(The) role of Transmembrane 4 L6 family member 5 in the development of zebrafish

다국어 초록 (Multilingual Abstract)

Transmembrane 4 L6 family member 5 (TM4SF5), a member of tetraspanin L6 superfamily, is a glycoprotein which has four transmembrane domains. TM4SF5 has been reported to be closely involved in tumorigenesis, being a signaling regulator during communication between tumor cells and tumor microenvironment via interaction with various proteins such as growth factor receptors, integrin receptors. It has been shown that TM4SF5 regulates important cellular processes like epithelial-to-mesenchymal transition (EMT), which is importantly functional for development of live fibrosis, tumorigenesis, and cell migration/invasion. In zebrafish, somitogenesis and myotome formation are the processes that a wide range of cellular processes including cell migration/adhesion, EMT and morphological changes occur simultaneously. However, the function of TM4SF5 during embryogenesis in which EMT and cell migration/adhesion are crucial remains unknown. Here we investigated the function of TM4SF5 in zebrafish embryogenesis.
Expression of tm4sf5 mRNA was elevated during somitogenesis, and deteced in porsterior somites. After 1 dpf, tm4sf5 transcripts were detected in whole myotome. Knock-down of tm4sf5 using morpholino oligonuceotides resulted in an aberrant development of trunk and abnormal morphology of muscle fibers. The overall muscle mass was also reduced in tm4sf5 morphants. In addition, the knock-down of tm4sf5 altered expression of integrin alpha5, which is known to be expressed in muscle pioneer and necessary for somite boundary maintenance. Because the somite boundaries mature into myotome boundaries and necessary for proper morphogenesis of muscle fibers, we could infer that tm4sf5-dependentt integrin alpha5 expression or function is important for normal myotome formation. Taken together, these observations suggest that TM4SF5 plays a regulatory role in zebrafish embryogenesis, especially in myogenic processes, presumably via collaborations with integrin alpha5.

목차 (Table of Contents)

• ABSTRACT 1
• LIST OF FIGURES 4
• INTRODUCTION 5
• MATERIALS AND METHODS 9-16
• 1. Protein sequence analysis 9
• 2. Zebrafish strains and maintenance 9
• 3. RNA isolation and cDNA synthesis from zebrafish embryos 9
• 4. Polymerase chain reaction 10
• 5. In vitro transcription of digoxygenin-UTP labeled anti-sense RNA probes 11
• 6. Whole mount in situ hybridization 12
• 7. Whole mount in situ immunostaining 14
• 8. Anti-sense morpholino oligonucleotides 15
• 9. Cryosection 15
• 10. Microscopy 16
• RESULTS 17-35
• 1. Comparison of TM4SF5 protein sequence homology from zebrafish and other vertebrates 17
• 2. Expression of tm4sf5 during zebrafish development 19
• 3. Knock-down of tm4sf5 by antisense morpholino-oligonucleotides 25
• 4. Expression pattern of myogenic progenitor cell marker in control and tm4sf5 morphant embryos 28
• 5. Slow muscle and fast muscle development in control and tm4sf5 morphant embryos 31
• 6. Altered expression pattern of integrin α5 in tm4sf5 morphant embryos 33
• DISCUSSION 36
• REFERENCES 41
• ABSTRACT IN KOREAN 44