Zebrafish as a Novel Model System to Study the Function of Caveolae and Caveolin-1 in Organismal Biology

Caveolin-1 (Cav-1) was first identified as a marker protein for the purification of caveolae organelles.1–3 Subsequently, it was later determined that Cav-1 expression is essential for caveolae formation.4,5 Thus, Cav-1^(−/−)-deficient mice morphologically lack caveolae organelles. Surprisingly, these mice are viable and fertile.4,5 In striking contrast, zebrafish (Danio rerio) lacking Cav-1 display important developmental abnormalities and embryonic lethality. These novel findings by Fang et al are described and highlighted in this issue of The American Journal of Pathology.6

Caveolin-1 Isoforms: Structure and Tissue-Specific Expression Patterns

The gene encoding Cav-1 has the same organization, with three exons and two introns, in human,7 mouse,8 and zebrafish.6 This suggests an important and conserved role for Cav-1 in whole-organismal biology. In fact, sequence alignment reveals that the Cav-1 protein is highly evolutionarily conserved, from Caenorhabditis elegans to humans (Figure 1).

Figure 1.

Evolutionary conservation of the caveolin-1 (Cav-1) protein. Alignment of the Cav-1 protein sequences from C. elegans (accession no. Q94051), D. rerio (accession no. Q6YLH9), and Homo sapiens (accession no. Q2TNI1) was produced using CINEMA.25 Note that the β-isoform uses an internal methionine (M32 in humans; M34 in zebrafish) as an initiation codon, as indicated (α versus β). As such, only the α-isoform undergoes tyrosine phosphorylation [Y14 in humans and zebrafish; see asterisk], since the β-isoform lacks tyrosine 14. The positions of the caveolin-signature-sequence (SQ) and the caveolin-scaffolding-domain (CSD) are highlighted. Palmitoylated cysteine residues have been boxed, in the case of the human and zebrafish sequences. Color coding is as follows: white, hydrophobic residues; blue, positively charged residues; red, negatively charged residues; green, small hydrogen bonding residues; brown, glycine/proline residues; yellow, cysteine residues; and cyan, aromatic residues.

Interestingly, a single Cav-1 gene encodes two protein isoforms that differ slightly, only by their N-terminal sequence.9 More specifically, Cav-1α is a 178-amino acid protein, whereas Cav-1β is 147 amino acids and lacks the first 31 N-terminal residues of Cav-1α. These two Cav-1 isoforms have been shown to be translated from distinct mRNA species.10 Until now, the specific functional role of each Cav-1 isoform had not been clearly defined. Nonetheless, Cav-1α and -1β have different subcellular distributions, as demonstrated by recent studies.9,11,12 Moreover, Cav-1α has been shown to form caveolae more readily than Cav-1β.13

In zebrafish, Cav-1 mRNAs are detected during the very early stages of development. Late in development, the Cav-1α mRNA is the only isoform detectable in intestinal epithelium, whereas both Cav-1α and -1β mRNAs are produced in the heart, pharyngeal vasculature, notochord, somites, skin, and neuromast tissues. Interestingly, these data are similar to those obtained in Xenopus laevis.14

In the mouse, Cav-1α protein expression is detected early in the embryo (E15).12 Maximal expression is observed in the vasculature, the lungs, the kidneys, and the gut. Interestingly, in the lungs, Cav-1α expression first appears in endothelial cells. The importance of Cav-1 in the vasculature has also been highlighted by Bullejos et al, who observed high levels of Cav-1 mRNA in the developing ovaries but not testes.15 This difference is due to the formation of a more dense and more complex vascular network in the ovaries.15

Roles of Caveolin-1 during Development

In mice, Cav-1 expression does not appear to be as essential as in zebrafish, since its elimination is not lethal.16,17 However, its role in the vasculature and other tissues is clearly important, since its absence has been associated with many disease-related phenotypes, most notably in the lung, vasculature, heart, adipose tissue, and the mammary gland. However, the detailed developmental progression of the Cav-1-deficient mouse embryo has yet to be determined. For example, alterations observed in Cav-1-deficient murine lungs could result from developmental abnormalities.

In zebrafish, Cav-1 down-regulation, in the case of both isoforms (Cav-1α and Cav-1β), is associated with important defects occurring by 12 hours after fertilization. This time point is normally associated with a remarkable increase in Cav-1 mRNA levels. As expected, reductions in the Cav-1 protein are also associated with a major reduction in the number of caveolae.

One of the first proteins shown to associate with caveolae is actin.18,19 This “anchoring” interaction appears to be responsible, at least in part, for the extremely reduced mobility of caveolae at the cell surface.20 In addition, during cellular migration, Cav-1 has been shown to assume a polarized distribution in migrating endothelial cells.21,22 Moreover, it was also shown that this specific polarization during trans-migration requires the presence of the Tyr14 residue within Cav-1 for phosphorylation, since the distribution of other forms of Cav-1 (Cav-1α (Y14A) and Cav-1β) are not polarized.22

It is important to note that phosphorylation of Cav-1α at Tyr14 has been associated with its subcellular localization in close proximity to focal adhesions,23 as well as caveolae-mediated endocytosis.24 Interestingly, in zebrafish, a deficiency in Cav-1α cannot be rescued by a mutant form of Cav-1α (Y14F) that cannot undergo phosphorylation. In addition, Cav-1 deficiency is associated with severe disruption of the actin cytoskeleton. These findings suggest that Cav-1 plays a critical role in cell migration and/or endocytosis in zebrafish. Likewise, overexpression of the full-length Cav-1α isoform could not rescue the phenotype induced by the absence of the Cav-1β isoform, and visa versa. Taken together, these data suggest for the first time that Cav-1α and Cav-1β have nonoverlapping functions and that these differences may be related to the ability of the Cav-1α isoform to undergo tyrosine phosphorylation at residue 14.

Replacement of zebrafish Cav-1 by the corresponding human Cav-1 isoform could complement the phenotypes associated with the absence of each isoform. This finding further suggests that the function of the Cav-1 protein is highly conserved throughout evolution. In mammals, however, the absence of Cav-1 may not be as lethal as in zebrafish because of the existence of redundant compensatory mechanisms.

Conclusions

Clearly, the mouse and human systems are more complicated than zebrafish. However, the zebrafish model of development will provide, for the first time, a genetically tractable system to perform rapid and detailed mutagenesis of both Cav-1α and Cav-1β isoforms. As such, the zebrafish system is a new experimental tool for investigators to directly dissect the relationship between the primary structure of Cav-1 and its essential developmental and whole organismal functions.