Pitx2 Expression in the Ocular Mesoderm is Required for Extraocular Muscle Development and Eyelid Closure

Abstract

Purpose: : The homeobox gene Pitx2 is expressed in the mesoderm and neural crest during eye development, and the loss of Pitx2 results in severe defects in the tissues derived from these lineages. Loss of Pitx2 function in neural crest recapitulates many but not all features of the global knockout. In particular, the extraocular muscles are unaffected in the Pitx2 neural crest knockout, but completely absent in the global knockout. To test the hypothesis that Pitx2 is required in mesoderm for specification of extraocular muscles, we created a mesoderm specific knockout of Pitx2 and analyzed the resulting phenotype.

Methods: : Gene targeting was used to create a conditional Pitx2 allele (Pitx2^flox) by introducing loxP sites flanking the critical fifth exon of Pitx2. Mice carrying the Pitx2^flox allele and the Rosa26 Cre reporter allele were mated to mice carrying a Pitx2^null allele and a Cre transgene that is expressed in the cranial mesoderm. The resulting mesoderm specific Pitx2 knockout (Pitx2–mko) embryos were compared to wildtype littermates on the basis of histology and gene expression.

Results: : Pitx2–mko embryos have absent or severely reduced extraocular muscles and a corresponding loss of the muscle–specific markers myogenin and myosin heavy chain. Pitx2–mko embryos survive longer and are less dysmorphic than global knockout animals, which allowed us to uncover a previously unrecognized requirement for Pitx2 in eyelid development. The mutant eyelid primordia have normal histology but fail to close at embryonic day e16.5. A subset of Pitx2–mko embryos exhibits retinal coloboma.

Conclusions: : Pitx2 is required in mesoderm for specification of extraocular muscles through a mechanism that includes activation of early muscle–specific transcriptional factor genes. The eyelid closure defect suggests that a PITX2–dependent extrinsic signal in mesoderm is required for morphogenetic movement of the eyelid epithelium. We are analyzing additional molecular markers to determine the precise underlying mechanism. The presence of coloboma in a subset of Pitx2–mko embryos suggests PITX2–dependent extrinsic factors in mesoderm are also required for closure of the optic fissure, but the underlying mechanism and the source of the reduced penetrance remain to be determined.

Keywords: extraocular muscles: development • eyelid • transcription factors