Caveolae are small flask-shaped invaginations of the plasma membrane. Discovered over fifty years ago, we are only now beginning to understand the role of these structures. Caveolins are integral membrane proteins associated with caveolae. Caveolin-1 and -2 are found in most cell types while caveolin-3 is generally muscle specific. Caveolins have been associated with diseases such as muscular dystrophy and cancer suggesting that understanding the role of caveolins may answer some fundamental questions about these diseases.
Chapter 2 addresses the role of caveolin-3 in the mouse. Sequence analysis of the promoter region of caveolin-3 mouse genomic DNA revealed similarities to the human sequence. Putative regulatory elements were identified within the 5' sequence of the caveolin-3 gene, including a conserved myogenin binding site required for skeletal muscle regulation and a Nkx2.5 binding site that may be required for cardiac expression. Restriction mapping allowed for the construction of two targeting vectors that were injected into mouse ES cells to produce a mouse line that has no caveolin-3 expression. This chapter also addresses the role of caveolin-3 in muscle development in the zebrafish, Danio rerio. We employed an antisense oligonucleotide approach (morpholino) in order to knockdown the expression of caveolin-3. Loss of caveolin-3 expression results in disruption to the normal muscle architecture with isolated muscle fibres displaying major disruption to the contractile apparatus. Previous studies addressing the role of caveolin-3 in myotube fusion has been controversial. These studies suggest that caveolin-3 is important in myotube fusion as reduction of caveolin- 3 expression results in an increase in the number of mononucleate muscle fibres.
Expression analysis of caveolin-3 in the developing zebrafish uncovered some novel findings; caveolin-3 was expressed very early in development, in the slow muscle precursor cells, the adaxial cells. This expression precedes the expression of elements of the contractile apparatus of the muscle. Expression was also discovered in a non-muscle cell type, the notochord, suggesting novel regulatory elements and functions in the zebrafish.
In chapter 3 of this thesis, expression of caveolin-1 was examined in the zebrafish embryo. Caveolin-1 was expressed in major signalling tissues of the zebrafish embryo including the tailbud, Kupffer's vesicle, and the notochord. Expression was also discovered in the heart, periderm and neuromasts of the posterior and anterior lateral line. Loss of expression of the alpha isoform of caveolin-1 using antisense oligonucleotides resulted in disruption to the lateral line neuromasts, sensory organs important in schooling and prey capture in the fish. Loss of caveolin-1 expression also resulted in reduction of heart rates suggesting that caveolin-1 alpha has an important role in cardiac development in the zebrafish.
In summary this work demonstrates the value of different model systems to explore the roles of different genes. The zebrafish, in particular, has proven itself a new and exciting model system in which to further our understanding of the caveolin family members in the developmental system.