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ptk7 mutant zebrafish models of congenital and idiopathic scoliosis implicate dysregulated Wnt signalling in disease.

Hayes M, Gao X, Yu LX, Paria N, Henkelman RM, Wise CA, Ciruna B - Nat Commun (2014)

Bottom Line: We identify a novel sequence variant within a single IS patient that disrupts PTK7 function, consistent with a role for dysregulated Wnt activity in disease pathogenesis.Furthermore, we demonstrate that embryonic loss-of-gene function in maternal-zygotic ptk7 mutants (MZptk7) leads to vertebral anomalies associated with CS.Our data suggest novel molecular origins of, and genetic links between, congenital and idiopathic forms of disease.

View Article: PubMed Central - PubMed

Affiliation: 1] Program in Developmental &Stem Cell Biology, The Hospital for Sick Children, 686 Bay Street, PGCRL 15-9712, Toronto, Ontario, Canada M5G 0A4 [2] Department of Molecular Genetics, The University of Toronto, Toronto, Ontario, Canada M5S 1A8.

ABSTRACT
Scoliosis is a complex genetic disorder of the musculoskeletal system, characterized by three-dimensional rotation of the spine. Curvatures caused by malformed vertebrae (congenital scoliosis (CS)) are apparent at birth. Spinal curvatures with no underlying vertebral abnormality (idiopathic scoliosis (IS)) most commonly manifest during adolescence. The genetic and biological mechanisms responsible for IS remain poorly understood due largely to limited experimental models. Here we describe zygotic ptk7 (Zptk7) mutant zebrafish, deficient in a critical regulator of Wnt signalling, as the first genetically defined developmental model of IS. We identify a novel sequence variant within a single IS patient that disrupts PTK7 function, consistent with a role for dysregulated Wnt activity in disease pathogenesis. Furthermore, we demonstrate that embryonic loss-of-gene function in maternal-zygotic ptk7 mutants (MZptk7) leads to vertebral anomalies associated with CS. Our data suggest novel molecular origins of, and genetic links between, congenital and idiopathic forms of disease.

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Related in: MedlinePlus

Zygotic ptk7 mutants do not display vertebral patterning abnormalities but do show vertebral ‘wedging’ reminiscent of human IS.(a–f) Live calcein staining of (a,b) 4 mm, (c,d) 4.5 mm and (e,f) 6.0 mm (a,c,e) ptk7 heterozygote and (b,d,f) zygotic ptk7 (Zptk7) mutant larvae. All scale bars, 0.5 mm. (g,h) Lateral views of caudal vertebrae from three-dimensional microCT renderings of adult (g) ptk7 heterozygote and (h) Zptk7 mutant zebrafish. Ptk7 mutant vertebrae display a wedge-shaped morphology at the apex of curves. (i,j) Graphs depicting dorsal:ventral and left:right length ratios of trunk and tail vertebrae in ptk7 heterozygote (i; n=3) and Zptk7 mutant (j; n=12) zebrafish. To characterize vertebral shape changes, we measured the length of individual thoracic and tail vertebrae along the dorsal, ventral, left and right sides. We calculated dorsal:ventral and left:right length ratios to assess possible length asymmetries in the dorso-ventral and medio-lateral planes, respectively. Horizontal dotted lines demarcate arbitrary±0.1, determined by the maximum deviation from 1 in ptk7 heterozygote controls and used to illustrate the deviation of Zptk7 vertebrae from normal. Deviation from 1±0.1 is evident at multiple vertebrae in three dimensions along the entire length of the spine. Type 1–4 Zptk7 mutants are grouped by point colour, and each individual fish is represented by a unique point shape and colour. The graph’s horizontal axis spans anterior (A) to posterior (P), from the first abdominal vertebrae to the last caudal vertebrae. The Weberian and caudal tail vertebrae were excluded from our analysis. (k,l) Maximum intensity projections (MIP) in the cranio–caudal direction from high-resolution micro-computed tomography (microCT) of (k) ptk7/+ and (l) Zptk7 mutant zebrafish. Dorsal is up and ventral is down. (m) Quantification of maximum pixel intensity corrected for background intensity to infer approximate bone density in ptk7/+ (n=3) and Zptk7 (n=12) zebrafish. Maximum pixel intensities of the vertebrae were extracted from MIPs made from 10 sequential microCT slices. Three locations from trunk to tail were quantified and averaged for each fish. Error bars represent standard deviation. NS, not significant.
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f2: Zygotic ptk7 mutants do not display vertebral patterning abnormalities but do show vertebral ‘wedging’ reminiscent of human IS.(a–f) Live calcein staining of (a,b) 4 mm, (c,d) 4.5 mm and (e,f) 6.0 mm (a,c,e) ptk7 heterozygote and (b,d,f) zygotic ptk7 (Zptk7) mutant larvae. All scale bars, 0.5 mm. (g,h) Lateral views of caudal vertebrae from three-dimensional microCT renderings of adult (g) ptk7 heterozygote and (h) Zptk7 mutant zebrafish. Ptk7 mutant vertebrae display a wedge-shaped morphology at the apex of curves. (i,j) Graphs depicting dorsal:ventral and left:right length ratios of trunk and tail vertebrae in ptk7 heterozygote (i; n=3) and Zptk7 mutant (j; n=12) zebrafish. To characterize vertebral shape changes, we measured the length of individual thoracic and tail vertebrae along the dorsal, ventral, left and right sides. We calculated dorsal:ventral and left:right length ratios to assess possible length asymmetries in the dorso-ventral and medio-lateral planes, respectively. Horizontal dotted lines demarcate arbitrary±0.1, determined by the maximum deviation from 1 in ptk7 heterozygote controls and used to illustrate the deviation of Zptk7 vertebrae from normal. Deviation from 1±0.1 is evident at multiple vertebrae in three dimensions along the entire length of the spine. Type 1–4 Zptk7 mutants are grouped by point colour, and each individual fish is represented by a unique point shape and colour. The graph’s horizontal axis spans anterior (A) to posterior (P), from the first abdominal vertebrae to the last caudal vertebrae. The Weberian and caudal tail vertebrae were excluded from our analysis. (k,l) Maximum intensity projections (MIP) in the cranio–caudal direction from high-resolution micro-computed tomography (microCT) of (k) ptk7/+ and (l) Zptk7 mutant zebrafish. Dorsal is up and ventral is down. (m) Quantification of maximum pixel intensity corrected for background intensity to infer approximate bone density in ptk7/+ (n=3) and Zptk7 (n=12) zebrafish. Maximum pixel intensities of the vertebrae were extracted from MIPs made from 10 sequential microCT slices. Three locations from trunk to tail were quantified and averaged for each fish. Error bars represent standard deviation. NS, not significant.

