progenitor cell populations, controlling binary cell fate decisions and regulating boundary formation [21]. Hes1, encoding a transcriptional repressor, plays central roles in cell proliferation and differentiation processes in multiple cell types and is required to maintain progenitor cells in an undifferentiated state [22–26]. Recently the A17-Myf5-nlacZ-T55 (T55) transgene has been shown to be expressed in the SHF and OFT of the developing mouse heart as a result of an integration site position effect [27]. We have localized the T55 transgene integration site upstream of Hes1 and show that the transgene is expressed in a subset of Hes1 expression sites, including pharyngeal mesoderm of the SHF. Although Hes1 has not been previously implicated in heart development, mice lacking Hes1 display arterial pole alignment anomalies, including overriding aorta and ventricular septal defects, common components of congenital heart disease in man. These defects are preceded by impaired deployment of cells of the SHF and cardiac neural crest revealing a requirement for Hes1 during OFT development. Results Characterization of the T55 transgene integration site The Myf5 A17-nlacZ-T55 (T55) transgene is expressed in the SHF and OFT as a result of an integration site position effect [27]. In order to identify new genes expressed in the SHF, we characterized the transgene integration site. Using fluorescent in situ hybridization (FISH) the insertion site was mapped to chromosome 16 B2–B4 (Fig. 1A). An inverse PCR approach with primers in the 59 region of the nlacZ reporter gene was used to isolate a transgene-integration site junction fragment containing 256 bp of flanking sequence (Fig. 1B). Southern blot and PCR analysis confirmed the presence of this junction specifically in DNA from mice carrying the T55 transgene (Fig. 1C, data not shown). Hes1 in Heart Development A nucleotide BLAST search using the 256 bp flanking sequence revealed that this sequence mapped to chromosome 16 B2, consistent with the FISH localization (Fig. 1D). The insertion site junction lies between two genes, Optic Atrophy 1 (Opa1), encoding a mitochondrial dynamin-related GTPase, and Hairy/Enhancer of Split 1 (Hes1), encoding a basic-helix-loop-helix containing transcriptional repressor, situated 258 kb and 224 kb from the isolated sequence respectively. Opa1 and Hes1 are part of a 4 gene synteny block centered on Hes1 that is conserved in zebrafish. The expression profiles of Hes1 and Opa1, together with those of 4 EST sequences and 2 predicted genes mapping between Hes1 and Opa1, were evaluated by wholemount in situ hybridization at E9.5 (Fig. 2). Hes1 transcripts showed a regionalized expression profile overlapping in distribution with the T55 transgene (Fig. 2A, Fig. 3), including pharyngeal, forelimb, tail, intersomitic, neural tube, midbrain and nasal ectoderm expression sites (Fig. 2A). In contrast, Opa1 transcripts were broadly expressed in the embryo and riboprobes detecting EST sequences between Opa1 and Hes1, including the predicted genes 9530020007RIK and GM1968, revealed either low-level expression in the anterior region of the embryo or no expression (Fig. 2B). Together, these results suggest that Hes1 may be the endogenous target of the cis-regulatory sequences trapped by the T55 transgene. Additional gene expression studies were carried out to test this hypothesis. The distribution of Hes1 transcripts was compared with that of nlacZ, together with b-galactosidase expression, in T55 embryos at E10.5 and E12.5. At E10.5, T55 and Hes1 expression overlapped in pharyngeal epithelia, the pericardial region and the forelimb (Figs 3A, 4A). T55 transcripts accumulated in a subset of Hes1 expression sites in the central nervous system, including specific populations of neurons in the brain and ventral neural tube (Fig. 3A). At E12.5, expression of the transgene and Hes1 was observed in the interdigital region of the developing limb-buds and pulmonary endoderm (Fig. 3B). These experiments revealed that Figure 1. Molecular characterization of the T55 transgene integration site. (A) Fluorescent in situ hybridization to metaphase chromosomes prepared from T55 splenocytes showing transgene localization to chromosome 16 B2–B4. (B) Map of the T55 integration site (top) and endogenous locus (bottom) showing the structure of the 39 end of the transgene array (black box, A17 Myf5 enhancer; blue box, nlacZ reporter gene) and the position of the inverse PCR primers (red arrowheads); B, BamHI; Bg, BglII. Also shown are the flanking sequence isolated by inverse PCR (red) and the Southern blot probe (grey) used to identify the predicted 5.5 kb BamHI fragment in transgenic DNA in addition to a 3.8 kb wildtype fragment (C). (D) Ensembl map of the genomic region surrounding the integration site (red arrow) on chromosome 16, showing the position of the flanking genes Opa1 and Hes1 and intermediate gene predictions based on EST alignments. doi:10.1371/journal.pone.0006267.g001 PLoS ONE | www.plosone.org 2 July 2009 | Volume 4 | Issue 7 | e6267