Zebrafish in neurodevelopmental disorders studies: Genetic models and pathological involvement of microglia

Abstract

Neurodevelopmental disorders (NDDs) are a group of brain disorders with a neonatal or early childhood onset and are lifelong. Various factors including genetics, and environmental and immune‐related risk factors have been associated with NDDs. Given the complex nature of these disorders, multiple animal models have been used to investigate their aetiology and underlying cellular and molecular mechanisms. Recently, zebrafish have attracted great attention as an emerging model for studying NDDs. In addition to their easy maintenance, short developmental cycle, ex utero embryonic evolution, and optical clarity, zebrafish have successfully recapitulated phenotypes seen in human genetic disorders. This review explores the growing role of zebrafish in NDD research, by summarizing recently developed zebrafish genetic models for autism spectrum disorder, schizophrenia, and cerebral palsy. We then explore the potential of zebrafish as a model for studying NDDs linked to immune system dysfunction.

The relevance of the pathogenic (de novo or inherited) mutations found in patients with neurodevelopmental disorders can be investigated using zebrafish as an animal model, by functional screening of the target genes and phenotypic assessments during both early development and adulthood.

Abbreviations

ASD

autism spectrum disorder

NDD

neurodevelopmental disorder

What this paper adds

• Zebrafish present a complementary model for investigating dysfunctional pathways underlying neurodevelopmental disorders (NDDs).

• Their unique characteristics provide exceptional settings to study microglia's role in NDDs.

NDDs are an etiologically heterogeneous group of brain disorders that arise from a disturbance in the brain development and manifest themselves in the form of cognitive, communicative, or psycho‐behavioural impairment. ¹ , ² These disorders have been primarily described as ‘disorders usually first diagnosed in infancy, childhood, or adolescence’, pointing towards a prenatal or early postnatal origin of the disruptive events that prevent proper brain development. ³ NDDs encompass a broad range of conditions from the well‐known, best studied disorders like autism spectrum disorder (ASD), intellectual disability, schizophrenia, attention‐deficit/hyperactivity disorder, and cerebral palsy (CP), to recently discovered rare genetic disorders such as SLC6A1 disease, Lessel‐Kreienkamp syndrome, RNU4‐2 syndrome, and LRRC7‐associated NDDs. ⁴ , ⁵ , ⁶ , ⁷ , ⁸

Advances in genome‐wide association studies, large‐scale whole‐exome sequencing, and, more recently, whole genome sequencing have enabled the identification of high‐confidence NDDs risk genes in both coding and non‐coding regions of the genome. These findings highlighted that genetically defined NDDs account for a substantial proportion of NDD cases, ⁸ , ⁹ , ¹⁰ , ¹¹ , ¹² given the involvement of numerous genes in regulating brain function. These include genes encoding cell adhesion molecules (e.g. NEUROLIGIN‐NEUREXIN), signalling proteins (e.g. SYNGAP), synaptic proteins (e.g. SHANK), small noncoding ribonucleic acids (e.g. RNU4‐2), and proteins involved in ribonucleic acid metabolism (e.g. AGO2). ⁵ , ⁸ , ¹³

Nevertheless, while genetic alterations are strongly linked to the onset of NDDs, the influence of environmental and immune risk factors cannot be ignored as, in many cases, the interplay of multiple risk factors complicates the identification of the exact cause of these disorders. ¹ , ¹⁴

To date, a variety of in vitro and in vivo approaches have been employed to study the aetiology and pathological mechanisms underlying NDDs and their associated comorbidities. While the mouse remains the most extensively used animal model, smaller organisms such as the fruit fly and zebrafish have recently emerged as valuable tools for rapidly screening multiple candidate genes and exploring the complex and heterogeneous nature of NDDs. ¹⁵ In this review, we specifically emphasize the contributions of the zebrafish model to NDD research.

ZEBRAFISH

The zebrafish (Danio rerio), a tropical freshwater fish, was first introduced by George Streisinger into biological research in the 1970s. As non‐mammalian vertebrates, zebrafish share significant physiological and genetic similarities with humans. Approximately 70% of human coding genes, including most disease‐associated genes, have a direct functional ortholog in zebrafish. ¹⁶ , ¹⁷ The degree of evolutionary conservation of the non‐coding human genes is, however, lower in zebrafish. ¹⁸

