RESEARCH ARTICLE 1137

Development 137, 1137-1147 (2010) doi:10.1242/dev.045534 © 2010. Published by The Company of Biologists Ltd b-catenin deficiency causes DiGeorge syndrome-like through regulation of Tbx1 Sung-Ho Huh and David M. Ornitz*

SUMMARY DiGeorge syndrome (DGS) is a common genetic disease characterized by pharyngeal apparatus malformations and defects in cardiovascular, craniofacial and glandular development. TBX1 is the most likely candidate disease-causing gene and is located within a 22q11.2 chromosomal deletion that is associated with most cases of DGS. Here, we show that canonical Wnt–b-catenin signaling negatively regulates Tbx1 expression and that mesenchymal inactivation of b-catenin (Ctnnb1) in mice caused abnormalities within the DGS phenotypic spectrum, including great vessel malformations, hypoplastic pulmonary and aortic arch arteries, cardiac malformations, micrognathia, thymus hypoplasia and mislocalization of the parathyroid gland. In a heterozygous Fgf8 or Tbx1 genetic background, ectopic activation of Wnt–b-catenin signaling caused an increased incidence and severity of DGS- like phenotypes. Additionally, reducing the gene dosage of Fgf8 rescued pharyngeal arch artery defects caused by loss of Ctnnb1. These findings identify Wnt–b-catenin signaling as a crucial upstream regulator of a Tbx1–Fgf8 signaling pathway and suggest that factors that affect Wnt–b-catenin signaling could modify the incidence and severity of DGS.

KEY WORDS: b-catenin, Tbx1, Fgf8, Pharyngeal arch, DiGeorge syndrome

INTRODUCTION is expressed in the pharyngeal arch endoderm, core mesoderm, DiGeorge syndrome (DGS) is one of the most common genetic anterior heart field and head mesenchyme, but is absent in neural disorders with an incidence of 1 in 4000 live births. More than 90% crest-derived mesenchyme (Chapman et al., 1996; Torres-Juan et al., of DGS cases are associated with hemizygous deletion of 2007; Vitelli et al., 2002a). chromosome 22q11.2 (Lindsay, 2001; Scambler, 2000). Among the In PA development, Tbx1 regulates fibroblast growth factor genes in this region, loss of the Tbx1 transcription factor is thought (Fgf) signaling by regulating the expression of Fgf8 and fibroblast to be the major etiology of DGS phenotypes in humans (Baldini, growth factor receptor 1 (Fgfr1) (Hu et al., 2004; Park et al., 2003). The spectrum of DGS pathologies includes defects in 2006). Tbx1 and Fgf8 compound heterozygotes result in more pharyngeal arch artery formation and/or remodeling, cardiac severe phenotypes than Tbx1 heterozygotes, indicating that these outflow tract and ventricular and/or atrial septal defects, thymus and genes interact genetically (Vitelli et al., 2002b). Sonic hedgehog parathyroid aplasia/hypoplasia and craniofacial anomalies (Lindsay, (Shh) promotes Tbx1 expression in the PA region through a Fox 2001; Scambler, 2000). All of these phenotypes are caused by transcription factor-dependent mechanism (Garg et al., 2001; malformation of a transient embryonic structure called the Yamagishi et al., 2003). pharyngeal apparatus (Wurdak et al., 2006). The pharyngeal Wnt proteins are highly conserved, secreted, cysteine-rich apparatus comprises pharyngeal arches (PAs) and pharyngeal glycoproteins that bind to frizzled (Fzd) receptors. In vertebrates, 19 pouches. PAs are composed of ectoderm, endoderm, neural crest- Wnt and 10 Fzd genes have been identified. Activation of Wnt derived mesenchyme and core mesoderm. Coordinated interaction signaling results in increased cytosolic b-catenin (Ctnnb1). among all of these cell types is necessary to form the tissues derived Translocation of b-catenin to the nucleus allows interactions with from the PAs. transcription factors in the T-cell factor/lymphocyte-enhancing In mice, heterozygosity of Tbx1 results in minor cardiovascular factor (Tcf/Lef) family and regulates the transcription of numerous