Background: Microdeletion of chromosome 22q11 is associated with significant developmental anomalies, including disruption of the cardiac outflow tract, thymic/parathyroid aplasia and cleft palate. Amongst the genes within this region, TBX1 is a major candidate for many of these developmental defects. Targeted deletion of Tbx1 in the mouse has provided significant insight into the function of this transcription factor during early development of the cardiac and pharyngeal systems. However, less is known about its role during palatogenesis. To assess the influence of Tbx1 function on gene expression profile within the developing palate we performed a microarray screen using total RNA isolated from the secondary palate of E13.5 mouse embryos wild type, heterozygous and mutant for Tbx1. Results: Expression-level filtering and statistical analysis revealed a total of 577 genes differentially expressed across genotypes. Data were clustered into 3 groups based on comparison between genotypes. Group A was composed of differentially expressed genes in mutant compared to wild type (n = 89); Group B included differentially expressed genes in heterozygous compared to wild type (n = 400) and Group C included differentially expressed genes in mutant compared to heterozygous (n = 88). High-throughput quantitative real-time PCR (RT-PCR) confirmed a total of 27 genes significantly changed between wild type and mutant; and 27 genes between heterozygote and mutant. Amongst these, the majority were present in both groups A and C (26 genes). Associations existed with hypertrophic cardiomyopathy, cardiac muscle contraction, dilated cardiomyopathy, focal adhesion, tight junction and calcium signalling pathways. No significant differences in gene expression were found between wild type and heterozygous palatal shelves. Conclusions: Significant differences in gene expression profile within the secondary palate of wild type and mutant embryosisconsistent withaprimary rolefor Tbx1 during palatogenesis. Keywords: Palatogenesis, Cleft palate, Microarray, 22q11.2DS, DiGeorge syndrome Background 192430), conotruncal anomaly face (CAFS or Takao syn- 22q11.2 deletion syndrome (22q11.2DS) is the most drome; MIM 217095) and isolated outflow tract (OFT) common human microdeletion  occurring with a defects of the heart [5–9]. These conditions are charac- prevalence of 1:4000 and incidence ranging from terized predominantly by the presence of congenital 1:2000–6395 [2–4]. This microdeletion is associated heart defects, thymic and parathyroid hypoplasia, and with several syndromic conditions including DiGeorge craniofacial dysmorphism, including oro-facial clefting (DGS; MIM 188400), velocardiofacial (VCFS; MIM that predominates as isolated cleft palate, micrognathia and (less commonly) dental defects [10–13]. The most common deletions are phenotypically indistinguishable from each other and consist of either a 3 Mb segment * Correspondence: email@example.com Centre for Craniofacial Development and Regeneration, King’s College spanning the low copy repeats (LCR) A-D (around 85% London Dental Institute, Floor 27, Guy’s Tower, London SE1 9RT, UK of cases); or a smaller 1.5 Mb deletion that spans LCR Department of Orthodontics, King’s College London Dental Institute, London, UK Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Zoupa et al. BMC Genomics (2018) 19:429 Page 2 of 20 A-B seen in around 15% of cases [14–16]. A less com- septum superiorly and primary palate anteriorly, com- mon LCR C-D deletion of the typical 22q11.2DS region pleting separation of the nasal and oral cavities [44–46]. has also been identified, which is associated with a In the developing mouse embryo, Tbx1 is expressed in much-reduced prevalence of cardiac malformations and epithelium of the palatal shelves throughout palatogen- oro-facial clefting [17–19]. 22q11.2DS is a contiguous esis from embryonic day (E)12.5–15.5 . The etio- gene and haploinsufficient syndrome with at least 30 dif- logical basis of the cleft palate phenotype in Tbx1 ferent genes potentially contributing to the characteristic mutants is not fully understood but has been associated clinical features [20, 21]. Amongst the genes identified with abnormal palatal shelf elevation, possibly due to a as candidates for the development of 22q11.2DS, combination of increased tongue height, decreased pal- T-Box 1 (TBX1), which encodes a T-Box-containing atal shelf width, perturbed cell proliferation and transcription factor is recognised as a major determinant apoptosis . In addition, inappropriate fusion between through its location within the 22q11 critical region the palatal shelf epithelium and tongue has also been [21–23], expression in organs affected within the clinical described in this mutant, associated with spectrum [24–27] and observations that loss of Tbx1 hyper-proliferation and disrupted differentiation . function in mouse recapitulates the clinical findings seen More recently, confocal image analysis has found only in many DGS subjects [23, 28–31]. Supporting this, subtle differences in levels of proliferation within mesen- TBX1 mutation has been identified in a sporadic case of chyme of the palatal shelves between wild-type and mu- DGS  and Tbx1 haploinsufficiency results in the tant until the later stages of palatogenesis; although most characteristic phenotypes related to developmental significant differences in mesenchymal cell orientation defects in the embryonic pharyngeal apparatus [32, 33]. were found in mutant shelves, which might contribute DGS is also referred to as the III-IV pharyngeal pouch to the cleft phenotype . syndrome, as the pharyngeal pouches and their associ- We are interested in further defining the role of Tbx1 ated blood vessels are the structures most commonly af- during the process of murine palatogenesis. Specifically, fected [23, 30]. Apart from the aortic arch, thymus and we have investigated regulation of this transcription fac- parathyroid gland defects, Tbx1 murine models also tor in the secondary palate and carried out a manifest craniofacial anomalies that arise from develop- functionally-based microarray using the Tbx1 mouse mental defects associated with pharyngeal arches I and model. We compared total RNA isolated from dissected II [23, 34, 35]. Indeed, conditional mutant models have secondary palatal shelves derived from E13.5 wild type +/− −/− revealed a tissue-specific requirement and a dose sensi- (WT), Tbx1 (heterozygous) and Tbx1 (mutant) em- tivity for Tbx1 during murine pharyngeal development bryos and clustered the data into three groups based on [20, 36–38]. comparison between the three genotypes. Microarray The majority of 22q11.2DS individuals have a character- analysis demonstrated that in the absence of functional istic craniofacial morphology including lateral displace- Tbx1, significant changes occur in the expression profile ment of the inner canthi, swollen eyelids, small mouth, of numerous genes in mutant versus WT and mutant hypoplastic mandible, flat nasal bridge and square nose versus heterozygous groups. The most significant path- [39–41]. Cleft palate (including submucous cleft) is also ways affected in both groups were the hypertrophic car- present in approximately 10% of subjects . Morpho- diomyopathy, cardiac muscle contraction, dilated logical studies to assess embryonic malformations in vari- cardiomyopathy, focal