Abstract
Purpose: :
PITX2 is a homeodomain transcription factor that was shown to be involved in Axenfeld-Rieger syndrome when mutated. During ocular development, Pitx2 is expressed in the neural crest-derived periocular mesenchyme that makes a major contribution to the developing anterior segment structures. FOXD3 is a forkhead transcription factor that is active during embryonic development and displays both repression and activation capabilities. Foxd3 is expressed in premigratory neural crest cells in vertebrates and in the cranial neural crest region in zebrafish. A possible contribution of FOXD3 to ocular development has not yet been examined. We are interested to investigate a possibility of FOXD3 and PITX2 being part of the same pathway during ocular development. Towards this goal, we obtained two zebrafish lines (Znomics, Inc.) carrying insertions into pitx2 or foxd3 regions and undertaken their genetic and phenotypic characterization.
Methods: :
Fish were genotyped using tail tissue and genomic PCR with viral insert as well as gene-specific primers to confirm the insertion site and to identify heterozygote carriers. Heterozygote carriers were mated and embryos were genotyped using the same approach. The progeny was inspected for phenotypic defects at 1 to 6 days of development. Histology and in situ hybridization will be performed using standard protocols.
Results: :
The presence of insertions into pitx2 and foxd3 regions was confirmed for both lines: foxd3 insertion interrupts coding region while the pitx2 insertion is located within the intron. The foxd3 homozygous embryos demonstrated visible defects in the jaw and overall head development. This is consistent with previously reported data. Some foxd3 homozygous embryos showed alterations in eye size and appearance. The pitx2 homozygous embryos appeared phenotypically normal. In order to examine adult stages, embryos were raised and adult fish are now available for testing. Expression analysis of pitx2 and foxd3 transcripts as well as adult fish phenotype analysis will be presented at the meeting.
Conclusions: :
Zebrafish lines carrying insertions into pitx2 and foxd3 regions were obtained from Znomics, Inc. and the insertion sites were confirmed. Phenotypic characterization of mutant embryos demonstrated multiple defects in the developing head structures for foxd3 mutants and absence of any visible anomalies for pitx2 embryos carrying an insert. Studies of adult phenotypes are ongoing.
Keywords: gene/expression • genetics • in situ hybridization