Analysis Tools
cellular
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• immunohistochemistry of embryonic fibroblasts, femoral articular cartilage, and skin shows fibrillin 2 microfibrils in discrete foci adjacent to nuclei rather than in extracellular matrix, and in situ hybridization of E13.5 eyes shows intracellular fibrillin 2 expression in cells of the anterior eye, particularly in the non-pigmented ciliary region, rather than the normal extracellular expression in the periocular mesenchyme and the anterior region of the eye between the neural retina and lens
• endoplasmic reticulum stress was found in regions where fibrilin 2 inclusions are found, including the anterior developing ciiary margin of the developing eye
• quantitative label-free mass-spectrometry of culture medium from E13.5 embryonic fibroblasts shows reduced secretion of many extracellular proteins, including collagens, periostin, and folistatin-like 1, and immunoblotting for intracellular levels of collagen type VI alpha 1 shows increased intracellular retention
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growth/size/body
small ears
(
J:203031
)
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• even smaller than in heterozygotes
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• homozygotes have a reduced body weight as early as 4 days of age, the earliest age assessed, and this persists with the average weight being less than half that of wildtype controls at 21 days of age
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limbs/digits/tail
syndactyly
(
J:203031
)
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• hindlimb syndactyly restricted to digits 2, 3, and 4, in all homozygotes ranges in severity from simple soft tissue syndactyly to osseous fusions of the entire phalanges of these digits
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skeleton
vision/eye
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• the ciliary body is consistently absent in both heterozygotes and homozygotes, with the anterior retina at E14.5 having diminished canonical Wnt signalling and the non-pigmented ciliary at E15.5 being smaller than normal, but the iris appears to form normally
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small lens
(
J:203031
)
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• abnormal ocular development appears to originate in defects of the developing ciliary epithelium
• increased numbers of caspase 3 positive apoptotic cells are found in the developing retinas at embryonic days 13.5 and 16.5
• fibrillin 2 staining is found within the cells throughout the developing eye rather than in the usual extracellular locations, consistent with failed secretion of this extracellular matrix protein, and this intracellular staining is strongest in the anterior neural retina and adjacent retinal pigment epithelium
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• histology of the anterior retina at embryonic day E15.5 shows a reduction in non-pigmented ciliary body tissue
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• although the initial description of this mutation indicated anophthalmia, dissection reveals microphthalmia
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• significant rosetting of the neural retina cell layers, with the rosettes composed primarily of disorganised rod photoreceptors
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• ectopic ganglion cells are found in multiple regions of the retina
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• a reduced number of inner nuclear layer cells is found by VSX2 antibody staining
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• immunohistochemistry shows decreased calbindin in the outer plexiform layer indicating a disruption to the projections emanating from horizontal cells
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• at 21 days of age rhodopsin is localised to punctate regions of the retina, not the outer segment of the retina, and normal lamination is disrupted
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• absence of vitreous in the anterior chamber and between the lens and retina
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• transmission electron microscopy of eye scleral cells shows fibrillin 2 positive inclusions within the lumen of the enlarged rough enodplasmic reticulum, organized into thick fibril-like aggregates, with apparent structural periodicity
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nervous system
hearing/vestibular/ear
small ears
(
J:203031
)
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• even smaller than in heterozygotes
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craniofacial
small ears
(
J:203031
)
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• even smaller than in heterozygotes
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