Developmental Biology 403 (2015) 22–29

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Mesodermal expression of Moz is necessary for cardiac septum development Hannah K. Vanyai a,b, Tim Thomas a,b,n,1, Anne K. Voss a,b,n,1 a b

Walter and Eliza Hall Institute of Medical Research, Melbourne 3052, Victoria, Australia Department of Medical Biology, The University of Melbourne, Melbourne 3052, Victoria, Australia

art ic l e i nf o

a b s t r a c t

Article history: Received 2 September 2014 Received in revised form 9 April 2015 Accepted 10 April 2015 Available online 23 April 2015

Ventricular septal defects (VSDs) are the most commonly occurring congenital heart defect. They are regularly associated with complex syndromes, including DiGeorge syndrome and Holt–Oram syndrome, which are characterised by haploinsufficiency for the T-box transcription factors TBX1 and TBX5, respectively. The histone acetyltransferase monocytic leukaemia zinc finger protein, MOZ (MYST3/ KAT6A), is required for the expression of the Tbx1 and Tbx5 genes. Homozygous loss of MOZ results in DiGeorge syndrome-like defects including VSD. The Moz gene is expressed in the ectodermal, mesodermal and endodermal aspects of the developing pharyngeal apparatus and heart; however it is unclear in which of these tissues MOZ is required for heart development. The role of MOZ in the activation of Tbx1 would suggest a requirement for MOZ in the mesoderm, because deletion of Tbx1 in the mesoderm causes VSDs. Here, we investigated the tissue-specific requirements for MOZ in the mesoderm. We demonstrate that Mesp1-cre-mediated deletion of Moz results in high penetrance of VSDs and overriding aorta and a significant decrease in MOZ-dependant Tbx1 and Tbx5 expression. Together, our data suggest that the molecular pathogenesis of VSDs in Moz germline mutant mice is due to loss of MOZ-dependant activation of mesodermal Tbx1 and Tbx5 expression. & 2015 Elsevier Inc. All rights reserved.

Keywords: MOZ KAT6A MYST3 Tbx1 Tbx5 Cardiac septal defect

Introduction Congenital heart disease (CHD) accounts for approximately one-third of all congenital disorders (Dolk et al., 2011). Ventricular septal defects (VSDs) are the most common CHD at
2.62 per 1000 births (van der Linde et al., 2011). VSDs can occur in isolation or as part of complex developmental syndromes, such as DiGeorge syndrome (velo-cardio-facial/22q11.2 deletion syndrome) and Holt–Oram syndrome. DiGeorge syndrome is caused by a heterozygous microdeletion at chromosomal region 22q11.2 (de la Chapelle et al., 1981; Driscoll et al., 1992a, 1992b; Scambler et al., 1991). The T-box transcription factor encoding gene, TBX1, is central to the deletion interval and in rare cases DiGeorge syndrome is caused by heterozygous mutations in TBX1 alone (Yagi et al., 2003; Paylor et al., 2006; Torres-Juan et al., 2007; Zweier et al., 2007). Mice heterozygous for Tbx1 display a number of characteristic DiGeorge syndrome (DGS)-like anomalies (Jerome and Papaioannou, 2001; Lindsay et al., 2001; Merscher n Correspondence to: Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Melbourne 3052, Victoria, Australia. E-mail addresses: [email protected] (T. Thomas), [email protected] (A.K. Voss). 1 These authors contributed equally and share senior authorship.

http://dx.doi.org/10.1016/j.ydbio.2015.04.011 0012-1606/& 2015 Elsevier Inc. All rights reserved.

