To gain further insight into the mechanisms underlying
Pitx2 gene dose effects on extraocular muscle development, we initiated a comparison of global gene expression in eye primordia isolated from E12.5
Pitx2 +/+,
Pitx2 +/−, and
Pitx2 −/− embryos (
n = 4 per genotype). These genotypes correspond to 100%, 50%, and 0% of the normal PITX2 function levels, respectively. RMA, a robust and well-established methodology, was used to quantify signal intensity and normalize signals.
40 Normalized data were then analyzed using the robust two-step FDR-CI method to identify statistically significant (
P < 1) differentially expressed genes.
41 42 This approach is now well-established.
43 Hierarchical clustering of the top 200 differentially expressed genes revealed striking patterns. Three distinct patterns of gene expression were observed based on their response to
Pitx2 gene dose
(Fig. 3) . Expression of genes in set A decreased in parallel with
Pitx2 gene dose, indicating that these genes are dose sensitive and activation correlates positively with PITX2 protein levels. Expression of genes in set B was reduced in eyes of
Pitx2 −/− but not
Pitx2 +/− embryos, indicating that these genes are not affected by
Pitx2 gene dose. Finally, expression of genes in set C increased with decreasing
Pitx2 gene dose, indicating that these genes are dose sensitive, but that activation correlates negatively with increasing PITX2 protein levels. Because whole eye primordia were used as the RNA source for these experiments, differentially expressed genes represent both myogenic and nonmyogenic functions. We next evaluated each gene in sets A, B, and C to identify those that could be confirmed as muscle-specific based on previously published results or publicly available expression data. In all, 50 muscle-specific, dosage-sensitive genes were identified
(Table 1) . Consistent with the decrease of extraocular muscle morphogenesis in response to
Pitx2 gene dose, expression of all the muscle-specific genes decreased in parallel with
Pitx2 gene dose (
Fig. 3 ; set A). No muscle-specific genes were identified in set C, whose expression increases with decreasing
Pitx2 gene dose.