Zebrafish are highly efficient for research because of their rapid generation time, high fertility, and cost‐effective maintenance. Their ex utero development allows for easy embryonic and larval manipulation, while their optically transparent developmental stages provide a unique opportunity to observe organ formation in a living vertebrate. The ease of transgenesis, along with the growing number of fluorescent reporter zebrafish lines, enables real‐time visualization of fluorescently labelled cell behaviour inside intact tissues. Additionally, zebrafish can be genetically manipulated, allowing researchers to screen phenotypes at various developmental stages. Common gene inactivation techniques include transcription activators like effector nucleases, zinc finger nucleases, and, most notably, CRISPR‐associated protein 9, which enables the generation of bi‐allelic knockouts in the F0 animals. Transient gene silencing can also be achieved using small, modified oligonucleotides called morpholinos, while target gene overexpression is possible through messenger ribonucleic acid microinjection. ¹⁹ , ²⁰ , ²¹

Several characteristics make zebrafish an excellent complementary model to traditional mammalian models in neurodevelopmental and neurodegenerative disease research, as well as in neurobehavioral studies (Table 1). ¹⁹ , ²⁰ , ²² , ²³ , ²⁴ These include structural and functional similarities between their brain and that of mammals, neurobehavioral parallels with humans, sensitivity to key neurotropic drugs, and the ability to conduct large‐scale, high throughput behavioural analyses even at early developmental stages. ²⁵ , ²⁶ This model organism has been successfully used to study a range of neurobehavioral conditions, including social anxiety, aggression, hyperactivity, bipolar disorder, avoidance and exploration behaviours, defensive response, fear conditioning, learning and memory, cognition, schizophrenia, attention‐deficit/hyperactivity disorder, epilepsy, and the effects of neuroactive drugs. ²⁶ , ²⁷ Notably, in some cases, zebrafish models exhibit behavioural phenotypes that more closely resemble human conditions compared to mouse models. For instance, a zebrafish model of epilepsy with a GABRA1 loss‐of‐function mutation displays fully penetrant generalized seizures at juvenile stages, closely mirroring the human phenotype, whereas the corresponding homozygous knockout mouse model only exhibits a tremor phenotype ²⁸ (Figure 1).

TABLE 1.

Advantages and limitations of zebrafish as an animal model in brain studies.

Advantages	Limitations
Structural and functional similarities with mammalian brain	Lack of some key brain structures, such as cortex
Fully sequenced genome that contains the orthologs of 60% to 80% of all genes in humans, including over 82% of disease‐associated genes	Duplicated copies of some human genes
Small size, easy breeding and maintenance, high fertility, and rapid life cycle	Different levels of sexual dimorphism due to the absence of sex chromosomes
Ex utero embryonic development and optical transparency of early stages
Real‐time imaging of developmental stages
Genetically modifiable and traceable
Amenable to behavioural studies, even during early developmental stages

FIGURE 1.

Modelling epilepsy in zebrafish. Motion tracks of 5 weeks post‐fertilization epileptic gabra1^‐/‐ and control gabra1^+/+ fish, 1 second before (–1 sec), at the time of (0 sec), and several seconds after the light exposure (+50 sec). Green circles highlight uncontrolled body movements of the epileptic fish immediately after the light exposure, and the start of a freezing phase characterized by total lack of movement after around 1 minute of seizure, all the while the control fish remains almost unresponsive to light. In this experiment, ZebraBox apparatus was used for motion tracking of zebrafish.

The zebrafish genome underwent a teleost‐specific whole genome duplication event during evolution, leading to a higher number of duplicated genes compared to other vertebrates. As a result, interpreting the phenotypic effects of human genes in this model can be challenging, as gene duplication can produce varying functional outcomes. ¹⁶ , ¹⁷ To fully assess the impact of a gene on a phenotype, both copies of a duplicated gene with complementary functions often need to be deleted or mutated. However, zebrafish can also be particularly useful for uncovering new gene functions in cases of subfunctionalization, where the two duplicated copies partition the ancestral gene's functions. This can be especially advantageous for studying genes that are essential for survival in mice, where a complete knockout would be lethal. In zebrafish, the presence of duplicated genes allows for the investigation of partial loss‐of‐function effects without causing embryonic lethality, thereby revealing previously unknown roles of these genes in development and disease. Additionally, criteria such as the conservation of the primary gene sequence, expression patterns, and the retention of key protein domains can help identify the duplicated gene copy most closely resembling its human counterpart. ²⁹

