Zebrafish as a Model Organism for Congenital Hydrocephalus: Characteristics and Insights

Zebrafish models for ophthalmology

Diseases of the photoreceptor cells can lead to irreversible vision loss and blindness in humans; however, effective therapies for these patients are lacking. The development of suitable animal models of cone disease is challenging because many of the popular model animals used are nocturnal, which means that they are rod-dominant visual, and their cones are dissimilar to human cones. However, zebrafish, whose retina is rich in cone cells, provide an opportunity to mimic photoreceptor disease in diurnal vertebrates. ²³ Aged zebrafish retinas (>30 months) exhibited age-related changes similar to those observed in humans. ²⁴ Zebrafish develop many pathologies and various types of photoreceptor diseases (developmental, rod, cone, and mixed) resembling macular degeneration. ²⁵ Compared to other model organisms, zebrafish are better adapted for investigating single-cell transcriptomics (SCT) during initial development. Many SCT studies (not including zebrafish) that analyze ocular components are limited to very few or only one developmental stage because of the adverse effects of postmortem extraction. ²⁶ However, zebrafish can overcome these difficulties due to their high reproductive rate and rapid initial development (the basic components of the eye develop within 5 days after fertilization). ²⁷ Besides, they are well-suited for multi-timepoint SCT analyses and subsequent functional gene investigations of vertebrate development. ²⁸ Consequently, zebrafish offer distinct advantages for research in the field of ophthalmology due to the conservation of their eye morphology, physiological tissues, and regulatory signaling pathways.^28–30 The applications of zebrafish ophthalmology disease models are summarized in Table 1. As animal models for ophthalmology, zebrafish models with a single copy of a functionally conserved gene accurately reproduce the human phenotype, such as the pde6c or rho mutant lines. Although some lines have phenotypes that differ from humans (for example, the cep290 mutation causes cone-specific degeneration in a zebrafish model, whereas the CEP290 mutation causes Leber congenital amaurosis in humans), animal models are intended to accurately reproduce human disease manifestations and to provide insights into photoreceptor function. Therefore, zebrafish provide unique opportunities to elucidate the molecular mechanisms underlying photoreceptor disease and degeneration.

Zebrafish models for toxicology

Zebrafish are widely used for toxicology research from molecular mechanisms to organismal phenotype and behavior, particularly in the aspects of organ system toxicology,^31,32 investigating mechanisms of action, ecotoxicity, environmental toxicants, chemical toxicity screening, and drug screening. Toxicological outcomes in zebrafish are easy to establish, including LC₅₀, morphological and physiological parameters that indicate acute toxicity or death, such as embryo coagulation, nondetachment of the tail, lack of somite formation, absence of heartbeat, the curvature of the spine (scoliosis), pericardial and yolk sac edema, and alterations in the swimming bladder. ³³ Besides, zebrafish embryos and larvae are transparent. In some transgenic zebrafish lines, fluorescent proteins are activated in the presence of contaminants or environmental stressors, making them easy to observe (Table 1).^34,35

Zebrafish models for developmental biology

As a model organism, zebrafish have close developmental trajectories of many systems with humans, and the formation of some structures is similar to that of mammals (such as the aortic arch). Therefore, many researchers have used zebrafish as a tool in various developmental biology studies, particularly in the fields of the heart, eye morphogenesis, nervous system, and skeleton. ³⁶ Bergmann et al. developed a new transgenic zebrafish strain (NBT: GCaMP3) that stably expressed a fluorescent calcium reporter gene in all major brain regions of larval brains up to 21 days of age, which can be used to characterize the receptor field of optic tectum neurons in the late larval stage. ³⁷ In the field of neurodevelopmental disorders, zebrafish were used to test prenatal risk factors for autism spectrum disorder and intellectual disability. ³⁸ The advantages of zebrafish in heart research can also be used to establish human congenital heart disease models. ³⁹ Because of the transparency of zebrafish embryos and larvae, morphological changes in the cardiovascular system during development can be visualized by transmission light or fluorescence imaging (Table 1). These studies revealed the potential use of zebrafish models to effectively evaluate novel genetic variants, contributing to the early identification of pathogenic factors and accelerating disease diagnosis and treatment.

