Essential role of lysyl oxidases in notochord development

Abstract

Recent studies reveal a critical role for copper in the development of the zebrafish notochord, suggesting that specific cuproenzymes are required for the structural integrity of the notochord sheath. We now demonstrate that beta-aminopropionitrile, a known inhibitor of the copper-dependent lysyl oxidases, causes notochord distortion in the zebrafish embryo identical to that seen in copper deficiency. Characterization of the zebrafish lysyl oxidase genes reveals eight unique sequences, several of which are expressed in the developing notochord. Specific gene knockdown demonstrates that loss of loxl1 results in notochord distortion, and that loxl1 and loxl5b have overlapping roles in notochord formation. Interestingly, while notochord abnormalities are not observed following partial knockdown of loxl1 or loxl5b alone, in each case this markedly sensitizes developing embryos to notochord distortion if copper availability is diminished. Likewise, partial knockdown of the lysyl oxidase substrate col2a1 results in notochord distortion when combined with reduced copper availability or partial knockdown of loxl1 or loxl5b. These data reveal a complex interplay of gene expression and nutrient availability critical to notochord development. They also provide insight into specific genetic and nutritional factors that may play a role in the pathogenesis of structural birth defects of the axial skeleton.

Fig. 1

β-aminopropionitrile recapitulates the notochord phenotype of copper deficiency. Wild-type embryos were incubated in vehicle (A), 10 μM neocuproine (B), or 10 mM β-aminopropionitrile supplemented with 10 μM CuCl₂ (C). At 48 hpf, the notochord (arrowheads) is distorted in embryos treated with neocuproine (B) and β-aminopropionitrile (C); however, melanocytes (arrows) are present after β-aminopropionitrile (C) but not neocuproine treatment (B), demonstrating that β-aminopropionitrile does not act through copper chelation.

Fig. 2

Lysyl oxidase family members in zebrafish. (A) The zebrafish genome encodes eight distinct lysyl oxidases, all of which contain a conserved copper binding domain depicted in yellow. Scavenger receptor, cysteine-rich domains (SRCR) of unknown function are in blue and are truncated in Loxl3a and Loxl3b (SR with strikethrough). (B) Phylogenetic tree depicting the evolutionary relationship between human and zebrafish lysyl oxidases. HsCD163 is used as an outgroup, and bootstrap values over 100 replicates are noted. Loxl5a and Loxl5b (green) represent new additions to the lysyl oxidase family that closely resemble LOX and LOXL1 in structure. Zebrafish encode orthologues to all human lysyl oxidase genes except LOXL4.

Fig. 3

Four lysyl oxidases are expressed throughout the developing zebrafish notochord by in situ hybridization. (A–D) loxl1; (E–H) loxl2b; (I–L) loxl3b; and (M–P) loxl5b. Stages of development are 5 somites (A,E,I,M); 10 somites (B,F,J,N); 15 somites (C,G,K,O); and 20 somites (D,H,L,P). loxl1 and loxl5b are robustly expressed at the caudal notochord tip (arrowheads) while loxl5b is also expressed anterior to the notochord (asterix).

Fig. 4

Lysyl oxidases are expressed by notochord vacuolar cells. (A–D) Frozen cross-sections of 15 somite embryos were obtained after in situ hybridization with the following probes: (A) loxl1; (B) loxl2b; (C) loxl3b; and (D) loxl5b. The notochord is indicated (arrowheads). Hypochord staining of loxl1 (arrows) suggests a role for this family member in extracellular matrix crosslinking near the developing aorta. (E) Cartoon demonstrating the location of the hypochord (h) and floorplate (f), which are closely apposed to the notochord (n). Lysyl oxidases are not expressed in the floorplate.

Fig. 5

Morpholino knockdown of loxl1 or loxl1 and loxl5b together results in notochord distortion. Wild-type embryos were injected with morpholinos (MO) to notochord-expressed lysyl oxidases and photographed at 24 hpf. (A) 12 ng standard control; (B) 6 ng loxl1; (C) 3.7 ng loxl2b; (D) 12 ng loxl3b; (E) 7.4 ng loxl5b; (F) 2.4 ng loxl1 and 5 ng loxl5b. The notochord (arrowheads) is strikingly distorted after knockdown of loxl1 (B), and caudal vein edema develops with the loxl5b morpholino (E, arrows). Combining doses of loxl1 and loxl5b morpholino that do not cause notochord distortion alone also recapitulates the notochord phenotype seen with neocuproine and β-aminopropionitrile treatment (F). This distortion is specifically rescued by co-injection of mRNA encoding either loxl1 (H) or loxl5b (I), but not control sequence (G).

Fig. 6

Partial knockdown of loxl5b sensitizes embryos to notochord distortion in the presence of suboptimal copper nutrition (A–D) or disruption of col2a1 (E,F). Wild-type embryos were injected with 5 ng of control morpholino (A,C) or loxl5b morpholino (B,D), and incubated with (C,D) or without (A,B) 2 μM neocuproine starting at 3 hpf. Embryos injected with loxl5b morpholino develop notochord distortion (arrowhead) in the context of diminished copper availability (D). Embryos injected with 7.4 ng of col2a1 morpholino (E) are sensitized to develop notochord distortion (arrowhead) upon co-injection of 5 ng of loxl5b morpholino (F), demonstrating a genetic interaction between col2a1 and loxl5b. Photographs were obtained at 30 hpf (A–D) and 24 hpf (E,F).

Fig. 7

Expression of col2a1 mRNA by notochord vacuolar cells persists late following lysyl oxidase inhibition. Wild-type embryos were injected with 7.4 ng control morpholino (A), incubated in 10 μM neocuproine (B), incubated in 10 mM β-aminopropionitrile supplemented with 10 μM CuCl₂ (C), or injected with 3.7 ng each of loxl1 and loxl5b morpholino (D). In situ hybridization was carried out at 24 hpf, and frozen sections confirmed persistent col2a1 expression following lysyl oxidase inhibition (B–D, arrowhead), as well as floorplate and hypochord staining (A–D, arrows).

Fig. 8

Electron micrographs of the notochord sheath from cross-sections of 30 hpf embryos: (A) 7.4 ng control morpholino; (B) 10 μM neocuproine; (C) 10 mM β-aminopropionitrile; (D) 3.7 ng each of loxl1 and loxl5b morpholino. Sheath components include a basal lamina (I), fibrillar layer (II), and granular layer (III). All three layers are preserved after lysyl oxidase inhibition.

Fig. 9

Model of pathways involved in notochord formation. (A) The cuproenzymes Loxl1 and Loxl5b crosslink collagen II in the notochord sheath, which suppresses vacuolar cell expression of col2a1 as the notochord differentiates. (B) Polymorphisms at multiple genetic loci interact with environmental factors to cause disease. As a result, what is normally considered adequate nutrition may in fact be suboptimal during a specific developmental window. The gene atp7a encodes a transporter required for copper uptake (Mendelsohn et al., 2006).