Supplemental Information MOZ Regulates the Tbx1 Locus, and Moz Mutation Partially Phenocopies DiGeorge Syndrome


Inventory of Supplemental Information


Supplemental data


Supplemental Experimental Procedures

Table, related to Experimental Procedures: cDNA templates for cDNA and cRNA probes. 

Table, related to Experimental Procedures:

Primers used for RT-qPCR experiments. (E-H) Schematic drawing of the aortic arch defects in Moz Δ/Δ mutant pups at E18.5. (E) Wild type vs. (F) the most common abnormality, interrupted aortic arch type B, which occurred in 14 of 19 Moz Δ/Δ mutant pups examined. Among these, three had an abnormal right descending aorta as depicted in (G). The remaining 5 of 19 pups had less severe abnormalities with abnormal origin of the RSA, abnormally large diameters and lack of wall structure development in the large blood vessels as seen in (H). None of 28 Moz Δ/+ heterozygous and wild type, littermate control pups exhibited abnormalities of the aortic arch. 

Table, related to Experimental Procedures: Primers used for qPCR in ChIP experiments


Supplemental References


Supplemental data

(I-N) The cardiovascular defects in Moz Δ/Δ mutant pups. E18.5 Moz +/+ (I) and Moz Δ/Δ (J) H&E stained transverse sections of hearts. Compared to Fig. 1N,O, note the milder ventricular septal defect (arrow in J vs. I). (K, L) Overview of ink-injected large vasculature of E9.5 Moz +/+ (K) and Moz Δ/Δ (L), which appear similar at E9.5 as opposed to the differences seen at E10.5 (Fig. 1). (M, N) Right lateral view of resin-injected large vasculature of E10.5 Moz +/+ (M) and Moz Δ/Δ (N). Note that, in contrast to the absence of the 4th pharyngeal arch artery on the left body side in the Moz Δ/Δ mutants (Fig. 1), the 4th pharyngeal arch artery is present on the right body side Moz Δ/Δ (N), albeit thinner than in the wild type control (M). Moz ∆/∆ Tbx1-Tg hearts at five ventro-dorsal levels. Note the large, medium and small ventricular septal defects in the Moz ∆/∆ mutant hearts (arrows) and the absence of ventricular septal defects in the Moz ∆/∆ Tbx1-Tg hearts. The penetrance of ventricular septal defects in the Moz ∆/∆ mutants is 95% (Table 1). 

Supplemental Experimental Procedures

cDNA templates for cDNA and cRNA probes. 

Gene


Provided by

Ref. 

Tbx1

V. E. Papaiaonnou (Chapman et al., 1996) Tbx2 
V. E. Papaiaonnou (Chapman et al., 1996) Tbx3 
V. E. Papaiaonnou (Chapman et al., 1996) Tbx4 
V. E. Papaiaonnou (Chapman et al., 1996) Tbx5 
V. E. Papaiaonnou (Chapman et al., 1996) Hsp90β (Voss et al., 2000) Primers used for RT-qPCR experiments. 

Gene


GeneBank accession number


Forward


Reverse

Corresponding to exons* *qPCR product encompasses FOXA2/FOXC1/FOXC2 response element as published (Yamagishi et al., 2003). 
Tbx1 NM_011532 GTGGACCCTCGAAAAGACAG CCTACCAGAATCACCGGATC 4-5 Tbx2 NM_009324 AGATGGTCATCACCAAGTCC CGATGTCCATTAGCAGGATG 1-2 Tbx5 NM_011537 ACACATCGTGAAAGCAGACG TAACTCCAGGTCATCACTGC 6-7 Tbx20 NM_00120505 AGCTCTCCTCTCGAGCCAAT GCACATGATGATTTCTCCACAA 1-2 Chd7 NM_0010814417 GGAGAACCCTGAGTTTGCTG ACCACAGGTCCAGTGAGGAG Exons 37-38 Foxa2 NM_010446 GAGCCGTGAAGATGGAAGG CATGTTGCTCACGGAAGAGTAG Exons 2-3 Foxc1 NM_008592 GCCAGAGCTCCCTCTACAGC CTGCAGGTTGCAGTGGTAAG Exon 1 Foxc2 NM_013519 CATGCAGGCGCGTTACTC CCGCCCGGTAGTAGTTTTG Exon 1 Gata4 NM_008092 CTGGAAGACACCCCAATCTC GTAGTGTCCCGTCCCATCTC Exons 1-2 Gnb1l NM_001081682 GCTCTGCTGGGAAGGTACTG GCAATTCCAGGATTGGTGAG

Figure S1 ,

 S1 Figure S1, related to Figure 1: Craniofacial, palate, aortic arch and cardiac abnormalities in Moz mutant neonates and embryos. 