et al., 2001). Major clinical features of the syndrome include conotruncal cardiac anomaly and craniofacial defects (Botto et al., 2003; Kobrynski and Sullivan, 2007). Approximately 80% of DGS patients are born with CHDs, including VSDs and malformations of the cardiac outflow tract (OFT) (McDonald-McGinn and Sullivan, 2011). Holt–Oram syndrome is caused by haploinsufficiency of another T-box factor gene, TBX5 (Basson et al., 1997; Li et al., 1997). Patients display upper limb abnormalities and more than 85% present with CHDs, including atrial and ventricular septal defects and OFT malformations. Tbx5 heterozygous mice model Holt– Oram syndrome (Bruneau et al., 2001; Koshiba-Takeuchi et al., 2006; Mori et al., 2006). We have previously reported that the chromatin modifier monocytic leukaemia zinc finger protein (MOZ/MYST3/KAT6A) is essential for normal activation of the Tbx1 and Tbx5 genes in vivo (Voss et al., 2012b). MOZ is a member of the MYST family of histone acetyltransferases (Thomas and Voss, 2007; Voss and Thomas, 2009) and was first identified in chromosomal translocations causing acute myeloid leukaemia (Borrow et al., 1996). MOZ is required for histone 3 lysine 9 acetylation (H3K9ac) at Hox genes and lack of MOZ results in inadequate Hox gene expression and an extensive anterior homeotic transformation (Voss et al., 2009). Importantly, Moz homozygous mutant mice phenocopy DiGeorge syndrome. They display craniofacial defects and hyperplastic or

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absent thymus with 100% penetrance, as well as CHDs, including aortic arch defects (74% penetrance) and VSDs (95% penetrance; Voss et al., 2012b). In this context, MOZ is required for H3K9ac and expression of the Tbx1 and Tbx5 genes and transgenic overexpression of Tbx1 rescues the VSDs in homozygous Moz mutant mice (Voss et al., 2012b). Here, we focus on the mesoderm-specific role of MOZ in heart and pharyngeal apparatus development. Development of the structures affected in DiGeorge syndrome is known to be exquisitely sensitive to time- and tissue-specific expression of Tbx1 (Aggarwal et al., 2010; Liao et al., 2004; Zhang and Baldini, 2008; Zhang et al., 2005, 2006). Tbx1 is expressed in the ectoderm, endoderm and mesoderm, but not in neural crest (Chapman et al., 1996). The VSDs caused by Tbx1 mutation have been traced to the mesodermal expression domain (Zhang et al., 2006). Using mesoderm posterior 1 (Mesp1-cre) promoter driven cre-recombinase to delete the Mozlox conditional mutant allele, we determined the tissue-specific requirements for MOZ within the mesoderm, where both Tbx1 and Tbx5 are expressed. We found that cardiac septum development requires mesodermal MOZ and that expression of both Tbx1 and Tbx5 is decreased after Mesp1-cre deletion of Moz.

Results and discussion The Mozlox allele (Voss et al., 2009) incorporates loxP sites flanking exons 3–7, deletion of which results in complete loss of function. The Mesp1-cre knock-in allele (Mesp1-creKI) is active in the anterior mesoderm from E6.5 onwards (Saga et al., 1999; Fig. 1A). The deletion efficiency of Mesp1-creKI was quantified by assessing the level of residual Mozlox allele in the heart. The functional Mozlox allele was reduced to approximately 20% of the level of undeleted littermate controls (Fig. 1B). Though these nondeleted cells likely included some Mesp1-negative cells present in

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the heart (Kitajima et al., 2006), cells which escaped recombination of Mozlox alleles may also have contributed to the residual 20% Mozlox signal in Mozlox/lox;Mesp1-cre þ /KI hearts. Mesoderm-deleted Mozlox/lox;Mesp1-cre þ /KI animals crossed with Mozlox/lox mice without the Mesp1-creKI allele (Mozlox/lox;Mesp1 þ / þ ) produced offspring of both parental genotypes. However, the Moz mesoderm-deleted animals were significantly and progressively underrepresented compared to the expected 1:1 ratio (Fig. 1C), showing that mesoderm deletion of Moz resulted in lethality over an extended period of time. Nevertheless, approximately 40% of the Mozlox/lox;Mesp1-cre þ /KI animals survived and were outwardly normal and fertile. Approximately two-thirds of the losses of Mozlox/lox;Mesp1-cre þ /KI animals occurred before postnatal day (PN)7, whereas the remaining deaths (or terminal disease¼ethical endpoint) occurred between weaning and sexual maturity at 6 weeks of age (Fig. 1D). The latter were associated with respiratory distress, weight loss and a hunched posture. Together our data suggest that mesoderm-specific deletion of Moz causes lethality during three distinct periods, (a) during foetal development before E18.5 (
26%), (b) between E18.5 and PN7 (
14%) and (c) between weaning and sexual maturity (
19%), resulting in a total loss of
60% of all anterior mesoderm Moz deleted animals. Moz germline mutation causes foetal or perinatal death. Moz–/– DGS-like anomalies include cleft palate and severe cardiovascular abnormalities. We therefore examined litters of Mozlox/lox;Mesp1-cre þ /þ females crossed with Mozlox/lox;Mesp1-cre þ /KI males for DGS-like defects at E18.5. The majority of Moz mesoderm-deleted pups (23/25) had aortic arches indistinguishable from littermate controls (34/34; Fig. 2A–B′). However, two Mozlox/lox;Mesp1-cre þ /KI pups (8%) exhibited aortic arch defects, namely one case of right-sided descending aorta with aberrant origin of the right subclavian artery from the pulmonary trunk (Fig. 2C,C′) and one instance of right-sided aortic arch. These