Autism spectrum disorder in zebrafish

ASD is one of the most extensively studied groups of NDDs. Key characteristics of ASD include problems with social communication and interaction, and restricted or repetitive behaviours. It affects 1% to 2% of the population, with males being four times more likely to be impacted than females. ³⁰ Both environmental and immune factors, as well as genetic risk factors, have been reported to influence brain development and contribute to ASD. Among these, pathogenic mutations in genes encoding synaptic proteins, such as SHANK3, which regulate synapse formation and function, are strongly associated with the disorder. ¹³ However, despite advances in identifying high confidence ASD risk genes, the exact mechanisms by which dysfunctional genes contribute to autism phenotype remain unclear. ³¹

In zebrafish, deficiency in shank3b (one of the SHANK3 orthologs) causes morphological defects, repetitive swimming behaviour, and reduced social interactions, all of which resemble autism‐like features observed in patient. ³² Additionally, the knockdown of shank3a, or syngap1b, which encodes another synaptic protein, results in significant neuronal cell death, delays in brain development, seizure‐like behaviours, and motor deficits. ³³ Knocking down chd8, which encodes chromodomain helicase DNA binding protein 8, also results in increased cell proliferation in the brain, macrocephaly, and disruption in gastrointestinal motility, mirroring the phenotypes seen in patients. ³⁴ , ³⁵

Microduplications and microdeletions in a single copy of the human 16p11.2 chromosomal region are tightly associated with NDDs. ³¹ Zebrafish possess a set of highly active 16p11.2 homolog genes on chromosomes 3 and 12, which are crucial for central nervous system (CNS) development. Knockdown of these genes in zebrafish results in a loss‐of‐function phenotype, with malformation of brain structures. ³⁶ One such gene, kctd13, when overexpressed in zebrafish, causes microcephaly, while its disruption results in macrocephaly due to decreased and increased brain cell proliferation respectively. ³⁷ Additionally, disrupting the orthologs of two other ASD risk genes, CNTNAP2 and DYRK1A, in zebrafish leads to altered brain functions. cntnap2 mutant zebrafish show forebrain GABAergic deficits, a lower seizure threshold, and nighttime hyperactivity, while loss of dyrk1aa function causes behavioural and social impairments resembling ASD phenotypes in humans. ³⁸ , ³⁹

Schizophrenia in zebrafish

Schizophrenia is a developmental neuropsychiatric disorder with a prevalence of around 1%. Symptoms appear gradually, typically from young adulthood. They include cognitive impairment, memory deficit, delusions, hallucinations, and social withdrawal. In addition to neurotransmitter imbalance and environmental or immune risk factors, genetic determinants including common variants, copy number variants, and rare protein‐coding variants also play a significant role in schizophrenia pathogenesis. ⁴⁰ , ⁴¹ , ⁴² However, the mechanisms through which these variants contribute to the disorder are still poorly understood.

Integrating genome‐wide association studies with ribonucleic acid sequencing data from postmortem brain samples of individuals with schizophrenia led to the identification of a set of putative causal genes, whose functional relevance was assessed in vivo by manipulating their expression levels in zebrafish embryos. Of the five candidate genes tested, three (furin, tsnare1, or cntn4) were found to influence zebrafish brain development, resulting in distinct head size phenotypes that were linked to alterations in cell proliferation or apoptosis. ⁴³

In adult zebrafish, loss of function of the orthologous schizophrenia risk gene znf536 is associated with abnormal brain anatomy, particularly affecting the cerebellum, as well as reduced anxiety‐like behaviour and social interactions. At the larval stage, knockout zebrafish also exhibit growth deficiencies due to a diminished ability to compete for food, compared to their wild‐type siblings. ⁴⁴ Knockdown of mapk3, an ortholog of a novel SCZ risk gene within the 16p11.2 interval, induced microcephaly in zebrafish. ⁴⁵ Additionally, two de novo mutations (R1117X and R539W) in SHANK3, primarily associated with ASD, have been linked to the early adulthood onset of schizophrenia, suggesting a shared molecular pathway underlying both disorders. Knocking down both shank3a and shank3b in zebrafish resulted in smaller head and trunk sizes and impaired touch‐evoked responses. ⁴² , ⁴⁶ Furthermore, disrupting disc1, the ortholog of a schizophrenia risk gene involved in brain development and neurogenesis in a WNT signalling‐dependent mechanism, caused the formation of a U‐shaped body with truncated tail and early brain morphology abnormalities. ⁴⁷

