The Mediator Complex Subunit Med10 Regulates Heart Valve Formation in Zebraﬁsh by Controlling Tbx2b-Mediated Has2 Expression and Cardiac Jelly Formation

Biochemical and Biophysical Research Communications 477 (2016) 581e588

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Biochemical and Biophysical Research Communications

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The mediator complex subunit Med10 regulates heart valve formation in zebraﬁsh by controlling Tbx2b-mediated Has2 expression and cardiac jelly formation

* Steffen Just a, , 1,Soﬁa Hirth a, 1, Ina M. Berger a, 1, Mark C. Fishman c, * Wolfgang Rottbauer b, a Molecular Cardiology, Department of Internal Medicine II, University of Ulm, Ulm, Germany b Department of Internal Medicine II, University of Ulm, Ulm, Germany c Novartis Institutes for BioMedical Research, Cambridge, MA, USA article info abstract

Article history: In search for novel key regulators of cardiac valve formation, we isolated the zebrafish cardiac valve Received 7 June 2016 mutant ping pong (png).Wefind that an insertional promoter mutation within the zebrafish mediator Accepted 17 June 2016 complex subunit 10 (med10) gene is leading to impaired heart valve formation. Expression of the T-box Available online 22 June 2016 transcription factor 2b (Tbx2b), known to be essential in cardiac valve development, is severely reduced in png mutant hearts. We demonstrate here that transient reconstitution of Tbx2b expression rescues AV Keywords: canal development in png mutant zebrafish. By contrast, overexpression of Forkhead box N4 (Foxn4), a Med10 known upstream regulator of Tbx2b, is not capable to reconstitute tbx2b expression and heart valve Tbx2b fi Foxn4 formation in Med10-de cient png mutant hearts. Interestingly, hyaluronan synthase 2(has2), a known Has2 downstream target of Tbx2 and producer of hyaluronan (HA) - a major ECM component of the cardiac Valve formation jelly and critical for proper heart valve development - is completely absent in ping pong mutant hearts. Zebrafish We propose here a rather unique role of Med10 in orchestrating cardiac valve formation by mediating Foxn4 dependent tbx2b transcription, expression of Has2 and subsequently proper development of the cardiac jelly. © 2016 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction transcriptional activators and repressors. To convey the regulatory information from these transcriptional activators or repressors to The cardiac atrioventricular canal (AVC) ensures highly the basal transcription machinery, the multiprotein Mediator specialized functions, including cardiac chamber demarcation, AV complex (Med) serves as a connecting bridge between initiating conduction and the formation of endocardial cushions. Endocardial and accomplishing elements. In this context, the Mediator complex cushions are cells of the AVC that undergo an epithelial-to- physically links transcription factors bound to DNA to the RNA mesenchymal transition (EMT) and are remodelled into func- polymerase II and the basal transcription machinery. In vertebrates, tional cardiac valves during late cardiogenesis [1]. The molecular the Med complex consists of more than 30 protein subunits, sub- underpinnings that orchestrate vertebrate heart valve formation divided into a head, middle, tail and regulatory domain [2]. are only poorly deciphered yet. Whereas several Med subunits such as Trap80 or Med6 are indis- Temporo-spatial control of transcription is of central impor- pensable for general gene transcription, other subunits including tance during organogenesis and is mainly orchestrated by Med10, Med12 or Med13 are dispensable for general transcription but are involved in the regulation of specific gene programs, also in the heart [3e5]. Nevertheless, the role of the Mediator complex during cardiac development and particularly heart valve formation * Corresponding authors. Department of Internal Medicine II, Albert-Einstein- is only poorly understood. Allee 23, 89081 Ulm, Germany. In search for key-regulators of cardiac valve formation, we iso- E-mail addresses: [email protected] (S. Just), wolfgang.rottbauer@ fi m683 uniklinik-ulm.de (W. Rottbauer). lated the zebra sh heart valve mutant ping pong (png ) [6].We 1 Equal contribution. demonstrate that defective valve formation in png is caused by a http://dx.doi.org/10.1016/j.bbrc.2016.06.088 0006-291X/© 2016 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 582 S. Just et al. / Biochemical and Biophysical Research Communications 477 (2016) 581e588 promoter mutation within the mediator complex subunit 10 a large-scale ENU-mutagenesis screen the recessive, embryonic (med10) gene leading to reduction of med10 transcription. We find lethal zebrafish mutant ping pong (pngm683)(Fig. 1AeD) [6]. During that tbx2b expression in the AVC of png mutant hearts is absent and the first 48 h post fertilization (hpf), the png heart jogs leftwards, that reintroduction of Tbx2b rescues endocardial cushion devel- loops, myocardial and endocardial cell layers develop properly, and opment. Additionally, ectopic expression of foxn4, a known tran- atrial and ventricular cardiomyocytes express chamber-specific scriptional regulator of Tbx2b, is not capable to reconstitute tbx2b myosin heavy chains (Fig. 1EeH; Movies 1 and 2, Fig. S1AeH). expression in the AVC of png mutants, implying that Med10 might Usually, at 72 hpf, cardiac chambers in wild-types are demarcated act as a mediator of Foxn4 signals to guarantee proper tbx2b by the AV ring and the endocardial cushions, specialized cells that expression in the AV myocardium. As a consequence, hyaluronan give rise to the AV valves (Fig. 1F). By contrast, in png mutants heart synthase 2, a known downstream target