Fig. 1. Mesp1-cre deletion of Moz causes lethality between E18.5 and sexual maturity. (A) Schematic representation of the Mesp1 expression pattern (purple) and lineage of Mesp1-cre-expressing cells (blue) in the anterior mesoderm, after Saga et al. (2000) and Saga et al. (1999). Extraembryonic mesoderm domain stippled (E7.5). (B) Q-PCR quantification of Mesp1-cre-mediated deletion of the Mozlox allele in mesoderm-rich heart tissue at E9.5, E10.5 and E12.5, normalised to the reference gene Hsp90ab1 and relative to Mozlox/lox;Mesp1-cre þ / þ tissues. Data are displayed as mean 7 standard error of the mean (s.e.m.) and were analysed by unpaired two-sided t-test. (C) Genotype distribution among offspring from Mozlox/lox;Mesp1-cre þ /KI x Mozlox/lox;Mesp1-cre þ / þ crosses. (D) Survival curve of Moz Mesp1-cre-deleted mice and controls. *data between E18.5 and PN7 inferred based on percentage of Mozlox/lox;Mesp1-cre þ /KI animals at both of those ages.

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Fig. 2. Aortic arch defects in E18.5 Moz mesoderm-deleted mutant pups. (A–C) Macroscopic view of the great vessels at E18.5 and (A′–C′) traced. Note the mispatterned aortic arch in a subset of Mozlox/lox;Mesp1-cre þ /KI pups (C,C′) compared with unaffected Mozlox/lox;Mesp1-cre þ /KI pups (B,B′) and Mozlox/lox;Mesp1-cre þ / þ littermate controls (A, A′). (D–H) Thymus (D–F) and ventral view of the palate after removal of the lower jaw (G,H) in genotypes indicated above. Representative images are shown. AA, ascending aorta; DA*, abnormal ductus arteriosus; DeA, descending aorta; HP, SP hard and soft palate; LCCA, RCCA left and right common carotid artery; LL, RL, left and right lobe of the thymus; LSCA, RSCA left and right subclavian artery; aRSCA*, aberrant origin of the right subclavian artery; PT, pulmonary trunk; rDeA*, descending aorta abnormally descending on the right. Scale bar ¼1150 mm (A–C), 1100 mm (D–F) and 1660 mm (G,H).

aortic arch defects occur primarily due to abnormal development of either the left (right-sided aortic arch) or right (aberrant origin of the right subclavian artery) fourth pharyngeal arch artery (Lindsay and Baldini, 2001), a hallmark of Tbx1-dependant DGS-like anomalies of the aortic arch (Jerome and Papaioannou, 2001; Lindsay et al., 2001; Merscher et al., 2001; Randall et al., 2009). Whilst aortic arch defects would contribute to loss of Moz mesoderm-deleted animals soon after birth, they do not account for loss of
60% of these animals by 10 weeks of age. Examination of the thymus and palate revealed no DGS-like abnormalities of these structures in either genotype (Fig. 2D–H). Given the high incidence of VSDs in Moz germline mutant animals (95%), we examined haematoxylin and eosin (H&E)-stained serial sections of E18.5 hearts. 21 of 22 Mozlox/lox;Mesp1-cre þ /KI hearts displayed VSDs, compared with none in the control hearts

