Supplementary Fig. 1

Supplementary Fig. 4

CMFDG labelling and analysis of Tbx1LacZ cell enrichment. Example of CMFDG labelling of an E9.5 Df1/Tbx1^lacZ embryo separated from the rest of the litter by phenotype. X-gal staining represents expression of the Tbx1^lacZ allele. This labelling procedure is also carried out on the Tbx1^+/lacZ;Df1/+;wt pool of embryos. RT-PCR analysis indicates that the wild type Tbx1 allele is present only in the FDG-positive cell population containing Df1/+ and Tbx1^+/lacZ cells. The Tbx1LacZ allele is only present in FDG-positive cell populations and represents Df1/Tbx1^lacZ and Tbx1^+/lacZ cells.

Intracardiac ink injection of E10.5 Hes1 mutants. (A) In wild type embryos, bilateral pairs of caudal PAAs R3–R6 and L3–L6 are present at E10.5. (B) A proportion of Hes1^+/− mutants have hypoplasia of the fourth arch artery (arrow) and persistent first and second arch arteries (C). Hes1^−/− mutants also exhibit fourth arch artery aplasia (arrow in D), persistent second arch arteries and aberrant branching of the arch arteries (arrow in E and inset) and sixth arch artery hypoplasia (arrow in F). L indicates left; R, right.

22q11DS-like defects in E15.5 Hes1^−/− mutants. (A–D) Great vessel defects in E15.5 Hes1^−/− embryos. (A) Normal wild type (wt) great vessel morphology. (B–D) Hes1^−/− (–/–) mutants with RAA and right-sided ductus arteriosus (B), IAA-B and RAA (C), and I-RSA (D). (E) In wt embryos, the right subclavian artery branches off the right common carotid. (F) Hes1^−/− mutant in which the right subclavian branches off the pulmonary trunk. (G–L) OFT defects and VSD in Hes1^−/− mutants. (G, H) Transverse MRI scans. (I–L) Transverse histological sections. (G) In wild type embryos the interventricular septum separates the right and left ventricles. (H) Hes1^−/− mutants exhibit a VSD (dark staining represents blood). (I, J) Consecutive transverse sections of a wild type embryo in which the pulmonary trunk arises from the right ventricle (I) and the aorta joins the left ventricle (J). (K, L) Consecutive sections of a Hes1^−/− embryo with double outlet right ventricle in which both the aorta and pulmonary trunk arise from the right ventricle. (M, N) Transverse MRI sections of the palate at E15.5. (M) In wild type embryos the palatal shelves are fused. (N) Hes1^−/− mutants exhibit cleft palate where the palatal shelves do not meet in the midline. (O, P) Transverse MRI sections of the thymic lobes which are missing in Hes1^−/− mutants (P). ao indicates aorta; aoa, aortic arch; da, ductus arteriosus; ivs, interventricular septum; lcc, left common carotid; lsca, left subclavian artery; lv, left ventricle; ps, palatal shelf; pt, pulmonary trunk; rcc, right common carotid; rsca, right subclavian artery; rv, right ventricle; t, tongue; th, thymic lobe; vsd, ventricular septal defect. Asterisks indicate missing segments.

Proliferation defect in Hes1 mutants. (A–B) Immunohistochemistry with anti-phospho-Histone H3 (PH3) antibody in paraffin sections of E8.5 WT (A) and Hes1^−/− (B) embryos showing a decrease in PH3-positive cells within the surface ectoderm of Hes1^−/− mutants (arrowhead). (C–D) Immunohistochemistry with anti-Ki67 antibody in sections of E8.5 WT (C) and Hes1^−/− (D) embryos showing an increase in the number of Ki67-negative cells in Hes1^−/− embryos (arrowhead). (E) Graph comparing the percentage of PH3 positive cells and Ki67 negative cells in the pharyngeal ectoderm of WT embryos and Hes1^−/− mutants. A significant decrease in PH3 positive cells (p < 0.05) and increase in Ki67 negative cells (p < 0.05, Student's t-test) is observed in Hes1^−/− mutants.

Supplementary Fig. 2

Expression of Hes1 in wild type and Tbx1^−/− embryos. (A–C) Tbx1LacZ expression at E9.5. (A) Whole embryo, (B) coronal section and (C) transverse section of Tbx1^+/LacZ embryo. (D) Transverse section illustrating Hes1 expression at E9.5 by in situ hybridisation. (E–H) Whole mount in situ hybridisation (E–F) and corresponding transverse sections (G–H) showing Hes1 expression is diminished in Tbx1^{−/−−/−} embryos. a indicates arch; ov, otic vesicle. (I) Overlapping expression of Hes1 and Tbx1 as detected by immunofluorescence staining of transverse cryosections of wild type E8.5 embryos. Pharyngeal mesenchyme is indicated by a black arrow, endoderm by a white arrowhead, mesoderm with a red arrowhead and ectoderm with a yellow arrowhead.

Hes1 mutants exhibit loss of vascular smooth muscle markers in the developing PAAs. (A, B) Immunofluorescence on coronal E10.5 embryo sections for the endothelial marker, Endomucin (Endo). Endo staining is present in wild type (A) and Hes1^−/− embryos with aplastic/non-patent 4th PAAs (arrow in B). (C, D) Immunofluorescence on coronal E11.5 embryo sections for α-SMA. Strong α-SMA expression surrounding the 3 rd, 4th and 6th PAA in wild types (C) is lost in the hypo/aplastic left 3 rd and 4th PAA (red and white arrows in D, respectively) of Hes1^−/− mutant embryos. The contralateral vessels, which have formed in this embryo, have normal α-SMA staining.

22q11DS-like defects in E10.5 and E15.5 Hes1 conditional mutants. (A–F) ROSAR26R reporter crosses. (A–C) Hes1^−/fl;Wnt1Cre x ROSAR26R and (D–F) Hes1^−/fl;Ap2αCre x ROSAR26R crosses following X-gal staining. (A) E9.5 whole mount image, (B) sagittal section of (A) showing strong recombination in the neural crest of first and second pharyngeal arches (black arrows), and (C) transverse section of (A) showing recombination in the neural crest cells at the second heart field level (white arrow). (D) E9.5 whole mount image, (E) sagittal section of (D) showing strong recombination in the neural crest and ectoderm of first and second pharyngeal arches (black arrows) and (F) transverse section of (D) showing recombination in the ectoderm (black arrow) and neural crest cells (white arrow). (G–I) Great vessel defects in E15.5 Hes1^−/fl;Ap2αCre embryos. (G) Normal Hes1^+/fl (wild type) great vessel morphology. (H–I) Hes1^−/fl;Ap2αCre (Hes1 null in the ectoderm and neural crest) mutants with aberrant branching of the RCC (H) and I-RSA (I). (J) Intracardiac ink injection of E10.5 Hes1^−/fl;Ap2αCre mutants displaying persistent second arch arteries (R2, L2) and unilateral third arch artery aplasia (arrow). (K–O) Thymic defects in E15.5 Hes1^−/fl;Ap2αCre embryos. (K) Normal thymic lobe morphology in Hes1^+/fl (wild type) embryos. (L–M) Hypoplastic lobes (L) and a single, hypoplastic lobe (M) observed in Hes1^−/fl;Ap2αCre mutants. (N–O) Transverse section of the thymic lobes in Hes1^−/fl;Ap2αCre embryos showing normal development (N) and aplasia (O). aoa indicates the aortic arch; lcc, left common carotid; oft, outflow tract; rcc, right common carotid; rsca, right subclavian artery; rv, right ventricle; t, thymic lobe. Asterisks indicate missing or duplicated segments.