of Tbx2, is absent in png chambers are not separated by an AV ring and no endocardial hearts leading to loss of hyaluronic acid (HA) production in the cushion cells are visible (Fig. 1H; Fig. S1I, J; Movies 3 and 4). cardiac jelly and thereby the defective endocardial cushion Supplementary data related to this article can be found online at development. http://dx.doi.org/10.1016/j.bbrc.2016.06.088. To define the cardiac defects in png mutants, we first examined 2. Methods the expression of the myocardial chamber marker Atrial Natriuretic Factor (Anf). Usually, anf is expressed in both cardiac chambers but 2.1. Zebrafish Strains, Histology, Immunostaining/-Blotting and in is absent from AVC in wild-types. We find anf distributed in both situ hybridization heart chambers and the AVC of png mutants (Fig. 1I, J), suggesting that AV myocardium is not properly specified. Upon Bmp4 stimuli Care and breeding of zebrafish Danio rerio was as described [7]. endocardial cells usually accumulate at the AVC, increasingly ex- The study was performed after appropriate institutional approvals, press Notch1b, and then migrate into the AV jelly to undergo EMT which conform to EU Directive 2010/63/EU. Pictures and movies [11]. Thus, to evaluate if altered Bmp4 expression in png mutant were recorded 24, 48 and 72 hpf. Embryos were fixed in 4% PFA and hearts affects endocardial notch1b expression, we assayed bmp4 embedded in JB-4 (Polysciences, Inc.). 5 mm sections were cut, dried and notch1b distribution and find them misexpressed in myocardial and stained with H&E. Immunostaining of Dent’s fixed embryos and endocardial cells of the atrium and the ventricle, respectively was performed using distinct myosin heavy chains antibodies (Fig. S2AeD). Therefore, to analyze if loss of regular Notch1b (MF20 and S46). In situ hybridization, hyaluronic acid (HA) staining expression leads to impaired initiation of EMT in the png AVC, we and Western Blotting were performed as described [7e9]. assessed the expression of secreted phosphoprotein 1 (spp1), a well- established marker for activated AV endocardial cells that migrate 2.2. Injection procedures, LiCl treatment and quantitative real-time into the extracellular matrix and then undergo EMT [12,13].As PCR shown in Fig. 1K, L, spp1 RNA is completely absent from the AVC of png mutant hearts at 72 hpf, implicating impaired initiation of EMT Morpholinos were directed against the translational start site of endocardial AVC cells to be responsible for the AVC defect. (MO1-med10 ¼ 50-CGAGGTTATCAAACTTCTCCGCCAT-30), the splice donor site of exon 1 (MO2-med10 ¼ 50-GATTTTAACTCA- CAGTTTCTGGTTG-30) of the zmed10 gene and against the donor site 3.2. Ping pong (pngm683) encodes the zebrafish mediator complex of exon 2 (MO-foxn4 ¼ 50-TCAAAGGAAATCACCCCCTACCTGT-30)of subunit 10 (Med10) the zfoxn4 gene. A standard control oligonucleotide (MO-control) (GENETOOLS, LLC) was injected at the same concentration as a To identify the ENU-induced png mutation, we performed a negative control. Sense-capped RNA was synthesized using the positional walk. By a genome-wide study of segregation of micro- mMESSAGE mMACHINE system (Ambion) from pCS2zmed10, satellite markers using bulked segregant analysis, we mapped the pCS2zfoxn4, pCS2ztbx2b, and pCS2ndr2 [7]. To induce Wnt png mutation between the microsatellite markers Z9059 and signaling, the established heat-shock inducible transgenic line Z11872 on chromosome 19. Genetic fine mapping and genotyping Tg(hsp70l:wnt8a-GFP) was used [10]. Embryos were treated with of 1497 png mutant embryos revealed two open reading frames LiCl (0.1 M and 0.15 M) from 36 hpf to 48 hpf. qRT-PCR was per- within the critical mutation interval encoding for the zebrafish formed according to standard protocols with the SYBR-Green Mediator complex subunit 10 (Med10; AAW56465) and UBE2QL1 method (Thermo Scientific) using an ABI 7000. cDNA was gener- (ENSDARG00000079276) (Fig. 1M). By sequencing of wild-type and ated from 2 mg RNA of 48 hpf png mutants and wild-types using png mutant cDNA, no alterations of the coding sequences of these oligo (dT) primer and SuperScript II reverse transcriptase genes were identified. To assess if altered Med10 or UBE2QL1 (Invitrogen). transcription is the molecular cause of the png mutant phenotype, we next assayed transcript levels of both genes by quantitative real- 2.3. Statistical analyses time PCR in wild-types and png mutants. We find significantly reduced zmed10 transcript levels in png mutants compared to wild- Unpaired two tailed students’ t-test was performed to assess type littermates, whereas UBE2QL1 mRNA levels are unaltered in statistical significance of biological replicates of the quantitative png mutants (Fig. 1N). Sequencing of the zmed10 promoter region data. For the qRT-PCR data t-test was performed to the delta Ct reveals an 1193 bp insertion located 110 bp upstream of the tran- values. For each replicate, pooled samples (50 embryos) were scriptional start site of the med10 gene (Fig. 1M). Interestingly, the analyzed. identified ping pong promoter mutation is identical to the one isolated in tennis match (ten) zebrafish mutants [5], which also 3. Results display defective AV canal formation. To substantiate our finding that impaired AVC development in 3.1. Zebrafish ping pong mutants display impaired AVC png mutants is due to altered Med10 function, we performed development Morpholino-mediated med10 knockdown studies as well as gene rescue experiments and find that impaired Med10 transcription In search for key-regulators of AVC development, we isolated in indeed accounts for the png mutant phenotype (Suppl. Fig. 3). S. Just et al. / Biochemical and Biophysical Research Communications 477 (2016) 581e588 583

