Knockdown of Zebrafish Lumican Gene (zlum) Causes Scleral Thinning and Increased Size of Scleral Coats

Lung-Kun Yeh,Chia-Yang Liu,Winston W.-Y Kao,Chang-Jen Huang,Fung-Rong Hu,Chung-Liang Chien,I-Jong Wang

doi:10.1074/jbc.m109.043679

Abstract

The lumican gene (lum), which encodes one of the major keratan sulfate proteoglycans (KSPGs) in the vertebrate cornea and sclera, has been linked to axial myopia in humans. In this study, we chose zebrafish (Danio rerio) as an animal model to elucidate the role of lumican in the development of axial myopia. The zebrafish lumican gene (zlum) spans approximately 4.6 kb of the zebrafish genome. Like human (hLUM) and mouse (mlum), zlum consists of three exons, two introns, and a TATA box-less promoter at the 5'-flanking region of the transcription initiation site. Sequence analysis of the cDNA predicts that zLum encodes 344 amino acids. zLum shares 51% amino acid sequence identity with human lumican. Similar to hLUM and mlum, zlum mRNA is expressed in the eye and many other tissues, such as brain, muscle, and liver as well. Transgenic zebrafish harboring an enhanced GFP reporter gene construct downstream of a 1.7-kb zlum 5'-flanking region displayed enhanced GFP expression in the cornea and sclera, as well as throughout the body. Down-regulation of zlum expression by antisense zlum morpholinos manifested ocular enlargement resembling axial myopia due to disruption of the collagen fibril arrangement in the sclera and resulted in scleral thinning. Administration of muscarinic receptor antagonists, e.g. atropine and pirenzepine, effectively subdued the ocular enlargement caused by morpholinos in in vivo zebrafish larvae assays. The observation suggests that zebrafish can be used as an in vivo model for screening compounds in treating myopia.

Highlights

Myopia is a very common ocular disorder, which is characterized by excessive elongation of the eyeball
We have demonstrated that zebrafish lumican has all the structural features of small leucine-rich proteoglycans (SLRP), i.e. a central domain of nine leucine-rich repeats flanked by N- and C-terminal domains with conserved cysteine residues [45]
B, diameters of collagen fibril were analyzed in the corneal stroma, anterior and posterior sclera of 12 dpf-old-wild type, and zlum-MO-injected group

Summary

Introduction

Myopia is a very common ocular disorder, which is characterized by excessive elongation of the eyeball. Stroma, anterior sclera, and posterior sclera from six zlum-MO The entire genomic DNA sequence of zlum is shown in and six wild type zebrafish at each 7- and 12-dpf stage were supplemental Fig. 1.

Results

Conclusion