defects, whereas Tbx1-null mice display the most severe features genes implicated in proliferation, differentiation and other cellular characteristic of DGS (Guris et al., 2001; Jerome and Papaioannou, processes (Clevers, 2006). Limited genetic evidence suggests that 2001; Lindsay et al., 2001; Merscher et al., 2001). Transgenic mice Wnt–b-catenin signaling might be involved in pharyngeal apparatus which have an additional human TBX1 gene and patients which have development. Inactivation of Wnt1 and Wnt3, as well as conditional a that stabilizes the TBX1 protein also develop DGS deletion of b-catenin in neural crest-derived cells, results in neural phenotypes (Liao et al., 2004; Torres-Juan et al., 2007; Zweier et al., crest defects, including components of the first PA and cardiac 2007). This suggests that the amount of TBX1 protein is crucial for outflow tract (Brault et al., 2001). A recent study indicates that normal development and that either loss or gain of TBX1 can cause inactivation of b-catenin in anterior heart field progenitors, using DGS phenotypes. Consistent with a role in PA development, Tbx1 conditional targeting genes that are expressed early in development (e.g. Isl1-Cre, SM22-Cre and Mef2c-Cre), causes defects in the outflow tract and right ventricle by inhibiting the proliferation of Department of Developmental Biology, Washington University School of Medicine, Isl1-positive anterior heart field progenitor cells (Ai et al., 2007; St Louis, MO, USA. Cohen et al., 2007; Kwon et al., 2007; Lin et al., 2007; Qyang et al., 2007). Also, constitutive activation of -catenin signaling in anterior *Author for correspondence ([email protected]) b heart field progenitor cells causes enhanced progenitor cell

Accepted 25 January 2010 proliferation and inhibition of differentiation (Ai et al., 2007; Cohen DEVELOPMENT 1138 RESEARCH ARTICLE Development 137 (7) et al., 2007; Qyang et al., 2007). The involvement of Wnt–b-catenin to the manufacturer’s instructions. Results were graphed as relative signaling in pharyngeal apparatus development is suggested by the expression compared with control, where control was scaled to 1. At least expression of b-catenin in PA mesenchyme. three independent dissections were used for each analysis. Here, we show that canonical Wnt–b-catenin signaling is active India ink injection in PA mesenchyme, where it functions to negatively regulate Mouse embryos at various stages were dissected in PBS and injected with expression of Tbx1 and downstream signaling pathways, including India ink by intra-cardiac perfusion using custom-made glass micropipettes. Fgf signaling [Fgf8, Fgfr1 and Pea3 (Etv4 – Mouse Genome Samples were fixed in 10% formalin, washed in PBS, dehydrated in a series Informatics)] and Gcm2. Mesenchymal deletion of b-catenin of graded methanol and cleared using BABB solution (1 benzyl alcohol to disrupts PA artery remodeling and neural crest cell differentiation, 2 benzyl benzoate). Samples were photographed on an Olympus SZX12 leading to abnormalities in the great vessels. Other consequences of stereo microscope. All staining patterns are representative of at least three samples. loss of b-catenin include craniofacial defects, thymic hypoplasia and detachment and mislocalization of the parathyroid gland. Wholemount immunohistochemistry Complementary gain-of-function studies show opposite effects on Embryos were isolated and fixed in 4% PFA overnight at 4°C. Samples were Tbx1 expression and downstream signaling but surprisingly similar washed and dehydrated in a series of graded methanol and stored in 100% DGS-like phenotypes. These findings indicate that Wnt–b-catenin methanol at –20°C until used. Samples were rehydrated and treated with signaling is a crucial upstream factor that regulates the level of Tbx1 H2O2 overnight at 4°C. Samples were washed with PBT, blocked with and downstream