Regulation of Motility of Myogenic Cells in Filling Limb Muscle Anlagen by Pitx2

Abstract

Cells of the ventrolateral dermomyotome delaminate and migrate into the limb buds where they give rise to all muscles of the limbs. The migratory cells proliferate and form myoblasts, which withdraw from the cell cycle to become terminally differentiated myocytes. The myogenic lineage colonizes pre-patterned regions to form muscle anlagen as muscle fibers are assembled. The regulatory mechanisms that control the later steps of this myogenic program are not well understood. The homeodomain transcription factor Pitx2 is expressed specifically in the muscle lineage from the migration of precursors to adult muscle. Ablation of Pitx2 results in distortion, rather than loss, of limb muscle anlagen, suggesting that its function becomes critical during the colonization of, and/or fiber assembly in, the anlagen. Microarrays were used to identify changes in gene expression in flow-sorted migratory muscle precursors, labeled by Lbx1EGFP/+, which resulted from the loss of Pitx2. Very few genes showed changes in expression. Many small-fold, yet significant, changes were observed in genes encoding cytoskeletal and adhesion proteins which play a role in cell motility. Myogenic cells from genetically-tagged mice were cultured and subjected to live cell-tracking analysis using time-lapse imaging. Myogenic cells lacking Pitx2 were smaller, more symmetrical, and had more actin bundling. They also migrated about half of the total distance and velocity. Decreased motility may prevent myogenic cells from filling pre-patterned regions of the limb bud in a timely manner. Altered shape may prevent proper assembly of higher-order fibers within anlagen. Pitx2 therefore appears to regulate muscle anlagen development by appropriately balancing expression of cytoskeletal and adhesion molecules.

Prediction of gene network models in limb muscle precursors

“…All four genes are involved in regulating cell motility and expression of surface receptors to allow for homophilic cell recognition of the muscle precursors. We have shown that Pitx2 regulates cytoskeletal, adhesion, and signaling genes in migratory muscle precursor cells (Campbell et al, 2012). Proper regulation of the cytoskeleton allows migratory cells to form protrusions in directions of positive signals in order to explore the extracellular environment.…”

“…We analyzed microarrays from E12.5 mouse forelimb tissue enriched for Lbx1 + muscle progenitor cells (Campbell et al, 2012). These data have been deposited in NCBI’s Gene Expression Omnibus (Edgar et al, 2002) and are accessible through GEO Series accession number GSE31945 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE31945).…”

“…Analysis of Lbx1 + muscle progenitor cells isolated from E12.5 Pitx2 mutant forelimbs has revealed that a small group of cytoskeletal, adhesion, and signaling genes are potential targets for regulation by Pitx2 . Muscle progenitors of Pitx2 mutants were smaller, more symmetrical, had increased actin bundling, and decreased motility preventing myogenic cells from filling limb bud anlagen (Campbell et al, 2012). …”

Mapping the chromatin state dynamics in myoblasts

“…Correlation studies were performed for the sequences marked with different signatures. Differential enrichment of histone marks and Pol II were observed around a number of Pitx2 target genes identified in Lbx1 EGF/+ migratory myoblasts (Campbell et al, 2012b). Quantitative real-time PCR assays (qPCR) assessed gene expression and revealed that the H3K27me3 chromatin mark best predicted relative gene expression.…”

Identification of the GTPase-activating protein DEP domain containing 1B (DEPDC1B) as a transcriptional target of Pitx2

“…It has been demonstrated that Pitx2 can regulate the expression of a group of genes such as PLOD (procollagen lysyl hydroxylase), Dlx2, ANF (atrial natriuretic factor), cyclin D1/D2 , p21, MYC , Amelogenin ( Amel ), and the T-box genes at the transcriptional level [9-16]. Through regulation of its downstream target genes, Pitx2 is implicated in controlling various biological processes such as cell migration and cell proliferation [12-14, 17]. In particular, fate-mapping studies in mice have shown that Pitx2 is required for the regulation of cell migration during craniofacial and hypothalamus development [18, 19].…”