expression programs that support cardiomyocyte dedifferentiation and proliferation are driven by changes in chromatin accessibility, which are controlled by transcription factors like AP-1.^156,157 They are also used to model liver injury, providing insights into the cellular and molecular mechanisms of liver regeneration with potential applications for human therapies. Studies indicate that within seven days post-surgery, zebrafish can recover their liver mass to normal levels, often exceeding the original size before fine-tuning back to baseline over the following week.¹⁵⁸ The regeneration process in zebrafish involves several key signaling pathways. For instance, the BMP and FGF signaling pathways are crucial during the early stages of liver regeneration. Additionally, hormone activity, ribosome biogenesis, and antioxidant activity also play a part in liver regeneration.¹⁵⁹ Zebrafish biliary epithelial cells can transdifferentiate into hepatocytes, a process regulated by signaling pathways such as MAPK, PI3K, and mTOR.¹⁶⁰

Zebrafish can regenerate parts of their central nervous system, including the retina and spinal cord, facilitated by Müller glia and radial glial cells. Upon retinal injury, Müller glia dedifferentiate, re-enter the cell cycle, and produce neuronal progenitor cells (NPCs) that proliferate and differentiate into the lost neurons. Key factors such as Midkine-a and transcriptional regulators like Bromodomain (Brd).^161–163 Zebrafish can regenerate axonal tracts and entire tissues in the CNS, making them a valuable model for studying CNS injuries and diseases. The absence of small leucine-rich proteoglycans (SLRPs), which are known to hinder axon regeneration in mammals, allows zebrafish to recover functional capabilities more effectively after CNS damage.¹⁶⁴ Additionally, their fin regeneration model helps researchers understand the stages of tissue regeneration, including wound healing and blastema formation. Zebrafish fin regeneration begins with the formation of a blastema at the injury site, consisting of mesenchymal progenitor cells derived from dedifferentiated osteoblasts and other cell types. A protective wound epithelium forms over the injury, ensuring the undifferentiated state of blastemal cells.¹⁶⁵ The regeneration process balances proliferation and differentiation, with osteoblasts migrating to the injury site to form new bone structures. Several signaling pathways, including Neuregulin-1 (NRG1), Retinoic Acid, NF-κB, and Wnt signaling, regulate this process. Ultimately, the regenerated fin restores its size and morphology, influenced by positional memory within blastemal cells, which guides accurate reconstruction.^166,167