signaling molecules that are important for PA blocking solution (2% skim milk, 0.1% Triton X-100 in PBS) and incubated with a primary antibody overnight at 4°C. Samples were washed with PBT development. five times and incubated with secondary antibody conjugated with HRP overnight at 4°C. After secondary antibody incubation, samples were MATERIALS AND METHODS washed five times with PBT and developed using HRP substrate (Vector Mice Laboratories). Samples were photographed on an Olympus SZX12 stereo F/F F(DEx3)/+ lacZ/+ All mouse strains, including Ctnnb1 , Ctnnb1 , Fgf8 , microscope. Tbx1–/+, Dermo1-Cre (Twist2-Cre – Mouse Genome Informatics), Wnt1- Cre and ROSA26 reporter (R26R), have been previously described (Brault Wholemount in situ hybridization et al., 2001; Danielian et al., 1998; Grieshammer et al., 2005; Harada et Embryos were dissected in diethylpyrocarbonate (DEPC)-treated PBS and al., 1999; Jerome and Papaioannou, 2001; Soriano, 1999; Sosic et al., fixed in 4% PFA. After washing, samples were dehydrated in methanol. 2003). To inactivate Ctnnb1 in PAmesenchyme, Dermo1-Cre; Ctnnb1F/+ Samples were rehydrated and washed with hybridization solution, and mice were mated with Ctnnb1F/F mice to generate mice with the genotype incubated overnight with digoxigenin-labeled RNA probes. After washing, Ctnnb1F/F; Dermo1-Cre. These mice are referred to as Ctnnb1Dermo1-Cre. samples were incubated with anti-digoxigenin antibody conjugated with Control mice were of the genotype Ctnnb1F/F or Ctnnb1F/+; Dermo1-Cre. alkaline phosphatase (Roche) and the color reaction was performed using To inactivate Ctnnb1 in neural crest cells, Wnt1-Cre; Ctnnb1F/+ mice alkaline phosphate substrate (Roche). Samples were photographed on an were mated with Ctnnb1F/F mice to generate mice with the genotype Olympus SZX12 stereo microscope. Ctnnb1F/F; Wnt1-Cre. These mice are referred to as Ctnnb1Wnt1-Cre. Control mice were of the genotype Ctnnb1F/F or Ctnnb1F/+; Wnt1-Cre. To Immunofluorescent staining Sections (8 m) from R26RDermo1-Cre embryos were incubated with anti- - ectopically activate Ctnnb1, Ctnnb1F(DEx3)/+ mice were mated with  b galactosidase antibody (Abcam, #ab9361, 1:500) and anti-neurofilament Dermo1-Cre mice to generate mice with a genotype of Ctnnb1F(DEx3)/+; antibody (Developmental Studies Hybridoma Bank, 2H3, 1:500). Alexa Dermo1-Cre. These mice are referred to as Ctnnb1(DEX3)Dermo1-Cre. 555-conjugated anti-mouse secondary antibody (Invitrogen, 1:200) and Control mice were of the genotype Ctnnb1F(DEx3)/+ or Dermo1-Cre. For Alexa 488 anti-chicken secondary antibody (Invitrogen, 1:200) were used detecting expression of Cre recombinase, Dermo1-Cre mice were crossed to visualize the staining pattern. Samples were photographed on an Olympus with R26R mice. All mice were maintained on 129SV/J-C57B6/J mixed FV500 confocal microscope. genetic background. Skeletal preparation b-galactosidase staining E16.5 embryos were fixed for 24 hours in ethanol and 24 hours in acetone. Embryos were dissected in ice-cold PBS and fixed overnight in Mirsky’s Embryos were skinned and then stained with 0.3% Alcian Blue and 0.1% Fixative (National Diagnostics). Samples were washed three times in PBT Alizarin Red for 24 hours, rinsed with H2O and incubated in 1% KOH/20% (PBS, 0.1% Tween-20) and incubated in staining solution (2 mM MgCl2, 35 glycerol solution. Samples were cleared in a series of glycerol solutions mM potassium ferrocyanide, 35 mM potassium ferricyanide, 1 mg/mg X- (50%, 80% and 100%) for 1 week each and then stored in 100% glycerol. Gal in PBT) at 37°C until color reaction was apparent. Samples were washed Samples were photographed on an Olympus SZX12 stereo microscope. in PBT, fixed in 10% formalin and imaged under a dissecting microscope. Samples were then soaked in 30% sucrose overnight, embedded and frozen in OCT solution for cryosectioning. Sections (12 m) were