from reduced neural crest cell numbers. Alternatively, epithelial- mesenchymal transition of OFT endothelial cells, known to be regulated by Notch signalling [9], may be defective in Hes12/2 embryos, possibly due to a later role of Hes1 in the OFT itself. Dissecting the requirement for Hes1 in different cell-types by conditional mutagenesis will determine the relative importance of different sites of Hes1 expression (including pharyngeal mesoderm, endoderm, ectoderm, neural crest and endothelial cells) during OFT morphogenesis. The early defect in OFT elongation is likely to underlie the subsequent ventriculoarterial alignment defects observed in fetal Hes12/2 hearts. Intriguingly, a dextraposed aorta and ventricular septal defect are observed in less than half of mutant embryos whereas a shorter OFT was observed in all mutant embryos scored at E10.5. Given that there is no loss of Hes12/2 embryos between E10.5 and E15.5, this suggests that there is a degree of phenotypic recovery. The embryonic OFT obtains its maximal length at E10.5 and subsequently becomes incorporated into the ventricular outlets and non-myocardial base of the great arteries; OFT length in Hes12/2 embryos may be on the borderline of that required to achieve normal alignment. Overriding aorta and ventricular septal defects are common components of human congenital heart defects. Defects in OFT development in human patients carrying JAGGED1 or NOTCH2 mutations or in mice carrying mutations in Notch ligands, receptors or target genes demonstrate the importance of this intercellular signaling pathway in arterial pole development [13– 20]. Notch signaling has also been implicated in neural crest and valve development [9,12,16]. The regulation of SHF progenitor cell deployment during heart tube extension may be an additional mechanism by which Notch intercellular signaling controls heart development. Future investigation of the upstream control of Hes1 expression in the SHF will test this hypothesis. Materials and Methods Mice A17-Myf5-nlacZ-T55 (T55) and Mlc1v-nlacZ-24 transgenic mice have been previously described [3,27]. Hes1+/2 mice were kindly provided by Franc¸ois Guillemot (NIMR, UK) and maintained on a CD1 outbred background [23]. DNA isolated from tail-tips or yolk sacs was genotyped for Hes1 alleles as described [33]. Mlc1v- nlacZ-24 and A17-Myf5-nlacZ-T55 transgenic mice were genotyped by X-gal staining or by PCR [3,27]. Mouse care and procedures were in accordance with guidelines approved by the Departmental Direction of Veterinary Services of the French Ministry of Agriculture. Isolation of a transgene integration site junction fragment Inverse PCR was performed using SphI and BglII digested T55 hemizygous tail-tip DNA, as described [3]. Primers A and B were used for inverse PCR using the Expand PCR system (Roche). A 2.6 kb PCR product containing 256 bp of flanking sequence was subcloned using the pGemT-Easy vector (Promega). DNA sequences were aligned using NCBI BLAST, DNA Strider (Release 1.4) and the Ensembl mouse genome browser. Southern blot mapping was carried out with a 770 bp probe generated by PCR using primers C and D that detects a 3.8 kb wildtype fragment and a 5.5 kb fragment subsequent to transgene integration in BamHI digested genomic DNA. PCR validation was carried out using primers E, F and G. Primers E and F generate a 500 bp product from the wildtype locus and F and G a 374 bp product after transgene integration. Hes1 in Heart Development Oligonucleotide primer sequences A 59-AAAACGACGGGATCATCGCGAGCCATGC-39 B 59-ACTGGAGGCTGAAGTTCAGATGTGCGGC-39 C 59-GGTGGCAGCAGTAATCCATGATCTTG-39 D 59-TGTCAAATCAGATCTCTCCAGATGCC-39 E 59-CCTGCTGCTTTGGACAATGC-39 F 59-AAGTCGTTGGAGACCTTGTC-39 G 59-ACCGCTGGATAACGACATTG-39 Wholemount analysis, histology and immunochemistry Embryos were dated taking the day of the plug as embryonic day (E) 0.5. Hearts were dissected at E15.5 and analyzed using a Zeiss Lumar stereo dissecting microscope; selected hearts were subsequently analyzed after cryostat or paraffin embedding and sectioning using standard techniques. Sections were stained with haemotoxilin and eosin or treated for 15 min with antigen unmasking solution (Vector) prior to immunohistochemistry using standard protocols. Primary antibodies used were: b-galactosidase (1/300, Cappel), MF20 (1/20, DSHB), AP-2a (1/25, DSHB clone 3B5), a-actinin (1/200, DSHB), p27kip1 (1/200, Cell Signaling), phospho-Histone H3 (1/400, Upstate), Ki67 (1/25, Dako), Isl1 (1/ 100, DSHB clones 402D6 and 394D5) and Caspase 3 (1/100, Cell Signaling). Secondary antibodies used were: anti-mouse Alexa 488 (Interchim), anti-mouse Cy3 (Jackson), anti-rat biotin (Jackson), anti-rabbit Alexa 488 (Interchim) and anti-rabbit Cy3 (Jackson). All secondary antibodies were used at a 1/200 dilution. The anti- rat biotinylated secondary was amplified using the Renaissance TSA Fluorescence System (Perkin Elmer), according to the manufacturer’s instructions. Slides were counterstained with Hoechst and observed using an ApoTome microscope (Zeiss). Mesenchymal Ap-2a positive nuclei were scored at E9.5 in 5 or more 7 mm sections, 14 mm apart, from 3 mutant and 3 control embryos. X-gal staining was carried out as described [3]; embryos were collected and fixed for 10 min in 4% paraformaldehyde, extensively washed in 16PBS and stained for 5 hours at 37uC in a solution containing 4 mg/ml of X-gal. After staining, the samples were washed in PBS, post-fixed and observed under a Zeiss Lumar stereomicroscope. For each experiment a minimum of 3 embryos of each genotype was scored. OFT length and angle were measured from stereotyped ventral views of X-gal stained Mlc1v- nlacZ-24 embryos at E9.5 (24–26 somites) and E10.5 (30–35 somites) using Metamorph software. Immunoblotting Protein lysates were obtained from microdissected E8.5 hearts and ventral pharyngeal regions of control and mutant embryos. Proteins were separated by SDS-PAGE and transferred to Hybond-C extra membranes (Amersham Biosciences) prior to blocking with 5% dry milk for 2 hours. Membranes were incubated overnight at 4uC with primary antibodies (anti-actin, diluted 1/2000, Sigma; anti-p27kip1, diluted 1/1000, Cell Signaling Technology) followed by chemiluminescent detection (Western Lightening, Enhanced Luminol reagent, Perkin Elmer). Quantification of immunoblots was carried out using Image Quant TL software (Amersham Biosciences). In situ hybridization Whole mount in-situ hybridization was performed as previously described [3]. The Hes1 riboprobe was synthesised by T7 RNA polymerase from a 553 bp template amplified by PCR using primers Hes1F and Hes1R: PLoS ONE | www.plosone.org 9 July 2009 | Volume 4 | Issue 7 | e6267