Abstract
Purpose::
Pitx2 is the only known genetic regulator of extraocular muscle (EOM) development, but little else is understood about the mechanisms underlying EOM development. The purpose of the current experiments was to test several potential mechanisms of Pitx2 function in EOM development including the role of Pitx2 in EOM specification and differentiation as well as the role of Pitx2 in the survival and proliferation of EOM myoblasts.
Methods::
Pitx2null and Pax7LacZ mice were mated to generate timed pregnancies and the resulting embryos were analyzed on the basis of histology and gene expression. Markers for cell death and proliferation were also examined. Bioinformatics was used to look for conserved PITX2 binding sites within the promoters and enhancers of the muscle regulatory factors (MRFs ) Myf5, MyoD, and Myog. Luciferase assays are currently being used to test the functionality of these sites.
Results::
PITX2 is expressed in EOM primordia two days prior to the expression of PAX7, MYOD, and MYOG in wildtype mice. Pitx2null/null embryos completely lack EOMs and expression of PAX7 and the MRFs in the cranial mesoderm is absent. Conversely, embryos lacking Pax7 function have EOMs with normal expression of PITX2, confirming the genetic hierarchy suggested by the Pitx2 mutant mice. Bioinformatics analysis of known or proposed MRF promoters and enhancers has identified predicted PITX2 binding sites that are conserved between mouse and human, which are currently being tested for functionality in vitro. EOM precursor cells are greatly reduced in embryos lacking Pitx2 function at e11.5, so changes in cell death and proliferation earlier in development are being investigated as the potential mechanism.
Conclusions::
Pitx2 is genetically upstream of Pax7, Myf5, MyoD, and Myog in EOM development, making it the earliest known activator of EOM specification. Available evidence suggests that Pitx2 may directly activate the expression of these genes. Pitx2 may also be required for the expansion or survival of the EOM myoblasts. While Pax7 may play a role in EOM development, it is not essential, which further highlights the importance of Pitx2.
Keywords: extraocular muscles: development • genetics • development