Essential role for the alpha 1 chain of type VIII collagen in zebrafish notochord formation

Abstract

Several zebrafish mutants identified in large-scale forward genetic screens exhibit notochord distortion. We now report the cloning and further characterization of one such mutant, gulliver(m208) (gul(m208)). The notochord defect in gul(m208) mutants is exacerbated under conditions of copper depletion or lysyl oxidase cuproenzyme inhibition that are without a notochord effect on wild-type embryos. The gul(m208) phenotype results from a missense mutation in the gene encoding Col8a1, a lysyl oxidase substrate, and morpholino knockdown of col8a1 recapitulates the notochord distortion observed in gul(m208) mutants. Of interest, the amino acid mutated in gul(m208) Col8a1 is highly conserved, and the equivalent substitution in a closely related human protein, COL10A1, causes Schmid metaphyseal chondrodysplasia. Taken together, the data identify a new protein essential for notochord morphogenesis, extend our understanding of gene-nutrient interactions in early development, and suggest that human mutations in COL8A1 may cause structural birth defects.

Fig. 1. gul^m208 mutants are sensitized to lysyl oxidase inhibition

Clutches from gul^m208/+ intercrosses were incubated in vehicle (A, B), the copper chelator neocuproine (2 μM) (C, D), or the lysyl oxidase inhibitor β-aminopropionitrile (1 mM) (E, F). The notochord is normal in wild-type embryos treated with vehicle, neocuproine, or β-aminopropionitrile (A, C, E). Notochords of gul^m208 mutants incubated in neocuproine and β-aminopropionitrile (D, F, arrowheads) are substantially more distorted than mutants incubated in vehicle (B, arrowheads). Insets show notochord at higher magnification. Embryos were treated with PTU to inhibit melanin pigmentation and photographed at 30 hpf.

Fig. 2. Electron microscopy reveals notochord abnormalities in gul^m208 mutants

(A-E) Transmission electron micrographs of truncal cross-sections from embryos at 24 hpf. (A) Notochord sheath and vacuolated notochord cell of a wild-type embryo, with small areas of protein accumulation visible in the rough endoplasmic reticulum (arrow). (B) Notochord sheath and vacuolated notochord cell of a gul^m208 mutant with large circular aggregates of protein in the rough endoplasmic reticulum (arrow). (C) Notochord and hypochord of a gul^m208 mutant with large circular aggregrates of protein in the hypochord (red arrows) and notochord (yellow arrow). (D) Notochord sheath of a wild-type embryo with inner (i), medial (m), and outer (o) layers indicated. Small areas of protein accumulation are visible in the rough endoplasmic reticulum (red arrow). (E) Notochord sheath of a gul^m208 mutant where the collagen fibrils in the medial (m) layer appear disorganized. A large circular aggregate of protein is visible in the rough endoplasmic reticulum (red arrow). Not = vacuolated notochord cell; Hyp = hypochord.

Fig. 3. The gul^m208 mutation disrupts the col8a1 gene

(A) The gul^m208 lesion was meiotically mapped to a centromeric region of chromosome 9 bounded by markers zK46K9 and zK229B18. The number of recombinants at each marker is noted. According to the physical genome assembly (Zv7), 26 genes are located within the critical region, including the alpha 1 chain of type VIII collagen (col8a1). (B) Sequencing of wild-type and gul^m208 mutant cDNAs revealed a missense mutation in gul^m208 mutant col8a1 that changes a tyrosine to a histidine. (C) Structure of the predicted 711-amino acid zebrafish Col8a1 protein. NC1 and NC2 are two non-collagenous domains. The Y628H substitution present in gul^m208 mutants (arrow) is located within the C1q-like domain (grey) of NC1. (D) Protein sequence alignment of N-terminal portions of select C1q-like domains. Conserved residues, including Y628 (arrow) are shaded yellow. Sequence numbering refers to the zebrafish Col8a1 sequence (DrCol8a1).

Fig. 4. col8a1 expression is consistent with the gul^m208 phenotype

(A-I) Whole-mount in situ hybridization for col8a1 was performed on wild-type embryos at various developmental stages. (A) Lateral view of a 5-somite embryo with col8a1 expression in the vacuolated notochord cells (arrow). (B-D) Lateral views of 10- (B), 15- (C), or 20- (D) somite embryos with col8a1 expression in the notochord (arrow), floorplate (black arrowhead), hypochord (white arrowhead), and prechordal plate region (asterix). (E) Lateral view of an embryo at 24 hpf with col8a1 expression in the floorplate (black arrowhead), hypochord (white arrowhead), and prechordal plate region (asterix). (F) Dorsal view of an embryo at 24 hpf with col8a1 expression in the caudal somites (arrowheads). (G) Lateral view of an embryo at 30 hpf with col8a1 expression in the floorplate (black arrowhead) and hypochord (white arrowhead). (H) Dorsal view of an embryo at 48 hpf with col8a1 expression in jaw cartilages (arrows). (I) Lateral view of an embryo at 48 hpf with col8a1 expression in jaw cartilages (arrows). (J) RT-PCR for col8a1 at the indicated developmental stages. As controls, maternally-expressed 5′-AMP-activated protein kinase catalytic subunit alpha-1 (ampka1) and zygotically-expressed spadetail (spt) were amplified in parallel (Griffin et al., 1998; Wagner et al., 2004). The fold induction of col8a1 relative to ampka1 is noted. Unfert. = unfertilized; NA = not applicable.

Fig. 5. The Y628H substitution prevents Col8a1 trimerization

(A) Constructs encoding wild-type (lane 1) or gul^m208 mutant (lane 2) Col8a1 were transcribed and translated in the presence of radiolabelled methionine, and the products analyzed as described under Experimental Procedures. Col8a1 monomers and trimers are indicated. Co-transcription and translation of half the amounts of wild-type and mutant construct together does not prevent trimerization (lane 3). The approximate location of molecular weight markers (kDa) is noted. (B) RT-PCR to assess the splice status of xbp1 in wild-type embryos and gul^m208 mutants at 24 hpf. Splicing of xbp1 is not observed in gul^m208 mutants but occurs after incubation of wild-type embryos in tunicamycin. U = unspliced; S = spliced. Amplification of the gene spt was performed in parallel to confirm that equal amounts of cDNA were used for each condition.

Fig. 6. Morpholino knockdown of col8a1 recapitulates the gul^m208 phenotype

(A) Wild-type embryos injected with 12 ng of control morpholino exhibit normal morphology. (B) Embryos injected with 2.4 ng of a splice morpholino targeting col8a1 develop notochord distortion (arrowheads) characteristic of gul^m208 mutants. (C) RT-PCR at 24 hpf demonstrating a dose-dependent reduction in col8a1 transcript after morpholino injection. (D) Embryos injected with 12 ng of a splice morpholino targeting col8a1 develop notochord distortion (arrowheads) that is worse than in gul^m208 mutants. Insets show notochord at higher magnification. All embryos were treated with PTU to inhibit melanin pigmentation and photographed at 30 hpf.

Fig. 7. Model of Col8a1 assembly in wild-type and gul^m208 mutant embryos

Col8a1 monomer containing the Y628H mutation is unable to fold, preventing trimer formation and higher-order assembly that occurs in the absence of the mutation. The Y628H mutation is located within the NC1 domain of Col8a1 (asterisk).