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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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Maternal-zygotic ptk7 mutant embryos display segmentation defects.(a,b) Whole-mount in situ hybridization (WISH) of somite patterning gene expression at the 18–20 somite stage. All images are posterior views with the embryonic tail bud pointing towards the bottom of the field of view. WT and MZptk7 mutant embryos stained for (a) her7, (b) dlc expression. The expression domains of these markers is expanded within MZptk7 mutants consistent with PSM morphogenetic defects24. Arrowhead in a highlights anterior domain asymmetry in one MZptk7 mutant embryo. Scale bars, 50 μm. (c,d) myoD (somite) staining in (c) WT and (d) MZptk7 embryos to visualize segmentation during somitogenesis. MZptk7 display defects in the regular spacing and morphology of the somites at this stage. Scale bars, 100 μm. (e,f) Lateral view of WISH for Xirp2a expression to mark somite boarders41 in (e) WT and (f) MZptk7 mutant embryos at 40 hpf. The horizontal brackets and asterisks highlight examples of somite fusions. Defects in the shape and size of the somites are also apparent in MZptk7 mutants. Scale bars, 100 μm. (g,h) pax9 expression in (g) WT and (h) MZptk7 mutant embryos to visualize sclerotome. Scale bars, 100 μm.
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f6: Maternal-zygotic ptk7 mutant embryos display segmentation defects.(a,b) Whole-mount in situ hybridization (WISH) of somite patterning gene expression at the 18–20 somite stage. All images are posterior views with the embryonic tail bud pointing towards the bottom of the field of view. WT and MZptk7 mutant embryos stained for (a) her7, (b) dlc expression. The expression domains of these markers is expanded within MZptk7 mutants consistent with PSM morphogenetic defects24. Arrowhead in a highlights anterior domain asymmetry in one MZptk7 mutant embryo. Scale bars, 50 μm. (c,d) myoD (somite) staining in (c) WT and (d) MZptk7 embryos to visualize segmentation during somitogenesis. MZptk7 display defects in the regular spacing and morphology of the somites at this stage. Scale bars, 100 μm. (e,f) Lateral view of WISH for Xirp2a expression to mark somite boarders41 in (e) WT and (f) MZptk7 mutant embryos at 40 hpf. The horizontal brackets and asterisks highlight examples of somite fusions. Defects in the shape and size of the somites are also apparent in MZptk7 mutants. Scale bars, 100 μm. (g,h) pax9 expression in (g) WT and (h) MZptk7 mutant embryos to visualize sclerotome. Scale bars, 100 μm.

Mentions: CVMs are a hallmark of CS, and are thought to arise during early embryogenesis as a result of abnormal segmentation and somite formation339. To determine whether segmentation is disrupted in MZptk7 mutant embryos, we performed in situ hybridization of her7 and dlc (direct reporters of the Notch segmentation mechanism and nascent somite differentiation)40 within the PSM of 18–20 somite-staged MZptk7 mutant embryos, looking for possible irregularities in gene expression. The PSM domain was broader and expanded relative to total body length in MZptk7 mutants, consistent with a role for Ptk7 in tail-bud patterning and morphogenesis24. Of note, although her7 (n=24) and dlc (n=18) gene expression appeared to oscillate normally in MZptk7 mutant embryos (Fig. 6a,b), occasional irregularities in the symmetry of gene expression were observed (n=9/24 in her7; Fig. 6a), indicating possible defects in somite patterning. Strikingly, myoD and Xirp2a (ref. 41) expression analysis revealed severe defects in the regular morphology and spacing of the somites in MZptk7 mutants, as well as somite fusions (Fig. 6c–f). Visualization of pax9 gene expression also revealed abnormalities in the regular pattern of sclerotome in MZptk7 mutants (Fig. 6g,h), consistent with abnormal somite patterning.


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)

Maternal-zygotic ptk7 mutant embryos display segmentation defects.(a,b) Whole-mount in situ hybridization (WISH) of somite patterning gene expression at the 18–20 somite stage. All images are posterior views with the embryonic tail bud pointing towards the bottom of the field of view. WT and MZptk7 mutant embryos stained for (a) her7, (b) dlc expression. The expression domains of these markers is expanded within MZptk7 mutants consistent with PSM morphogenetic defects24. Arrowhead in a highlights anterior domain asymmetry in one MZptk7 mutant embryo. Scale bars, 50 μm. (c,d) myoD (somite) staining in (c) WT and (d) MZptk7 embryos to visualize segmentation during somitogenesis. MZptk7 display defects in the regular spacing and morphology of the somites at this stage. Scale bars, 100 μm. (e,f) Lateral view of WISH for Xirp2a expression to mark somite boarders41 in (e) WT and (f) MZptk7 mutant embryos at 40 hpf. The horizontal brackets and asterisks highlight examples of somite fusions. Defects in the shape and size of the somites are also apparent in MZptk7 mutants. Scale bars, 100 μm. (g,h) pax9 expression in (g) WT and (h) MZptk7 mutant embryos to visualize sclerotome. Scale bars, 100 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4155517&req=5

f6: Maternal-zygotic ptk7 mutant embryos display segmentation defects.(a,b) Whole-mount in situ hybridization (WISH) of somite patterning gene expression at the 18–20 somite stage. All images are posterior views with the embryonic tail bud pointing towards the bottom of the field of view. WT and MZptk7 mutant embryos stained for (a) her7, (b) dlc expression. The expression domains of these markers is expanded within MZptk7 mutants consistent with PSM morphogenetic defects24. Arrowhead in a highlights anterior domain asymmetry in one MZptk7 mutant embryo. Scale bars, 50 μm. (c,d) myoD (somite) staining in (c) WT and (d) MZptk7 embryos to visualize segmentation during somitogenesis. MZptk7 display defects in the regular spacing and morphology of the somites at this stage. Scale bars, 100 μm. (e,f) Lateral view of WISH for Xirp2a expression to mark somite boarders41 in (e) WT and (f) MZptk7 mutant embryos at 40 hpf. The horizontal brackets and asterisks highlight examples of somite fusions. Defects in the shape and size of the somites are also apparent in MZptk7 mutants. Scale bars, 100 μm. (g,h) pax9 expression in (g) WT and (h) MZptk7 mutant embryos to visualize sclerotome. Scale bars, 100 μm.
Mentions: CVMs are a hallmark of CS, and are thought to arise during early embryogenesis as a result of abnormal segmentation and somite formation339. To determine whether segmentation is disrupted in MZptk7 mutant embryos, we performed in situ hybridization of her7 and dlc (direct reporters of the Notch segmentation mechanism and nascent somite differentiation)40 within the PSM of 18–20 somite-staged MZptk7 mutant embryos, looking for possible irregularities in gene expression. The PSM domain was broader and expanded relative to total body length in MZptk7 mutants, consistent with a role for Ptk7 in tail-bud patterning and morphogenesis24. Of note, although her7 (n=24) and dlc (n=18) gene expression appeared to oscillate normally in MZptk7 mutant embryos (Fig. 6a,b), occasional irregularities in the symmetry of gene expression were observed (n=9/24 in her7; Fig. 6a), indicating possible defects in somite patterning. Strikingly, myoD and Xirp2a (ref. 41) expression analysis revealed severe defects in the regular morphology and spacing of the somites in MZptk7 mutants, as well as somite fusions (Fig. 6c–f). Visualization of pax9 gene expression also revealed abnormalities in the regular pattern of sclerotome in MZptk7 mutants (Fig. 6g,h), consistent with abnormal somite patterning.

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