In situ hybridization of a col15a1 antisense RNA probe on whole mount zebrafish embryos from 10 to 48 hpf. (A, B) at 10 hpf, col15a1 expression is exclusively restricted to the notochord. (C, D) at 15 hpf, expression decreases in notochord. An intense labelling remains in the caudal part (arrowhead). (E, F) at 24 hpf, col15a1 expression is vanished in notochord, except in the caudal part. A weak signal of hybridization is also observed in the head region and in otic vesicles (arrow). Insert shows the signal in the caudal notochord (arrow) and in the chordo-neural hinge (arrowhead). (G, H) at 36 hpf, a weak signal appears in pectoral fins. (I, J) at 48hpf, a more intense col15a1 expression is detected in brain, in otic vesicle and in pectoral fins. Magnification: A–D: x 350; E–J: x 300

Immunostaining of frozen cross-section of 48 hpf wild type (A) and Col15a1-MO (B) zebrafish embryos using antibodies against mouse collagen XV NC8 domain. (A) In wild type, collagen XV is deposited around the notochord in the peri-notochordal sheath. A labelling is also observed in the skin. (B) Collagen XV immunostaining is completely extinguished in collagen XV morphants attesting for the morpholino efficiency. Note that staining persists in skin. Magnification: x 400.

48 hpf embryos injected with anti-sense Col15a1 morpholinos (B) are almost similar to the embryos injected with Col15a1-Ms (A) except they are slightly shorter. Histological analysis of collagen XV morphants. Transversal sections of wild-type (C), Col15a1-Ms (D) and Col15a1-MO injected embryos at 48hpf. In Col15a1-MO, the peri-notochordal sheath is thinner compared to wild-type and control embryos. In 48 hpf collagen XV morphants, the muscular tissue is disorganized and horizontal myosepta are no visible. Neural tube and vessels are normal. (F, G) Lateral views of wild-type embryos (F) and Col15a1-MO embryos (G) at 48 hpf stained with antibodies to dystrophin to reveal the shape of myotomes. Contrary to wild type embryos (F), morphants (G) exhibit a U-shape of the myotomes. Magnification: A: x 200; B: x 200; C–E: x 400; F, G: x 300.

The lack of collagen XV affects the nototochord differentiation. (A) In 24 hpf wild type embryos, the notochord visualized by Nomarsky microscopy shows cells with large vacuoles. (B) In collagen XV morphants, these vacuoles are smaller. Whole mount in situ hybridization of 24 hpf (C, E and inserts in G, H) and 30 hpf (G, H) wild-type (C, E, G) and col15a1-MO (D, F, H) zebrafish embryos with Ehh (C, D) and Col2a1 (E–H) probes. In Col15a1-MO, a persistent strong expression of the early chordamesoderm marker, ehh (D) and of Col2a1 (F, H) is observed in notochord. At 24 hpf, ehh expression (C) disappears and Col2a1 expression (E) is restricted to the floor plate (arrow) and the hypochord (arrowhead) n wild type embryos. (I, J) Laminin immunostaining in wild-type (I) and col15a1-MO (J) embryos. In morphants, the basement membrane is more diffuse and exhibits several staining interruptions. In figures A–F and I, J, the embryo head is on the left and the dorsal side on the top. Magnification: A, B: x 600; C–F: x 300; G, H: x 900; I, J: x 400.

