Molecular analysis and characterization of zebrafish keratocan (zKera) gene.
Corneal small leucine-rich proteoglycans play a pivotal role in maintaining corneal transparency and function. In this study, we isolated and characterized the zebrafish (Danio rerio) keratocan (zKera) gene. The human keratocan sequence was used to search zebrafish homologues. The zKera full-length genomic DNA and cDNA were generated via PCR of zebrafish genomic DNA and reverse transcription-PCR of total zebrafish eye RNA, respectively. The zKera spanning 3.5 kilobase pairs consists of two exons and one intron and a TATA-less promoter. The zKera encodes 341 amino acids with 59% identity to its human counterpart and 57% identity to that of mouse keratocan. Like mouse and chick keratocan, zKera mRNA is selectively expressed in the adult cornea; however, during embryonic development, zKera mRNA is expressed in both the brain and the cornea. Interestingly, it is expressed mainly in corneal epithelium but also in the stroma. A pseudogene was proved by introducing a zKera promoter-driven enhanced green fluorescence protein reporter gene into fertilized zebrafish eggs. Using morpholino-antisense against zKera to knock down zKera resulted in a lethal phenotype due to massive caspase-dependent apoptosis, which was noted by a significant increase of active caspase-3 and caspase-8 in the developing forebrain area, including the eyes. This is different from mouse, for which keratocan-deficient mice are viable. Taken together, our data indicate that mammalian keratocan is conserved in zebrafish in terms of gene structure, expression pattern, and promoter function.
Yeh
Lung-Kun
LK
Department of Anatomy and Cell Biology, College of Medicine, National Taiwan University, Taipei 100, Taiwan; Department of Ophthalmology, Chang-Gung Memorial Hospital (Linko), Chang-Gung University College of Medicine, Taoyuan 333, Taiwan.
Liu
Chia-Yang
CY
Department of Ophthalmology, Cincinnati, Ohio 45267-0838.
Chien
Chung-Liang
CL
Department of Anatomy and Cell Biology, College of Medicine, National Taiwan University, Taipei 100, Taiwan.
Converse
Richard L
RL
Department of Ophthalmology, Cincinnati, Ohio 45267-0838.
Kao
Winston W-Y
WW
Department of Ophthalmology, Cincinnati, Ohio 45267-0838; Department of Cell Biology, Neuroscience, and Anatomy, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0838.
Chen
Muh-Shy
MS
Department of Ophthalmology, National Taiwan University Hospital, Taipei 100, Taiwan.
Hu
Fung-Rong
FR
Department of Ophthalmology, National Taiwan University Hospital, Taipei 100, Taiwan.
Hsieh
Fon-Jou
FJ
Department of Obstetrics and Gynecology, National Taiwan University Hospital, Taipei 100, Taiwan.
Wang
I-Jong
IJ
Department of Ophthalmology, National Taiwan University Hospital, Taipei 100, Taiwan. Electronic address: ijong@ha.mc.ntu.edu.tw.
D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D053148:Caspase 3 / Q000378:metabolism; D003315:Cornea / Q000254:growth & development / Q000378:metabolism / Q000648:ultrastructure; D018507:Gene Expression Regulation, Developmental; D006801:Humans; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D020287:In Situ Nick-End Labeling; D051379:Mice; D018345:Mice, Knockout; D046529:Microscopy, Electron, Transmission; D008969:Molecular Sequence Data; D010802:Phylogeny; D011401:Promoter Regions, Genetic / Q000235:genetics; D011509:Proteoglycans / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D015027:Zebrafish / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D029961:Zebrafish Proteins / Q000145:classification / Q000235:genetics / Q000378:metabolism*
C529479:Kera protein, zebrafish; D011509:Proteoglycans; D029961:Zebrafish Proteins; D053148:Caspase 3