Zebrafish models for aberrant cognitive disease

Zebrafish possess unique characteristics as a model organism for neurological research. ⁴⁰ Although the central nervous system of zebrafish originates from different neurogenic processes, it shares similar tissue organization with mammals, displaying the same main brain structures, such as the forebrain, midbrain, hindbrain, and spinal cord (Fig. 1A and Table 1). Additionally, at the cellular level, zebrafish and mammals exhibit parallels, exemplified by astrocytes, ependyma cells, and cerebellar Purkinje cells (Table 1).^51–53 In zebrafish, the observation of hydrocephalus is notably more straightforward, allowing for confirmation through multiple indicators, such as ventricle transparency and volume and magnetic resonance imaging. ⁵⁴ Currently, a range of imaging techniques have been employed to investigate the pathophysiology of brain-related disorders in zebrafish. These techniques include fluorescence microscopy, laser scanning microscopy, transmission electron microscopy, and high-throughput imaging systems.^55–57

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FIG. 1.

Pathogenesis of zebrafish CH. (A) Main brain structures and direction of the CSF of zebrafish. Different colors represent different brain structures in the picture, and the blue arrow indicates the direction of CSF flow from the telencephalon to the rhombencephalon. (B) Ciliary abnormalities caused by different genetic mutations in zebrafish with CH. pard3 leads to abnormal orientation. fler, ⁴¹ ttll6, ⁴¹ ofd1, ⁴² ift46 ⁴³ and wtip ⁴⁴ mutation shorten the cilia, while fler leads to the defect of tube B and ofd1 leads to the absence of central microtube. (C) Ciliary motility impairment caused by different gene mutations in zebrafish with congenital hydrocephalus. dyx1c, ⁴⁵ wtip, ⁴⁴ ccp5, ⁴⁶ and ttc4 (−9c, −36, and 39c) lead to impaired ciliary motility, while calaxin ⁴⁷ triggers irregular ciliary beating and inversion. (D) Blood–brain barrier disruption with claudin-5 ⁴⁸ caused by VE-cadherin/β-catenin or VE-cadherin/FoxO1 pathways in zebrafish with congenital hydrocephalus. (E) Dysplasia of choroid plexus epithelial cells in zebrafish with congenital hydrocephalus caused by wt1, ⁴⁹ ecrg4, ⁵⁰ and many other genes. The related references are listed in Table 3. CH, congenital hydrocephalus; ChP, choroid plexus; CSF, cerebrospinal fluid; Di, diencephalon; Me, mesencephalon; Rh, rhombencephalon; Tel, telencephalon.

Besides, many behavioral tests that have been used to study cognitive phenotypes in rodents, such as the open-field test, the light-dark box, and the T- or Y-maze, have been successfully applied to zebrafish. ⁵⁸ Zebrafish can recognize geometric figures and form spatial memories. ⁵⁹ These novel findings and breakthroughs indicate the potential of zebrafish for studying aberrant cognitive and neurodegenerative diseases. To date, several orthologs of major disease-associated genes have been found in zebrafish, including Alzheimer’s disease, attention deficit hyperactivity disorder, epilepsy, Huntington’s disease, Parkinson’s disease, schizophrenia, and others (Table 2). Transgene, knockdown, knockout, or overexpression of mutants of these genes suggests that they have conserved functions in the development and survival of aberrant cognitive disease and neurodegenerative diseases.

Hydrocephalus in zebrafish and human

Zebrafish serve as a potential model organism for studying hydrocephalus. The hydrocephalus phenotype in zebrafish typically manifests as ventricular enlargement, head swelling, reduced eye size, decreased pigmentation, weakened blood circulation, and pericardial edema. These phenotypes can be observed in the early developmental stages (20 hpf) and worsen over time. Detection methods include microscopy, gene expression analysis (such as in situ hybridization or qPCR), advanced imaging techniques (MRI or CT), and behavioral tests to assess neurological function. ⁵⁹ The pathophysiology of hydrocephalus in zebrafish is similar to that in humans, involving disruption of CSF circulation, absorption, or overproduction. ⁸⁵ At the cellular and developmental levels, ependymal cells in zebrafish and humans play crucial roles in maintaining CSF flow and ventricular structure, with ciliary dysfunction leading to CSF flow abnormalities. Rapid development and transparent embryos make zebrafish ideal for studying the dynamic changes in hydrocephalus during development, providing insights into the mechanisms and potential therapeutic approaches.

