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First published online 5 June 2007
doi: 10.1242/jcs.003830

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Caveolin-1 is required for lateral line neuromast and notochord development

¹ Institute for Molecular Bioscience, University of Queensland, Brisbane 4072, Australia
² Centre for Microscopy and Microanalysis, University of Queensland, Brisbane 4072, Australia
³ School of Biomedical Sciences, University of Queensland, Brisbane 4072, Australia
⁴ Institute of Neuroscience, University of Oregon, Eugene, OR 97403, USA

View larger version (118K): [in this window] [in a new window]   Fig. 1. cav1 localises to key signalling tissues in the developing zebrafish. Developmental expression pattern of cav1 was analysed by whole-mount mRNA in situ hybridisation. Anterior is to the left and dorsal to the top unless otherwise stated. (A) Dorsal view of a 12-hour embryo where cav1 expression is detected in the notochord (N). (B) At the same time cav1 expression can be seen in the epithelial lining of Kupffer's vesicle (posterior view; asterisk). (C-E) Between 16-19 hours cav1 expression is very high in a cluster of cells within the tailbud (tb) (C, 16 hours, arrow; D, 17 hours), notochord and epidermis. (F) At 36 hours, cav1 expression is high in the periderm but excluded from the migrating primordium of the lateral line (mp). (G) A magnified view of the periderm at 36 hours shows expression around the nuclei. By 72 hours, expression is apparent in the neuromasts (arrowheads, I,J,L,N), the heart (h, H) the branchial arches (ventral view, asterisk, I) and in the pronephric ducts (arrows, L). (K) At 48 hours expression can be seen in a region near the intersomite borders, probably the intersegmental vessels (arrow). (M) By 6 days, cav1 expression in the heart is restricted to the ventricle (arrow). Bars, 250 µm (A, also for D and E) (C,F,J-N); 25 µm (B); 10 µm (G); 50 µm (H).

View larger version (79K): [in this window] [in a new window]   Fig. 2. Zebrafish, chick and mouse notochords express caveolin. (A-E) Ultrastructural analysis of caveolae in the notochord during embryonic development. (A) 30-hour zebrafish embryo showing the notochord at low magnification. Arrowheads indicate the sheath surrounding the notochord cells. The notochord cells contain large vacuoles (v) surrounded by a thin layer of cytoplasm. The three boxed areas show the cell-cell contact regions within the notochord where the highest density of caveolae are found. (B) Peripheral area of the notochord of a 48-hour embryo showing several cell-cell contact regions densely covered in caveolae. Four different cells are indicated (1-4). Caveolae in cell 2 are indicated by dots; red dots indicate caveolae on the plasma membrane oriented towards the centre of the notochord (in contact with cell 3), yellow dots indicate caveolae on the cell surface in contact with cell 4. Note the difference in density on the two surfaces of the cell with more caveolae oriented towards the centre of the notochord away from the sheath (s). (C,E) Central area of 48-hour embryos showing high density of caveolae close to longitudinally running filaments (small arrowheads in C). Three different cells are indicated in C (1-3). Note the putative desmosomes (large arrowheads). (D) 24-hour embryos show a lower density of caveolae. (F,G) Paraffin-embedded sections from zebrafish (72 hours, F; inset shows different focal plane), chicken (Stage 24, G), and mouse (E10.5, H) labelled for caveolin; sc, spinal cord. Caveolin expression is high in the notochord of all three species. Bars, 10 µm (A); 1 µm (B); 500 nm (D,E); 50 µm (F-H).

View larger version (209K): [in this window] [in a new window]   Fig. 3. Caveolae interact with intermediate filaments in the notochord. (A) Thin sections of 3-day post fertilisation embryos showing caveolae within the cell-cell contact regions of the notochord. Caveolae (c) at the plasma membrane contact a distinct layer of filaments on the cytoplasmic side (f indicates layer of filaments). Frequent connections (arrowheads) between caveolae and filaments are apparent (boxed areas in A shown in lower panels). (B) Images from a tomogram of the notochord (see supplementary material Movie 1). (C) Images of same areas boxed in B with arrowheads indicating the same areas in different sections. Connections between caveolae and filaments are apparent. (D) Paraffin embedded 72-hour zebrafish sections labelled with a pan-cytokeratin antibody indicating expression in the notochord. Boxed regions demonstrate regions of interest in panel E and F with panel E showing cells in the periphery of the notochord, and panel F showing a central region. (E) In the peripheral regions, caveolin is associated with the plasma membrane between cells (arrows) and keratin is associated with bundles of fibres (arrowhead). (F) In the central strut-like regions of intercellular contact, cytokeratin fibres are found on either side of caveolin-labelled caveolae. Immunogold labelling of 3-day zebrafish sections show caveolin (small gold, arrows) in close proximity to cytokeratin (large gold, arrowheads). Note that the notochord is particularly hard to preserve in frozen sections owing to the large `empty' vacuoles and extremely thin bridges of cytoplasm in between. S, sheath; PM, plasma membrane. Bars, 200 nm (A,B); 100 nm (inset A,C); 100 µm (D); 100 nm (E,F).