Fig. 1. Effects of the png mutation on heart development. (AeD) Wild-type (wt) (A) and png mutant (C) embryos and close-ups of the wt (B) and png (D) hearts at 72 hpf. (E, G) MF20 and S46 immunostainings reveal that chamber speciﬁcation and differentiation is not altered in png mutants (E). (F, H) H&E staining of sections of wt (F) and png (H) hearts at 72 hpf. png mutant heart chambers are not separated by an AV ring and no endocardial cushions (EC) develop at the AVC, whereas png myocardial (my) and endocardial (en) cell layers are clearly deﬁned. (I, J) anf is misexpressed in AV myocardial cells of png hearts (I), whereas anf is absent from wt AV myocardium (J) (AVC, white arrow). (K, L) Spp1 is expressed in AVC and OFT endocardium of wt embryos at 72 hpf (K), whereas spp1 expression is absent from the AVC of png hearts (L) (Schematic descriptions: I0,J0,K0,L0). (M) Integrated genetic and physical map of the png locus on chromosome 19. The png promoter mutation (1193bp insertion) is indicated (red). (N) The png mutation leads to severely diminished med10 transcription (relative expression 0.074 ± 0.039, n ¼ 7, P < 0.001), whereas mRNA levels of UBE2QL1 are unaltered (relative expression 0.93 ± 0.18, n ¼ 3, ns).

