International Journal of Marine Science, 2024, Vol.14, No.5, 332-340 http://www.aquapublisher.com/index.php/ijms 335 genes essential for cilia formation, such as IFT46, results in multiple phenotypes associated with ciliopathies, including kidney cysts, pericardial edema, and ventral axis curvature. These defects are characterized by shortened and abnormal cilia in various tissues, such as the kidney and spinal canal (Lee et al., 2015; 2018). The overexpression of the ciliary membrane protein Arl13b in zebrafish leads to an increase in ciliary length, which disrupts motility in motile cilia but retains signaling capacity in immotile primary cilia. This suggests that Arl13b plays a crucial role in ciliary membrane extension and length regulation (Lu et al., 2015). 4.2 Disruption of intraflagellar transport (IFT) Intraflagellar transport (IFT) is essential for the assembly and maintenance of cilia by transporting proteins along the axoneme. Disruption of IFT can lead to severe ciliopathies. For instance, mutations in IFT74 result in ciliogenesis defects and slow photoreceptor degeneration in zebrafish, highlighting the critical role of IFT proteins in maintaining ciliary structure and function (Luo et al., 2021). Similarly, IFT52 mutations impair the assembly of the IFT-B complex and its localization to cilia, leading to decreased cilia length and associated ciliopathies such as short-rib thoracic dysplasia and congenital anomalies of the kidney and urinary tract (Dupont et al., 2019). The deletion of WDR31, along with RP2 and ELMOD1, results in the accumulation of IFT complex B components and kinesin KIF17 in cilia, indicating their role in regulating IFT and BBSome trafficking (Cevik et al., 2023). 4.3 Protein networks regulating ciliary maintenance The maintenance of ciliary structure and function is regulated by complex protein networks. IFT proteins, such as IFT46 and IFT74, are crucial for ciliary development and maintenance. In zebrafish, IFT46 is expressed in various ciliated tissues and is localized to the basal body. Knockdown of IFT46 results in multiple ciliopathy-associated phenotypes, demonstrating its essential role in ciliary development 18. Additionally, the Hippo pathway effector YAP1 is modulated by IFT complex B proteins (IFT88, IFT54, and IFT20) during cardiogenesis in zebrafish, highlighting a noncanonical role for IFT proteins in regulating ciliary-related pathways (Peralta et al., 2020). Moreover, endothelial cilia play a critical role in maintaining vascular integrity during zebrafish development, with Hedgehog signaling being a major mechanism involved in this process (Kallakuri et al., 2015). 5 Observational Techniques in Zebrafish Ciliary Research 5.1 High-resolution imaging of cilia High-resolution imaging techniques are crucial for studying the intricate structures and functions of cilia in zebrafish. Transmission electron microscopy (TEM) and electron tomography (ET) have been employed to analyze the axonemal structure of cilia in zebrafish, providing detailed insights into their morphology. For instance, TEM and ET were used to characterize the cilia in the olfactory pit (OP) and the left-right organizer (LRO) of zebrafish embryos, revealing structural similarities to human respiratory cilia. Additionally, a new protocol involving glycol methacrylate (GMA) embedding has been developed to preserve fluorescent signals in zebrafish embryos, allowing for high-resolution imaging of internal structures without the need for antibodies. This method is particularly useful when advanced imaging technology is not readily available. 5.2 Genetic markers for ciliary studies Genetic markers are essential tools for identifying and studying cilia in zebrafish. The identification of cilia-specific markers, such as Nephrocystin-3 (NPHp3), has facilitated the visualization of cilia dynamics in vivo. A transgenic zebrafish line expressing a fusion protein of NPHp3 and mCherry under the β-actin promoter has been developed, enabling efficient labeling of cilia in multiple cell types from embryonic stages to adulthood without causing developmental or physiological defects (Zhang et al., 2022). This transgenic line allows for live imaging of ciliary dynamics and the trafficking of cilia proteins, providing valuable insights into the roles of these organelles. 5.3 Use of fluorescent reporters in live zebrafish embryos Fluorescent reporters are widely used in zebrafish research to study ciliary function and dynamics in live embryos. The transparency of zebrafish embryos and larvae allows for non-invasive imaging during their rapid development. Fluorescent reporter molecules, such as those used in transgenic lines expressing fluorescent proteins under specific promoters, enable in vivo imaging of cilia and other cellular structures (Tonelli et al., 2020). For example,
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