Cerebral palsy in zebrafish

CP is another group of NDDs characterized by movement and posture impairments, affecting approximately 0.2% of live births worldwide. The primary cause of CP is damage to the developing brain, typically occurring before or during birth, although postnatal events contribute to at least 10% of cases. While the initial damage is stationary, manifestations change with time, and the growth of the brain and development of the CNS may lead to the changes in the lesion. ⁴⁸ CP is often diagnosed later, typically between 3 to 5 years of age, despite early signs of the disorder. Common comorbidities associated with CP include intellectual disability, epilepsy, speech impairments, vision and hearing deficiency, and ASD. ⁴⁹ , ⁵⁰ , ⁵¹

Several factors, including low birthweight, preterm birth, birth asphyxia, and prenatal infection‐related immune factors, are major contributors to CP. However, the significant role of genetic factors is becoming increasingly apparent through genome‐wide association studies and next‐generation sequencing technologies. ⁵² , ⁵³ The potential pathogenicity of a copy number variation causing the deletion of pdcd6ip was investigated in vivo using zebrafish. Knockdown of this gene led to morphological defects, severe motor control impairment, microcephaly, cardiac oedema, and inability to swim in a straight line, highlighting PDCD6IP as a potential risk gene for NDDs. ⁴⁹

The first zebrafish model of AP‐4‐deficiency, a known CP‐associated risk gene, was generated by silencing ap4s1, resulting in decreased head size, CNS deformities, cardiac oedema, a curved tail, and significant locomotor impairment. ⁵⁴ Similarly, the pathogenicity of AGAP1, a regulator of endosomal trafficking with recurrent genetic variations in CP cases, was assessed in vivo using agap1 zebrafish morphants. These animals displayed developmental delays, a curved tail, reduced startle and escape responses, and reduced motility, supporting a potential role for disrupted AGAP1 in the pathology of neurological disorders. ⁵⁰

Taken together, these data highlight zebrafish as a valuable model for investigating the significance of novel risk genes and exploring the functional mechanisms of known genes associated with NDDs. However, as previously mentioned, genetics is only part of the complex puzzle of NDD risk factors. While research over the past decades has primarily focused on neurons and their networks, recent studies have revealed the involvement of other cellular players in the pathophysiology of NDDs. Among these, non‐neuronal brain cells, known as glia, have emerged as key contributors. Glial cells, which include astrocytes, oligodendrocytes, and microglia, make up about half of the brain's total cell population. While they do not directly participate in neuronal communications, they play crucial roles in developmental processes essential for proper brain maturation and function. ⁵⁵ , ⁵⁶

As we explore the increasingly recognized roles of the neuroimmune compartment in brain development, we will focus on microglia, the brain‐resident immune cells that protect neurons from pathogenic events and the consequences of injury by clearing pathogens and cellular debris and will summarize the growing body of evidence linking these cells to the aetiology of NDDs.

MICROGLIA

As described by Rio‐Hortega in 1932, microglia are a unique type of cell uniformly distributed throughout the brain and spinal cord, featuring short extensions from their small cell bodies, that are capable of migration, proliferation, and phagocytosis. ⁵⁷ Microglia constitute around 15% of the total glial cell population in the CNS. ⁵⁸ , ⁵⁹ They are the first responders in the CNS's immune system and play indispensable roles in brain development, homeostasis, and recovery after injury and pathogenic insults. ⁶ , ⁶⁰ Unlike neurons, astrocytes, and oligodendrocytes, which derive from the neuroectoderm, microglia originate from myeloid cells of the hematopoietic system. ⁶¹ During early development, primitive macrophages that arise from the extra embryonic yolk sac invade the CNS and differentiate locally into microglia. In humans, the first cells showing microglia progenitor features appear at around 3 weeks, ⁶² while in mice, the seeding of the parenchyma begins around embryonic day 9.5. ⁶³ Microglia then maintain a stable population throughout life, without needing input from bone marrow‐derived progenitors, owing to their self‐renewal and local proliferation capacity. ⁶⁴ , ⁶⁵

Remarkably, the early developmental steps of microglia are conserved between zebrafish and mammals at both cellular and molecular levels. In zebrafish, as in mice, microglia arise from yolk sac‐derived primitive macrophages early in development, in a process dependent on pu.1 and irf8. ⁶⁶ , ⁶⁷ Similar to their mammalian counterparts, the maintenance of zebrafish microglia relies on Csf1r signalling which is essential for proper macrophage development, ⁶⁸ , ⁶⁹ , ⁷⁰ , ⁷¹ and their identity is regulated by transforming growth factor beta (Wittamer lab, manuscript in preparation). These features make the transparent zebrafish embryo an invaluable tool for tracking microglial dynamics and associated events in a vertebrate model, without disrupting the homeostatic state of the CNS ⁷² (Figure 2).

