Hes1 in Heart Development Figure 2. Comparison of transgene and integration site gene expression profiles. (A) E9.5 embryos after X-gal staining (T55 embryo, left) or in situ hybridization with a lacZ (T55 embryo, middle) or Hes1 riboprobe (right). Overlapping expression sites include the ventral pharyngeal region (ph), forelimb (fl), ventral neural tube (cns), segmental intersomitic region (s), midbrain (mb), tail region (t) and nasal ectoderm (ne). Asterisk indicates lacZ riboprobe trapping in the otic vesicle. (B) Expression profiles of Opa1 and two predicted genes mapping to the intergenic region at E9.5. doi:10.1371/journal.pone.0006267.g002 the T55 transgene and Hes1 are co-expressed in a subset of Hes1 expression sites. Hes1 is expressed in the second heart field In T55 embryos, b-galactosidase activity is detected in the superior wall of the OFT and contiguous pericardial mesoderm at E10.5 [27]; nlacZ transcripts in T55 positive embryos accumulate in pericardial mesoderm but were not observed in the myocardial OFT wall. b-galactosidase activity in the OFT wall is thus the result of either extremely low-level transcription or b-galactosidase perdurance from prior transcription in OFT progenitor cells (Fig. 4A). At this timepoint Hes1 transcripts accumulate in pericardial mesoderm; after extensive staining, Hes1 transcript accumulation was detected in cells in the superior wall of the OFT (Fig. 4A). Hes1 transcripts also accumulate at low-level in cells likely to correspond to cardiac neural crest cells in the distal OFT; T55 transgene expression is also observed in these cells (Fig. 4A). Analysis of earlier developmental timepoints revealed that the T55 transgene is expressed in splanchnic mesoderm dorsal to the heart tube at E8.5 (Fig. 4B) [27]. Endogenous Hes1 transcripts were observed in the dorsal pericardial wall and in mesoderm lateral to the pharynx, in addition to pharyngeal epithelia and neuroecto- derm. The expression of Hes1 in pharyngeal mesoderm overlaps with that of Fgf10; however, the mesodermal expression domain of Fgf10 extends further, both in a posterior direction and into cranial mesoderm, than that of Hes1 (Fig. 4C). Hes1 is therefore coexpressed with Fgf10 in pharyngeal mesoderm dorsal to the early heart tube, including the region of the SHF. Heart defects in Hes1 mutant mice Hes1 mutant mice die during late fetal development with defects affecting neural, eye, pancreas, pituitary and lung development, among other organs [22–26]. This phenotype reflects the critical role of Hes1 in maintaining progenitor cell populations and regulating differentiation during organogenesis [22]. In order to investigate whether Hes1 is required for SHF development, we analyzed cardiac development in Hes1 mutant mice. SHF defects affect OFT formation and arterial pole alignment [1,4,7] and embryos were scored at E15.5 for abnormalities in ventriculoar- terial alignment. During normal development, the ascending aorta obtains an independent connection with the left ventricle and the pulmonary trunk with the right ventricle. 21/127 Hes12/2 embryos were recovered (expected 32/127; p.0.05)) and 8/21 (38%) mutant embryos displayed an externally visible dextraposed ascending aorta; this phenotype was not observed in Hes1+/2 or Hes1+/+ embryos (Fig. 5A). Histological sections of selected embryos revealed that the aorta failed to obtain a dorsal position with respect to the pulmonary trunk resulting in a ventricular septal defect and overriding aorta (observed in 3 out of 4 Hes12/2 hearts with a dextraposed aorta that were sectioned). The extent to which the aorta overrides the right ventricle was ,50% in the hearts scored (Fig. 5). Overriding aorta and ventricular septal defects were not observed in 1 of the 4 sectioned hearts with a dextraposed aorta or in 6 additional Hes12/2 hearts without an externally visible dextraposed aorta. No defects were observed on histological analysis of 5 Hes1+/2 and 5 Hes1+/+ hearts. Loss of Hes1 therefore results in defects in normal alignment of the aorta and left ventricle during cardiac remodeling in a significant fraction of mutant embryos. The etiology of this defect was analyzed by examining Hes12/2 embryos at earlier stages of heart development. In order to visualize SHF progenitor cells and OFT development in mutant embryos, we crossed Hes1+/2 mice with the Mlc1v-nlacZ-24 transgenic line, in which transgene integration upstream of the PLoS ONE | www.plosone.org 3 July 2009 | Volume 4 | Issue 7 | e6267