Research article
The role of cldnh during the early retinal development in zebrafish

https://doi.org/10.1016/j.exer.2020.108207Get rights and content

Highlights

  • Claudin-h is required for normal development of neural retina and its vessels.

  • Claudin-h affects the density and the lumenization of the hyaloid vessels.

  • Claudin-h affects the lamination and the thickness of the neural retina.

  • Claudin-h affects the expression of atoh7, pcdh17, crx, neurod1, insm1a and sox9b.

  • Claudin-h affects the expression of hlx1 and myl7.

Abstract

Claudin-3, an integral component of tight junction, has recently been shown to be expressed in retinal ganglion cells, retinal pigment cells, and retinal vascular endothelial cells. However, the role of claudin-3 in the development of the neural retina and its vessels remains undefined. This study aimed to investigate the role of zebrafish claudin-h (cldnh), the closest ortholog of mouse and human claudin-3, in the development of the neural retina and its vessels. Cldnh levels in green fluorescent protein transgenic zebrafish were genetically manipulated by cldnh morpholino oligonucleotide (MO) and cldnh mRNA to investigate gene function. The expression of cldnh was analyzed using polymerase chain reaction and immunofluorescence staining. The altered morphological, cellular and molecular events in the cldnh MO-morphant eyes were detected using hematoxylin-eosin staining, fluorescent dye injection, confocal in vivo imaging, BrdU labeling, TUNEL assay, RNA sequencing, and Western blot. We demonstrated that the cldnh protein was expressed in the neural retina and the hyaloid vessel which is the predecessor of the retinal vessel in zebrafish. Cldnh knockdown delayed lamination of the neural retina and reduced its thickness, which might be associated with the downregulation of the retinal development-related genes of atoh7, pcdh17, crx, neurod1, insm1a, sox9b and cdh11, and the upregulation of the cell cycle and apoptosis-associated genes of tp53, cdkn1a and casp8. Cldnh knockdown also reduced the density and interrupted the lumenization of the hyaloid vessels, which might be owing to the downregulation of the vessel formation-related genes of hlx1 and myl7. In conclusion, cldnh was required for the normal development of the neural retina and its vessels in zebrafish, providing a basis for elucidating its role in the pathogenesis of retinal vascular or inflammatory diseases.

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).

References (43)

  • J.A. Phipps et al.

    The renin-angiotensin system and the retinal neurovascular unit: a role in vascular regulation and disease

    Exp. Eye Res.

    (2019)
  • Y.C. Shen et al.

    Zebrafish cone-rod (crx) homeobox gene promotes retinogenesis

    Dev. Biol.

    (2004)
  • B. Strilic et al.

    The molecular basis of vascular lumen formation in the developing mouse aorta

    Dev. Cell

    (2009)
  • K. Xu et al.

    Blood vessel tubulogenesis requires Rasip1 regulation of GTPase signaling

    Dev. Cell

    (2011)
  • H. Yokoi et al.

    Expression profiling of zebrafish sox9 mutants reveals that Sox9 is required for retinal differentiation

    Dev. Biol.

    (2009)
  • P. Zou et al.

    A conserved role of αA-crystallin in the development of the zebrafish embryonic lens

    Exp. Eye Res.

    (2015)
  • Y. Alvarez et al.

    Genetic determinants of hyaloid and retinal vasculature in zebrafish

    BMC Dev. Biol.

    (2007)
  • P. Berndt et al.

    Tight junction proteins at the blood-brain barrier: far more than claudin-5

    Cell. Mol. Life Sci.

    (2019)
  • M. Buttmann et al.

    Atorvastatin partially prevents an inflammatory barrier breakdown of cultured human brain endothelial cells at a pharmacologically relevant concentration

    J. Neurochem.

    (2007)
  • M. Cao et al.

    Metalloproteinase Adamts16 is required for proper closure of the optic fissure

    Invest. Ophthalmol. Vis. Sci.

    (2018)
  • Y. Chen et al.

    Protocadherin-17 function in Zebrafish retinal development

    Dev Neurobiol

    (2013)
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