FIGURE 2.

Fluorescently labelled neurons and microglia in the transparent brain of larval zebrafish.

Microglia function and dysfunction: Implications for neurodevelopmental disorders

In the developing brain, microglia actively control the size of the neural precursor cell pool by balancing cell survival and elimination, achieved through the production of soluble factors and by inducing programmed cell death and/or phagocytosis, respectively. Microglia are also involved in guiding the migration, maturation, and differentiation of neuronal progenitors into functional neurons. ⁷³ , ⁷⁴ Additionally, they facilitate synaptic pruning, a vital process for the formation of neuronal circuits. ⁷⁵ , ⁷⁶ Microglia are also critical for quickly repairing lesions that form at fetal cortical boundaries because of physiological morphogenetic stress, helping to maintain structural integrity as the brain grows. ⁷⁷ Beyond their interactions with neurons, microglia also communicate with glial cells during brain development, and emerging evidence is beginning to reveal their roles in astrogliogenesis and oligodendrogenesis in vivo. ⁶ , ⁷⁸

Given the crucial role of microglia in CNS development and maintenance, it is not surprising that their absence or dysfunction can contribute to neurological disorders. Evidence of this comes from recent findings of rare disorders caused by a complete lack of microglia due to homozygous or bi‐allelic mutations in CSF1R, leading to severe developmental brain defects such as agenesis of the corpus callosum and enlarged ventricles. ⁷⁹ , ⁸⁰ Additionally, heterozygous mutations in CSF1R and TREM2, another microglia‐associated gene, cause leukoencephalopathy with axonal spheroids (a neurodegenerative disease) and Nasu‐Hakola disease, respectively. ⁸¹ , ⁸² Interestingly, various environmental factors can also impact microglia function, potentially leading to neurological disorders. For example, maternal immune activation can trigger fetal microglia to undergo morphological and phenotypic changes, potentially causing harmful effects on the offspring's brain. ⁸³ Other risk factors, such as air pollution and prenatal stress, have also been shown to disrupt microglial homeostasis, further increasing the risk of NDDs. ⁸⁴ , ⁸⁵

Microglia dysfunction in ASD

Among the various functions of microglia in the developing brain, synaptic pruning has recently gained significant attention, particularly because of its dysregulation being linked to certain forms of NDDs, which may arise from an imbalance between excitatory and inhibitory synaptic signalling. Notably, core microglia genes such as CX3CR1, TREM2, P2Y12, and complement component C4 play key roles in the synaptic pruning process. ⁶

Emerging evidence suggests that microglia contribute to the pathology of ASD and schizophrenia, mainly by acting as a dysfunctional synaptic modulator. ASD is often associated with increased spine density, likely due to insufficient synaptic pruning by microglia. ⁸⁶ Postmortem brain studies in patients with ASD revealed significantly lower densities of ramified microglia and reduced levels of TREM2 protein. ⁶ , ³⁰ Similarly, disrupted microglia‐mediated synapse elimination in Trem2‐deficient mice leads to autism‐like behaviour, ⁸⁷ while ASD‐related features are also observed in Cx3cr1‐deficient mouse models with impaired synaptic remodelling. ⁸⁸ In zebrafish, loss‐of‐function mutations in two ASD‐associated genes, dyrk1a and scn1lab, lead to elevated microglia numbers, suggesting a mechanism where dysregulated neurogenesis and synaptic signalling may alter microglia density in the brain. ⁸⁹ Furthermore, mutations in MEPC2, a gene expressed throughout the CNS, are associated with impaired microglial activity that contributes to the pathogenesis of Rett syndrome, an ASD‐associated NDD. ⁹⁰ While the specific microglial phenotype in mecp2‐null zebrafish remains to be fully investigated, these animals show abnormalities in locomotor activity and thigmotaxis, ⁹¹ along with a dysregulated cytokine profile, all of which resemble Rett syndrome features seen in patients. ⁹²