co-stained with  RESULTS Nuclear Fast Red solution, washed with PBS and tap water, dehydrated in a series of ethanol and xylene, mounted and photographed with a Zeiss Canonical Wnt–b-catenin signaling is active in Axioplan 2 microscope. All staining patterns are representative of at least pharyngeal mesenchyme three samples. The involvement of Wnt–b-catenin signaling in PA development is not well established. To investigate whether Wnt–b-catenin RNA isolation, cDNA synthesis and quantitative RT-PCR analysis signaling is involved in PA development, we examined canonical E9.5 and E10.5 embryos were dissected by cutting from the first PAto the Wnt–b-catenin signaling using the TOPGAL reporter mouse line start of the forearm (the PA region excluding heart and outflow tract) and (DasGupta and Fuchs, 1999). At E9.5, TOPGAL b-galactosidase total RNA from this region was isolated using the RNeasy Kit (Qiagen) activity was first found in core mesenchyme of the PAs and in following the manufacturer’s instructions. cDNA was synthesized using Ј the SuperScript II First-Strand cDNA Synthesis Kit (Invitrogen). pharyngeal mesoderm (Fig. 1A,A ). This finding was in agreement Quantitative RT-PCR was performed on an ABI 7500 Thermocycler using with other canonical Wnt–b-catenin reporters, such as BATGAL and TaqMan probes for Tbx1 (ABI, Mm00448948_m1) and Fgf8 (ABI, Tcf/Lef-lacZ transgenic lines published by other groups (Cohen et Mm00438921_m1). Gapdh (ABI, Mm99999915_g1) was used to al., 2007; Lin et al., 2007). Canonical Wnt–b-catenin signaling was

normalize samples. Amplification and analysis were performed according also known to be activated in the cardiac outflow tract as early as DEVELOPMENT b-catenin regulation of pharyngeal arch development RESEARCH ARTICLE 1139

E8.5 with continued activity through E10.5 (Cohen et al., 2007; Lin Inactivation of mesenchymal b-catenin results in et al., 2007). These observations indicate that canonical Wnt–b- DGS-like phenotypes catenin signaling is continuously activated in PA mesenchyme To investigate the function of canonical Wnt–b-catenin signaling in during PA development. PA mesenchyme, we examined conditional knockout mice in which a floxed allele of Ctnnb1 was inactivated with Dermo1-Cre. The Dermo1-Cre allele expresses Cre recombinase in most mesenchymal regions. To examine Dermo1-Cre activity in pharyngeal mesenchyme, we crossed Dermo1-Cre mice with ROSA26 reporter (R26R) mice (Soriano, 1999) to generate Dermo1- Cre; R26R embryos (referred to as R26RDermo1-Cre). In R26RDermo1-Cre embryos, Cre recombinase activity using R26R-b- galactosidase staining was detected as early as E9.5 in PA core mesenchyme, head mesenchyme and pharyngeal mesenchyme (Fig. 1B). However, at this stage, Cre recombinase activity was absent from mesenchyme that originated from neural crest-derived cells. At E10.5, b-galactosidase activity was detected throughout pharyngeal mesenchyme regions, including mesenchyme that originated from neural crest (Fig. 1C-E). Dermo1-Cre was then used to inactivate a floxed allele of b-catenin by generating Dermo1-Cre; Ctnnb1F/F embryos (referred to as Ctnnb1Dermo1-Cre). Immunohistochemistry was performed to determine the extent to which Wnt–b-catenin was deleted in tissue expressing Cre recombinase. At E10.5, expression of b-catenin was markedly reduced in PA mesenchyme of Ctnnb1Dermo1-Cre embryos, although its expression was retained in pharyngeal arch epithelia (Fig. 1F,G). Beginning at E12.5, edema in the head and back, and hemorrhage around PA structures, was evident in Ctnnb1Dermo1-Cre embryos (see Fig. S1A,B in the supplementary material). This persisted through E17.5 (see Fig. S1C-F in the supplementary material; data not shown). Control embryos did not show any edema or hemorrhage at these stages of development. Local edema and hemorrhage indicated that Ctnnb1Dermo1-Cre embryos developed