Research articleThe role of cldnh during the early retinal development in zebrafish
Introduction
Transmembrane protein claudins are the major structural components of tight junction in epithelia and endothelia, and play a critical role in the permeability of epithelial and endothelial cells (Gunzel and Yu, 2013). Claudin family comprises 27 members in mice and humans (Mineta et al., 2011), which are functionally divided into barrier-forming ones (claudin-1, -3, -5, -11, −14, and −18) and channel-forming ones (claudin-2, -10a, and −10b, −15, and −17) (Gunzel and Yu, 2013; Rosenthal et al., 2017). Claudin-3 (cldn3) is expressed in a variety of epithelia, such as the epithelia of lung, intestine, liver, kidney, urinary tract, mammary glands, skin, and pigmented retina (Gunzel and Yu, 2013; Peng et al., 2010; Rosenthal et al., 2017). Cldn3 is also expressed in the endothelial cells of the blood-brain barrier (BBB) (Buttmann et al., 2007; Coisne et al., 2005; Liebner et al., 2008; Parikh et al., 2015; Shawahna et al., 2017; Wolburg et al., 2003) and the inner blood-retinal barrier (iBRB) (Xu et al., 2005), and plays an important role in the formation and maintenance of the BBB. The iBRB isolates the neural retina from the blood circulation, maintaining a stable microenvironment for normal retinal function. The breakdown of the iBRB is a common pathological condition in various retinal vascular or inflammatory diseases, such as retinopathy of prematurity, diabetic retinopathy, retinal vein occlusion and uveitis (Erickson et al., 2007; Klaassen et al., 2013). Besides, cldn3 has also been detected in retinal ganglion cells (Luo et al., 2011). Thus far, whether cldn3 plays a role in regulating the retinal development remains unknown.
Zebrafish claudin-h (cldnh; 1880 bp) is the closest ortholog of mouse and human cldn3, encoding a 23 kDa protein (Zhang et al., 2012). The high homology of cldnh protein among zebrafish, mouse and human indicates its conservation across these species. In addition, the transparency of zebrafish embryos allows the visualization of vessel formation. Hyaloid vessels in zebrafish develop into retinal vessels without regression (Xie et al., 2010), and adhere to the retinal inner limiting membrane by 30 day post fertilization (dpf) (Alvarez et al., 2007). Moreover, the hyaloid vessels of zebrafish form the iBRB as early as 3 dpf (Xie et al., 2010). It is feasible to use the hyaloid vessels of zebrafish to study the iBRB formation during the retinal development. Hence, we used transgenic zebrafish that specifically expresses green fluorescent protein (GFP) in the vascular endothelial cells to investigate the role of cldnh in the development of the neural retina and its vessels.
Section snippets
Zebrafish
The transgenic Tg (flk1:EGFP) zebrafish line which specifically expresses GFP in the vascular endothelial cells was provided by the Zebrafish Model Animal Laboratory of Sun Yat-Sen University. The protocols were approved by the Institutional Animal Care and Use Committee of Zhongshan Ophthalmic Center (protocol number 2018-049) and all experiments were performed in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.
Immunofluorescence staining
The eyes of zebrafish embryos at 24,
Spatiotemporal expression of the cldnh mRNA and protein in the developing zebrafish eye
To determine the expression of the cldnh mRNA and protein during zebrafish eye development, quantitative polymerase chain reaction (qPCR) and immunofluorescence staining were performed. From 24 to 72 hpf, the expression level of the cldnh mRNA peaked at 30 hpf and then gradually decreased (Fig. 1A). The cldnh protein was absent in the zebrafish eye at 24 and 30 hpf (Fig. 2A1-B4), started to be expressed at the lens, unlaminated neural retina, and RPE at 36 hpf (Fig. 2C1-4), and was evidently
Discussion
Cldn3 has been reported to be important in the formation and maintenance of the BBB (Berndt et al., 2019; Wolburg et al., 2003) and the expression of cldn3 in cerebral capillaries is down-regulated in pathological conditions of hypoxia, ischemia (Berndt et al., 2019; Wolburg et al., 2003) and inflammation (Berndt et al., 2019; Wolburg et al., 2003). However, less is known about the role of cldn3 in the development of iBRB and neural retina. The protein level of cldn3 is reduced under conditions
Declaration of competing interest
None.
Acknowledgments
We thank Shulan Yang and Xiaoling Guo of Sun Yat-Sen University for providing the Tg(flk1:EGFP) line and excellent zebrafish husbandry. This work was supported by the National Natural Science Foundation of China to Y.L (81770971); and the Natural Science Foundation of Guangdong Province, China to Y.L (2017A030313787).
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