adhesion, calcium signalling and ous Tbx1 genotypes also reveal the presence of cleft palate tight junction pathways. High-throughput quantitative in Tbx1-overexpressing mice [42, 43]. Therefore, both loss RT-PCR validation confirmed significant variation be- and gain of Tbx1 function can lead to the development of tween WT and mutant in the expression of 26 individual a cleft phenotype. genes. We discuss these findings within the context of The palate is divided anatomically into primary and murine secondary palatogenesis. secondary regions with the secondary palate composed of both hard and soft tissues. Embryologically, the sec- Results ondary palate is derived from the paired maxillary pro- Regulation of Tbx1 in the developing secondary palate cesses of pharyngeal arch I, which gives rise to the Tbx1 transcriptional activity is present in epithelium of palatal shelves. During palatogenesis, these shelves are the secondary palate shelves throughout the processes of initially situated bilaterally adjacent to the developing growth, elevation and fusion (Additional file 1)and Tbx1 tongue; however, progressive growth and elevation re- mutant mice have a fully penetrant cleft palate [23, 30, 31]. sults in them positioning themselves above the tongue, We are interested in further defining the function of this with further medial growth leading to fusion with their transcription factor during palatogenesis at the molecular counterpart along the midline to create a single continu- level and first sought to understand how Tbx1 transcrip- ous palate. The palatal shelves also fuse with the nasal tion might be regulated in the palatal shelf epithelium. We Zoupa et al. BMC Genomics (2018) 19:429 Page 3 of 20 began by investigating the effect of abrogating either Sonic receptor 2b (Fgfr2b), regulates cell proliferation in the hedgehog (Shh) or Fibroblast growth factor (Fgf) signaling mesenchyme . Whilst Shh also negatively regulates in palatal shelf explants as there are potential associations Bmp4 in the mesenchyme, which is itself upstream of between these signaling networks and Tbx1 function in Fgf10 . Tbx1 interacts with a number of these mole- the developing palate. Shh is also expressed in the palatal cules during embryogenesis, being directly upstream of epithelium and lies upstream of Tbx1 in the pharyngeal Fgf10 in the early heart field [28, 56]; negatively modu- endoderm ; whilst Fgf signaling can maintain Tbx1 ex- lating Bmp4 through the binding of Smad1 in cardio- pression in early odontogenic epithelium . Specifically, myocytes  and being downstream of Shh in E13.5 secondary palatal shelves were isolated and cultured endoderm of the early pharynx . Within the palate for 24 h in the presence of either the Shh antagonist cyclo- itself, it has been variously suggested that Tbx1 nega- pamine or the Fgf receptor inhibitor SU4502. Interestingly, tively regulates Fgf10 and Bmp4, whilst positively regu- whilst an absence of Shh signaling did not affect Tbx1 lating Fgf8 and Pax9, although there is currently not a transcription, loss of Fgf signaling resulted in a loss of consensus on these findings [47, 48]. Tbx1 activity in the palatal epithelium after 24 h of culture Although we could find no evidence that Tbx1 is (Fig. 1a-g). These results place Tbx1 downstream of Fgf downstream of Shh signaling in the palatal epithelium, signaling during early palatogenesis and in contrast to the there is considerable overlap of expression. We therefore pharyngeal region, loss of Shh does not affect Tbx1. investigated known targets of Shh within palatal shelves WT and mutant for Tbx1 using in situ hybridization. Altered gene expression in the secondary palate of Tbx1 Interestingly, we found no significant differences in ex- mutant mice pression of Shh, Fgf10 and Fgfr2b between WT and mu- It is known that Shh, Fgf and Bone morphogenetic pro- tant (Fig. 2a-f). However, whilst Fgf8 expression was also tein (Bmp) signaling pathways are important during nor- normal in the mutant shelves (Fig. 2g-h), Bmp4 and mal development of the palate [51–53]; in particular, paired-box 9 (Pax9) were slightly up and downregulated, reciprocal signaling between epithelial Shh and mesen- respectively in the posterior region of the secondary pal- chymal Fgf10, mediated through fibroblast growth factor ate (Fig. 2i-l). These apparent changes in Bmp4 and Fig. 1 Regulation of Tbx1 expression in the early secondary palate. Wholemount in situ hybridization on palatal shelf explants cultured for 24 h in the presence or absence of the Shh inhibitor cyclopamine and the Fgf receptor inhibitor SU5402. a Tbx1 is expressed in the palatal shelf epithelium and first molar tooth germ (arrowed); (b) in the absence of Shh signaling, Tbx1 is maintained; (c) in the absence of Fgf signaling, Tbx1 is lost; (d) Shh signaling is active in the developing palate and first molar (arrowed) as shown by expression of the Shh transcriptional target patched1 (Ptch1); (e) in the presence of cyclopamine Ptch1 transcription is lost; (f) Fgf signaling is active in the developing palate and first molar (arrowed), as shown by expression of the Fgf transcriptional target sprouty2 (Spry2); (g) in the presence of SU4502 Spry2 is lost. Lines mark the medial edge of the palatal shelf Zoupa et al. BMC Genomics (2018) 19:429 Page 4 of 20 Fig. 2 Signaling interactions during development of the secondary palate in WT and Tbx1 mutant embryos. Section in situ hybridization demonstrating the expression of key signaling molecules. a, b Shh;(c, d) Fgf10;(e, f) Fgfr2b;(g, h) Fgf8;(i, j) Bmp4;(k, l) Pax9 Pax9 expression in the mutant might simply be a func- to be differentially expressed in the mutant compared to tion of altered numbers of cells expressing these genes heterozygote palate (adj. p < 0.1, fold change 1.3). in the palate mesenchyme, particularly as the Tbx1 do- Amongst these, 11 genes were upregulated, whereas 77 main within the palatal epithelium does not completely were downregulated (Table 1). In Group A, from the 89 overlie those of Bmp4 or Pax9 in the mesenchyme . genes that were searched, 9 Kyoto Encyclopedia of However, given the evidence of retarded growth in Tbx1 Genes and Genomes (KEGG) pathways were identified mutant palatal shelves [47, 48] if an alteration in cell (Fig. 3a). The most statistically enriched pathways (adj. number is responsible for any of these changes, it would p < 0.1) were all associated with cardiac muscle physi- seem to be more likely for Pax9. ology and included hypertrophic cardiomyopathy, car- diac muscle contraction, dilated cardiomyopathy, Microarray analysis arrhythmogenic right ventricular cardiomyopathy and To further identify potential transcriptional target genes vascular smooth muscle contraction. Other pathways of Tbx1 implicated in palatogenesis, microarray analysis included phagosome and focal adhesion, tight junc- was carried out using cDNA transcribed from total RNA tion and calcium signaling pathways and Alzheimer's derived from the dissected secondary palatal shelves of disease (Additional file 3). In Group C, from the 88 +/+ +/− −/− E13.5 Tbx1 ; Tbx1 and Tbx1 embryos (n = 3 for genes that were searched, 10 KEGG pathways were each genotype). identified (Fig. 3b). The most statistically enriched After normalization and filtering of microarray data, pathways (adj. p < 0.1) were all also associated with