Genomic profiling reveals Pitx2 controls expression of mature extraocular muscle contraction-related genes

Abstract

Purpose: To assess the influence of the Pitx2 transcription factor on the global gene expression profile of extraocular muscle (EOM) of mice.

Methods: Mice with a conditional knockout of Pitx2, designated Pitx2(Δflox/Δflox) and their control littermates Pitx2(flox/flox), were used. RNA was isolated from EOM obtained at 3, 6, and 12 weeks of age and processed for microarray-based profiling. Pairwise comparisons were performed between mice of the same age and differentially expressed gene lists were generated. Select genes from the profile were validated using real-time quantitative polymerase chain reaction and protein immunoblot. Ultrastructural analysis was performed to evaluate EOM sarcomeric structure.

Results: The number of differentially expressed genes was relatively small. Eleven upregulated and 23 downregulated transcripts were identified common to all three age groups in the Pitx2-deficient extraocular muscle compared with littermate controls. These fell into a range of categories including muscle-specific structural genes, transcription factors, and ion channels. The differentially expressed genes were primarily related to muscle contraction. We verified by protein and ultrastructural analysis that myomesin 2 was expressed in the Pitx2-deficient mice, and this was associated with development of M lines evident in their orbital region.

Conclusions: The global transcript expression analysis uncovered that Pitx2 primarily regulates a relatively select number of genes associated with muscle contraction. Pitx2 loss led to the development of M line structures, a feature more typical of other skeletal muscle.

Figure 1.

Hierarchical clustering of the gene probes identified as differentially expressed between extraocular muscles of Pitx2^{Δflox/Δflox} and Pitx2^flox/flox mice at 3 weeks, 6 weeks, and 3 months of age. Transcripts identified are those at the intersection of data obtained with the MAS and RMA algorithms (Affymetrix). The three independent replicates of each group are represented. The scale at the top right denotes normalized expression levels (red, high expression; blue, low expression).

Figure 2.

Venn diagram showing the numbers of differentially expressed transcripts between Pitx2^{Δflox/Δflox} and Pitx2^flox/flox EOM shared by or unique to the three time points.

Figure 3.

Validation of myomesin 2 expression in Pitx2^{Δflox/Δflox} extraocular muscle. Western blot analysis (A) showed that myomesin 2 is not expressed in Pitx2^flox/flox extraocular muscle but readily detected in the Pitx2^{Δflox/Δflox} extraocular muscle. This upregulation is unique to extraocular muscle as myomesin 2 is expressed in both extensor digitorum longus and heart at comparable levels between Pitx2^{Δflox/Δflox} and Pitx2^flox/flox mice. Duplicate samples of EOM tissues from two different mice of both Pitx2^{Δflox/Δflox} and Pitx2^flox/flox genotypes were used for Western blot analysis. Electron microscopy showed that sarcomeres of the extraocular muscles from the Pitx2^flox/flox mice (B) do not have M-lines, whereas that from Pitx2^{Δflox/Δflox} mice (C) now have M-lines.

Figure 4.

Schematic representation of key gene expression alterations that are associated with the conditional knockout of Pitx2 in EOM. The transcript alterations are consistent with the physiologic alterations that would produce a muscle that has greater contractile force and speed, which is consistent with previous physiologic studies (see Discussion section for details).