Cilia are hair-like structures that extend from the cell surface and are unique in eukaryotic organisms. They consist of a matrix, axoneme, and ciliary membrane. The axoneme, which is mainly composed of microtubules, serves as the main structure of cilia. ⁸⁶ Cilia can be categorized into two forms: motile and nonmotile cilia. Motile cilia move in a regular rhythm and direction, while nonmotile cilia serve as sensory organelles. Numerous studies have suggested that cilia dysfunction can disrupt the circulation of CSF, potentially contributing to the development of CH. Ciliary defects are primarily caused by genetic mutations. Presently, several zebrafish models with CH phenotypes are designed based on ciliary abnormalities (Fig. 1B, C). Here, we summarized these zebrafish models with ciliary defects and classified them into two subtypes: ciliogenesis and cilia movement defects (Table 3).

Zebrafish model with CH caused by ciliogenesis defects

Ciliary generation defects can be broadly classified into two categories: alterations in ciliary morphology and number. Currently, there is an increasing emphasis on investigating the pathological repercussions arising from alterations in ciliary morphology (Fig. 1B).

Kramer-Zucker and his colleagues initially explored the impact of ciliary morphology on the organ development and intraorgan fluid dynamics of pronephros, brain, and Kupffer’s vesicles in zebrafish. Subsequently, they identified that defects in ciliary morphology or motility could also contribute to the onset of CH. ¹¹⁹ Subsequently, researchers discovered that mutations in zebrafish genes, such as exoc5/sec10, ⁸⁷ cdc42,^88,89 and ift46, ⁴³ were associated with truncated or absent cilia in the choroid plexus, ultimately contributing to hydrocephalus. Knockout of the wdr16 gene disrupts osmotic regulation and brain water balance, causing severe hydrocephalus, which is also correlated with cilia. ²⁰ Afterward, research has delved deeper, shifting the focus toward the study of the structural components of cilia, particularly microtubules. In 2010, E. Hong elucidated that, during neurogenesis, microtubules and the polarity protein collaboratively regulate the positioning of centrosomes. Pard3 deficiency in zebrafish can induce aberrant cilia growth and orientation, eventually leading to the development of CH. ⁹⁰ This study revealed a novel molecular mechanism associated with the pathogenesis of CH and provided valuable insights for developing novel zebrafish models of this condition. Notably, in contrast to the other models, there were nearly no complications in the pard3 mutation CH zebrafish models. In 2020, pertinent research further advanced to the protein subunit level. The knockout of chmp4b, which codes for a subunit with the function of endosomal sorting complexes, was found to interfere with cilia assembly. It even triggers the fragmentation of existing cilia, resulting in the onset of hydrocephalus. ⁹²

In zebrafish, besides the fact that several gene defects related to ciliary generation have been identified as causative factors for CH, other studies have revealed a correlation between the number of cilia and CH. In studies on the correlation between microtubule protein polyglutamylation and cilia formation in zebrafish, knockout of ttll6 (responsible for microtubule protein polyglutamylation) was observed to induce CH. ⁴¹ This study substantiates the significance of polyglutamylation of microtubule proteins in cilia generation and motility. Then, N. Pathak and colleagues extended this investigation and conducted a more detailed analysis of the expression patterns of ccp during zebrafish development. Their findings revealed that among the ccp family, ccp1 expression is confined to the nervous system. Only the knockout of the ccp5 gene led to elevated glutamylation of cilia microtubule protein, triggering ciliary dysfunction and ultimately manifesting as a CH phenotype. ⁴⁶