View larger version (91K): [in this window] [in a new window]   Fig. 4. Injection of cav1-MOs downregulates Cav1 expression. Embryos were injected with control MO (A,C,D,G,H) or cav1-MO1 (I,J) or cav1-MO2 (B,E,F). At 48 hours, control MO injected embryos appear normal (A), whereas embryos injected with cav1-MO2 are curled with disrupted notochords and tails (B). At 28 hours, control MO injected embryos have a normal notochord (C), which is also seen at 48 hours (D). In cav1-MO2 injected embryos the notochord appears `bubbly' and undulating (E) and the undulating notochord is still apparent at 48 hours (F). When embryos are labelled for caveolin with the pan-caveolin antibody at 2 days post fertilisation, control MO injected embryos have normal Cav1 labelling at the cell surface (G) with normal Cav3 labelling in muscle (H), but when embryos are injected with cav1-MO1 at 1.5 ng/embryo, the labelling at the junction of these cells is diminished, although some weak intracellular labelling remains (I). Staining in the muscle is unaffected by cav1-MO1 (J compared with H). (K) Western blot analysis of cav1-MO1 and cav1-MO2 48 hours post injection labelled with the Transduction Laboratories polyclonal caveolin antibody show complete downregulation of Cav1 at 3 ng/embryo as indicated by loss of the band corresponding to caveolin (seen in the control MO lane), 10 µg protein was loaded in each lane and Coomassie Blue staining of the membrane shows equal protein loading. (L) Injection of cav1-MO2 causes the body length to be reduced. The few straight embryos were measured to get fish length (cav1-MO2, n=38; control MO n=37). Error bars are standard deviation. P values were determined using two-tailed t-test for samples with equal variance. Shorter lengths are also observed in curved embryos and with cav1-MO1. (M) Injection of cav1-MO1 at 1.5 ng/embryo causes a reduced number of neuromasts at 72 hours along the posterior lateral line (n=71) compared with control embryos. Neuromasts were identified by DASPEI labelling. (N) Loss of both Cav1 and Cav3 results in a more dramatic phenotype where small rounded cells are still evident at 48 hours. Bars, 250 µm (A,B); 50 µm (C-F,N); 20 µm (G-J).

View larger version (122K): [in this window] [in a new window]   Fig. 5. Downregulation of cav1 disrupts normal notochord development. (A-F) Analysis of notochords in 30-hour embryos injected with control MO (A,C,E) and cav1-MO2 (B,D,F). Control MO injected embryos have large vacuoles and a tight undulating sheath (A,C). cav1-MO2 injected embryos have small vacuoles with a large amount of cellular material around the edge of the notochord (B). The sheath (s) appears to be much wider and more disorganised in cav1-MO2 injected embryos (D) compared with control MO injected embryos (C). cav1-MO2 injected embryos have many fewer caveolae and large spaces between cell membranes (F) compared with the controls (E). (G) Loss of both Cav1 and Cav3 results in a more dramatic downregulation of caveolae number. Arrowheads, membranes of individual cells; v, vacuole. Bars, 10 µm (A,B); 1 µm (C-G).

View larger version (70K): [in this window] [in a new window]   Fig. 6. cav1-MO disrupts development of neuromasts in the posterior lateral line. (A) Projected stack of confocal sections of zebrafish embryos labelled with caveolin antibody shows labelling around the neuromasts near the eye (e) at 72 hours. Cav1 whole-mount in situ hybridisation was carried out and sections of 72-hour embryos were examined. (B) cav1 mRNA is found throughout the neuromast. (C) Scanning electron micrograph of a 72-hour control zebrafish embryo with normal neuromasts. DASPEI labelling (D-F) and scanning electron microscopy (G-J) reveal that 72-hour control MO injected embryos have seven to eight neuromasts along their posterior lateral line (D) but when they are injected with a morpholino targeted to cav1 at 1.5 ng/embryo (cav1-MO1), the number of neuromasts are severely reduced (E). (F) The result was confirmed with the second morpholino to cav1 (cav1-MO2). (G-J) The number of neuromasts in the posterior lateral line after cav1 MO injection was also shown to be reduced (H,J) compared with WT embryos (G,I). I and J are magnifications of the boxed areas of G and H to demonstrate the loss of neuromasts in the lateral line. h, head. Bars, 20 µm (A-C); 250 µm (D-H); 100 µm (I,J).