3.3. Defective Wnt and Nodal signaling does not account for to analyze if enhanced Wnt signaling accounts for defective AV impaired AV valve formation in png valve development in png mutants, we overexpressed Wnt8, known to effectively activate canonical Wnt signaling, using the Lin et al. demonstrated that Morpholino-mediated knock-down transgenic zebrafish line Tg(hsp70l:wnt8a-GFP) that expresses of med10 leads to defective tail bud formation due to enhanced Wnt wnt8a-GFP under control of the heat-shock inducible hsp70l pro- and diminished Nodal signaling [5]. Interestingly, neither tennis moter in wild-type embryos. However, activation of Wnt signaling match nor png zebrafish mutants show defective tail bud formation, using established heat-shock protocols [10] does not interfere with implying both lines to be hypomorphic. Nevertheless, whether the development of the AVC as observed in png mutants altered Wnt and Nodal signaling also accounts for the AVC canal (Fig. 2AeB0; Fig. S4A, B). Similarly, induction of Wnt signaling by defect in tennis match or png mutants is not known yet [5]. Hence, LiCl does also not induce AVC defects in sensitized heterozygous 584 S. Just et al. / Biochemical and Biophysical Research Communications 477 (2016) 581e588 png embryos (Fig. S4CeF), implicating that enhanced Wnt signaling overexpression of nodal related protein 2 (cyclops, ndr2) mRNA does does not account for the AVC defects in png mutants. Vice versa, not suppress the png mutant AVC phenotype (Fig. 2CeE). These increasing Nodal activity in homozygous png mutants by transient findings indicate that the png mutant heart valve phenotype seems

Fig. 2. Defective Tbx2b and not Wnt or Nodal signaling accounts for defective AVC development in png.(A,A0,B,B0) Heat-shock induced overexpression of Wnt8 to activate canonical Wnt signaling using the transgenic line Tg(hsp70l:wnt8a-GFP). Overexpression of wnt8 in wt does not phenocopy the png heart phenotype (n ¼ 35, 3 independent experiments). (C, D) Ectopic expression of ndr2 in png mutants cannot rescue the heart phenotype (n ¼ 29, 3 independent experiments). (E) Efficient expression of myc-tagged ndr2 in png mutants was demonstrated by Western-Blot. (FeI) In contrast to wt (F, H), tbx2b staining is absent in AVC of png mutants at 72 hpf (G, I). (J, L) tbx2b expression is reconstituted in med10 RNA-injected png mutants (L), but not in controls (J). (K, M) Accordingly, anf is misexpressed in AV myocardial cells of KCl-injected png mutants (K), whereas anf is absent from rescued AV myocardium of med10 RNA-injected png mutants (M) (white arrow, AVC). S. Just et al. / Biochemical and Biophysical Research Communications 477 (2016) 581e588 585 neither mediated by enhanced Wnt nor reduced Nodal signaling. mutants, suggesting a specific role for Med10 in the regulation of Tbx2b expression in the AV myocardium. Hence, to further examine whether loss of Tbx2b expression in the AVC is indeed the molec- 3.4. Foxn4-Med10-Tbx2b signaling is disrupted in png mutant ular cause for defective AVC development in png mutants, we hearts injected 320 pg tbx2b-mRNA into png mutants and find that 19.70 ± 2.32% (P < 0.001) of homozygous png mutants show The T-box transcriptional regulator Tbx2 was found to suppress properly developed AVC accompanied by the formation of endo- expression in the myocardium of the AVC [14] to guarantee proper cardial cushions (Fig. 3AeE), suggesting that Med10 regulates AVC AVC development [15]. Thus, to evaluate whether altered Tbx2b development mainly via the control of Tbx2b expression. signaling accounts for the misexpression of anf and the structural In slip jig mutant zebrafish, forkhead box transcription factor 4 AVC defects in png mutants, we assayed expression of tbx2b and (Foxn4) was found to regulate tbx2b transcription in AVC [15]. find that in png mutant hearts tbx2b mRNA is absent in the AVC Hence, to assess if similar to the situation in slip jig mutants, ectopic (Fig. 2FeI). Next, to assess if ectopic expression of Med10 can expression of foxn4 can reconstitute tbx2b expression and thereby reconstitute the expression of tbx2b thereby suppressing the endocardial cushion formation in png mutants, we injected up to development of AVC defects in png mutants, we injected 800 pg of 320 pg of foxn4-mRNA into png mutants. However, we find that med10-mRNA into 1-cell stage png mutant embryos. As shown in foxn4-mRNA injection does not lead to re-expression of tbx2b in the Fig. 2JeM, ectopic expression of med10-mRNA reconstitutes both, AVC and reconstitution of AV valve formation in png mutants regular tbx2b and anf expression in AV myocardial cells of png

