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N.S. Zinkevich, D.V. Bosenko, R. Tyler, E.V. Semina; Studies of Zebrafish pitx2 Demonstrate Conservation of Expression and Function With the Human Gene . Invest. Ophthalmol. Vis. Sci. 2006;47(13):5631.
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PITX2 is a homeodomain transcription factor associated with Axenfeld–Rieger syndrome in humans, autosomal dominant disorder that is characterized by anterior segment defects, glaucoma, umbilical abnormalities and tooth dysgenesis. In order to evaluate usefulness of zebrafish model for studies of the human condition, we performed detailed expression analysis of zebrafish pitx2 gene during early and late embryonic development with a particular focus on tissues related to the human phenotype. Also, we analyzed role of zebrafish pitx2 during eye development by expressing the pitx2 dominant–negative form under control of an ocular enhancer. In addition to this, we plan to evaluate phenotypes associated with morpholino–mediated knockdowns of pitx2 isoforms.
Localization of pitx2 mRNA was detected by in situ hybridization using digoxigenin–labeled antisense riboprobes on 24– to 120–hours post fertilization whole–mount embryos. We also studied expression of zebrafish pitx2 in embryos and adult fish by RT–PCR. Transient transgenics were created by injection of plasmid constructs into 1–2 cell embryos and phenotypes were evaluated by gross examination and histology.
RT–PCR experiments identified pitx2 transcripts in zebrafish embryos starting from 3–hpf as well as in adult fish. By in situ hybridization experiments, pitx2 mRNA was found to be localized in the developing brain, periocular mesenchyme, anterior segment of the developing eye, cartilage of pharyngeal arches, somites, and urogenital system. Transient transgenics injected with pitx2 dominant–negative form under control of an ocular enhancer demonstrated malformed eye with small pupil.
Zebrafish pitx2 gene demonstrated expression pattern that is conserved with human and mice. Zebrafish is a viable system for modeling a human disease. These experiments demonstarated that pitx2 function is conserved in vertebrate development and therefore zebrafish represents a useful model for studying anomalies associated with pitx2–deficiencies.
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