Hes1 in Heart Development Figure 3. Comparison of T55 and Hes1 expression during embryonic development. (A) E10.5 wholemount embryos (left) and cryosections after X-gal staining (top) or in situ hybridization with a lacZ (T55 embryo, middle) or Hes1 riboprobe (bottom). The transgene is expressed at a subset of sites of Hes1 expression including the forelimb (fl), pharyngeal region (including ectoderm, arrowheads), neural tube (nt), including a specific population of neurons in the ventral neural tube (arrows) and diencephalon (d); ph, pharynx. Asterisk indicates probe trapping in the otic vesicle. (B) E12.5 forelimb (left) and lung (right) after X-gal staining (top) and nlacZ (middle) or Hes1 (bottom) in situ hybridization, showing similar transgene and Hes1 expression in the interdigital region (arrows) and pulmonary epithelium (arrowheads). Scale bar: 200 mm. doi:10.1371/journal.pone.0006267.g003 Fgf10 locus results in expression in pharyngeal mesoderm, including cells of the SHF, OFT and right ventricle (Fig. 6A) [3]. We analyzed OFT morphology at two different days of development (E9.5 and E10.5) and measured OFT length and the angle between the proximal and distal regions of the OFT (Fig. 6B). OFT length and angle in Hes12/2 embryos was indistinguishable from that of control littermates at E9.5. In contrast, at E10.5, we observed a significantly increased angle between the proximal and distal regions of the OFT compared to somite-matched control littermates; moreover, OFT length was concomitantly decreased (Fig. 6B). These results demonstrate that the Hes12/2 OFT is shorter and straighter than that of control littermates at midgestation. Loss of Hes1 impairs SHF proliferation without altering differentiation The decrease in OFT length in Hes12/2 embryos is suggestive of a defect in SHF development. Hes1 is known to maintain progenitor cell populations in the developing embryo through the regulation of differentiation and proliferation [22–26]. We therefore analyzed SHF differentiation at E9.5; no precocious accumulation of sarcomeric myosin heavy chain or a-actinin was observed in splanchnic mesodermal SHF cells in the dorsal pericardial wall of Hes12/2 embryos at this stage (Fig. 7A). The SHF contributes to both myocardium and smooth muscle at the arterial pole of the heart [2]. Normal myocardial and smooth muscle differentiation at the base of the great arteries were observed in mutant hearts at E12.5 using SM22a and a-actinin immunochemistry (data not shown). Proliferation within the pharyngeal region of Hes12/2 embryos was evaluated by scoring for the proliferation markers phospho- Histone H3 and Ki67, revealing M-phase and all proliferating cells respectively. SHF progenitors were identified in the dorsal pericardial wall and pharyngeal mesenchyme at E8.5 using Isl1 or b-galactosidase immunochemistry in Mlc1v-nlacZ-24 embryos. A reduction in the number of phospho-Histone H3 positive (mitotic) Mlc1v-nlacZ-24 positive cells was observed, from 13.7% in wildtype embryos to 5.7% in Hes12/2 embryos (p,0.001; Fig. 7B, 7C). Moreover, 11.7% of Mlc1v-nlacZ-24 positive cells were Ki67- negative (non-proliferating) in mutant embryos compared to 5.7% in wildtype embryos (p,0.001; data not shown). A reduction in proliferating cell numbers was also observed in pharyngeal endoderm from 9% in wildtype embryos to 5% in mutant embryos (p,0.001). These results suggest that cell cycle progres- sion in the SHF and pharyngeal endoderm is impaired in mutant embryos. In contrast, no differences in the percentage of phospho- Histone H3 or Ki67 positive cells were observed in the linear heart tube of Hes12/2 embryos at the same E8.5 timepoint. Equivalent low numbers of Caspase 3-positive cells were found in the pharyngeal region of wildtype and mutant embryos, suggesting that apoptosis levels are unaltered in the absence of Hes1 (data not shown). Hes1 has been shown to control proliferation through the transcriptional repression of cyclin-dependent kinase inhibitors including p27kip1 and p57kip2 [28,29]. We investigated p27kip1 distribution in the SHF of Hes12/2 embryos and observed an expansion of p27kip1 positive cells in SHF cells in the dorsal pericardial wall proximal to the dorsal mesocardium in 3/4 PLoS ONE | www.plosone.org 4 July 2009 | Volume 4 | Issue 7 | e6267