cardiovascular problems. Therefore, we analyzed the cardiovascular system of Ctnnb1Dermo1-Cre embryos and compared them with control embryos (Table 1). To examine great vessel morphology, India ink was injected intracardially and visualized at E16.5. In control embryos, the innominate and left common carotid artery normally branch off the aortic arch in close proximity (Fig. 2A; see Fig. S2A in the supplementary material). In Ctnnb1Dermo1-Cre embryos, the distance widens (arrow in Fig. 2B; see also Fig. S2B,C in the Fig. 1. Dermo1-Cre-mediated recombination during PA supplementary material) and the right subclavian artery originates development. (A)TOPGAL b-galactosidase expression was observed in aberrantly from the descending aorta (arrowhead in Fig. 2B; see the core mesenchyme of pharyngeal arch (PA)1 and PA2 and in also Fig. S2B,C in the supplementary material). Aortic arch pharyngeal mesenchyme at E9.5. b-galactosidase expression was also hypoplasia was also evident in Ctnnb1Dermo1-Cre embryos (see Fig. detected in the endocardial cushion in cells undergoing epithelial S2B, dashed arrow, in the supplementary material). The (endocardial)-mesenchymal transition. (AЈ)Magnified view of A showing Dermo1-Cre Dermo1- pulmonary artery branches were hypoplastic in Ctnnb1 TOPGAL expression in PA core mesenchyme (arrows). (B)E9.5 R26R embryos compared with control embryos (Fig. 2C,D; see also Fig. Cre embryo showing trace b-galactosidase activity in pharyngeal mesenchyme (arrow). R26RDermo1-Cre was also active in the endocardial S2C in the supplementary material). The heart of control embryos cushion (arrowhead). However, Dermo1-Cre was not active in neural showed normal septation of the ventricle and the atrium and crest-derived mesenchyme in the PAs at this stage. (C)E10.5 R26RDermo1- alignment of the aortic arch and pulmonary trunk (Fig. 2E,G). Dermo1-Cre Cre embryos showing b-galactosidase activity in all PA mesenchymal cells However, Ctnnb1 embryos contained ventricular and including neural crest-derived mesenchyme. No Cre activity was found in atrial septal defects with 100% penetrance (Fig. 2F; Table 1). the myocardium. (D-G)Co-immunohistochemistry showing Ctnnb1Dermo1-Cre embryos also showed other cardiac defects, b-galactosidase (green) and b-catenin expression (red) in PA mesenchyme including overriding aorta (53%), double-outlet right ventricle Dermo1-Cre at E10.5. (D,E) R26R embryos showing that b-galactosidase (27%) and persistent truncus arteriosus (6.7%; Fig. 2E-H; Table1; expression is restricted to PA mesenchyme and does not overlap with data not shown). In addition to these cardiovascular defects, 100% DAPI-stained (blue) nuclei in ectoderm and endoderm (arrow). of Ctnnb1Dermo1-Cre embryos showed a small mandible (F)Ctnnb1+/+ embryos show staining for b-catenin in PA mesenchyme, (micrognathia; arrowhead in Fig. 2I,J,O,P; Table 1), low-set small ectoderm and endoderm. (G)In Ctnnb1Dermo1-Cre mice, b-catenin expression was decreased in PA mesenchyme. h, heart; hm, xxx ? xxx; ov, external ears (arrow in Fig. 2I,J; Table 1), cleft palate (Table 1; otic vesicle; pa1, first pharyngeal arch; pa2, second pharyngeal arch; pm; data not shown), a detached and hypoplastic thymus (Fig. 2K,L), pharyngeal mesenchyme. Scale bar: 400m in C; 200m in AЈ,B,D-G. mislocalized parathyroid glands (next to the thymus; Fig. 2M,N), DEVELOPMENT 1140 RESEARCH ARTICLE Development 137 (7)