Additionally, studies have revealed that mutation of wtip, encoding the LIM domain protein WTIP, can result in the functional impairment of ccp1 and ccp5, culminating in a decrease in ciliated cells. ⁴⁴ These studies have established a framework for identifying ciliary genes pertinent to the nervous system.^120,121 Furthermore, studies have pointed out that the combined knockout of the calb2a and calb2b genes, transient knockdown of cdc42, sec10, dcdc2, and ccdc28b, as well as reducing the expression of nherf1a/slc9a3r1, collectively lead to the diminished genesis of cilia and severe CH in zebrafish.^88,93–96 Among these, the dcdc2 knockout and low expression of nherf1a/slc9a3r1 are related to the classical Wnt signaling pathway, which is important in the development of CH. Overexpression of dcdc2 inhibits β-catenin in the Wnt pathway and modulates the pathogenesis of hydrocephalus. Similarly, nherf1a/slc9a3r1 also functions through the Wnt/PCP pathway.

Despite the conventional concept linking CH to cilia defects disrupting the flow of CSF, recent research indicates an additional correlation between brain ventricular anomalies and motile cilia development. Specifically, gmnc and foxj1a mediate the transition from solitary ciliated cells to multiciliated cells, resulting in an enlarged brain ventricle and CH. ⁹⁸

Although the above studies have successfully constructed a zebrafish model of CH (except for the model of E. Hong), hydrocephalus only appears as a complication of other prominent conditions (such as kidney cysts, asymmetrical malformations, curved bodies, and others). Consequently, the CH zebrafish model designed for ciliary generation defects is in its nascent stage. To address these multifaceted complications, it is necessary to find a specific gene exclusively affecting the brain cilia to refine CH zebrafish model organisms.

Zebrafish model with CH caused by cilia movement defects

In most circumstances, genes affecting cilia generation have adverse effects on cilia movement. For instance, wtip, gmnc, and ccp5 reduce cilia number, morphology, and motility. Except for the mutations and abnormalities mentioned above, alterations in cilia number, structure, and the absence of basal roots can all impact ciliary motility (Fig. 1C). In zebrafish, four tetratricopeptide repeat-containing (TTC) proteins (ttc4, -9c, -30, -36, and -39c encoded) are crucial for cilia formation and motility. ¹²² Defects in these proteins can cause CH. Notably, hydrocephalus, attributed to impaired ciliary motility, always correlates with obstruction of CSF flow.

However, alterations in ciliary motility do not come just from the three factors mentioned above. Various genes governing ciliary motility still exist. For instance, the knockout of the dynein arm protein-encoding gene dnaaf3 in zebrafish disrupts the assembly of dynein arms and ciliary motility, ultimately leading to the development of hydrocephalus. ¹⁰⁰ In 2013, the study conducted by G. Chandrasekar and his team investigated the tetrapeptide repeat domain-containing protein DYX1C1, a factor associated with rodent neuronal migration. Researchers have discovered that dyx1c1 is expressed across multiple ciliated tissues in zebrafish. Zebrafish with dyx1c1 mutations exhibit deficiency in both the outer and inner arms, resulting in ciliary motility disorders and subsequent CH. ⁴⁵ Similarly, within the same year, the research highlighted that the knockout of two genes, bbs1 and nphp7.2, both influencing ciliary motility in zebrafish, leads to hydrocephalus. ¹⁰¹ Furthermore, in 2019, it was found that the calaxin mutation in zebrafish triggers irregular ciliary beating and inversion, inducing abnormal CSF circulation and, consequently, CH. ⁴⁷

Regrettably, CH zebrafish models developed based on ciliary motility disorders still face challenges because these genes are hard to localize precisely in the brain. Consequently, these models may also lead to other typical diseases in other systems (such as kidney cysts), where cilia also play a significant role. These facts make research focusing on CH difficult.