Mentions: To determine whether observed scoliosis resulted from CVMs, vertebrae formation was imaged in Zptk7 mutant and control animals using calcein stain (a vital, fluorescent calcium-binding chromophore)32. Larvae were analyzed at 4, 4.5 and 6.0 mm standard length. No defects in vertebral patterning or formation were observed in Zptk7 mutants (n=33) compared with ptk7/+ siblings (n=40; Fig. 2a–f). High-resolution micron-scale computed tomography (microCT) was then performed to further examine spinal curvature and vertebral structure in Zptk7 mutant zebrafish. Three-dimensional skeletal reconstructions of 12 Zptk7 mutant adults and 3 ptk7/+ siblings revealed the profound late-onset curvatures typical of mutant fish and confirmed that abnormal vertebral patterning is not a general aspect of the Zptk7 mutant phenotype (Fig. 1i,m; Supplementary Movies 1–5).


ptk7 mutant zebrafish models of congenital and idiopathic scoliosis implicate dysregulated Wnt signalling in disease.

Hayes M, Gao X, Yu LX, Paria N, Henkelman RM, Wise CA, Ciruna B - Nat Commun (2014)

Zygotic ptk7 mutants do not display vertebral patterning abnormalities but do show vertebral ‘wedging’ reminiscent of human IS.(a–f) Live calcein staining of (a,b) 4 mm, (c,d) 4.5 mm and (e,f) 6.0 mm (a,c,e) ptk7 heterozygote and (b,d,f) zygotic ptk7 (Zptk7) mutant larvae. All scale bars, 0.5 mm. (g,h) Lateral views of caudal vertebrae from three-dimensional microCT renderings of adult (g) ptk7 heterozygote and (h) Zptk7 mutant zebrafish. Ptk7 mutant vertebrae display a wedge-shaped morphology at the apex of curves. (i,j) Graphs depicting dorsal:ventral and left:right length ratios of trunk and tail vertebrae in ptk7 heterozygote (i; n=3) and Zptk7 mutant (j; n=12) zebrafish. To characterize vertebral shape changes, we measured the length of individual thoracic and tail vertebrae along the dorsal, ventral, left and right sides. We calculated dorsal:ventral and left:right length ratios to assess possible length asymmetries in the dorso-ventral and medio-lateral planes, respectively. Horizontal dotted lines demarcate arbitrary±0.1, determined by the maximum deviation from 1 in ptk7 heterozygote controls and used to illustrate the deviation of Zptk7 vertebrae from normal. Deviation from 1±0.1 is evident at multiple vertebrae in three dimensions along the entire length of the spine. Type 1–4 Zptk7 mutants are grouped by point colour, and each individual fish is represented by a unique point shape and colour. The graph’s horizontal axis spans anterior (A) to posterior (P), from the first abdominal vertebrae to the last caudal vertebrae. The Weberian and caudal tail vertebrae were excluded from our analysis. (k,l) Maximum intensity projections (MIP) in the cranio–caudal direction from high-resolution micro-computed tomography (microCT) of (k) ptk7/+ and (l) Zptk7 mutant zebrafish. Dorsal is up and ventral is down. (m) Quantification of maximum pixel intensity corrected for background intensity to infer approximate bone density in ptk7/+ (n=3) and Zptk7 (n=12) zebrafish. Maximum pixel intensities of the vertebrae were extracted from MIPs made from 10 sequential microCT slices. Three locations from trunk to tail were quantified and averaged for each fish. Error bars represent standard deviation. NS, not significant.
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f2: Zygotic ptk7 mutants do not display vertebral patterning abnormalities but do show vertebral ‘wedging’ reminiscent of human IS.(a–f) Live calcein staining of (a,b) 4 mm, (c,d) 4.5 mm and (e,f) 6.0 mm (a,c,e) ptk7 heterozygote and (b,d,f) zygotic ptk7 (Zptk7) mutant larvae. All scale bars, 0.5 mm. (g,h) Lateral views of caudal vertebrae from three-dimensional microCT renderings of adult (g) ptk7 heterozygote and (h) Zptk7 mutant zebrafish. Ptk7 mutant vertebrae display a wedge-shaped morphology at the apex of curves. (i,j) Graphs depicting dorsal:ventral and left:right length ratios of trunk and tail vertebrae in ptk7 heterozygote (i; n=3) and Zptk7 mutant (j; n=12) zebrafish. To characterize vertebral shape changes, we measured the length of individual thoracic and tail vertebrae along the