Prediction of regulatory networks in mouse abdominal wall

Abstract

Sequence specific transcription factors are essential for pattern formation and cell differentiation processes in mammals. The formation of the abdominal wall depends on a flawless merge of several developmental fields in time and space. The absence of Pitx2 leads to an open abdominal wall in mice, while mutations in humans result in umbilical defects, suggesting that a single homeobox transcription factor coordinates the formation and patterning of this anatomical structure. Gene expression analysis from abdominal tissue including the abdominal wall after removal of the major organs, of wild type, Pitx2 heterozygote and mutant mice, at embryonic day 10.5, identified 275 genes with altered expression levels. Pitx2 target genes were clustered using the "David Bioinformatics Functional Annotation Tool" web application, which bins genes according to gene ontology (GO) key word enrichment. This provided a way to both narrow the target gene list and to start identifying potential gene families regulated by Pitx2. Target genes in the most enriched bins were further analyzed for the presence and the evolutionary conservation of Pitx2 consensus binding sequence, TAATCY, on the -20 kb, intronic and coding gene sequences. Twenty Pitx2 target genes that passed all the above criteria were classified as genes involved in cell transport and growth. Data from these studies suggest that Pitx2 acts as an inhibitor of protein transport and cell apoptosis contributing to the open body wall phenotype. This work provides the framework to which the developmental network leading to abdominal wall syndromes can be built.

Figure 1. Evolutionary Conserved Pitx2 binding Sites

Comparative analysis of Pitx2 consensus binding sites in the Pitx2 target genes. Genomic consensus sequences 20kb upstream of the transcriptional start site were mapped against the aligned sequences in the top twenty Pitx2 target genes. Red dots indicate a predicted Pitx2 binding site. Vertical red dots indicate the number of species the motif was found.

Figure 2. Pitx2 Target Genes Visualized with Cytoscape

Each circle represents an individual gene with Pitx2 as the core regulator. Red and green lines represent repressed and activated Pitx2 target genes respectively. The length of the lines represents the strength of their relationships. (A) Representation of target gene relations to Pitx2 based on the sum of binding sites and number of species conserved from Table 2. Genes with a greater net number of sites and species are mapped closer to the Pitx2 core. (B) Representation of target gene relations to Pitx2 based on fold change. The greater the fold change the closer the mapping of the gene to Pitx2. It appears that Pitx2 is a general repressor for the majority of our target genes.

Figure 3. Predicted Pitx2 Network Model in Abdominal Wall

BioTapestry was used to create a model of the Pitx2 regulatory network in the abdominal wall. Genes within the nested regions are identified as different gene regulatory components. Red and green links represent repressed and activated interactions from pitx2 as found in this study, while blue links represent interactions identified from literature.