The lack of collagen XV disrupts notochordal basement membrane and prevents normal muscle striation. Transmission electron micrographs of the notochord (A, B) and of the myotome of 48 hpf control (A, C, E) and morphant (B, D, F–I) zebrafish. (A) In wild type and in control embryos, basement membrane surrounding the notochord (no) is constituted by three different layers: a true basement membrane (bm, arrow), a dense fibrous layer (dl) that surrounds the notochord (black bar), and an outermost fibrous layer (ol) parallel to the notochord (white bar). (B) In Col15a1-MO embryos, the basement membrane is highly disorganized. In the dense fibrous layer (dl), collagen fibers are not properly arranged. The outermost fibrous layer (ol) is less dense and more diffuse compared to control. (C, E) In 48 hpf control embryos, myofibrils (my) are completely developed and the cytoplasm is overgrown with myofibrils. As observed in the longitudinal section (E) muscle striation is complete. (D, F–H) In Col15a1-MO embryos, the number of myofibrils (my) is low. Myofibrils are preferentially located in the cortical area of the cells (C) and often present an orthogonal arrangement (G). (F–H) Muscle striation (s) is more or less visible depending on the severity of the morphotype. (I) Multivesicular vesicles which represent a sign of muscle degeneration are observed. Bar: 2 μm.

Images extracted from video-tape recordings showing the loss of motility of zebrafish embryos injected with Col15a1 morpholinos (B) compared to control embryos injected with Col15a1-Ms morpholinos. (A) Wild type embryos rotate and show twitch movements of the tail. (B) In contrast, Col15a1 embryos show only few short contractions.

The lack of collagen XV disrupts slow and fast muscle patterning. (A, B) Lateral views of the myotomal segments of 48 hpf wild-type (A) and Col15a1-MO (B) embryos, revealing the slow and fast fibers stained with phalloïdin-FITC (arrows). (B) Similar views of morphants reveal an important disorganization of myofibrils. (C) 48 hpf wild-type and (D) Col15a1-MO smhc-GFP transgenic embryos, showing the organization of the slow fibers. Note that the slow fibers are collapsed in the Col15a1-MO embryos and that the nuclei are less visible. (E, F) Medial views of wild-type (E) and Col15a1-MO (F) embryos, revealing the fast fibers stained with mAb F310. At 48 hpf, only few Col15a1-MO fast fibers are observed, and large areas of the muscle are not stained (arrows). (G, H) Lateral views of 48 hpf wild-type (G) and Col15a1-MO (H) staining with β-catenin antibodies show that the lack of collagen XV does not lead to a decrease in muscle cell number. Magnification: A–H: x 300.

Transverse sections through the trunk of 24 hpf zebrafish embryos labelled with 4D9 antibody to identify muscle pioneers (arrows) and Medial Fast Fibers (MFFs, arrowheads). (A) In wild-type embryo sections, muscle pioneers and MFFs are located at the horizontal myosepta level. (B) Sections of Col15a1-MO embryo show an increased MFF number (n = 60/65) whereas the muscle pioneer distribution and number are normal. Magnification: x 600.

Ptc expression pattern in Col15a1-MO (B, D, G, H) compared to the wild-type (A, C, E, F) embryos at 9 hpf (A, B) and 24 hpf (C–H). (A, B) Similar ptc1 expression is detected at 9 hpf in adaxial cells (arrow) and neural plate (asterisk) of wild-type (A) and Col15a1-MO (B) embryos. (C, E, F) Ptc1 expression in myotomes of 24 hpf wild-type embryos in lateral (C), and dorsal views (E), and in cross-sections of the trunk region (F). (D, G, H) Expansion of the ptc1 expression domain in the myotomes of Col15a1-MO embryos at 24 hpf. (D) Lateral view, (G) Dorsal view, (H) Cross-section. Magnification: A, B: x 200; C–E, G: x 400; F, H: x 500.

Projection pattern of primary CaP and MiP motorneurons in the wild-type (A) and Col15a1-MO (B) embryos. (A, B) Lateral views of confocal scans of 24 hpf whole mount embryos stained with znp-1 antibody. Ventral motor nerve extends shorter or forms extended branched axons in B. Magnification: A, B: x 400.

Transmission electron microscopy (A, B, E, F) and histological (C, D) micrographs of Col15a1-MO (A, C–F) and control (B) embryos at 48 hpf showing that the lack of collagen XV does not alter the development of neural tube (A, B), digestive tractus (C) and pronephretic ducts (D–F) (c: cilia; nt: neural tube; pd: pronephretic ducts).