FIGURE SUMMARY
Title

b3galt6 Knock-Out Zebrafish Recapitulate β3GalT6-Deficiency Disorders in Human and Reveal a Trisaccharide Proteoglycan Linkage Region

Authors
Delbaere, S., De Clercq, A., Mizumoto, S., Noborn, F., Bek, J.W., Alluyn, L., Gistelinck, C., Syx, D., Salmon, P.L., Coucke, P.J., Larson, G., Yamada, S., Willaert, A., Malfait, F.
Source
Full text @ Front Cell Dev Biol

Schematic representation of the GAG biosynthesis and the evolutionary conservation of the zebrafish and human b3galt6/B3GALT6 gene and B3galt6/β3GalT6 protein. (A) Simplified schematic representation of the PG linkage and GAGs biosynthesis. PG core proteins are synthesized in the endoplasmic reticulum and undergo further modification in the Golgi apparatus. First, a tetrasaccharide linkage region is synthesized that originates from the addition of a Xyl onto a serine residue of the core protein. Subsequent addition of two Gals and one GlcA residues completes this process. Depending on the addition of GlcNAc or GalNAc to the terminal GlcA residue of the linkage region, HS or CS/DS PGs will be formed, respectively. The elongation of the GAG chain continues by repeated addition of uronic acid and amino sugar residues. The GAG chain is then further modified by epimerization and sulfation (not shown). The main genes encoding for the enzymes involved in the linkage region biosynthesis are indicated in bold and biallelic mutations in these genes result in different linkeropathies. Disorders resulting from deficiency of the enzymes are indicated in black boxes. The deficient enzyme (β3GalT6) and the related disorder studied here are depicted in red (e.g., spEDS-B3GALT6 and SEMD-JL1). (B) Schematic representation of the genomic structure of the H. sapiens B3GALT6 (NM_080605.4) and D. rerio b3galt6 (NM_001045225.1) genes. The white and black boxes represent the untranslated regions and the single B3GALT6/b3galt6 exon, respectively. The zebrafish b3galt6 gene resides on chromosome 11, spans a region of 1.06 kb and has not been duplicated during teleost evolution. (C) Amino acid (AA) sequence alignment (Clustal Omega) between H. sapiens β3GalT6 (Uniprot: Q96L58) and D. rerio B3galt6 (Uniprot: Q256Z7). The luminal galactosyltransferase domain is highlighted in gray. Conservation of AA sequences is shown below the alignment: “*” residues identical in all sequences in the alignment; “:” conserved substitutions; “.” semi-conserved substitutions; “space” no conservation. (D) Schematic representation of the cmg20 allele in b3galt6 mutants. b3galt6 consists of three parts, a cytosolic domain (AA 1-11), a transmembrane domain (TM, AA 12-34) and a luminal domain (AA 35-335). The location of the CRISPR/Cas9-generated indel variant is indicated by a scissor symbol and the resulting premature termination codon (PTC) is depicted by a red star. (E) RT-qPCR analysis of relative b3galt6 mRNA expression levels in b3galt6–/– adult zebrafish (0.597 ± 0.03919) (n = 5) compared to WT siblings (1 ± 0.05395) (n = 6). Data are expressed as mean ± SEM. A Mann-Whitney U test was used to determine significance, **P < 0.01.

EXPRESSION / LABELING:
Gene:
Fish:
Anatomical Term:
Stage: Adult
PHENOTYPE:
Fish:
Observed In:
Stage: Adult

The identification of a trisaccharide linkage region in the b3galt6–/– zebrafish. (A,B) MS2 fragment mass spectra of CS-glycopeptides derived from biglycan (UniProt: A8BBH0) showed different linkage regions in WT compared to the b3galt6–/– zebrafish. (A) The peptide (DQEEGSAVEPYKPEHPTCPFGCR) in WT zebrafish was modified with a residual hexasaccharide structure, corresponding to the canonical tetrasaccharide linkage region (m/z 1281.79; 3+). The hexasaccharide structure was modified with one sulfate group (SO 3-) at the subterminal GalNAc residue and one phosphate group (HPO3-) at the xylose residue (Supplementary Figure S7). (B) The peptide (DQEEGSAVEPYKPEHPTCPFGCR) in b3galt6–/– zebrafish was modified with a residual pentasaccharide structure, corresponding to a non-canonical trisaccharide linkage region (m/z 1227.78; 3+). The pentasaccharide structure was modified with two phosphate/sulfate groups. Their exact positioning and identity could not be determined due to low intensities of corresponding ion peaks, and the positions are therefore assigned in analogy of the WT structure.