Zebrafish model with CH caused by BBB disruption

The BBB serves as a semi-permeable protective partition that segregates circulating blood from the brain microenvironment. Its composition comprises three layers from the inside out: tightly connected brain capillary endothelial cells, continuous basement membrane, and end feet of neuroglial cells. The BBB prevents harmful substances from entering the brain microenvironment. Studies have demonstrated that disruption of the BBB, including increased permeability, is associated with the development of hydrocephalus (Table 3). Some genes are related to BBB development.

Claudin-5a, encoded by cldn5a (claudin-5), is a tight junction protein specifically located in the brain that is notably abundant at the BBB in zebrafish (Fig. 1D). Phosphorylation plays a pivotal role in both its expression and function by regulating claudin-5 expression or the protein itself. Several phosphorylation sites and activation mechanisms have been revealed to impair the integrity and tightness of endothelial cells. ⁴⁸ Foxo1 and β-catenin inhibit the transcription of claudin-5 by binding to its promoter. Cldn5a gene expression can be regulated by VE-cadherin. VE-cadherin expression triggers Akt activation, subsequently leading to the phosphorylation of FoxO1. This phosphorylation limits β-catenin translocation to the nucleus, thereby alleviating its inhibitory effect on claudin-5 transcription. ¹²³ In 2010, a zebrafish model of CH was developed by inactivating VE-cadherin. ¹²⁴ This manipulation resulted in reduced claudin-5 expression, thereby causing BBB abnormalities and the emergence of hydrocephalus. Expansion of the zebrafish embryonic brain before the establishment of the embryonic BBB is essential. However, an investigation demonstrated that mutations in claudin5a prevented this procedure. The reduced brain ventricular volume was caused by a decrease in paracellular tightness of the cerebral–ventricular barrier.

Moreover, this discovery established a foundation for comprehending the collaborative influence of claudin-5a and Na⁺/K⁺-ATPase during ventricular expansion. ¹²⁵ Zebrafish claudin-5a serves as a direct homolog of human CLDN5. No additional complications have arisen from zebrafish model organisms by claudin-5 modification. ¹²⁶ Therefore, developing a CH zebrafish model using claudin-5a as a specific molecule is promising for further research.

The choroid plexus is also a crucial component of the BBB (Fig. 1E). The Wilms tumor suppressor gene WT1 in humans encodes a zinc-finger transcription factor. Furthermore, its paralogs in zebrafish, wt1a and wtlb, are necessary for normal choroid plexus formation and function, which explains why wt1a deficiency causes CH in zebrafish. ⁴⁹ Similarly, human esophageal cancer-related gene-4 (ECRG4) and its protein Aurrin are associated with choroid plexus epithelial cell development. Knockout of the ecrg4 gene can induce a dose-dependent CH zebrafish model, and this phenotype can be reversed by coinjection of antisense morpholinos with ecrg4 mRNA. ⁵⁰ Moreover, components of the classical Notch signaling pathway, such as notch1b, deltaA, and deltaD, can also mediate choroid plexus-related disorders and cause hydrocephalus in zebrafish. ¹²⁷

The CH zebrafish models mentioned above, based on the BBB, exhibit relatively high specificity. However, the lifespan of the zebrafish is limited. For instance, wt1a mutant zebrafish survive for <10 days. Furthermore, many studies have focused on zebrafish embryos, leaving uncertainties regarding the feasibility of surviving into adulthood for further investigative purposes. Accordingly, it reminds us that the lifespan should also be considered when developing CH zebrafish models.

Zebrafish model with CH caused by brain ventricular system abnormalities

The brain ventricular system (BVS), surrounded by ependymal cells, is formed by interconnected cavities filled with CSF. BVS is vital for regulating neurogenesis, brain function, and internal homeostasis. Dysregulation of the BVS can lead to CH.^8,128,129 Brain ventricular expansion in hydrocephalus is primarily caused by an imbalance between the production and reabsorption of CSF or obstruction of its flow. Moreover, patients with CH manifest additional structural brain anomalies, suggesting that hydrocephalus may not solely arise from CSF homeostasis disruption but could also be due to the abnormal development of the brain parenchyma. ¹³⁰ Current research indicates that BVS-related genes are associated with embryonic brain development, ventricular structure, brain vascularization, cell adhesion, and brain epithelial cells (Table 3).

