Hes1 in Heart Development Figure 7. Impact of loss of Hes1 on differentiation and proliferation in the second heart field. (A) Immunochemistry with anti-MHC (left), anti-a-actinin (right) and anti-b-galactosidase antibodies in transverse sections through the caudal pharyngeal region of an E9.5 embryo carrying the Mlc1v-nlacZ-24 transgene. Note the b-galactosidase positive a-actinin and MF20 negative cells in the dorsal pericardial wall (arrowheads). Nuclei are labeled with Hoechst (blue). (B) Immunochemistry with anti-phospho-Histone H3 and anti-b-galactosidase antibodies in paraffin sections of E8.5 Hes1+/+ and Hes12/2 embryos carrying the Mlc1v-nlacZ-24 transgene. (C) Histogram comparing the percentage of phospho-Histone H3 positive nuclei in pharyngeal endoderm, myocardium and SHF of Hes1+/+ and Hes12/2 embryos. A decrease in phospho-Histone H3 positive nuclei is observed in Hes12/2 SHF and endoderm (p,0.001, Student’s t-test). (D) Immunochemistry with an anti-p27kip1 antibody in paraffin sections at E8.5. Note that p27kip1 is expanded in the SHF of Hes12/2 hearts (arrowheads). Nuclei are labeled with Hoechst (blue). (E) Western blot of microdissected heart and ventral pharyngeal regions of Hes1+/+ and Hes12/2 embryos showing elevated p27kip1 protein levels in the absence of Hes1. Scale bars (A): 50 mm; (B): 100 mm; (D) 50 mm. doi:10.1371/journal.pone.0006267.g007 regulatory sequences trapped by the transgene. The integration site lies within a region of conserved synteny between mouse and zebrafish that may correspond to a genomic regulatory block, such as have been proposed to contain conserved non-coding elements targeting developmental regulatory genes [30]. Our data suggest that regulatory elements within this block control the spatiotem- poral expression pattern of Hes1. Comparison of T55 and Hes1 expression profiles suggest that the transgene has trapped only a subset of the cis-regulatory elements mediating Hes1 activation. In contrast, sites of b-galactosidase accumulation but not Hes1 expression may result from inherent differences in transcript versus b-galactosidase protein stability. Further differences may arise from the fact that Hes1 is highly regulated both transcrip- tionally and post-transcriptionally; oscillations in Hes1 protein levels play a critical role in measuring developmental time in unsegmented paraxial mesoderm [21]. b-galactosidase activity in a subregion of OFT myocardium may thus in part provide a readout of prior heterogeneity in Hes1 expression in SHF progenitor cells. Hes1 is known to be a downstream component of the Notch signaling pathway and a critical regulator of multiple stem cell populations in the developing embryo [22]. Accelerated differen- tiation in Hes1 mutant embryos results in depletion of progenitor cell populations and a failure of late differentiating cell types [22– 26]. Here we show that Hes1 is required for normal deployment of SHF progenitor cells. However, we did not observe accelerated PLoS ONE | www.plosone.org 7 July 2009 | Volume 4 | Issue 7 | e6267