Developmental delay and morphological abnormalities in 20 dpf b3galt6–/– zebrafish. (A) Weight, standard length and Fulton’s condition factor of WT and b3galt6–/– zebrafish larvae at 20 dpf (n = 10). (B) External phenotype of the head of juvenile WT and b3galt6–/– sibling zebrafish. All images are oriented anterior to the left, posterior to the right, dorsal to the top and ventral to the bottom. (1-1’)b3galt6–/– zebrafish display a rounder frontal region (black line), shorter parietal/occipital region (white horizontal line) and a malformed posterior edge of the opercular apparatus (dotted black line), compared to WT control. (2-2’) Representative images of cartilage AB staining of the head. The olfactory region (indicated by “o” in the WT and arrowhead in the b3galt6–/– picture) and the otic region (indicated by white asterisk) show delayed development of cartilage elements in b3galt6–/– zebrafish as compared to WT. (3-3’) Representative images of AR mineral staining of the head. The olfactory region (indicated by “o”) and the otic region (indicated by asterisk) show severely delayed mineralization in b3galt6–/– zebrafish larvae as compared to WT. (4-4’) Representative images of AB staining of the caudal fin. The modified associated elements show developmental delay in b3galt6–/– zebrafish as compared to WT. E, epural; HS PU2, haemal spine of preural 2; HS PU3, haemal spine of preural 3; Hy1–5, hypural 1–5; NS PU2, neural spine preural 2; opc, opistural cartilage; Ph, parhypural (5-5’) Representative images of AR mineral staining of the caudal fin. Caudal vertebrae and their associated elements show no delay in ossification. Malformation or absence of caudal fin associated elements (block arrow indicates a neural arch; arrowhead indicates a missing haemal arch), and malformation of uroneural (uro) and vestigial neural arches and spines (vna/s) are observed in b3galt6–/– zebrafish; hypurals have a bent appearance. Scale bars: images of the head 500 μm, images of the caudal fin 200 μm.

Characterization of the external phenotype of adult b3galt6–/– zebrafish. (A)b3galt6–/– zebrafish (n = 16) had a significantly shorter standard length (SL) compared to their WT siblings (n = 17). The weight (W) was significantly lower in the mutant zebrafish. The Fulton’s condition factor [K = 100*(W/SL3)] was not significantly different between b3galt6–/– zebrafish and WT siblings. Data are expressed as box plots with min-to-max whiskers on which each individual data point is plotted. An unpaired t-test with Welch’s correction was used to determine significance. ****P < 0.0001, ns: not significant. (B) Adult b3galt6–/– zebrafish exhibit morphological abnormalities compared to their WT siblings; b3galt6–/– zebrafish display interruptions of the blue horizontal stripes (white arrows), smaller fins (arrowhead), aberrations of the head shape (black line), of the shape of the opercular apparatus (white line indicates the posterior edge) and of the pectoral fin (black line and arrow indicate the fin rays at the base of the fin, white line shows the outline of the fin).

Alizarin red stained mineralized bone in WT and b3galt6–/– adult zebrafish. The mineralized bones in the skeleton were studied in 4 months old WT and mutant zebrafish after AR mineral staining. (A)b3galt6–/– zebrafish display malformation of the cranium, vertebral column and caudal fin. (A1-1’) The cranium shows an underdeveloped olfactory region (black line) and a smaller compacted opercular bone (asterisk). (A2-2’) The vertebral column shows extra intramembranous outgrowths located where the distal tip of the neural and haemal arches fuse to a neural and haemal spine, respectively (arrowheads). (A3-3’) In the caudal fin fusion of preural vertebrae is observed (asterisk). Associated elements show intramembranous outgrowths (arrowhead) and the presence of extra bony elements (diamond). The distal tip of the hypurals is sometimes split (transparent arrowhead). Structures indicated in the WT caudal fin: Hy1-5, hypural 1-5, Ph, parhypural. (B) Details of mineralized structures in b3galt6–/– zebrafish. (B1-1’) The fifth ceratobranchial arch and its teeth are smaller in KO zebrafish. (B2-2’) The proximal (PR) and distal radials (DR) have irregular shapes in mutant zebrafish. Proximal (asterisk) and distal radials are sometimes fused. The bracket indicates a fusion between a proximal and distal radial, and the first fin ray (FR). (B3-3’) Ribs also show extra intramembranous bone (black arrowhead). (B4-4’) More mineralization was observed at the growth zone (bracket) of the scales in mutant zebrafish (arrowhead). Scale bars = 500 μm.

