Site-1 protease is required for cartilage development in zebrafish

Abstract

gonzo (goz) is a zebrafish mutant with defects in cartilage formation. The goz phenotype comprises cartilage matrix defects and irregular chondrocyte morphology. Expression of endoderm, mesoderm, and cartilage marker genes is, however, normal, indicating a defect in chondrocyte morphogenesis. The mutated gene responsible for the goz phenotype, identified by positional cloning and confirmed by phosphomorpholino knockdown, encodes zebrafish site-1 protease (s1p). S1P has been shown to process and activate sterol regulatory element-binding proteins (SREBPs), which regulate expression of key enzymes of lipid biosynthesis or transport. This finding is consistent with the abnormal distribution of lipids in goz embryos. Knockdown of site-2 protease, which is also involved in activation of SREBPs, results in similar lipid and cartilage phenotypes as S1P knockdown. However, knockdown of SREBP cleavage-activating protein, which forms a complex with SREBP and is essential for S1P cleavage, results only in lipid phenotypes, whereas cartilage appears normal. This indicates that the cartilage phenoptypes of goz are caused independently of the lipid defects.

Fig. 1.

Cartilage defects in goz. Alcian blue staining of sibling larvae (a, c, and e) and goz larvae (b, d, and f) at 5 dpf is shown. (a and b) Lateral view. (c and d) Ventral view of the head. (e and f) Enlargement of the branchial arches. Cartilage matrix staining is slightly reduced in goz mutants. All cartilage elements, including Meckel's cartilage (arrows in c and d), are smaller in the mutant. Tissue anterior to the eyes is missing (bars in c and d). The columnar arrangement of chondrocytes in the branchial arches is disrupted in goz mutants (arrows in e and f). (Scale bars are 200 μmin a–d and 50 μmin e and f.)

Fig. 2.

Proteoglycan phenotypes in goz. Wheat germ agglutinin staining of matrix proteoglycans in wild-type (WT) sibling larvae (a and c) and goz larvae (b and d) at 5 dpf. (a and b) Ventral view. (c and d) Enlargement of Meckel's cartilage. Proteoglycans are homogeneously stained in the cartilage matrix of sibling larvae (a and c). This staining is absent in the cartilage matrix of goz (arrow in d) where proteoglycans seem to accumulate ectopically (arrowheads in b and d). (Scale bars are 200 μm in a and b and 50 μm in c and d.)

Fig. 3.

Col II defects in goz. Immunohistological staining of Col II in wild-type (WT) sibling larvae (a, c, and e) and goz larvae (b, d, and f) is shown. (a and b) Ventral view of the head 5 dpf. (c and d) Enlargement of the trabeculae (tb) of the neurocranium (lateral view) at 2 dpf. (e and f) Enlargement of the notochord (n) in the tail at 2 dpf. Cartilage matrix is homogeneously stained in sibling larvae (a, c, and e), whereas abnormal protein aggregates can be seen in the cartilage matrix and around the notochord in goz larvae (d and f). (Scale bars are 200 μm in a and b and 50 μm in c–f.)

Fig. 4.

Positional cloning of the goz locus. Meiotic mapping using microsatellite markers (Middle) placed goz^tr6721 mutation on linkage group 18 (LG18) (Top) between markers z9194 and z7142, close to marker z24123. Single-nucleotide polymorphism markers are represented by gray ovals overlying genomic clones (solid lines in Middle). The number of recombinants between the different markers from 7,500 to 8,000 meioses is summarized at the bottom of Middle. (Bottom) Models of genes predicted in the critical interval (scale in kb). Exons are represented as vertical lines; direction of transcription is shown by arrowheads.

Fig. 5.

PMO phenocopy of goz phenotype. Shown are branchial arches of goz (b) or larvae injected with s1p (c) or s2p (d). PMOs show randomly arranged chondrocytes (arrows). Injection of SCAP PMOs resulted in wild-type cartilage (e), which is indistinguishable from injection controls (a). Larvae were stained with alcian blue at 5 dpf. (Scale bar is 50 μm.)

Fig. 6.

S1P. (A) S1P is synthesized as preproprotein and activated through sequential cleavage. A growth factor cytokine receptor (GFCR)-like motif is essential for this autocatalytic process (arrow in A). (B) Both goz alleles result in truncated proteins lacking intact GFCR-like motifs and are predicted to express only inactive S1P. An altered sequence caused by splice defects in goz^tr6721 is shown in red. The figure was adapted from Elagoz et al. (33).

Fig. 7.

Expression pattern of S1P. Whole-mount in situ hybridization with s1p in eight-cell stage (a), tailbud stage (b), two-somite stage (c), 20 hpf (d), and 34 hpf (e) zebrafish embryos. (a, c, and d) Lateral view. (b) Dorsal view. (e) Dorsolateral view. Both maternal (a) and zygotic (b–e) expression of s1p can be observed. Expression is pronounced in the midline at tailbud stage (arrow in b). Strong expression can be seen in the head (d, arrowhead) and the immature notochord (d, arrow) at 20 hpf. At 34 hpf (e), s1p is strongly expressed in the head, the fin buds (arrow), and the region of the endodermal pouches (arrowhead). (Scale bar is 200 μm.)

Fig. 8.

Lipid defects in zebrafish s1p mutants. Oil red O staining of 48 hpf zebrafish larvae. (a and b) Lateral view. (c–f) Ventral view. goz (b and d) and sibling (a and c) larvae and larvae injected with 6 ng of s2p2 (e) and 12 ng of SCAP1 (f) PMOs are shown. Strong lipid staining can be observed around the eye (arrow), the otic vesicle (arrowhead), and in the heart (h) of sibling larvae (a), in addition to the region of developing trabeculae (arrow in c). Both sibling and goz larvae show a strong lipid staining of the yolk (asterisks in a and b). Lipid deposit is severely reduced in all other tissues in goz larvae (b), especially around the eye (arrow) and the otic vesicle (arrowhead). No lipid can be detected around the developing trabeculae in goz (arrow in d). (e and f) Injection of s2p and SCAP PMOs results in the same reduction of lipid. (Scale bars are 200 μm in a and b and 100 μm in c–f.)

Fig. 9.

Expression pattern of xbp1 and grp78. Whole-mount in situ hybridization with xbp1 and grp78 at 48 hpf is shown from a lateral view. The expression patterns of xbp1 and grp78 are indistinguishable between goz (b, d, f, and h) and wild type (a, c, e, and g). (Scale bars are 200 μmin a, b, e, and f, 500 μm in c and d, and 400 μm in g and h.)