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G. Boisset, B. K. Polok, D. F. Schorderet; Pip5k3 Fleck Corneal Dystrophy-Linked Gene Characterization in Zebrafish. Invest. Ophthalmol. Vis. Sci. 2007;48(13):5699.
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Mutations in Pip5k3 gene are associated with François-Neetens Mouchetée Fleck Corneal Dystrophy (CFD) (Li et al. 2005). This rare autosomal dominant corneal dystrophy is characterized by small white flecks in the stroma which contain complex lipids. Pip5k3 is a well conserved gene and has been characterized in Human and Mouse but not in Zebrafish. In vitro studies show that this phosphoinositide 5-kinase family member is localized in the internal membranes, maintains vesicles integrity and regulates endosomes trafficking. It is also involved in insulin signaling by regulating GLUT4 vesicles translocation. Our aim is to characterize Pip5k3 expression in adult Zebrafish and during development.
Pip5k3 was amplified on 6-days-old larvae cDNA with primers designed from transcript predicted sequences found by database searches. Amplified fragments were cloned and sequenced. To analyse Pip5k3 expression in adult Zebrafish, Pip5k3 599 bp fragment was amplified from the cDNA of different organs. The eye expression was assessed by in situ hybridization on 12 µm eye cryosections. A 225 bp fragment was used as a template to synthesize a digoxigenin-labeled in situ RNA probe. Whole-mount in situ hybridization was performed on 24 hpf, 3dpf and 5dpf larvae using a 531 bp digoxigenin-labeled in situ RNA probe. Isoforms were analysed on 1 dpf, 5 dpf and 10 dpf larvae by enzymatic digestions of PCR amplified exons 2 and 7 containing a restriction site for HindIII and MslI respectively.
In Zebrafish the full coding sequence is 6303 bp long and consist in 42 exons and at least 3 splice variants. The first variant doesn't contain neither exon 2 nor exon 7, the second variant skips exon 7 and the third variant misses exon 2. The three isoforms are found at early stages of the development and the variant missing exon 7 isn't expressed at 10 dpf anymore.All the analysed organs of adult Zebrafish expressed Pip5k3. In situ hybridization on adult eye sections mainly shows a staining in the cornea, ganglion cell layer, inner nuclear layer and near the outer limiting membrane. Larvae have a strong staining at 5dpf in the region of the ear, brain and eye.
Even if Pip5k3 has a widespread expression in the adult fish, in the eye it seems to be localized in specific cell types. Larvae express this kinase at 5dpf and splice variants expression depends on larval stages. Further analyses are underway to elucidate the physiological role of Pip5k3 in Zebrafish eye.
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