comparison between mutant embryos and WT (Group cardiac muscle physiology, including hypertrophic and A), heterozygous and WT (Group B) and mutant versus dilated cardiomyopathy and arrhythmogenic right ven- heterozygous (Group C) were performed (adj. p < 0.1). tricular cardiomyopathy. Other pathways included The WebGestalt database was used to identify biological tight junction, calcium signalling, focal adhesion, neu- pathways associated with these differentially expressed roactive ligand-receptor interaction, phagosome and transcripts . In Group A, 89 genes were identified to Alzheimer’s disease pathways (Additional file 3). We be differentially expressed in mutant compared to WT were then interested to further identify the proportion (adj. p < 0.1, fold change 1.4). From these, 3 genes were of overlap amongst significantly differentially upregulated, whereas the majority (n = 86) were down- expressed genes between Groups A and C. (Fig. 4a regulated (Table 1). Group B includes differentially ). The two groups share 58 commonly expressed expressed genes arising from the comparison of hetero- genes (Table 2)whencomparedtoWT and heterozy- zygous and WT palates (n = 400, adj. p > 0.23). This gous; whereas 30 genes (Table 2) were uniquely ob- group list was not considered statistically significant (adj. served in Group A and 20 in Group C (Table 2;adj. p > 0.1) and therefore was not analysed further P < 0.1). The WebGestalt database was used to pro- (Additional file 2). In Group C, 88 genes were identified vide insights into the mechanism of regulation Zoupa et al. BMC Genomics (2018) 19:429 Page 5 of 20 +/+ +/− −/− Table 1 Group comparison of Tbx1 , Tbx1 and Tbx1 palatal shelves Gene ID Gene symbol Description logFC Fold Change Group A: Genes differentially expressed in mutant compared to WT palates 14,462 Gata3 GATA binding protein 3 1,10 2,15 66894 Wwp2 WW domain containing E3 ubiquitin protein ligase 2 0,64 1,55 20466 Sin3a transcriptional regulator, SIN3A (yeast) 0,45 1,37 27999 Fam3c family with sequence similarity 3, member C −0,43 −1,35 23,945 Mgll monoglyceride lipase −0,44 −1,36 22145 Tuba4a tubulin, alpha 4A −0,46 −1,38 23,945 Mgll monoglyceride lipase −0,46 −1,38 17286 Meox2 mesenchyme homeobox 2 − 0,48 − 1,39 227929 Cytip cytohesin 1 interacting protein −0,50 −1,41 21393 Tcap titin-cap −0,50 −1,42 13426 Dync1i1 dynein cytoplasmic 1 intermediate chain 1 −0,51 −1,42 231,633 Tmem119 transmembrane protein 119 −0,52 −1,43 21953 Tnni2 troponin I, skeletal, fast 2 −0,54 −1,46 27,273 Pdk4 pyruvate dehydrogenase kinase, isoenzyme 4 −0,54 −1,46 13,038 Ctsk cathepsin K −0,57 − 1,48 107765 Ankrd1 ankyrin repeat domain 1 (cardiac muscle) −0,57 −1,49 17533 Mrc1 mannose receptor, C type 1 −0,59 −1,50 50796 Dmrt1 doublesex and mab-3 related transcription factor 1 −0,59 −1,51 72713 Angptl1 angiopoietin-like 1 −0,61 −1,53 13346 Des desmin −0,67 −1,59 12862 Cox6a2 cytochrome c oxidase subunit VIa polypeptide 2 −0,69 −1,61 56437 Rrad Ras-related associated with diabetes −0,71 −1,64 12608 Cebpb CCAAT/enhancer binding protein (C/EBP), beta −0,71 −1,64 14066 F3 coagulation factor III −0,74 −1,67 50768 Dlc1 deleted in liver cancer 1 −0,74 − 1,67 12299 Cacng1 calcium channel, voltage-dependent, gamma subunit 1 −0,74 −1,67 76,757 Trdn triadin −0,76 −1,69 11475 Acta2 actin, alpha 2, smooth muscle, aorta −0,76 − 1,69 12292 Cacna1s calcium channel, voltage-dependent, L type, alpha 1S subunit −0,76 −1,70 56012 Pgam2 phosphoglycerate mutase 2 −0,79 −1,73 67951 Tubb6 tubulin, beta 6 class V −0,83 −1,78 11656 Alas2 aminolevulinic acid synthase 2, erythroid −0,84 − 1,80 19400 Rapsn receptor-associated protein of the synapse −0,85 −1,80 22004 Tpm2 tropomyosin 2, beta −0,86 −1,82 12575 Cdkn1a cyclin-dependent kinase inhibitor 1A (P21) −0,87 −1,83 17189 Mb myoglobin −0,88 −1,85 11609 Agtr2 angiotensin II receptor, type 2 −0,90 −1,86 21384 Tbx15 T-box 15 − 0,91 − 1,87 12955 Cryab crystallin, alpha B −0,92 −1,89 12955 Cryab crystallin, alpha B −0,92 −1,89 50795 Sh3bgr SH3-binding domain glutamic acid-rich protein −0,92 −1,89 17930 Myom2 myomesin 2 −0,95 −1,93 12180 Smyd1 SET and MYND domain containing 1 −0,96 −1,94 Zoupa et al. BMC Genomics (2018) 19:429 Page 6 of 20 +/+ +/− −/− Table 1 Group comparison of Tbx1 , Tbx1 and Tbx1 palatal shelves (Continued) Gene ID Gene symbol Description logFC Fold Change 59058 Bhlhe22 basic helix-loop-helix family, member e22 −0,96 −1,95 26465 Zfp146 zinc finger protein 146 −1,01 −2,01 12391 Cav3 caveolin 3 −1,02 −2,02 65086 Lpar3 lysophosphatidic acid receptor 3 −1,06 −2,09 170812 Ahsp alpha hemoglobin stabilizing protein −1,09 −2,13 14,077 Fabp3 fatty acid binding protein 3, muscle and heart −1,10 −2,15 11443 Chrnb1 cholinergic receptor, nicotinic, beta polypeptide 1 (muscle) −1,11 −2,16 17929 Myom1 myomesin 1 −1,14 −2,20 21953 Tnni2 troponin I, skeletal, fast 2 −1,16 −2,24 244954 Prss35 protease, serine 35 −1,19 −2,29 69253 Hspb2 heat shock protein 2 −1,20 −2,29 21957 Tnnt3 troponin T3, skeletal, fast −1,23 −2,35 14619 Gjb2 gap junction protein, beta 2 −1,24 −2,36 13009 Csrp3 cysteine and glycine-rich protein 3 −1,30 −2,46 12,350 Car3 carbonic anhydrase 3 −1,37 −2,59 56069 Il17b interleukin 17B −1,37 −2,59 11811 Apobec2 apolipoprotein B mRNA editing enzyme, catalytic polypeptide 2 −1,43 −2,69 11937 Atp2a1 ATPase, Ca++ transporting, cardiac muscle, fast twitch 1 −1,46 −2,76 66139 Tmem8c transmembrane protein 8C −1,48 −2,78 51801 Ramp1 receptor (calcitonin) activity modifying protein 1 −1,56 −2,94 24131 Ldb3 LIM domain binding 3 −1,56 −2,94 16545 Kera keratocan −1,81 −3,51 140781 Myh7 myosin, heavy polypeptide 7, cardiac muscle, beta −1,81 −3,51 21828 Thbs4 thrombospondin 4 −1,91 −3,75 13380 Dkk1 dickkopf homolog 1 (Xenopus laevis) − 1,94 −3,83 21955 Tnnt1 troponin T1, skeletal, slow −1,95 −3,87 58916 Myot myotilin −1,98 −3,95 17928 Myog myogenin −2,04 −4,12 21380 Tbx1 T-box 1 − 2,06 −4,16 53311 Mybph myosin binding protein H −2,06 −4,16 21952 Tnni1 troponin I, skeletal, slow 1 −2,26 −4,79 12,350 Car3 carbonic anhydrase 3 −2,31 −4,97 66402 Sln sarcolipin −2,40 −5,28 11472 Actn2 actinin alpha 2 −2,40 −5,29 17896 Myl4 myosin, light polypeptide 4 −2,44 −5,43 21956 Tnnt2 troponin T2, cardiac −2,53 −5,77 11464 Actc1 actin, alpha, cardiac muscle 1 −2,56 −5,90 66106 Smpx small muscle protein, X-linked −2,61 −6,11 21924 Tnnc1 troponin C, cardiac/slow skeletal −2,67 −6,35 17901 Myl1 myosin, light polypeptide 1 −2,76 −6,76 21925 Tnnc2 troponin C2, fast −2,77 −6,83 17907 Mylpf myosin light chain, phosphorylatable, fast skeletal muscle −2,88 −7,35 21956 Tnnt2 troponin T2, cardiac −2,92 −7,57 11459 Acta1 actin, alpha 1, skeletal muscle −3,10 −8,60 Zoupa et al. BMC Genomics (2018) 19:429 Page 7 of 20 +/+ +/− −/− Table 1 Group comparison of Tbx1 , Tbx1 and Tbx1 palatal shelves (Continued) Gene ID Gene symbol Description logFC Fold Change 17883 Myh3 myosin, heavy polypeptide 3, skeletal muscle, embryonic −3,22 −9,29 15,891 Ibsp integrin binding sialoprotein −3,51 −11,36 Group C: Genes differentially expressed in mutant compared to heterozygous palates 12,846 Comt catechol-O-methyltransferase 1,0 2,1 74,374 Clec16a C-type lectin domain family 16, member A 0,8 1,8 54153 Rasa4 RAS p21 protein activator 4 0,7 1,6 66894 Wwp2 WW domain containing E3 ubiquitin protein ligase 2 0,6 1,5 18,155 Pnoc prepronociceptin 0,6 1,5 56,538 Klk11 kallikrein related-peptidase 11 0,5 1,4 80904 Dtx3 deltex 3 homolog (Drosophila) 0,5 1,4 212,127 Proser1 proline and serine rich 1 0,5 1,4 108655 Foxp1 forkhead box P1 0,4 1,4 76501 Commd9 COMM domain containing 9 0,4 1,4 14809 Grik5 glutamate receptor, ionotropic, kainate 5 (gamma 2) 0,4 1,3 19280 Ptprs protein tyrosine phosphatase, receptor type, S −0,3 −1,3 18,008 Nes nestin −0,4 −1,3 27999 Fam3c family with sequence similarity 3, member C −0,4 −1,3 13426 Dync1i1 dynein cytoplasmic 1 intermediate chain 1 −0,4 −1,3 65114 Vps35 vacuolar protein sorting 35 −0,5 −1,4 21393 Tcap titin-cap −0,5 −1,4 17286 Meox2 mesenchyme homeobox 2 − 0,5 − 1,4 17286 Meox2 mesenchyme homeobox 2 − 0,5 − 1,4 72713 Angptl1 angiopoietin-like 1 −0,5 −1,4 67405 Nts neurotensin −0,6 − 1,5 11,303 Abca1 ATP-binding cassette, sub-family A (ABC1), member 1 −0,6 −1,5 21812 Tgfbr1 transforming growth factor, beta receptor I −0,6 −1,5 15,366 Hmmr hyaluronan mediated motility receptor (RHAMM) −0,6 −1,5 11,733 Ank1 ankyrin 1, erythroid −0,6 −1,5 21412 Tcf21 transcription factor 21 −0,6 −1,5 50796 Dmrt1 doublesex and mab-3 related transcription factor 1 −0,7 −1,6 12862 Cox6a2 cytochrome c oxidase subunit VIa polypeptide 2 −0,7 −1,6 50768 Dlc1 deleted in liver cancer 1 −0,7 −1,6 56437 Rrad Ras-related associated with diabetes −0,7 −1,6 56012 Pgam2 phosphoglycerate mutase 2 −0,7 −1,6 67951 Tubb6 tubulin, beta 6 class V −0,7 −1,6 11,870 Art1 ADP-ribosyltransferase 1 −0,7 −1,7 15375 Foxa1 forkhead box A1 −0,7 −1,7 11,475 Acta2 actin, alpha 2, smooth muscle, aorta −0,8 −1,7 12292 Cacna1s calcium channel, voltage-dependent, L type, alpha 1S subunit −0,8 −1,7 19400 Rapsn receptor-associated protein of the synapse −0,8 −1,7 80,882,479 Lrrn1 leucine rich repeat protein 1, neuronal −0,8 −1,7 17189 Mb myoglobin −0,8 −1,7 12299 Cacng1 calcium channel, voltage-dependent, gamma subunit 1 −0,8 −1,8 Zoupa et al. BMC Genomics (2018) 19:429 Page 8 of 20 +/+ +/− −/− Table 1 Group comparison of Tbx1 , Tbx1 and Tbx1 palatal shelves (Continued) Gene ID Gene symbol Description logFC Fold Change 12955 Cryab crystallin, alpha B −0,8 −1,8 11609 Agtr2 angiotensin II receptor, type 2 −0,9 −1,8 111,886,114 Cryab crystallin, alpha B −0,9 −1,8 17930 Myom2 myomesin 2 −0,9 −1,8 12180 Smyd1 SET and MYND domain containing 1 −0,9 −1,8 170812 Ahsp alpha hemoglobin stabilizing protein −0,9 −1,9 50795 Sh3bgr SH3-binding domain glutamic acid-rich protein −0,9 −1,9 14066 F3 coagulation factor III −0,9 −1,9 59058 Bhlhe22 basic helix-loop-helix family, member e22 −1,0 −2,0 12391 Cav3 caveolin 3 −1,0 −2,1 17929 Myom1 myomesin 1 −1,1 −2,1 26465 Zfp146 zinc finger protein 146 −1,1 −2,1 21384 Tbx15 T-box 15 − 1,1 −2,1 21384 Tbx15 T-box 15 − 1,1 −2,2 11443 Chrnb1 cholinergic receptor, nicotinic, beta polypeptide 1 (muscle) −1,1 −2,2 21953 Tnni2 troponin I, skeletal, fast 2 −1,2 −2,2 69253 Hspb2 heat shock protein 2 −1,2 −2,2 13009 Csrp3 cysteine and glycine-rich protein 3 −1,2 −2,3 21957 Tnnt3 troponin T3, skeletal, fast −1,3 −2,4 11937 Atp2a1 ATPase, Ca++ transporting, cardiac muscle, fast twitch 1 −1,3 −2,4 56069 Il17b interleukin 17B −1,3 −2,5 14619 Gjb2 gap junction protein, beta 2 −1,5 −2,8 11435 Chrna1 cholinergic receptor, nicotinic, alpha polypeptide 1 (muscle) −1,5 −2,8 11811 Apobec2 apolipoprotein B mRNA editing enzyme, catalytic polypeptide 2 −1,5 −2,9 24131 Ldb3 LIM domain binding 3 −1,6 −3,0 17927 Myod1 myogenic differentiation 1 −1,6 −3,1 66139 Tmem8c transmembrane protein 8C −1,7 −3,2 21828 Thbs4 thrombospondin 4 −1,8 −3,4 140781 Myh7 myosin, heavy polypeptide 7, cardiac muscle, beta −1,9 −3,7 58916 Myot myotilin −2,0 −3,9 87,201,087 Tnnt1 troponin T1, skeletal, slow −2,0 −3,9 17928 Myog myogenin −2,1 −4,3 53311 Mybph myosin binding protein H −2,2 −4,5 21952 Tnni1 troponin I, skeletal, slow 1 −2,4 −5,3 11,472 Actn2 actinin alpha 2 −2,4 −5,4 17896 Myl4 myosin, light polypeptide 4 −2,5 −5,5 66,402 Sln sarcolipin −2,5 −5,5 21,380 Tbx1 T-box 1 −2,6 −6,1 21956 Tnnt2 troponin T2, cardiac −2,6 −6,2 66106 Smpx small muscle protein, X-linked −2,7 −6,5 11464 Actc1 actin, alpha, cardiac muscle 1 −2,7 −6,5 92,760,598 Tnnc1 troponin C, cardiac/slow skeletal −2,7 −6,6 21925 Tnnc2 troponin C2, fast −2,8 −6,9 17901 Myl1 myosin, light polypeptide 1 −2,8 −7,2 Zoupa et al. BMC Genomics (2018) 19:429 Page 9 of 20 +/+ +/− −/− Table 1 Group comparison of Tbx1 , Tbx1 and Tbx1 palatal shelves (Continued) Gene ID Gene symbol Description logFC Fold Change 17907 Mylpf myosin light chain, phosphorylatable, fast skeletal muscle −3,0 −7,9 80,608,559 Tnnt2 troponin T2, cardiac −3,1 −8,5 11,459 Acta1 actin, alpha 1, skeletal muscle −3,1 −8,7 17883 Myh3 myosin, heavy polypeptide 3, skeletal muscle, embryonic −3,2 −9,3 Genes are listed based on fold change associated with these 58 common gene transcripts. Add- Confirmation of microarray data ing to the above approach, heat map and dendogram clus- For validation of the results obtained by microarray, tering of the commonly expressed genes, as well as RT-PCR was carried out using gene-specific primers uniquely expressed genes in Group A and Group C (n = (Applied Biosystems; Additional file 4) and the original 99 genes) revealed transcriptional homogenicity between RNA samples. In total, 27 genes from Group A and 28 genotypes (Fig. 4b). Genes upregulated in mutants clearly genes from Group C were selected for gene expression clustered together and were shown to be downregulated verification (Table 3). Changes in gene expression of these in heterozygote and WT samples (red asterisks in Fig. 4b). transcripts were normalized to that of ß-Actin. In In contrast, the downregulated transcriptome of mutant both groups, 27 genes were commonly expressed samples was shown to increase its expression in heterozy- (Table 3;Fig. 5a); Alas2 was uniquely present in gous and WT palates. Although statistical analysis re- Group A, whereas Ank1 and Chrna1 were uniquely vealed a non-significant expression pattern of Tbx1 present in Group C (Table 3; Fig. 5b). All genes heterozygous samples (adj. p values > 0.1), heat map re- tested were confirmed as being significantly changed vealed a similarity in gene expression pattern between het- between WT-mutant and heterozygote-mutant except erozygous and WT samples. for Ank1 (Group C; p = 0.102). In Group A, Rapsn, Fig. 3 a Pathway analysis of genes differentially expressed in the Tbx1 mutant secondary palate compared to WT (Group A); (b) pathway analysis of genes differentially expressed in the mutant secondary palate when compared to heterozygous (Group C): The pie chart depicts the number of assigned genes for each significantly enriched pathway. Data sets are illustrated as slices, the sizes of which are proportional to the number of genes implicated in each pathway. The ten pathways are listed and colour-coded on the right Zoupa et al. BMC Genomics (2018) 19:429 Page 10 of 20 Fig. 4 a Pairwise Venn diagram illustrating the comparison between gene sets from Tbx1 mutant secondary palate compared to WT (Group A) and Tbx1 mutant compared to heterozygous (Group C). The Venn diagram identified 58 common elements between Group A and Group C. Numbers in each section represent the number of genes. Transcripts utilized for the construction of the Venn diagram were statistically significant with adj. p values < 0.1; (b) heat map (hierarchical clustering) of commonly expressed genes in Groups A and C, as well as uniquely expressed genes in Group A and C. Hierarchical cluster of 99 genes