dorsal, ventral, left and right sides. We calculated dorsal:ventral and left:right length ratios to assess possible length asymmetries in the dorso-ventral and medio-lateral planes, respectively. Horizontal dotted lines demarcate arbitrary±0.1, determined by the maximum deviation from 1 in ptk7 heterozygote controls and used to illustrate the deviation of Zptk7 vertebrae from normal. Deviation from 1±0.1 is evident at multiple vertebrae in three dimensions along the entire length of the spine. Type 1–4 Zptk7 mutants are grouped by point colour, and each individual fish is represented by a unique point shape and colour. The graph’s horizontal axis spans anterior (A) to posterior (P), from the first abdominal vertebrae to the last caudal vertebrae. The Weberian and caudal tail vertebrae were excluded from our analysis. (k,l) Maximum intensity projections (MIP) in the cranio–caudal direction from high-resolution micro-computed tomography (microCT) of (k) ptk7/+ and (l) Zptk7 mutant zebrafish. Dorsal is up and ventral is down. (m) Quantification of maximum pixel intensity corrected for background intensity to infer approximate bone density in ptk7/+ (n=3) and Zptk7 (n=12) zebrafish. Maximum pixel intensities of the vertebrae were extracted from MIPs made from 10 sequential microCT slices. Three locations from trunk to tail were quantified and averaged for each fish. Error bars represent standard deviation. NS, not significant.
Mentions: To determine whether observed scoliosis resulted from CVMs, vertebrae formation was imaged in Zptk7 mutant and control animals using calcein stain (a vital, fluorescent calcium-binding chromophore)32. Larvae were analyzed at 4, 4.5 and 6.0 mm standard length. No defects in vertebral patterning or formation were observed in Zptk7 mutants (n=33) compared with ptk7/+ siblings (n=40; Fig. 2a–f). High-resolution micron-scale computed tomography (microCT) was then performed to further examine spinal curvature and vertebral structure in Zptk7 mutant zebrafish. Three-dimensional skeletal reconstructions of 12 Zptk7 mutant adults and 3 ptk7/+ siblings revealed the profound late-onset curvatures typical of mutant fish and confirmed that abnormal vertebral patterning is not a general aspect of the Zptk7 mutant phenotype (Fig. 1i,m; Supplementary Movies 1–5).

Bottom Line: We identify a novel sequence variant within a single IS patient that disrupts PTK7 function, consistent with a role for dysregulated Wnt activity in disease pathogenesis.Furthermore, we demonstrate that embryonic loss-of-gene function in maternal-zygotic ptk7 mutants (MZptk7) leads to vertebral anomalies associated with CS.Our data suggest novel molecular origins of, and genetic links between, congenital and idiopathic forms of disease.

View Article: PubMed Central - PubMed

Affiliation: 1] Program in Developmental &Stem Cell Biology, The Hospital for Sick Children, 686 Bay Street, PGCRL 15-9712, Toronto, Ontario, Canada M5G 0A4 [2] Department of Molecular Genetics, The University of Toronto, Toronto, Ontario, Canada M5S 1A8.

ABSTRACT
Scoliosis is a complex genetic disorder of the musculoskeletal system, characterized by three-dimensional rotation of the spine. Curvatures caused by malformed vertebrae (congenital scoliosis (CS)) are apparent at birth. Spinal curvatures with no underlying vertebral abnormality (idiopathic scoliosis (IS)) most commonly manifest during adolescence. The genetic and biological mechanisms responsible for IS remain poorly understood due largely to limited experimental models. Here we describe zygotic ptk7 (Zptk7) mutant zebrafish, deficient in a critical regulator of Wnt signalling, as the first genetically defined developmental model of IS. We identify a novel sequence variant within a single IS patient that disrupts PTK7 function, consistent with a role for dysregulated Wnt activity in disease pathogenesis. Furthermore, we demonstrate that embryonic loss-of-gene function in maternal-zygotic ptk7 mutants (MZptk7) leads to vertebral anomalies associated with CS. Our data suggest novel molecular origins of, and genetic links between, congenital and idiopathic forms of disease.

Show MeSH
Related in: MedlinePlus