The data provide essential new information toward understanding the complex interactions required for normal development and function of the mammalian eye. The results also underscore the importance of confirming neural crest and mesoderm fates in a model mammalian system. The complementary systems used in this study should be useful for studying the respective cell fates in other organ systems.

Heterozygous mutations in the homeobox gene, PITX2, result in ocular anterior segment defects and a high incidence of early-onset glaucoma. Pitx2 is expressed in both the neural crest and the mesoderm-derived precursors of the periocular mesenchyme. Complete loss of function in mice results in agenesis or severe disruption of periocular mesenchyme structures and extrinsic defects in early optic nerve development. However, the specific requirements for Pitx2 in neural crest versus mesoderm could not be determined using these mice, and only roles in the initial stages of eye development could be assessed due to early embryonic lethality. To determine the specific roles of Pitx2 in the neural crest precursor pool, we generated neural crest-specific Pitx2 knockout mice (Pitx2-ncko). Because Pitx2-nkco mice are viable, we also analyzed gene function in later eye development. Pitx2 is intrinsically required in neural crest for specification of corneal endothelium, corneal stroma and the sclera. Pitx2 function in neural crest is also required for normal development of ocular blood vessels. Pitx2-ncko mice exhibit a unique optic nerve phenotype in which the eyes are progressively displaced towards the midline until they are directly attached to the ventral hypothalamus. As Pitx2 is not expressed in the optic stalk, an essential function of PITX2 protein in neural crest is to regulate an extrinsic factor(s) required for development of the optic nerve. We propose a revised model of optic nerve development and new mechanisms that may underlie the etiology of glaucoma in Axenfeld-Rieger patients.

Local control of cell signaling activity and integration of inputs from multiple signaling pathways are central for normal development but the underlying mechanisms remain poorly understood. Here we show that Dkk2, encoding an antagonist of canonical Wnt signaling, is an essential downstream target of the PITX2 homeodomain transcription factor in neural crest during eye development. Canonical Wnt signaling is ectopically activated in central ocular surface ectoderm and underlying mesenchyme in Pitx2- and Dkk2-deficient mice. General ocular surface ectoderm identity is maintained during development in Dkk2-deficient mice but peripheral fates, including conjunctival goblet cells and eyelash follicles, are ectopically permitted within more central structures and eyelids are hypomorphic. Loss of DKK2 results in ectopic blood vessels within the periocular mesenchyme and PITX2 expression remains persistently high, providing evidence for a negative feedback loop. Collectively, these data suggest that activation of Dkk2 by PITX2 provides a mechanism to locally suppress canonical Wnt signaling activity during eye development, a paradigm that may be a model for achieving local or transient inhibition of pathway activity elsewhere during embryogenesis. We further propose a model placing PITX2 as an essential integration node between retinoic acid and canonical Wnt signaling during eye development.