found to be differentially expressed in the 3 mutant, 3 heterozygous and 3 WT palatal samples. Transcripts utilized for the construction of clustering were statistically significant with adj. p values < 0.1 except for heterozygous where adj. p values were > 0.1. Visual inspection of heat map and dendogram clustering of the 9 samples revealed that all triplicates of the same genotype clustered together. Upregulated genes in mutants clustered together (red asterisks on left) and their pattern of expression could be visibly compared top heterozygous and WT samples. Each row represents a specific gene, and each column represents each genotype of the samples analysed. The colour represents the expression level of the gene. Red represents high expression, while blue represents low expression. The expression levels are continuously mapped on the colour scale provided at the top left of the figure. The dendrogram at the top of the matrix provides the degree of similarity between examined groups assessing the similarity between expressed genes and samples used for comparison. Note the similarity in gene expression between WT and Tbx1 heterozygous transcripts Zoupa et al. BMC Genomics (2018) 19:429 Page 11 of 20 Table 2 Table of genes originate from the comparison of Group A and Group C lists Gene ID Gene symbol Description Fifty-eight commonly expressed gene set list from Group A and Group C comparison 16,545 Acta1 actin, alpha 1, skeletal muscle 11,475 Acta2 actin, alpha 2, smooth muscle, aorta 11,464 Actc1 actin, alpha, cardiac muscle 1 11,472 Actn2 actinin alpha 2 11,609 Agtr2 angiotensin II receptor, type 2 170,812 Ahsp alpha hemoglobin stabilizing protein 11,811 Apobec2 apolipoprotein B mRNA editing enzyme, catalytic polypeptide 2 11,937 Atp2a1 ATPase, Ca++ transporting, cardiac muscle, fast twitch 1 59,058 Bhlhe22 basic helix-loop-helix family, member e22 12,299 Cacng1 calcium channel, voltage-dependent, gamma subunit 1 12,299 Cav3 caveolin 3 11,443 Chrnb1 cholinergic receptor, nicotinic, beta polypeptide 1 (muscle) 12,862 Cox6a2 cytochrome c oxidase subunit VIa polypeptide 2 12,955 Cryab crystallin, alpha B 12,955 Cryab crystallin, alpha B 13,009 Csrp3 cysteine and glycine-rich protein 3 50,768 Dlc1 deleted in liver cancer 1 50,796 Dmrt1 doublesex and mab-3 related transcription factor 1 14,066 F3 coagulation factor III 14,619 Gjb2 gap junction protein, beta 2 69,253 Hspb2 heat shock protein 2 56,069 Il17b interleukin 17B 24,131 Ldb3 LIM domain binding 3 17,189 Mb myoglobin 17,286 Meox2 mesenchyme homeobox 2 53,311 Mybph myosin binding protein H 17,883 Myh3 myosin, heavy polypeptide 3, skeletal muscle, embryonic 140,781 Myh7 myosin, heavy polypeptide 7, cardiac muscle, beta 17,901 Myl1 myosin, light polypeptide 1 17,896 Myl4 myosin, light polypeptide 4 17,907 Mylpf myosin light chain, phosphorylatable, fast skeletal muscle 17,928 Myog myogenin 17,929 Myom1 myomesin 1 17,930 Myom2 myomesin 2 58,916 Myot myotilin 56,012 Pgam2 phosphoglycerate mutase 2 19,400 Rapsn receptor-associated protein of the synapse 56,437 Rrad Ras-related associated with diabetes 50,795 Sh3bgr SH3-binding domain glutamic acid-rich protein 66,402 Sln sarcolipin 66,106 Smpx small muscle protein, X-linked 12,180 Smyd1 myosin, heavy polypeptide 7, cardiac muscle, beta 6899 Tbx1 T-box 1 Zoupa et al. BMC Genomics (2018) 19:429 Page 12 of 20 Table 2 Table of genes originate from the comparison of Group A and Group C lists (Continued) Gene ID Gene symbol Description 12,384 Tbx15 T-box 15 21,393 Tcap titin-cap 21,828 Thbs4 thrombospondin 4 66,139 Tmem8c transmembrane protein 8C 21,924 Tnnc1 troponin C, cardiac/slow skeletal 21,925 Tnnc2 troponin C2, fast 21,952 Tnni1 troponin I, skeletal, slow 1 21,953 Tnni2 troponin I, skeletal, fast 2 21,955 Tnnt1 troponin T1, skeletal, slow 21,956 Tnnt2 troponin T2, cardiac 21,956 Tnnt2 troponin T2, cardiac 21,957 Tnnt3 troponin T3, skeletal, fast 67,951 Tubb6 tubulin, beta 6 class V 66,894 Wwp2 WW domain containing E3 ubiquitin protein ligase 2 26,465 Zfp146 zinc finger protein 146 Thirty uniquely expressed gene set of Group A 11,656 Alas2 aminolevulinic acid synthase 2, erythroid 72,713 Angptl1 angiopoietin-like 1 107,765 Ankrd1 ankyrin repeat domain 1 (cardiac muscle) 12,292 Cacna1s calcium channel, voltage-dependent, L type, alpha 1S subunit 12,350 Car3 carbonic anhydrase 3 12,350 Car3 carbonic anhydrase 3 12,575 Cdkn1a cyclin-dependent kinase inhibitor 1A (P21) 12,608 Cebpb CCAAT/enhancer binding protein (C/EBP), beta 13,038 Ctsk cathepsin K 227,929 Cytip cytohesin 1 interacting protein 13,346 Des desmin 13,380 Dkk1 dickkopf homolog 1 (Xenopus laevis) 13,426 Dync1i1 dynein cytoplasmic 1 intermediate chain 1 14,077 Fabp3 fatty acid binding protein 3, muscle and heart 27,999 Fam3c family with sequence similarity 3, member C 14,462 Gata3 GATA binding protein 3 15,891 Ibsp integrin binding sialoprotein 65,086 Lpar3 lysophosphatidic acid receptor 3 23,945 Mgll monoglyceride lipase 23,945 Mgll monoglyceride lipase 17,533 Mrc1 mannose receptor, C type 1 27,273 Pdk4 pyruvate dehydrogenase kinase, isoenzyme 4 244,954 Prss35 protease, serine 35 51,801 Ramp1 receptor (calcitonin) activity modifying protein 1 20,466 Sin3a transcriptional regulator, SIN3A (yeast) 231,633 Tmem119 transmembrane protein 119 21,953 Tnni2 troponin I, skeletal, fast 2 Zoupa et al. BMC Genomics (2018) 19:429 Page 13 of 20 Table 2 Table of genes originate from the comparison of Group A and Group C lists (Continued) Gene ID Gene symbol Description 22,004 Tpm2 tropomyosin 2, beta 76,757 Trdn triadin 22,145 Tuba4a tubulin, alpha 4A Twenty uniquely expressed gene set of Group C 11,303 Abca1 ATP-binding cassette, sub-family A (ABC1), member 1 11,733 Ank1 ankyrin 1, erythroid 11,870 Art1 ADP-ribosyltransferase 1 11,435 Chrna1 cholinergic receptor, nicotinic, alpha polypeptide 1 (muscle) 74,374 Clec16a C-type lectin domain family 16, member A 76,501 Commd9 COMM domain containing 9 12,846 Comt catechol-O-methyltransferase 80,904 Dtx3 deltex 3 homolog (Drosophila) 108,655 Foxp1 forkhead box P1 14,809 Grik5 glutamate receptor, ionotropic, kainate 5 (gamma 2) 56,538 Klk11 kallikrein related-peptidase 11 16,979 Lrrn1 leucine rich repeat protein 1, neuronal 17,286 Meox2 mesenchyme homeobox 2 17,927 Myod1 myogenic differentiation 1 67,405 Nts neurotensin 18,155 Pnoc prepronociceptin 212,127 Proser1 proline and serine rich 1 54,153 Rasa4 RAS p21 protein activator 4 21,384 Tbx15 T-box 15 21,812 Tgfbr1 transforming growth factor, beta receptor I Genes are listed alphabetically All genes described derived from the statistically significant groups (adj. p < 0.1) Sh3bgr, Tnnc2, Tnni2 and Tnnt2 were the most down- in the absence of Tbx1. We therefore focused our inves- regulated genes; whereas in Group C, these were tigations at E13.5, just prior to the period of rapid Csrp3, Sh3bgr, Sln, Tnnc2, Tnni2, Myh7 and Mylpf. growth and elevation . A key finding of this profile is the association between an absence of Tbx1 function and altered expression (pri- Discussion marily downregulation) in a number of muscle-related In the present study, functional explant assays and genes within the shelves of the secondary palate. Devel- microarray analysis of gene expression was carried out oping mononuclear and binucleate myofibril-containing in the palatal shelves of E13.5 mouse embryos WT, het- skeletal muscle cells are identifiable within the palatal