(0/4; Moz þ / þ ;Mesp1-cre þ /KI; Fig. 3A,E), suggesting that the VSDs observed in Moz germline deleted animals are caused by loss of mesodermal MOZ. Interestingly, the VSDs could be categorised as mild (a very small passage between the left and right ventricles, 2/22; Fig. 3B,F), moderate (a slightly larger disruption of the septum, 7/22; Fig. 3C,G) or severe (comprising an overriding aorta positioned above a major VSD, 12/22; Fig. 3D,H). One Moz mesoderm-deleted pup had no discernable VSD (summarised in Fig. 3I). Furthermore, three Moz mesoderm-deleted mutants displayed atrioventricular septal defects (AVSD; Fig. 3J–L). The VSD data suggest an explanation for the time course of death observed for the Mozlox/lox;Mesp1-cre þ /KI animals. A severe VSD with overriding aorta likely causes lethality, as the heart is unable to separate blood flow and thus oxygen supply to the

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Fig. 3. Cardiac septal defects in E18.5 Moz mesoderm-deleted mutant pups. H&E stained sections of hearts of Moz þ / þ ;Mesp1-cre þ /KI (A–D,J) and Mozlox/lox;Mesp1-cre þ /KI (E–H,K) E18.5 pups, overview (A,E) and higher magnification images (B–D,F–H,J,K). VSDs occurred as mild (arrows in F), moderate (arrowhead in G) or severe with overriding aorta (asterisk in E,H) in Mozlox/lox;Mesp1-cre þ /KI, but not in control, mice. (I) Summary of the incidence and severity of VSDs. (J,K) An AVSD (hash in K) was observed in some Mozlox/lox;Mesp1-cre þ /KI animals and never in the controls. (L) Summary of the incidence of AVSDs. Ao, aorta; DMP, dorsal mesenchymal protrusion; LA, RA, left and right atrium; LV, RV, left and right ventricle; VS, ventricular septum. Scale bar ¼580 mm (A,E) and 300 mm (B–D,F–H,J,K).

tissues is likely to become limiting. Indeed, the numbers roughly correspond: 55% of E18.5 pups had severe VSDs and 59% of Moz mesoderm deleted animals were lost, suggesting that most animals with moderate and mild VSDs survived. Chromatin-mediated mechanisms have been shown to be of great importance in the development of the cardiovascular systems (reviewed in Vallaster et al. (2012)). Both TBX1 and TBX5 aid in recruiting chromatin modifiers to their target genes to enable gene expression (Miller et al., 2008; Stoller et al., 2010). Interestingly, TBX5 also interacts directly with another MYST family histone acetyltransferase, TIP60 (Barron et al., 2005). Previously, we have identified Tbx1 and Tbx5 as direct targets of the histone acetyltransferase MOZ during embryonic development (Voss et al., 2012b). Given that the syndromes caused by haploinsufficiency of both these transcription factors involve VSDs, we hypothesised that the VSDs observed in Moz mesoderm-deleted animals may be due to compromise of MOZ-dependant, mesoderm-specific expression of Tbx1 and Tbx5.

Previous analysis of the relationship between Moz and Tbx1 and Tbx5 demonstrated that, in the absence of MOZ, H3K9ac at these genes and their mRNA levels were dramatically affected at E9.5 and E10.5 (Voss et al., 2012b). We therefore examined the expression of Tbx1 and Tbx5 by whole-mount in situ hybridisation at E9.5 and E10.5 and RT-qPCR analysis at E10.5 in Moz;Mesp1-credeleted embryos and controls. RNA/RNA in situ hybridisation revealed a generalised decrease in Tbx1 mRNA signal in Mozlox/lox;Mesp1-cre þ /KI embryos compared to control embryos at E9.5 (Fig. 4A,B) and E10.5 (Fig. 4E,F,E′,F ′). Germline deletion of Moz results in depletion of Tbx1 mRNA to an average of 50–60% wild type levels by RT-qPCR (Voss et al., 2012b). Here we determined that mesoderm-specific deletion of Moz reduced Tbx1 to 55% of control levels by RT-qPCR (Fig. 4I), suggesting that the requirement for MOZ in regulating Tbx1 expression is primarily or exclusively dependant on mesodermal expression of Moz. Tbx5 expression in Mozlox/lox;Mesp1-cre þ /KI E9.5 embryos was only slightly compromised compared to control embryos in the

