Chemical genetics suggests a critical role for lysyl oxidase in zebrafish notochord morphogenesis

Abstract

As a result of a chemical genetic screen for modulators of metalloprotease activity, we report that 2-mercaptopyridine-N-oxide induces a conspicuous undulating notochord defect in zebrafish embryos, a phenocopy of the leviathan mutant. The location of the chemically-induced wavy notochord correlated with the timing of application, thus defining a narrow chemical sensitivity window during segmentation stages. Microscopic observations revealed that notochord undulations appeared during the phase of notochord cell vacuolation and notochord elongation. Notochord cells become swollen as well as disorganized, while electron microscopy revealed disrupted organization of collagen fibrils in the surrounding sheath. We demonstrate by assay in zebrafish extracts that 2-mercaptopyridine-N-oxide inhibits lysyl oxidase. Thus, we provide insight into notochord morphogenesis and reveal novel compounds for lysyl oxidase inhibition. Taken together, these data underline the utility of small molecules for elucidating the dynamic mechanisms of early morphogenesis and provide a potential explanation for the recently established role of copper in zebrafish notochord formation.

Fig. 1. Structures of active compounds. 2-Mercaptopyridine-N-oxide 1 and sulfur derivatives (4 and 5), as well as thiosemicarbazones derived from cyclohexanone 2, n-butanal 3 and cyclopentanone 4 are shown.

Fig. 2. MCP 1-induced notochord deformation. Lateral views of 52 hpf wild-type embryos after treatment with 100 nM MCP 1. Note the prominent undulating deformations (B) of the notochord (n), contrasting with straight morphology of untreated sibling control (A).

Fig. 3. MCP 1-induced notochord and pigmentation phenotypes. Lateral views of 48 (a,c,e,g,j) and 72 hpf (b,d,f,h,k,i,l) embryos after treatment with thiosemicarbazones derived from cyclohexanone 2 (c,d), butanal 3 (e,f), cyclopentanone 4 (g–i) or MCP 1 (j–l) or untreated controls (a,b). All treated embryos were exposed to 100 µM of the compound, except (i) and (l) where the dose was only 5 µM. Note pronounced notochord (arrow) deformation induced by all 100 µM treatments and by 5 µM MCP 1, whereas lower dose of 2, 3 and 4 usually resulted in normal notochord development (shown for 4 (i), but similar for others). Note reduced melanisation of eyes (e) and melanocytes (*) under high dose treatments, with 48 hpf treated embryos usually lacking or having very reduced melanin (c,e,g,j), but with melanisation recovering at 72 hpf after treatment with 100 µM 2 (d), 3 (f), or 4 (h). Treatment with 5 µM 2–4 does not affect melanisation at 72 hpf (shown for 4 (i), but similar for others). MCP 1 treatment results in a stronger melanisation phenotype, with little or no recovery at 72 hpf in 100 µM (k) and moderate recovery at 72 hpf in 5 µM (l) treatment.

Fig. 4. Temporal control of notochord morphogenesis. The anterior boundary of the notochord defect (arrowhead) shifts in the posterior direction as the time of MCP 1 addition is delayed. 200 nM MCP 1 was added at (a) 12 hpf, (b) 16 hpf and (c) 20 hpf; all embryos were photographed at 48 hpf. (d) The relationship between the time of MCP 1 addition and the anterior boundary of the notochord defect is given as the adjacent somite number (mean ± s.d.). Embryos here were scored at 3 dpf. N for each time-point >10. (e) If MCP 1 added for a short time interval (12–15 hpf) and then removed, notochord defects are localised within restricted zone (demarcated by arrowheads), here shown in 3 dpf embryo. (f) If MCP 1 was present before 6 hpf, then removed at various subsequent time-points, the posterior boundary of the affected notochord region was shifted, although the rate of change was lower than for ‘ON graph’. N for each time-point >15.

Fig. 5. MCP 1 affects notochord morphology from late-somitogenesis stages. Lateral views of trunk of embryos treated with 200 nM MCP 1 (b,d,f,h) or left untreated (a,c,e,g) and photographed at 15 (a,b), 18 (c,d), 24 (e,f) and 36 (g,h) hpf. Insets show close-ups of notochord cells to show progressive enlargement of vacuoles. The notochord (n) appears rather normal at early stages, but rapidly becomes severely kinked during the phase of cell vacuolation (just detectable at 18 hpf (d), dramatic by 24 hpf (f)). Note in (g) and (h) the clear correlation between notochord distortion and incomplete rostrocaudal expansion.

Fig. 6. MCP 1 treatment disrupts notochord sheath differentiation. (a–d) col2α1 is expressed ectopically in the notochord of MCP 1-treated embryos. (a,c) Lateral views of untreated embryo at 24 hpf show lack of expression of col2α1 in notochord throughout trunk and most of tail, contrasting with strong expression in floorplate (arrow) and hypochord (arrowhead). (b,d) Sibling embryo treated with 100 µM MCP 1 shows strong ectopic expression of col2α1 in trunk notochord (*), as well as floorplate and hypochord expression. Identical phenotypes were seen with 2, 3 and 4 (data not shown). (e–h) Transmission electron micrographs of notochord sheath in 27 hpf control (e,g) and MCP 1-treated (100 nM, f,h) embryos. Note regular organization of collagen fibres in medial (m) and outer (o) layers of normal sheath, and disorganized fibre arrangements in MCP 1-treated fish. N: notochord cell. Scale bar, 200 nm (e,f); 100 nm (g,h).

Fig. 7. MCP 1 inhibits lysyl oxidase activity. Lysyl oxidase activity in crude fish extracts is measured in the absence and presence of a known lysyl oxidase inhibitor (β-APN) or MCP 1 or both combined. Data shown is representative of three separate experiments and activity is presented in relative fluorescent units with each data point as the mean of three triplicate measurements, error bars indicate standard deviation.