μCT analysis of adult WT and b3galt6–/– zebrafish. Quantitative μCT analysis of the vertebral column in five mutants vs. five WT zebrafish at the age of 4 months. The bone volume, tissue mineral density (TMD) and bone thickness were calculated for three parts of each vertebra: the vertebral centrum, the neural associated element (neural arch and neural spine) and the haemal associated element (haemal arch and haemal spine). Plots associated with a significant difference are colored in a lighter coloring scheme. The X-axis represents each vertebra along the anterior-posterior axis. The volume is significantly reduced for each part of the vertebra in b3galt6–/– zebrafish (P < 0.01, P < 0.001, and P < 0.000001, respectively). The thickness of the haemal (P < 0.01) and neural (P = 0.001) associated element is also significantly reduced in the mutants whereas the centrum thickness is not significantly reduced in b3galt6–/– zebrafish (P = 0.12). TMD is not significantly different between b3galt6–/– zebrafish and WT siblings when not normalized for standard length (P > 0.2). Data are presented as mean ± SEM. TMD, tissue mineral density; HA, hydroxyapatite.

PHENOTYPE:
Fish:
Observed In:
Stage: Adult

Collagen fibrillar architecture in 5 months old adult zebrafish. (A) Skin from 5 months old WT and b3galt6–/– adult zebrafish. The epidermis (Ep), the outer layer of the skin covering the scales (S) of zebrafish, appears thicker in b3galt6–/– zebrafish compared to the epidermis of WT zebrafish. A mature zebrafish scale consists of several layers. The limiting layer of the scale (LL) is rich in fine granules that in some areas are linearly arranged, forming dense layers. This matrix contrasts with that of the external layer(s) (EL) below. Structures resembling extrafibrillar crystals (arrowhead) are noted in the external layer of the mutant scale, which are rare in WT zebrafish sections. Upon investigation of the dermis of b3galt6–/– mutants, increased interfibrillar spaces are noted (arrows), which are absent in the dermis of WT adults. Scale bars = 500 nm. (B) Schematic representation and TEM images of the zebrafish intervertebral space. Schematic representation of the zebrafish intervertebral space. AC, autocentrum; B, bone; Cb, chordoblasts; Cc, chordocytes; ColII, type II collagen; E, elastin; iMC, immature collagen type I layer; MC, mature collagen type I layer. Starting from the central body axis, several recognizable structures are present in the intervertebral space along a proximo-distal axis: (i) notochord tissue consisting of chordocytes and chordoblasts, (ii) the notochord sheath consisting of a type II collagen layer and an external elastic membrane, (iii) and a connective tissue ligament consistent of a mature and an immature type I collagen layer. The mature type I collagen layer is organized in a typical plywood-like pattern and is continuous with the inner layer of the vertebral bone, i.e., the autocentrum, which also shows a plywood-like organization. The immature type I collagen layers consist of loose collagen fibers connecting the outer layer of vertebral bones. Notice the higher number of nuclei (N) in the b3galt6–/– zebrafish at the intervertebral space. Mature collagen (MC) displays a typical plywood-like organization, in WT zebrafish but not in comparable regions for b3galt6–/– mutant zebrafish. Cross section and longitudinal type I collagen regions are less clearly demarcated and smaller in immature collagen (iMC) from b3galt6–/– zebrafish compared to WT siblings. It is more difficult to pinpoint the different layers of the autocentrum (AC) in the KO compared to WT zebrafish. Bone (B) from the b3galt6–/– zebrafish shows electron dense structures (arrowhead). Scale bars = 10 μm for the intervertebral space and 1 μm for the other images.

Functional and structural analysis of adult WT and b3galt6–/– zebrafish muscle. (A) The critical swimming speed and (B) the endurance performance was determined for b3galt6–/– and length-matched WT zebrafish. (C) Semi-thin cross sections, stained with toluidine blue, demonstrate the presence of a thicker endomysium (arrowhead), located between the muscle fibers (Mf) at the horizontal myoseptum of b3galt6–/– zebrafish at the age of 5 months. (D) Transmission electron microscope structure of a sarcomere with indications of the Z and M line and A, I, and H band. The sarcomere length was determined at 5 different loci per semi-thin section, for 6 semi-thin sections origination from 3 different zebrafish per genotype (N = 30). A significant increase in sarcomere length for b3galt6–/– is noted. Furthermore, both the A and I band show an increase in length for the mutant zebrafish. Data are expressed as box plots with min-to-max whiskers on which each individual data point is plotted. A Mann-Whitney U test was used to determine significance. ****P < 0.0001, **P < 0.01.

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Acknowledgments
This image is the copyrighted work of the attributed author or publisher, and ZFIN has permission only to display this image to its users. Additional permissions should be obtained from the applicable author or publisher of the image. Full text @ Front Cell Dev Biol