Nestin is a type IV neurofilament protein. Zebrafish nestin is a direct homolog of the mammalian counterpart NES and is broadly expressed in the developing period of the nervous system. Studies have revealed that nestin knockdown in zebrafish embryos prompts developmental anomalies of the brain and ocular structures, leading to microcephaly and CH. During embryonic development, these zebrafish embryos manifest hindbrain and midbrain shrinkage symptoms, accompanied by ventricular enlargement. ¹⁰⁵ The effect of nestin on brain development is dose-dependent, implying the possibility of ameliorating embryonic microcephaly and CH by partially moderating nestin expression levels. This study also suggests the potential of developing an adult CH zebrafish model.

Previous studies elucidating the etiology of hydrocephalus have suggested that iron accumulation could influence the Wnt pathway. ¹³¹ The pantothenate kinase 2 gene (PANK2) in humans and its paralog pank2 in zebrafish are expressed in the brain, and their mutations are closely related to pantothenate kinase-associated neurodegeneration.^132–134 Notably, research reveals that pank2 is also vital for brain vascular formation, and the downregulation of pank2 causes an abnormal zebrafish brain structure and CH. ¹⁰⁶

The impact of L1CAM on the ventricular structure has been studied for a long time in humans. As early as 1995, researchers found that human L1CAM mutations can cause ventricular expansion, intellectual disability, corpus callosum agenesis, and hydrocephalus. ¹³⁵ Subsequent research illustrated that L1CAM LOF mutations can cause human X-linked hydrocephalus. ¹³⁶ The absence of these genes in Xenopus laevis and mice also causes hydrocephalus.^137,138 In zebrafish, l1cam exists in dual forms, namely l1cama and l1camb. Chl1a closely resembled l1cam. Adam10 codes a protease that orchestrates the detachment of the L1CAM protein from the cell surface by regulating the FNIII domain. ¹⁰⁸ Zebrafish chl1a demonstrates 46% homology to humans and governs ventricular system development and cell adhesion. Notably, chl1a-deficient zebrafish embryos have Reissner fiber formation deficiency, resulting in obvious hydrocephalus symptoms. ¹⁰⁷

Thioredoxin (encoded by thioredoxin 1) is a 12 kDa intracellular oxidoreductase. It plays a pivotal role in protecting cells and tissues against oxidative stress-induced apoptosis through its active center cysteine residue,^139,140 impending the proapoptotic function of signal-regulating kinase 1 via ubiquitination and degradation and regulating the transcription of angiogenic genes by blocking NF-κB activation.^141–143 Knockdown of thioredoxin 1 amplifies apoptosis in ventricular epithelial cells, culminating in hydrocephalus. However, mouse models reveal embryonic lethality upon this knockout. To establish a CH zebrafish model targeting thioredoxin 1, a survival-permitting strategy must be devised. Notably, environmental chemicals such as arsenic, cadmium, mercury, 1,1-bis-(4-chlorophenyl)−2,2,2-trichloroethane (DDT), and 2,3,7,8-tetrachlorodibenzo-p-dioxin affect thioredoxin expression by predominantly diminishing its mRNA levels. ¹⁰⁹ As a result, a zebrafish model for CH could be developed by diminishing thioredoxin expression via chemical exposure in zebrafish embryos.

Recent investigations have revealed the significance of voltage-gated potassium (Kv) channels in the emergence and maturation of BVS, which is attributed to their pivotal role in cell proliferation. ¹⁴⁴ Research has illuminated that silencing the zebrafish kcng4b, a homolog of the human gene encoding Kv6.4 subunit, results in the inhibition of neuroepithelial proliferation within the ventricles. This alteration is mediated by the modulation of Kv2.1 activity, which shifts neuroepithelial integrity. Therefore, ventricular expansion is induced. Consequently, zebrafish with kcng4b gene deficiency manifest the development of CH.^110,145,146