erozygous or mutant for Tbx1. This was prompted by shelves at E13  and findings of altered gene ex- the knowledge that Tbx1 is strongly expressed in epithe- pression are perhaps not surprising, given the essen- lium of the palatal shelves throughout palatogenesis, tial role of Tbx1 during the development of mutant embryos demonstrate cleft palate with complete branchiomeric musculature and somite-derived tongue penetrance [23, 24, 47, 48] and the findings that Tbx1 muscles [60–62] and detectable expression in adult has multiple potential roles during normal palatal shelf mouse muscle [63, 64]. In the embryo, Tbx1 activates elevation, elongation and adhesion [47, 48]. It is known the myogenic-determination genes myogenic factor 5 that several regulatory networks underlie signaling be- (Myf5) and myogenic differentiation (MyoD)inthe tween epithelium and mesenchyme during development mesodermal core of pharyngeal arches I and II . of the secondary palate and we sought to discover po- In addition, loss of Tbx1 results in impairment of the tential genetic pathways disrupted during palatogenesis onset of myogenic specification  and Tbx1 Zoupa et al. BMC Genomics (2018) 19:429 Page 14 of 20 Table 3 Validated genes from Groups A and C Gene ID Gene symbol Description Fold Change Group A Fold change Group C P Value Anova Validated genes commonly expressed in Groups A and C 69253 Hspb2 heat shock protein 2 −0.7 −0.94 0.0776 17907 Mylpf myosin light chain, phosphorylatable, fast skeletal muscle −1.1 − 1.15 0.053 140781 Myh7 myosin, heavy polypeptide 7, cardiac muscle, beta −0.97 −1.24 0.0472 50795 Sh3bgr SH3-binding domain glutamic acid-rich protein −1.77 −1.41 0.0433 66402 Sln sarcolipin −1.25 − 1.36 0.0373 12955 Cryab crystallin, alpha B −0.28 − 0.56 0.0332 11443 Chrnb1 cholinergic receptor, nicotinic, beta polypeptide 1 (muscle) −0.74 − 0.67 0.03 17929 Myom1 myomesin 1 −0.41 − 0.88 0.0299 12180 Smyd1 SET and MYND domain containing 1 −1.43 −0.79 0.0277 12299 Cacng1 calcium channel, voltage-dependent, gamma subunit 1 −0.91 − 0.69 0.0221 19400 Rapsn receptor-associated protein of the synapse −3.17 −1.08 0.0202 21925 Tnnc2 troponin C2, fast −1.75 − 1.28 0.0187 21384 Tbx15 T-box 15 − 1.24 − 0.59 0.0176 56437 Rrad Ras-related associated with diabetes −0.69 − 0.51 0.0168 12862 Cox6a2 cytochrome c oxidase subunit VIa polypeptide 2 −1.01 −0.94 0.0132 21828 Thbs4 thrombospondin 4 −1.16 −0.87 0.0103 21953 Tnni2 troponin I, skeletal, fast 2 −1.75 − 1.28 0.00946 11811 Apobec2 apolipoprotein B mRNA editing enzyme, −0.97 − 0.94 0.00473 catalytic polypeptide 2 11609 Agtr2 angiotensin II receptor, type 2 −0.56 − 0.53 0.00368 21956 Tnnt2 troponin T2, cardiac −1.67 − 1.01 0.00323 13009 Csrp3 cysteine and glycine-rich protein 3 −1.58 − 1.56 0.00302 67951 Tubb6 tubulin, beta 6 class V −0.03 − 0.59 0.00251 21955 Tnnt1 troponin T1, skeletal, slow −1.04 −0.82 0.00226 21380 Tbx1 T-box 1 − 0.80 −0.87 0.000242 14066 F3 coagulation factor III −0.81 − 0.52 0.000234 14619 Gjb2 gap junction protein, beta 2 −0.92 − 0.48 0.00000341 Gene ID Gene symbol Description Fold Change P Value (t-test) Validated gene uniquely expressed in Group A 11656 Alas2 aminolevulinic acid synthase 2, erythroid −0.65 0.0062 Validated genes uniquely expressed in Group C 11,733 Ank1 ankyrin 1, erythroid −0.21 0.102 11435 Chrna1 cholinergic receptor, nicotinic, alpha polypeptide 1 (muscle) −0.69 0.025 Genes are listed based on p value synergizes with the myogenic factor Myf5 for initiation of embryos might explain the absence of Myf5 and MyoD myogenic cell fate . Our array failed to identify vari- gene transcripts . In addition, both skeletal, smooth ation in Myf5 and MyoD, but verified downregulation of and non-muscle contractile systems have been identified Myf7 at E13.5 in mutant palatal shelves. This finding sug- and implicated in the process of normal palatal shelf ele- gests that Tbx1 functions upstream of myosin heavy chain vation [66, 67]. A number of the downregulated genes 7 (Myh7) during palatal shelf formation and just prior to identified have also been implicated in the process of skel- elevation, possibly as a myogenic factor. The presence of etal and cardiac muscle contraction (Tnni2, Tnnt1, Myh3, asymmetric expression patterns of myogenic regulatory Myom1, Tnnc2), which might reflect the lack of skeletal factors in early first arch-derived muscles of Tbx1 mutant myogenic determination. Interestingly, microarray analysis Zoupa et al. BMC Genomics (2018) 19:429 Page 15 of 20 Fig. 5 Quantitative reverse transcriptase polymerase chain reaction verification of genes identified in Groups A and C following the microarray analysis. a Common genes significantly changed between both WT-mutant and heterozygote-mutant; (b) genes significantly changed only between WT-mutant (Group A); (c) genes significantly changed only between heterozygote-mutant (Group C) + +/− of the early pharyngeal region of Df1/ ;Tbx1 embryos sites where fusion is required . In Tbx1 mutant mice, has previously demonstrated upregulation of Tnnc2 . aberrant oral adhesions between tongue and palatal It cannot be discounted that other intrinsic contractile shelves have been observed . In the present study, the systems might also be disrupted in the secondary palate of tight junction genes Myh3, Mylpf, Myh7 and Actn2 were Tbx1 mutant mice. Indeed, changes in expression levels downregulated in mutants at E13.5, suggesting a potential were also identified in genes associated with intracellular role for Tbx1 in the normal function of tight junctions calcium signaling (Atp2a1, Tnnc2, Cacna1s, Tnnc1), which present within the palatal shelf epithelium. is known to mediate a number of important physiological Comparison between WT-mutant and heterozygous-- processes of relevance to palatogenesis, including skeletal mutant shelves revealed 58 genes commonly expressed and smooth muscle contraction, apoptosis, cell motility in both groups. From these, 27 genes from Group A and and proliferation . 28 genes from Group C were selected for gene expres- After palatal shelf elevation, periderm cells joined by sion verification. Analysis revealed significant downregu- tight junctions are believed to function as a protective lation of 26 genes common to both groups (see Fig. layer, preventing aberrant adhesions and playing an im- 5a)with(Alas2)and (Ank1, Chrna1) individually portant role in mediating appropriate shelf adherence and downregulated in each group, respectively (see Fig. epithelial differentiation [70, 71]. Loss of periderm is re- 5b). Statistical analysis revealed significant downregu- quired at the tips of opposing palatal shelves and overall at lation of all genes tested through RT-PCR with the Zoupa et al. BMC Genomics (2018) 19:429 Page 16 of 20 exception of Ank1 (p = 0.102; see Fig. 5b). Pathway mesenchyme in the palate (Fig. 6). Indeed, the associa- analysis of these validated genes confirmed the associ- tions between Tbx1 function and muscle contraction ations between cardiac muscle contraction and cal- and calcium signaling, both activities that take place in cium signaling, but also suggested links with dilated the early mesenchyme, are consistent with this. In and hypertrophic cardiomyopathies. Although addition, Tbx1 seems to act co-operatively with Shh sig- 22q11.2DS is commonly associated with conotruncal naling in the palate, through the repression of Bmp4 and congenital