Microglia dysfunction in schizophrenia

Schizophrenia, in contrast, is characterized by excessive synapse elimination during adolescence and early adulthood. ⁹³ Postmortem cortical tissues from patients with schizophrenia show a significant reduction in synapse density, suggesting that dysregulated microglia‐mediated synaptic pruning may play a role in the pathology. This hypothesis is supported by in vitro studies indicating that microglia‐like cells derived from patients with schizophrenia display excessive engulfment of synaptic structures. ⁹⁴ Complement component C4, which promotes synapse elimination, has elevated levels in schizophrenia, and C4‐deficient mice show suppressed synaptic pruning. ³⁰ Additionally, C4 copy number variations have been observed in some patients with schizophrenia. ⁹⁵ Rare genetic variants of CX3CR1 are also associated with an increased risk of schizophrenia. ⁷⁸ Higher levels of pro‐inflammatory cytokines, resulting from prolonged microglial activation, have been observed in both blood and brain samples of patients with schizophrenia. ⁹⁶ Furthermore, the enrichment of certain schizophrenia‐associated genes in microglia reinforces the idea that microglia may be involved in the pathogenesis of schizophrenia. ⁹⁷

Microglia dysfunction in CP

Unlike ASD and schizophrenia, postmortem studies have identified sustained neuroinflammation as a key factor in white matter injury in CP, regardless of the initial cause of damage. Perinatal brain injury is rapidly followed by a glial response involving both microglia and astrocytes, with a peak in microglia accumulation at the lesion site occurring around 2 to 4 days post injury. However, the precise role of microglia in these conditions is not well understood. Research suggests that in the early stages of brain injury in newborn infants, microglia adopt a proinflammatory phenotype characterized by the release of TNF‐α and interleukin‐1β, which initially exacerbates white matter damage. Interestingly, although activated microglia may persist for days or even weeks, at later stages, their inflammation may also contribute to tissue regeneration and repair after injury. ⁹⁸ Whether these cells ultimately have a neuroprotective or neurotoxic effect in CP remains an open question.

Microglia‐associated neurodevelopmental disorders in zebrafish

Recently, zebrafish have played a key role in illustrating the involvement of dysfunctional microglia in RNASET2‐deficient leukodystrophy, a severe NDD characterized by motor impairment and cognitive decline. Unlike rodent models, rnaset2 mutant zebrafish replicate critical aspects of the human pathology, including hypoactivity at the larval stage, along with white matter lesions and neuroinflammation in the adult brain associated with atypical behaviour and reduced survival. Notably, it was discovered that microglia in rnaset2‐mutant embryos fail to clear cells undergoing neurodevelopmental apoptosis, leading to their accumulation. ⁹⁹ Replacing dysfunctional microglia with adult marrow‐derived macrophages in this model successfully restored the clearance of apoptotic brain cells, reduced neuroinflammation, and recovered motor activity in the mutant zebrafish. ¹⁰⁰

CONCLUDING REMARKS

As small non‐mammal vertebrates, zebrafish are increasingly used to study various human diseases and for rapid screening of genes and drugs. The ability of zebrafish models to replicate the effects of genetic alterations and mimic patient phenotypes, as partly discussed here, makes them a valuable tool for modelling developmental brain diseases. Disruption of genes linked to ASD and schizophrenia in zebrafish results in macrocephaly and microcephaly respectively, along with changes in apoptosis and cell proliferation similar to those seen in patients. Moreover, deficiencies in orthologs of CP risk genes lead to morphological defects and motor impairments in zebrafish. When it comes to the neuroimmune system, zebrafish provide a powerful model in which observing, tracking, and manipulating microglial cells during critical windows of brain development is possible, without disrupting brain homeostasis. This is especially important given the reactive nature of microglia, which complicates their study in petri dish cultures. As demonstrated with the rnaset2‐deficient leukodystrophy model, zebrafish have already provided valuable insights into how microglia contribute to NDDs. Although synaptic pruning in the context of NDDs has yet to be fully explored in this model, a recent study successfully documented microglia‐mediated engulfment of synapses in the zebrafish embryonic brain, highlighting the evolutionary conservation of this process. ¹⁰¹ This observation opens new possibilities for understanding microglia's role in disease aetiology. Despite some potential limitations, such as the abundance of duplicated genes compared to other vertebrates, ¹⁶ and possible discrepancies between morphants and mutants due to genetic compensation and transcriptional adaptation, ¹⁰² the unique characteristics of zebrafish make it a promising complementary model system for studying NDDs.

FUNDING INFORMATION

This work was supported by the Fonds de la Recherche Scientifique – FNRS under Grant #40013949.

CONFLICT OF INTEREST STATEMENT

None.

Hassani Nia F, Wittamer V. Zebrafish in neurodevelopmental disorders studies: Genetic models and pathological involvement of microglia. Dev Med Child Neurol. 2025;67:1257–1265. 10.1111/dmcn.16317

DATA AVAILABILITY STATEMENT

Data sharing not applicable to this article as no datasets were generated or analysed during the current study.