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Fig. 4. Decreased gene expression in Moz mesoderm-deleted embryos and direct binding of MOZ to the Tbx1 and Tbx5 locus. Whole-mount in situ hybridisation of E9.5 (A–D) or E10.5 (E–H and sectioned in E′–H′) embryos for Tbx1 (A,B,E,F,E′,F′) and Tbx5 mRNA (C,D,G,H,G′,H′). Tbx1 mRNA signal is broadly decreased in Moz mesoderm-deleted embryos at both time points (B,F,F′ compared with A,E,E′ arrow in F′ indicates decreased Tbx1 signal in paraxial mesoderm compared to E′). Note that this technique was not sensitive enough to detect Tbx1 expression in the secondary heart field. Tbx5 mRNA appears reduced in the DMP (arrow in D,C) but unaffected in the forelimb bud (arrowheads in C,D) at E9.5. Colour reaction was terminated prior to full development of the signal to reveal subtle changes in the DMP. At E10.5, Tbx5 signal is substantially depleted in the left ventricle (arrows in G,H), right atrium, second heart field and nascent pulmonary mesoderm (RA and arrow, respectively, in G′,H′) and only moderately affected in the forelimb (arrowhead in G,H). (I–J) RT-qPCR analysis of mRNA levels of Tbx1 and Tbx5 (I) and other genes important for heart and cardiac septum development (J) in E10.5 embryos. Legend in J also applies to I. (K,L) Detection of MOZ-V5 at Tbx1 and Tbx5 loci in vivo. Data are presented as enrichment over signal from V5-negative (Moz þ / þ ) tissue after normalisation to an internal IP intergenic control locus (K) or input (L). ‘_1’ and ‘_2’ indicates 1 and 0.49 kb upstream of the transcription start site (TSS) and 0.77 and 0.28 kb upstream the TSS, for Tbx1 and Tbx5, respectively. Note that the low enrichment at Tbx1 likely reflects the small, MOZ-V5-bound Tbx1 expression domain within the pharyngeal arches compared to the MOZ-V5-bound Tbx5 expression domain throughout the heart. Scale bar ¼380 mm (A–D,G,H), 500 mm (E,F) and 200 mm (E′–H′). Data are displayed as mean7 s.e.m. and were analysed by unpaired two-sided t-test (RT-qPCR) or 2-way ANOVA with genotype and genomic site within gene locus as the two independent factors (ChIP-qPCR); *p o0.05; **p o 0.01; ***p o0.001; n.s., not significant.

heart and surrounding region and not at all in the forelimb domain (Fig. 4C,D). However, one day later, at E10.5, there was a dramatic reduction in signal in the left ventricle and right atrium of Mozlox/lox;Mesp1-cre þ /KI embryos (Fig. 4G,H,G',H'), whereas Tbx5 mRNA signal in the forelimb was only moderately reduced

(Fig. 4H,G). RTqPCR showed Tbx5 mRNA levels at 75% control levels in Mozlox/lox;Mesp1-cre þ /KI embryos (Fig. 4I). Given that MESP1 is thought to directly regulate Tbx5 (Christoforou et al., 2013; Lindsley et al., 2008), we included the Mozlox/ þ ; Mesp1-cre þ /KI genotype to control for Mesp1 heterozygosity in the

