Pitx2 Function in Neural Crest Is Required for Specific Intrinsic and Extrinsic Functions During Eye Development

Abstract

Abstract: : Purpose: We have shown previously that the Pitx2 homeobox gene is expressed in neural crest and mesoderm during eye development. Global loss of Pitx2 results in defects to multiple chimeric eye structures arising from both precursor lineages. Therefore, it has been impossible to distinguish between neural crest– and mesoderm–specific functions of Pitx2 in the formation of distinct ocular tissues. The purpose of this study was to determine the neural crest–specific requirements for Pitx2 function during eye development. Methods: A conditional Pitx2 (flox) allele was generated by gene targeting in ES cells. Resulting Pitx2^flox mice were mated to mice expressing a Wnt1Cre transgene, specifically deleting Pitx2 in neural crest prior to PITX2 protein expression. Mutant eyes were examined morphologically, immunohistochemically, and molecularly to assess the developmental consequences loss of Pitx2 function specifically in neural crest during eye development. Results: Neural crest–specific deletion of Pitx2 results in agenesis of multiple ocular tissues arising totally or in part from neural crest, including corneal endoderm, corneal stroma, and sclera. Blood vessels are hypomorphic but NG2 expression demonstrates neural crest–derived mural cells are present. Extraocular muscles (EOM) are specified normally. The optic stalk does not extend normally, resulting in internally placed eyes that are directly attached to the ventral hypothalamus. The embryonic fissure does not involute and close. Based on PAX2 expression, initial specification of the optic stalk occurs and the defect arises subsequently. Neural ectoderm–derived pigmented epithelium is present at e12.5 but the pigment and PAX2/PAX6 expression boundaries are shifted into the optic cup, suggesting an early defect in RPE specification or proliferation. Ultimately, the anterior optic cup remains pigmented while the remainder of the eye is devoid of pigment. Conclusions: Pitx2 function is required for specification of multiple lineages from neural crest, likely through an intrinsic mechanism. Specification of EOM is likely to require Pitx2 function in mesoderm, since these muscles are missing in the complete absence of Pitx2 but are unaffected in neural crest knockout mice. Pitx2 also regulates extrinsic factors in neural crest that are required for normal development of the optic stalk and pigmented epithelium from neural ectoderm. These data provide important insights into the normal functions of Pitx2 in eye development and suggest mechanisms through which mutations in human PITX2 may lead to glaucoma.

Keywords: transgenics/knock-outs • transcription factors • genetics