Hes1 in Heart Development Figure 8. Loss of Hes1 impairs neural crest development. (A) In situ hybridization at E9.5 showing decreased Crabp1 transcript accumulation in the caudal pharyngeal region of Hes12/2 compared to Hes1+/+ embryos (arrowheads). (B) Immunochemistry with anti-AP-2a (red) and anti-b- galactosidase (green) antibodies in a transverse section through the caudal pharynx of an E9.5 embryo carrying the Mlc1v-nlacZ-24 transgene. (C) Histograms showing the percentage of b-galactosidase positive nuclei and the percentage of AP-2a positive nuclei in the pharyngeal region of Hes1+/+ and Hes12/2 embryos (left) and the numbers of total, b-galactosidase positive and AP-2a positive nuclei (right). Note the decrease in the number of AP- 2a positive cells in Hes12/2 embryos (p,0.001, Student’s t-test). (D) In situ hybridization at E11.5 showing decreased PlexinA2 transcript accumulation in the OFT of Hes12/2 compared to Hes1+/+ hearts (black arrowheads) in wholemount (top) and transverse sections (middle). Histological analysis reveals normal OFT cushion morphology in Hes12/2 embryos at E11.5 (bottom). Scale bars (B): 50 mm; (D): 200 mm. doi:10.1371/journal.pone.0006267.g008 differentiation in the SHF of Hes12/2 embryos. A possible explanation lies in the observation that Hey1 and Hey2 transcripts are also detectable in pharyngeal mesoderm (data not shown). Hes1 can heterodimerize with Hey1 to effect strong transcriptional repression and Hes1, Hes3 and Hes5 are known to have overlapping functions in the developing nervous system, uncov- ered by analysis of compound embryos [31,32]. Hes1 may therefore overlap in function with other members of the Hes and Hey gene families to regulate SHF development. In support of this argument, overriding aorta has been observed in embryos lacking Hey2 [17–20]. Hes1 also maintains progenitor cell populations in the developing embryo by regulating proliferation. In particular, Hes1 represses transcription of the CDK inhibitors p27kip1 and p57kip2 [28,29]. We observed that levels of the mitotic marker phospho-Histone H3 are reduced in the SHF of Hes1 mutant embryos at E8.5, whereas the number of Ki67-negative quiescent cells is increased. p27kip1 protein distribution at this stage reflects the low proliferative nature of differentiated cells in the linear heart tube. We observed that p27kip1 was expanded and upregulated in the dorsal pericardial wall of Hes1 mutant embryos, consistent with release from Hes1 mediated repression and providing new mechanistic insights into the regulation of SHF proliferation. In addition to reduced proliferation in the SHF, the Hes1 cardiac phenotype is likely to be compounded by the observation that less neural crest cells are found in the ventral pharyngeal region and distal OFT of mutant embryos. Hes12/2 embryos have defects in neural development that may affect the number of migrating neural crest cells [23] and low-level Hes1 transcript accumulation is observed in neural crest cells in the distal OFT. Alternatively, loss of endodermal or SHF signals may indirectly impact on neural crest cell numbers. Neural crest ablation in avian embryos results in impaired SHF deployment and a shortened OFT [6,8]; reduction of neural crest in the caudal pharynx could thus in turn influence SHF development in Hes12/2 embryos. OFT alignment defects in Hes1 mutant embryos have been independently observed by another group who, in addition, noted aortic arch artery abnormalities associated with a defective smooth muscle contribution at E11.5 (P. Scambler, personal communica- tion). Neural crest cell anomalies may therefore also contribute directly to the arterial pole phenotype of Hes1 mutant embryos. While we did not observe defects in great artery smooth muscle development, slight hypocellularity of OFT cushions could result PLoS ONE | www.plosone.org 8 July 2009 | Volume 4 | Issue 7 | e6267