heart defects, hypocalcemic dilated myocar- induction of Pax9. Interestingly, this co-operative activ- diopathy has also been described in association with ity would appear to be dependent upon Fgf signaling; this condition . RT-PCR validation of the micro- Shh in the epithelium is dependent upon reciprocal sig- array analysis demonstrated no significant changes in naling with Fgf10 in the mesenchyme  and our ex- gene expression between WT and heterozygous plant studies demonstrate that Tbx1 is also dependent shelves, consistent with the normal palatogenesis seen upon Fgf signaling. Although it is currently not known in heterozygous embryos . which Fgf ligand is required or whether this is within Tbx1 is known to regulate both Fgf8 and Fgf10 expres- the epithelium or mesenchyme, maintenance of epithe- sion in the early pharyngeal arches and cardiac outflow lial Tbx1 transcription is essential for normal palatogen- tract  and influence the spatial distribution of Fgf8 esis. Conditional loss of Tbx1 in either craniofacial and Bmp4 in the early mandible . It has also been mesenchyme  or mesoderm  does not result in suggested that Fgf8 is significantly downregulated in the cleft palate, in contrast to loss-of-function in the oral palatal shelf epithelium, whilst Fgf10 is upregulated in epithelium, which does . the mesenchyme at E13.5 in Tbx1 mutant embryos . However, we found no evidence of altered transcript Conclusions levels associated with these genes in our array. This We have conducted functional microarray analysis and same report also demonstrated diminished hyaluronic PCR validation of gene expression in the developing sec- acid (HA) in the palatal shelves of Tbx1 mutant mice ondary palate at E13.5 in the Tbx1 mutant embryo. Dif- and whilst we found no obvious genetic links to this ferentially regulated genes were detected in the absence finding within our array, HA has been shown to induce of this transcription factor. In the microarray, a total of matrix metalloproteinase 9 (MMP9), which was 89 genes demonstrated differential expression in Group downregulated. However, whilst some members of the A and 88 genes in Group C (adj. p < 0.1), whilst MMP family have been directly related to palatogenesis, high-throughput quantitative RT-PCR confirmed 27 at least in vitro; this did not include MMP9 . genes significantly changed between WT and mutant and In this microarray experiment, RNA was derived from 28 between heterozygote and mutant. Associations existed whole dissected palatal shelves and therefore no formal with cardiac muscle development, hypertrophic and di- distinction was made between changes in epithelial and lated cardiomyopathy, tight junction and calcium signal- mesenchymal gene activity. Tbx1 is localized to the pal- ing. These findings provide further evidence of a primary atal shelf epithelium at E13.5, but is clearly able to influ- role for Tbx1 during the process of palatogenesis. ence signaling activity between epithelium and Fig. 6 Molecular associations linking Tbx1 with Fgf and Shh signaling in the developing palate. Tbx1 in the palatal shelf epithelium is downstream of Fgf signaling, the ligand/s and source (epithelium/ mesenchyme) are currently unknown. Shh-Fgf10-Fgfr2b epithelial-mesenchymal reciprocal signaling  antagonizes Bmp4  and induces Pax9 indirectly through the induction of Osr2 [55, 82]. We and others  have demonstrated that Tbx1 acts to inhibit Bmp4 and induce Pax9. It has been suggested that Tbx1 activity is required for Fgf8 induction in the epithelium and Fgf10 inhibition in the mesenchyme ; however, we and others  have found no evidence of this Zoupa et al. BMC Genomics (2018) 19:429 Page 17 of 20 Methods total, 9 sets of RNA were collected, each derived from Mice paired secondary palatal shelves harvested from each Breeding mice were maintained in ventilated cages on an embryonic genotype (giving 3 samples from each alternating (12:12) light-dark cycle in the Biological genotype). Services Unit at King’s College London. Time-mated Tbx1 +/− embryos were generated by inter-crossing Tbx1 mice Microarray chip processing and data analysis on a C57/Bl6 background  such that noon of the day The expression profiling analysis was carried out at the on which vaginal plugs were detected was considered as Franklin-Wilkins Building Genomics Facility, King’s embryonic day (E) 0.5. Pregnant females were euthanized College London. Total RNA was reverse-transcribed and with cervical dislocation. cRNA generated using the MessageAmp II-Biotin Enhanced cRNA Amplification Kit (Ambion). cRNA tar- Explant culture gets were then hybridized to the Affymetrix Mouse Gene- Secondary palatal shelves were carefully micro-dissected Chip microarray (MOE430_A_2 GeneChip array), which from E13.5 WT embryos and cultured for 24 h in the is a single array containing 22,690 probe sets representing presence of cyclopamine or SU4502 as previously de- transcripts and variants from over 14,000 well character- scribed . Briefly, explants were cultured using a ized mouse genes. A single chip was used for each pair of modified Trowell technique at 37 °C in an atmosphere palatal shelves per genotype, with hybridization and scan- of 5% CO2 in serum-free Advanced DMEM/F12 (Gib- ning of array chips carried out according to recommended coBRL) supplemented with 20 U/ml penicillin and strepto- protocols (www.affymetrix.com). mycin (GibcoBRL), 10% Fetal Bovine Serum (GibcoBRL), Microarray data were analysed by the implementa- 50 mM transferrin (Sigma) and 150 μg/ml ascorbic acid tion of Bioconductor packages in the programming (Sigma). SU5402 (Calbiochem) was diluted in medium language R. Intensity values of every chip were from a 10 mM stock solution in DMSO and cyclopamine imported and evaluated with the packages affy, sim- (Sigma) was diluted from a 20 mg/ml stock solution in pleaffy and affyPLM. Pre-processing, normalization ethanol and added to the culture medium at a final and expression transformations were executed by the concentration of 75 μM for both inhibitors. A minimum function rma of the affy package . Gene expres- of (n = 6) palatal shelves were used for each experiment. sions were fitted to linear models and moderated t-statistics were calculated for specific comparisons In situ hybridisation using lmfit and eBayes functions of the limma pack- Wholemount digoxygenin and section S radioactive age . P-values were adjusted for multiple testing in situ hybridisation was carried out as previously de- with the Benjamini & Hochberg FDR method , scribed . Wholemount (n =6 palatal shelves) and implemented within the topTable function of the section (n = 3 embryos) images were photographed limma package. Venn diagram and heatmap showing using Leica or Zeiss Axioscop microscopes, respect- hierarchical clustering with complete linkage scaled ively. For radioactive in situ hybridisation, light and by genes were constructed using the packages darkfieldimagesweremergedin Adobe photoshop CS. VennDiagram and gplots respectively. Microarray Plasmid cDNA was kindly provided by the following datasets have been submitted to the Gene Expression investigators: Bmp4 (Brigid Hogan); Fgf8 (Ivor Mason); Omnibus (GEO) at NCBI (GSE37904). Fgf10; Fgfr2b (David Rice); Pax9 (Heiko Peters); Ptch1 (Matthew Scott); Shh (Andy McMahon); Sprty2 (M. Albert Basson), Tbx1 (Peter Scambler). Functional annotation of