The Pitx2 Transcription Factor: Role in Postnatal Extraocular Muscle

Abstract

Purpose:: The bicoid-like homeobox transcription factor Pitx2 gene was first cloned as the gene responsible for Rieger Syndrome, an autosomal dominant disorder characteristics of facial dimorphism, dental hyperplasia, ocular defects and umbilical protrusion. The Pitx2 gene plays an essential role in extraocular muscle (EOM) development but is also known to be expressed at high levels in mature EOM; however its function in the adult state is not known.

Methods:: We first evaluated pitx2 expression by RT-PCR, qPCR and immunohistochemistry. We then established a conditional mutant mouse line with an absence of Pitx2 in the postnatal EOM utilizing the Cre-loxP strategy and assessed muscle histology by standard methods as well as evaluated myosin isoform expression by qPCR and immunohistochemistry.

Results:: RT-PCR showed that all three alternatively spliced isoforms of Pitx2 (a, b and c) are expressed by EOM. QPCR showed that Pitx2a is the most abundantly expressed isoform and is 23 folds of that in extensor digitrorum longus and 91 fold of that in the cardiac muscle. Immunohistochemistry demonstrated differential expression of pitx2 among the EOM myonuclei. However, there was no obvious pattern to this expression, based on anatomic region (orbital versus global layer), proximity to the neuromuscular junction, or muscle fiber type. The EOM of the conditional mutant have no obvious histological abnormalities in the first weeks of life but have reduced slow myosin expression, and by 3 months slow myosin is significantly reduced by immunohistochemical assessment. Gene expression levels of alpha-cardiac, EOM specific and type 2x were reduced in comparison to wild-type controls. Other structural proteins such as myomesin, alpha-actin and troponin I also showed dramatic decrease of expression. Expression of myogenic transcription factors (myogenin, MyoD and myf5 were reduced between 10-20 fold.

Conclusions:: The investigation demonstrates the importance of Pitx2 in maintaining the mature EOM phenotype and a direct or indirect role in regulation of major muscle-specific gene expression in EOM.

Keywords: transcription factors • extraocular muscles: development