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Mesp1-cre-recombinase knock-in allele. These mesodermal Moz; Mesp1 double heterozygous animals displayed no compromise in Tbx5 expression (Fig. 4I), suggesting that the decrease in Tbx5 mRNA levels in Mozlox/lox;Mesp1-cre þ /KI embryos is primarily due to homozygous loss of mesodermal Moz expression. Our previous chromatin immunoprecipitation (ChIP) results in germline deleted Moz–/– embryos (Voss et al., 2012b) suggest that the reduction in Tbx1 and Tbx5 mRNA levels in Moz mesoderm-deleted embryos would be accompanied loss of MOZ-dependant H3K9ac at the Tbx1 and Tbx5 loci. In addition to Tbx1 and Tbx5, we examined the expression of the Pitx2, Gata4 and Nkx2.5 genes encoding transcription factors important for heart development, which, when mutated, result in cardiac septal defects in mice (Gittenberger-de Groot et al., 2014; Lin et al., 2012). Mutations in PITX2, GATA4 and NKX2.5 have been implicated in the pathogenesis of cardiac septal defects in humans (Gittenberger-de Groot et al., 2014; Lin et al., 2012). In addition, we examined Frzb mRNA, which encodes a secreted antagonist of WNT signalling that is required for atrioventricular cardiac cushion development in the chicken (Person et al., 2005). Furthermore, mutation of FRZB has been associated with one case of atrioventricular septal defect in humans (Ackerman et al., 2012). Interestingly, murine Frzb expression decreases in the absence of Tbx1 in vivo (Liao et al., 2008) and is upregulated when Tbx5 is overexpressed in mouse cardio fibroblasts (Zhou et al., 2012). Pitx2 is directly regulated by TBX1 in the secondary heart field (Nowotschin et al., 2006). In the absence of mesodermal Moz expression, Pitx2, Gata4 and Frzb mRNA levels were significantly reduced compared to controls (Fig. 4J). In contrast, expression of Nkx2.5 was not changed, demonstrating that the effect of loss of mesodermal MOZ has specific, rather than global, effects on genes important for heart and cardiac septal development. We previously reported the presence of the MOZ complex at the Tbx1 and Tbx5 loci, by performing ChIP using an antibody against ING5, a member of the MOZ complex, in the absence of a ChIP-grade antibody against MOZ. We have generated a knock-in mouse strain with a Moz allele that encodes full-length MOZ with a triple FLAG–V5–biotinylation signal sequence tag (FLAG–V5–BIO) fused to the carboxy-terminus (MOZ-V5). Unlike the Moz–/– mice, MozV5/V5 animals are viable and healthy, indicating that the MOZV5 fusion protein is functional. Anti-V5 ChIP was performed on Tbx1-enriched (microdissected first and second pharyngeal arches; Fig. 4K) and Tbx5-enriched tissue (sets of 3 pooled microdissected hearts; Fig. 4L) from E10.5 MozV5/V5 and Moz þ / þ embryos. MOZ-V5 enrichment at both loci over background in Moz þ / þ tissues was observed, providing evidence that the MOZ protein directly occupies the Tbx1 and Tbx5 locus. Our previous data have shown that, despite an
50% decrease in Tbx1 expression levels, germline-deleted Moz mutants display much higher penetrance of DGS-like defects than Tbx1 heterozygous animals, including 74% aortic arch defects compared to 11–38% in animals with various Tbx1 heterozygous alleles (summarised in Voss et al. (2012b)). This suggests that other MOZdependant genes in addition to Tbx1 contribute to the DGS-like anomalies in Moz germline-deleted mutants. For example, Tbx5 down-regulation is likely to contribute to the high incidence of VSDs. Moreover, transgenic overexpression of Tbx1 rescued the heart septal defects in germline Moz mutants, but not other DGSlike defects (Voss et al., 2012b). Interestingly, the low penetrance of aortic arch defects (8%) in Mozlox/lox;Mesp1-cre þ /KI models Tbx1 heterozygosity (11–38% penetrant) much more closely than Moz germline deletion (74% penetrant) does, suggesting that the TBX1independent mechanisms that contribute to the Moz germline mutant aortic arch phenotype may occur in tissues other than the mesoderm.

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Here we show that normal expression of the Tbx1 and Tbx5 genes is dependant on the histone acetyltransferase, MOZ, in the mesoderm. Compromise of this activation, by mesoderm-specific deletion of Moz, results in a high incidence of DGS-like VSDs.