Fig. 3. Ectopic expression of Tbx2b rescues the valve defect in png mutants. (AeE) Injection of tbx2b RNA into homozygous png mutants leads to the reconstitution of endocardial cushion development (19.70% ± 2.32%, n ¼ 101, P < 0.001, 4 independent experiments) (BeD), whereas injection of KCl has no effect (A). (D) H&E staining of sections of a tbx2b RNA- injected png heart at 72 hpf. Endocardial cushion development (black arrow) in png hearts is reconstituted. (E) In tbx2b RNA-injected png hearts, anf is only present in the ventricular and atrial myocardium and not in the AVC. (B, F-H) Injection of foxn4-RNA into homozygous png mutants does not rescue endocardial cushion development (n ¼ 87, 3 independent experiments). (G) H&E staining of sections of a foxn4 RNA-injected png heart at 72 hpf. No endocardial cushions are formed in foxn4 RNA-injected png hearts. (H) Tbx2b expression is not reconstituted in AV myocardial cells in png mutants injected with foxn4-RNA. (I, J) Injection of MO-foxn4 leads to a heart valve phenotype in 81.80% ± 10.33% (n ¼ 122, 4 independent experiments) and co-injection of med10-RNA and MO-foxn4 does not rescue the foxn4 morphant phenotype (80.01% ± 5.36, n ¼ 198, 4 independent experiments). (K, L) Injection of MO-foxn4 in wt (31.31% ± 28.61%, n ¼ 54, 4 independent experiments) and heterozygous png embryos (56.79% ± 31.36%, n ¼ 97, 4 independent experiments) revealed a sensitizing effect of Foxn4-depletion on heterozygous png embryos. 586 S. Just et al. / Biochemical and Biophysical Research Communications 477 (2016) 581e588

(Fig. 3FeH), implicating that Med10 either orchestrates Tbx2b mRNA level and distribution and find that in png mutants has2 is expression in the AVC in a rather Foxn4-independent manner, or completely absent in the AVC (Fig. 4AeD). Hence, to investigate if that Foxn4-mediated transcription of tbx2b strongly depends on loss of Has2 in png hearts leads to reduced HA levels in the cardiac intact Med10 function. Thus, we evaluated whether wild-type jelly, we performed HA staining of png mutants and find the med10-mRNA is able to rescue the AVC defect in Foxn4 mor- amount of HA in the cardiac jelly markedly reduced (Fig. 4E, F). phants. We find that ectopic expression of Med10 is unable to Next, to determine whether reduced HA levels in png mutant hearts reconstitute AVC development in Foxn4-deficient embryos (Fig. 3I, interfere with the development of the cardiac jelly, we co-stained J), implying that Med10 and Foxn4 signaling depend on each other. png xTg(fli1a:EGFP) transgenic zebrafish with Phalloidin and To ultimately prove whether Med10 and Foxn4 act in the same Dm-grasp (Alcam) and find by confocal sectioning that the area molecular pathway and depend on each other during heart valve between AV endocardium and myocardium is severely reduced in development, we performed sensitizing experiments in a hetero- png mutant hearts (Fig. S5A, B), suggesting that Med10-deficiency þ zygous png (png /-) background by reducing Foxn4 activity. To do interferes with the regular establishment of the cardiac jelly and so, we injected low doses of MO-foxn4 into heterozygous png em- that this leads to the inhibition of AV endocardial cell differentia- bryos and analyzed the resulting cardiac phenotype. After injection tion as shown by missing Dm-grasp-positive AV endocardial cells in of 2.4 ng MO-foxn4, 56.79% ± 31.36% of injected sensitized het- png mutant hearts (Fig. S5A, B). erozygous png embryos display a heart phenotype similar to the png phenotype accompanied by the absence of AV valves and severe regurgitation of blood between ventricle and atrium (Fig. 3K, 4. Discussion L). By contrast, only 31.31% ± 28.61% of injected homozygous wild- types develop heart valve defects (Fig. 3K, L). In search for signaling cascades that guide cardiac valve devel- Tbx2 is known to regulate the expression of hyaluronan synthase opment in vertebrates, we deciphered the molecular fundamentals fi fi 2(has2) responsible for the production of hyaluronan (HA), a major of the zebra sh AVC mutant ping pong and nd that the tran- ECM component of the cardiac jelly [16]. Interestingly, loss of Has2 scriptional Mediator complex subunit 10 (Med10) regulates the leads to severe cardiovascular abnormalities including the failure of development of the AV valves by mediating Foxn4 signals and transformation of cardiac endothelial into mesenchymal cells thereby controlling the expression of Tbx2b in AV myocardial cells (EMT) [16]. To evaluate whether defective Tbx2b signaling in png and the formation of the cardiac jelly. mutant hearts leads to the loss of has2 expression, we assayed has2 The Mediator complex (Med) integrates and transduces regulatory information from DNA-binding transcription factors to the