Mutations in the leucine-rich glioma inactivated 1 (LGI1) gene render individuals susceptible to hereditary epilepsy syndrome in human.^147–149 In zebrafish, there are two homologs of the LGI1 gene: lgi1a and lgi1b. Lgi1a anomalies are linked to epilepsy-like hyperactivity, concomitant with brain cell apoptosis,^150,151 while knockdown or mutation of the lgi1b gene leads to abnormal development. It does not induce epilepsy-like behavior; instead, lgi1b triggers brain cell apoptosis and brain volume reduction, resulting in enlarged ventricles and CH. ¹¹¹

BVS crucially regulates neurogenesis, brain function, and homeostasis. Dysregulation of the BVS can lead to CH, which is driven by CSF accumulation or developmental anomalies. Nestin deficiency in zebrafish demonstrates its importance in brain development and offers a potential therapeutic target for embryonic brain anomalies. The role of iron in CH via the Wnt pathway represents a novel etiological approach. Genes such as pank2, l1cam, and lgi1 influence the brain structure, providing insights into CH mechanisms. The impact of thioredoxin suggests a chemical-induced zebrafish model organism for CH, and Kv channels are linked to ventricular development. These findings enrich our understanding of CH and facilitate zebrafish model organism advancement.

Zebrafish model with CH caused by ions-related factors

The ion-transporter is crucial for the secretion of CSF and the dysregulation of the balance of Na⁺, Cl^-, and HCO³⁻, and then the secretion of CSF can also lead to hydrocephalus. In zebrafish, several ion-related gene defects or mutations have been identified as causative factors of CH (Table 3). Polycystin-2 (coded by pkd2) is a cation-permeable transient receptor potential ion channel that plays a pivotal role. During embryonic development, disruptions in pkd2 expression lead to its mislocalization on apical cell surfaces, culminating in hydrocephalus. This condition is rescued by human PKD2 mRNA expression.^152,153 Simultaneous disruption of the pkd1a and pkd1b genes, responsible for encoding the polycystic protein PKD1, leads to sustained upregulation of collagen mRNA expression. This alteration influences the secretion and assembly of the extracellular matrix, perturbing matrix integrity and ultimately resulting in CH.^99,154 Slc41a1 downregulation induces hydrocephalus in zebrafish but is accompanied by severe kidney lesions, which are also ions-related. ¹¹³ Na⁺/K⁺ ATPase is a transmembrane ion pump crucial for ion gradients and maintains basic cellular functions. In zebrafish, atp1a3, primarily expressed in neurons, exhibits two homologs with distinct brain expression patterns: atp1a3a and atp1a3b. Knockdown of atp1a3a and atp1a3b leads to membrane ion distribution imbalance, resulting in CSF accumulation, ventricular enlargement, and finally, hydrocephalus. ¹¹⁴

Zebrafish model with CH caused by other reasons

Arhgef7b/βPix knockdown results in the immature development of cerebral blood vessels in embryonic zebrafish, leading to hydrocephalus and intracranial-specific hemorrhage during early embryogenesis. This particular phenotype is attributed to specific βPix splice variants in the progression of CH. ⁸⁹ Both aberrant splicing of psen1 exon 8, a direct homolog of the human PSEN1 gene, and reduced PSEN1 protein activity in zebrafish can inhibit Notch signaling, resulting in obvious hydrocephalus.^115,155 HRG1 mutations perturb heme balance and influence CNS development and blood flow in humans. Transient hrg-1 knockdown in zebrafish leads to CH. ¹¹⁶ Mutation of the heat shock protein spg gene affects cell skeleton structure, axon growth, and neuron migration during early embryogenesis, leading to CH. ¹⁵⁶ Knockdown of ispd or gtdc2 as glycosyltransferase genes contributes to Walker–Warburg syndrome-associated hydrocephalus, which is potentially linked to brain integrity damage by muscle fiber formation defects and protein glycosylation.^117,118

Various gene defects and mutations in zebrafish can contribute to CH, making zebrafish a valuable model organism for studying genetic etiology and exploring potential therapeutic targets. It is prudent to acknowledge that although zebrafish is a widely employed model organism in biological investigations, additional endeavors should be made to establish a more human-related and stable model organism.