Materials and methods All animal experiments were approved by the Walter and Eliza Hall Institute Animal Ethics Committee and conducted in accordance with the Australian code of practise for the care and use of animals for scientific purposes. Mozlox (Voss et al., 2009), Mesp1-creKI (Saga et al., 1999) and MozV5 mice were maintained on the C57BL/6 background. The MozV5 allele was generated by inserting a FLAG–V5–BIO triple tag (Schwickert et al., 2014; Soler et al., 2010) into the endogenous Moz locus at the carboxy-terminus (MOZ-V5) using homologous recombination of a Moz-containing BAC in Escherichia coli, followed by targeting of the Moz locus in embryonic stem cells and germline chimera production. Noon of the day a vaginal plug was observed was considered embryonic day 0.5 (E0.5). Embryos and pups were collected at E9.5, E10.5, E12.5 and E18.5 and animals suffering respiratory distress or other illnesses were euthanised to minimise suffering (ethical endpoint in accordance with code of practise). Genomic DNA was extracted from E9.5, E10.5 and E12.5 isolated hearts to quantify the efficiency of conversion of the Mozlox allele to the Moz– deleted allele. qPCR was performed using the following primers targeted to Mozlox: 5′-ACCATGTGAGCACATGCATGC-3′; 5′ATAGGAACTTCATCAGTCAGGTA-3′. Hsp90ab1 was used as a reference gene to control for variation in genomic DNA input using the primers: 5′-AATTGACATCATCCCCAACC-3′, 5′-TCGTGCCAGACTTAGCAATG-3′. For mRNA analysis, E10.5 embryos were collected and their forelimb buds removed, to quantify more accurately the Tbx5 expression domains relevant to heart development. RT-qPCR primers against Tbx1, Tbx5, Pitx2, Gata4 and Nkx2-5 as well as the housekeeping control genes HSP90ab1 and Pgk1 and ChIP-qPCR primers Tbx1_1, Tbx1_2, Tbx5_1 and Tbx5_2 were previously described (Voss et al., 2012b). The following RT-qPCR primers were also used: Frzb, 5′-GGCTATGAAGACGAGGAACG-3′ and 5′-CCAAGGTGTCGGAGTTTCAT-3′ Psmb2, 5′GAGGGCAGTGGAGCTTCTTA-3′ and 5′-AGGTGGGCAGATTCAAGATG-3′ and MOZ-V5-unbound intergenic control for ChIP-qPCR, 5′-TGACAGAGGCTTGGAGTCCT-3′ and 5′-TCCTCTTCTGGTTCCCCTTT-3′. SYBRgreen technology (SensiMix™, Bioline, UK) on the LightCycler 480 (Roche, Switzerland) was used for qPCR, following cDNA preparation with Superscript IIIs (Life Technologies, USA) for RT-qPCR according to manufacturer's instructions. ChIP was performed as previously described (Voss et al., 2012a) with the following modifications: microdissected tissue was cross-linked for 10 min in 1% paraformaldehyde. Chromatin was sheared using the S220 Covaris sonicator (105 peak incident power; duty factor 7; 200 cycles/burst; 40 s; USA) and incubated in 1 ml ChIP dilution buffer (Voss et al., 2012a) with 20 ml of anti-V5-conjugated agarose beads (Sigma, A7345) precleared with 0.2% BSA in ChIP dilution buffer. The immunoprecipitated DNA was purified using ChIP DNA Clean & Concentrator kit (Zymo Research, D5205) according to manufacturer's instructions, before quantification by qPCR. Thymus and aortic arch defects were examined at E18.5 via microdissection and scored prior to genotyping. Palates were examined post-fixation in Bouin's fixative. Hearts were collected and fixed in 4% paraformaldehyde overnight before serial sectioning and staining with H&E. Whole-mount in situ hybridisation was carried out as previously described (Thomas et al., 2007) with antisense probes to Tbx1 and Tbx5 kindly provided by V.E. Papaiaonnou (Chapman et al., 1996). Statistical analyses were conducted using Stata/IC 10.0 (Stata Corporation, USA). Frequencies of expected and observed genotypes

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at E18.5, weaning and 10 weeks of age were examined by linear regression with Moz gene deletion status and age as two independent variables. The time course of deaths observed in Moz deleted animals after PN7 was examined in a Kaplan–Meier survivor function and incidence of deaths between genotypes were assessed by Pearson's chi-square test.

Author contributions HKV prepared the manuscript, performed the experiments and contributed to the development of the project. TT and AKV jointly devised and supervised the project and edited the manuscript.

Acknowledgements We thank Y. Saga for the Mesp1-cre mice, V.E. Papaiaonnou for cDNA probes, M. Busslinger and S.L. Nutt for the FLAG–V5–BIO triple tag and are grateful to F. Dabrowski, R. Cobb and M. Bergamasco for excellent technical support and to N. Downer for comments on the manuscript. This work was funded by Australian NHMRC Project grants 1010851, 1008699 and 1051078, NHMRC Research Fellowships (TT 1003435 and AKV 575512), Australian Postgraduate Award (HKV), Independent Research Institutes Infrastructure Support (IRIIS) Scheme from the Australian Government's National Health and Medical Research Council (NHMRC) and a Victorian State Government OIS (Operational Infrastructure Support) Grant.

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