Figure S2, related to Figure 3 :

 3 Figure S2, related to Figure 3: Expression of T-box genes in Moz Δ/Δ embryos. 


Figure S3, related to Figure 6 :

 6 Figure S3, related to Figure 6: Rescue of the Moz ∆/∆ mutant cardiac septal defect by a Tbx1 transgeneserial images of Moz ∆/∆ mutant and Tbx1 transgenic Moz ∆/∆ mutant hearts. 


Figure S1 :

 S1 Figure S1: Craniofacial, palate, aortic arch and cardiac abnormalities in Moz mutant neonates and embryos. E18.5 Moz +/+ (A, C), Moz Δ/Δ (B) and Moz -/-(D). (A, B) Sagittal, H&E stained section and (C, D) frontal view of the head. Note the absence of the palate (arrows B vs. A) and incomplete midline fusion of the face (arrow in D vs. C). 




  1b, mandibular portion of the 1st pharyngeal arch; 2, 2nd pharyngeal arch; 3, 4, 6, 3rd, 4th and 6th pharyngeal arch artery; Ao; aorta; dAo, dorsal aorta; DA, ductus arteriosus; FB, forebrain; He, heart; HB, hindbrain; LA, left atrium; LCCA, left common carotid artery; LSA, left subclavian artery; LPA, left pulmonary artery; LV, left ventricle; Mx, maxilla; Mb, mandible; MB, midbrain; NC, nasal cavity; OV, otic vesicle; PT, pulmonary trunk; RA, right atrium; RCCA, right common carotid artery; RSA, right subclavian artery; RV, right ventricle; SC, spinal cord; To, tongue; VS, ventricular septum. Scale bar equals 1340 µm in (A, B), 2.8 mm in (C, D), 590 µm in (I, J), 1300 µm in (K, L) and 180 µm in (M, N). 


Figure S2 :

 S2 Figure S2: Expression of T-box genes in Moz Δ/Δ embryos. E10.5 Moz +/+ (A, C, E) and Moz Δ/Δ (B, D, F) embryos. Whole-mount in situ hybridization for expression of Tbx3 (A-D), Tbx4 (E, F), and sense control in control embryos (G, H). Note the reduction in Tbx3 mRNA signal (dark purple; A vs. B, C vs. D). In contrast, Tbx4 mRNA signal is not reduced in the Moz Δ/Δ embryos (arrows in E, F). 1b, mandibular aspect of the 1st pharyngeal arch; 2, 3, 4, 2nd, 3rd and 4th pharyngeal arch; 4', region where the 4th pharyngeal arch should be present in the Moz Δ/Δ ; FL, forelimb bud; He, Heart; HL, hindlimb bud; OV, otic vesicle. Scale bar equals 1700 µm in (A, B), 470 µm in (C, D, G), 1860 µm in (E, F), 1480 µm (H). 


Figure S3 :

 S3 Figure S3: Rescue of the Moz ∆/∆ mutant cardiac septal defect by a Tbx1 transgeneserial images of Moz ∆/∆ mutant and Tbx1 transgenic Moz ∆/∆ mutant hearts. Images of haematoxylin and eosin stained paraffin serial sections of three E18.5 Moz ∆/∆ and three
Anne K Voss, Hannah K Vanyai, Caitlin Collin, Mathew P Dixon, Tamara S Mclennan, Bilal N Sheikh, Peter Scambler, Tim Thomas
Supplemental Information MOZ Regulates the Tbx1 Locus, and Moz Mutation Partially Phenocopies DiGeorge Syndrome