Hes1 in Heart Development Figure 4. Hes1 is expressed in the second heart field. (A) Comparison of transgene and Hes1 expression in right lateral views of hearts after X- gal staining and in situ hybridization at E10.5 (left three panels). X-gal positive cells are observed in the distal OFT wall (arrowhead) and pulmonary endoderm (arrow); nlacZ transcripts are observed in pulmonary endoderm but not the OFT. Low-level Hes1 transcript accumulation is observed in the distal OFT (black asterisk). Transverse sections (right two panels) show b-galactosidase and low-level Hes1 transcript accumulation in the pericardial region (white asterisks), superior OFT wall (arrow) and mesenchymal cells in the OFT (arrowhead). (B) At E8.5 X-gal and Hes1 positive cells are observed in ventral and right views in the pericardial wall (black arrowhead) and pharyngeal mesoderm (white arrowheads) where Fgf10 transcripts also accumulate. (C) E8.5 sections at the levels indicated in (B), showing Hes1 transcripts in neuroepithelium (ne), endoderm (e) and mesoderm (m) lateral and ventral to the pharynx, including the SHF (arrowheads), compared with Fgf10 expression. Note that caudally (level e) Hes1 and Fgf10 transcripts are observed in endoderm and mesoderm respectively. ht, heart tube. Scale bars: (A, C) 100 mm. doi:10.1371/journal.pone.0006267.g004 Hes12/2 hearts (Fig. 7D). Immunoblot analysis revealed a 2.15 fold increase in p27kip1 levels in microdissected ventral pharyngeal regions including the SHF in the absence of Hes1 (Fig. 7E). Together, these results suggest that loss of Hes1 perturbs proliferation, but not differentiation, of SHF progenitor cells, associated with elevated p27kip1levels. Cardiac neural crest cell defects in Hes1 mutant embryos Cardiac neural crest cells are critically required for OFT septation and normal SHF deployment [6]. In addition to the above defects in SHF development we observed a reduction in Crabp1 expression in the region of the 3rd and 4th–6th pharyngeal arches in Hes12/2 embryos at E9.5 (Fig. 8A). Transverse sections of the caudal pharynx at E9.5 revealed a reduction in AP-2a positive neural crest cells underlying the pharynx (Fig. 8B). Quantification of neural crest (AP-2a positive) and SHF (Mlc1v- nlacZ-24 b-galactosidase positive) nuclei revealed a significant decrease in neural crest cell numbers in mutant embryos compared to wildtype littermates (Fig. 8B, 8C). Subsequently, reduced PlexinA2 transcript accumulation was observed in the distal OFT of Hes12/2 embryos at E11.5 (Fig. 8D). Analysis of OFT cushion morphology at E11.5 revealed normal cushion development in the distal region of the OFT although slight hypocellularity was noted in proximal OFT cushions (Figs 8D, data not shown). Loss of Hes1 therefore affects two critical cell populations entering the arterial pole of the heart, the SHF and cardiac neural crest cells, leading to impaired heart tube elongation and arterial pole alignment defects at later stages of development. Discussion Here we identify Hes1 as a transcription factor encoding gene expressed in the SHF and show that Hes1 is required for normal OFT development. Characterization of the insertion site of the T55 transgene reveals that Hes1 is the likely endogenous target of PLoS ONE | www.plosone.org 5 July 2009 | Volume 4 | Issue 7 | e6267