Fig. 4. Defective Has2 signaling in png mutant hearts. (AeD) has2 staining is absent in AVC and OFT endocardium of png mutants at 48 (B) and 72 hpf (D). (E, F) Histological sections of wt (E) and png mutants (F) at 72 hpf stained for hyaluronic acid (HA) (brown) and counterstained with H&E. The amount of HA in the cardiac jelly of png mutants is severely reduced. S. Just et al. / Biochemical and Biophysical Research Communications 477 (2016) 581e588 587 basal RNA polymerase II (Pol II) machinery thereby serving as a Has2 expression and HA production and thereby in vertebrate AV connecting bridge between regulatory and accomplishing tran- valve formation. scriptional elements without direct DNA binding [17]. Not much is known about the functions of distinct Mediator subunits in verte- Disclosures brates. Med10 was shown to be dispensable for global transcriptional processes but found to be involved in formation of tail buds None. by regulating Wnt and Nodal signaling in zebrafish. Although a valve pathology was described in tennis match mutants, the role of Acknowledgments enhanced Wnt and impaired Nodal signals on AV valve development in tennis match embryos was not evaluated in this study [5]. We thank Nicole Trano, Jessica Rudloff, Katrin Vogt, Regine Baur, We find that neither transgenic overexpression of Wnt8 in wild- Karin Strele, Sabrina Diebold and Hedwig Frank for their excellent types nor increasing Wnt signaling in sensitized heterozygous technical assistance. This work was supported by Deutsche For- png embryos result in defective AVC formation, indicating that the schungsgemeinschaft (DFG) JU2859/2-1 (SJ) and the German Fed- AVC defect in png mutants is not due to over-activated Wnt eral Ministry of Education and Research (BMBF) (e:Med-SYMBOL- signaling as observed in APC mutant zebrafish [18]. Furthermore, HF grant #01ZX1407A) (SJ) as well as 01GS0108 (WR), 01GS0420 overexpression of Nodal related protein 2, known to activate Nodal (WR), 01GS0836 (WR) and 01GS1104 (WR) (NGFNplus), and signaling [19], is not capable to reconstitute valve formation in 01KU0901C (WR) Insight-DCM; the European Union (EU-Inheri- homozygous png mutants, indicating that impaired valve formation tance) (WR) and the Ministerium für Wissenschaft, Forschung und in Med10-deficient embryos is neither mediated by Wnt nor Nodal Kunst Baden-Württemberg (MWK) Juniorprofessurenprogramm signaling. (SJ). We find anfexpressed in png AVC myocardial cells, cells that are usually devoid of anf mRNA [20]. Tbx2 was shown to act as a Appendix A. Supplementary data transcriptional repressor in AV myocardium, inhibiting Anf transcription by forming a repressive ternary complex on the Anf pro- Supplementary data related to this article can be found at http:// moter together with Nkx-2.5 [21].Wefind that the expression of dx.doi.org/10.1016/j.bbrc.2016.06.088. tbx2b is absent in AV myocardial cells in png, explaining the misexpression of Anf in png AV myocardium. Chi et al. reported that Transparency document targeted ablation of tbx2b leads to malformations of the AVC accompanied by missing endocardial cushions [15]. These findings Transparency document related to this article can be found are consistent with our findings that png mutants also show online at http://dx.doi.org/10.1016/j.bbrc.2016.06.088. abrogated AVC development as a result of diminished tbx2b expression. 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