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

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 ϳ4.6 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. Downregulation 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.Myopia is a very common ocular disorder, which is characterized by excessive elongation of the eyeball. In Taiwan, the prevalence of myopia is about 84% of schoolchildren aged 16 -18, and the prevalence of high myopia (less than Ϫ6.0 D) at 18 years of age is 24% in girls and 18% in boys (1). In contrast, the prevalence of high myopia is much lower in Western countries, about 1% of the general population (2). These studies imply that genetic susceptibility of ethnic differences may account for the high prevalence of myopia in Taiwanese.The sclera contains a collagen-rich extracellular matrix that undergoes significant biochemical and biomechanical remodeling during the development of myopia (3). Linkage studies of high myopia have identified potential loci MYP1 (Xq28) and MYP3 (12q21-23); these loci are within and/or near the loci of the human genome containing several genes that encode small leucine-rich proteoglycans (SLRP), 3 i.e. biglycan (Xq27ter), decorin (12q21-22), lumican (12q21.3-22), and DSPG3 (12q21) (4 -9). Our previous study showed that certain variations (rs3759223 (C3 T)) of single nucleotide polymorphism in the lumican regulatory region may influence the promoter activities of lumican and affect fibrillogenesis in myopic eyes (10). Furthermore, Majava et al. (11) found that a novel single nucleotide polymorphism (c.893-105G3 A) of the lumican gene was associated with high myopia. Recently, our prospective case control study also showed that genetic variation in the regulatory domai...

Genome-wide gene expression in a patient with 15q13.3 homozygous microdeletion syndrome

“…30 A zebrafish LUM knockout model resulted in scleral thinning and ocular enlargement, among other changes. 31 Our patient has a markedly abnormal ERG with a depressed b-wave, a finding consistent with loss of TRPM1 and CSNB. [18][19][20][21] It is possible that the differential expression of LUM in the proband may be secondary to an indirect effect of CSNB.…”

Lumican is a major small leucine-rich proteoglycan (SLRP) in Atlantic cod (Gadus morhua L.) skeletal muscle

“…To the best of our knowledge, limited data on the SLRPs expression in teleost fish species exist, with the exception of work on zebrafish [1,2,20]. Interestingly, evidence for the expression of certain SLRPs (e.g.…”

“…zebrafish) in model systems is increasing. Proteoglycans such as the small, leucine-rich proteoglycans (SLRPs) may be significant contributors to fish health, growth and development as shown in zebrafish (Danio rerio) knock-out studies [1,2]. The SLRPs may also influence fillet texture as they are major collageninteracting molecules influencing collagen fibrillogenesis as well as being collagen network stabilizers [3].…”

Association between a Lumican Promoter Polymorphism and High Myopia in the Chinese Population: A Meta-Analysis of Case-Control Studies

“…Lumican regulates corneal inflammatory responses and promotes corneal epithelial wound healing [31,32,33,34]. The phenotype of the lumican-deficient mouse and zebrafish implicates lumican as a functional candidate gene for high myopia [19,21]. Several high-frequency single-nucleotide polymorphisms have been identified in lumican [22,26], some of which may contribute to the pathogenesis of high myopia [2,22,23,24,25,26]; however, other genetic association studies suggest that lumican polymorphisms are not associated with this condition [3,6,27,28].…”

“…Lumican-fibromodulin double-deficient mice manifest certain features of high myopia, including structural changes in the collagen fibrils of the sclera, thinning of the sclera, retinal detachment and increased ocular axial length [19]. Yeh et al [21] found that knockdown of zebrafish lumican by antisense morpholinos produced ocular enlargement resembling axial myopia. The ocular enlargement was caused by disruption of the collagen fibril arrangement in the sclera and resulted in scleral thinning.…”