differentially regulated gene sets In this study WEB-based GEne SeT AnaLysis Toolkit Tissue preparation and microarray analysis (WebGestalt, http://www.webgestalt.org/option.php, Secondary palatal shelves were carefully version 05/20/2014) was utilized to perform func- micro-dissected from E13.5 Tbx1 WT, heterozygous tional enrichment analysis on the data sets containing −/− or mutant embryos (3 embryos per genotype), stored genes from the Tbx1 versus WT shelves compari- −/− +/− as pairs from each embryo in RNAlater (Ambion) son (Group A), the Tbx1 versus Tbx1 shelves and then homogenized using a blunt 20-guage needle comparison (Group C) and the commonly expressed to an RNase-free syringe. Total RNA was extracted gene set of Group A and Group C. For each gene set, from homogenate derived from each shelf pair using WebGestalt used the hypergeometric test to evaluate an RNeasy Isolation Kit (Qiagen). RNA quality was functional enrichment against predefined categories checked using an Agilent Bioanalyzer and quantified collected from KEGG. Statistical analysis was per- with spectrophotometry (NanoDrop ND-1000). In formed according to the current default settings. Zoupa et al. BMC Genomics (2018) 19:429 Page 18 of 20 Validation with high throughput quantitative real-time Additional file 2: List of genes differentially expressed in WT compared RT-PCR and data analysis to heterozygous palates (n = 400) (Group B). (XLSX 33 kb) Candidate genes were validated with high-throughput Additional file 3: KEGG pathway analysis. (XLSX 11 kb) real time quantitative RT-PCR using the same nine Additional file 4: Quantitative RT-PCR primer/probe list. This table contains a complete list of the 63 primers/ probes used in the real-time total RNA samples from the microarray screen. RNA quantitative RT-PCR analysis of gene expression in the developing palate was converted to first-strand cDNA using the High of Tbx1 mice. (DOCX 90 kb) Capacity RNA-to-cDNA kit (Applied Biosystems). Real time PCR assays were identified using Applied Abbreviations Biosystems UmapIt tool to map microarray probeset 22q11.2DS: 22q11.2 deletion syndrome; Bmp: Bone morphogenetic protein; IDs to inventoried Taqman(r) assays. cDNA samples CAFS: Conotruncal anomaly face; DGS: DiGeorge syndrome; Fgf: Fibroblast growth factor; Fgfr2b: Fibroblast growth factor receptor 2b; KEGG: Kyoto and assay master mixes were combined on 384-well Encyclopedia of Genes and Genomes; Myf5: Myogenic factor 5; Myh7: Myosin real-time PCR plates (Applied Biosystems) using the heavy chain 7; MyoD: Myogenic differentiation; OFT: Outflow tract; Biomek FX liquid handling robot (Beckman Coulter). Pax9: Paired box 9; Ptch1: Patched 1; RT-PCR: Reverse Transcription Polymerase Chain Reaction; Shh: Sonic hedgehog; Smad: Mothers against A total of nine 384-well plates were used. Each cDNA decapentaplegic homologue; Spry2: Sprouty 2; TBX1: Transcription factor- sample was combined with each gene primer se- encoding T-Box 1; VCFS: Velocardiofacial syndrome; WT: Wild Type quence and replicated across four wells, giving four technical replicates for each PCR reaction. Each Acknowledgements 384-well plate contained a column for water (no-tem- The authors are grateful to Antonio Baldini and Peter Scambler for allowing access to the Tbx1 mouse line and Alex Huhn for expert mouse husbandry. plate control) and ß-Actin (house-keeping gene/en- dogenous control for data normalization) with a Funding 7900HT Quantitative PCR machine (Applied Biosys- This work was funded by a European Orthodontic Society Research Grant (to MTC). tems) used for the PCR reaction. The qPCR data was analysed using RQ manager (Applied Biosystems) and Availability of data and materials Microsoft Excel. The RQ manager uses CT values The microarray datasets generated and analysed during the current study are available in the Gene Expression Omnibus (GEO) repository at NCBI from the qPCR reaction along with normalisation of (GSE37904). https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE37904 the data to provide Relative Quantification (RQ) values (RQ = 2–ΔΔCT) for gene expression. For the Authors’ contributions 26 commonly expressed genes from Group A and C, MZ carried out the microarray analysis; in situ hybridization, analysed data ANOVA was used to detect statistically significant and wrote the manuscript; GMX carried out explant culture, in situ hybridization and wrote the manuscript; SB carried out RT-PCR analyses and differences in Relative Quantification group means be- analysed the data; IT performed bioinformatic analyses and critically revised tween WT, heterozygous and mutant genotypes. The the manuscript; MA supervised the microarray and validation analyses, differences in Relative Quantification for the uniquely analysed data and wrote the manuscript; MTC devised the experiments, analysed the data and wrote the manuscript. All authors have read and expressed Alas2 in Group A and Ank1, Chrna1 genes approved the manuscript. between the WT and the MUT (Group C) were ana- lysed by using t-test. All the above statistical analyses Ethics approval and graphs designs were performed in R. For the The welfare of animals used in research in the United Kingdom is protected by law. The Animal Scientific Procedures Act 1986 (ASPA) and Amendment graphs, the ggplot2 package was used (see Fig. 5). Regulations 2012 protects all animals used in procedures for scientific From the 29 genes selected from microarray analysis, purposes. This act is implemented by the Animals in Science Regulation Unit 28 individual genes showed significant changes in ex- (ASRU) of the United Kingdom Government Home Office. All animal work was approved by King’s College London Animal Welfare and Ethical Review pression levels in the mutant compared to WT and/or Body (AWERB) and carried out according to United Kingdom Government heterozygote (P value < 0.05), whereas only Agtr2 Home Office guidelines under project license number PPL70/7866. from Group C was shown to be non-statistically sig- nificant (P value = 0.102), in qPCR. Competing interests The authors declare that they have no competing interests. Additional files Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Additional file 1: Tbx1 lacZ reporter expression in the developing murine palate. (A) E12.5; (B) E13.5; (C) E14.5; (D) E15.5. Tbx1 is expressed in Author details epithelium of the primary (yellow arrowhead) and secondary palate Centre for Craniofacial Development and Regeneration, King’s College (white arrowhead) with expression persisting in these regions during the London Dental Institute, Floor 27, Guy’s Tower, London SE1 9RT, UK. process of fusion (orange and pink arrowheads, respectively). Expression Department of Orthodontics, King’s College London Dental Institute, is also seen in the maxillary incisor tooth germs (green arrowhead), London, UK. Division of Development and Gene Expression, Institute of maxillary molar tooth germs (red arrowhead) and palatal rugae (black Molecular Biology and BiotechnologyFoundation for Research & Technology, arrows). (TIF 2146 kb) Crete, Greece. Genomics Centre, King’s College London, London, UK. Zoupa et al. BMC Genomics (2018) 19:429 Page 19 of 20 Received: 1 March 2018 Accepted: 11 May 2018 chromosomal region homologous to the mouse Tbx1 gene. 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Published: Jun 4, 2018