Table1. DiGeorge syndrome-like phenotypes in Ctnnb1Dermo1-Cre embryos Cardiovascular Gland Craniofacial Genotype SCAD AAD OA DORV PTA VSD/ASD TD PD CP MG EED Control 0/14 0/14 0/24 0/24 0/24 0/24 0/14 0/14 0/14 0/14 0/14 CKO# 8/8 8/8 8/15 4/15 1/15 15/15 8/8 8/8 8/8 8/8 8/8 #CKO, Ctnnb1Dermo1-Cre; SCAD, subclavian artery defect; AAD, aortic artery defect; OA, overriding aorta; DORV, double-outlet right ventricle; PTA, persistent truncus arteriosus; VSD, ventricular septal defect; ASD, atrial septal defect; TD, thymus defect; PD, parathyroid gland defect; CP, cleft palate; MG, micrognathia; EED, external ear defect. poorly calcified craniofacial bones and a missing tympanic ring R26RDermo1-Cre embryos. Neurofilament-positive neuronal cells were (arrow in Fig. 2O,P). This repertoire of phenotypes (Table 1) is negative for b-galactosidase, indicating that the neuronal component within the spectrum of those of DGS. of neural crest cells does not activate Dermo1-Cre expression (Fig. 4E-G). This is consistent with Dermo1-Cre activation in PA Mesenchymal Ctnnb1 is necessary for PA artery mesenchyme and neural crest-derived mesenchyme after neural development crest cell migration. To determine whether Ctnnb1 functions earlier Many defects in the great vessels originate from defects in early in neural crest cell development, Wnt1-Cre was used to inactivate PA artery formation and/or remodeling (Graham, 2003). To Ctnnb1 in neural crest cells beginning at E8.5 of development. examine the origin of the great vessel anomalies in Consistent with previous reports, mice with the genotype Ctnnb1F/F; Ctnnb1Dermo1-Cre embryos, India ink injection was used to visualize Wnt1-Cre (referred to as Ctnnb1Wnt1-Cre) showed neural crest the PA arteries in E10.5-E13.5 embryos. At E10.5, there was no differentiation defects (Brault et al., 2001) that were similar to those difference in Ctnnb1Dermo1-Cre embryos compared with controls (Fig. seen in Ctnnb1Dermo1-Cre embryos (Fig. 4A,B,H,I). 3A,B). At this time, a dorsal aorta joins the PA arteries dorsally. At As previously observed, Ctnnb1Wnt1-Cre embryos showed E11.5, in control embryos, the dorsal aorta between the third and persistent truncus arteriosus accompanied by ventricular septal fourth PA arteries normally became hypoplastic and completely defects (see Fig. S3 in the supplementary material) (Kioussi et al., regressed by E13.5 (arrow in Fig. 3C,E). However, in Ctnnb1Dermo1- 2002). To determine whether Wnt–b-catenin signaling within the Cre embryos, the dorsal aorta between the third and fourth PA arteries neural crest is required for normal PA artery development and remained dilated (at E11.5) and still patent (at E13.5) (arrow in Fig. remodeling, India ink was injected to visualize the dorsal aorta. This 3D,F). These data indicate that Ctnnb1Dermo1-Cre embryos have analysis indicated that Ctnnb1Wnt1-Cre embryos had normal PA artery defects in PA artery remodeling. formation and remodeling compared with controls at E11.5 and E12.5 (Fig. 3G-J). These results suggest that the PA artery and Deletion of Ctnnb1 in mesenchyme causes cell neuronal phenotypes result from defects in surrounding tissue and non-autonomous defects in neural crest that loss of Ctnnb1 in neural crest-derived cells does not directly differentiation affect PA artery and neuronal development in Ctnnb1Dermo1-Cre Neural crest cells contribute significantly to craniofacial and embryos. cardiovascular development by migrating through and populating the PAs (Jiang et al., 2000). Deletion of several important molecules, Cell death during PA artery remodeling is such as Tgfb type II receptor and smoothened, in the neural crest cell decreased in Ctnnb1Dermo1-Cre embryos lineage, causes DGS-like phenotypes (Goddeeris et al., 2007; In PA artery remodeling, apoptotic cells are observed in Wurdak et al., 2005). To examine neural crest-derived cell migration mesenchyme surrounding regressed PA arteries (Yashiro et al., and development, control and Ctnnb1Dermo1-Cre embryos were 2007). To identify if this is also the case in the dorsal aorta, E12 stained for neurofilament to identify components of the peripheral embryos were stained using the TUNEL assay. Apoptotic cell death nervous system (PNS). Compared with controls, Ctnnb1Dermo1-Cre was evident in surrounding mesenchyme proximal to the dorsal embryos showed hypoplasia of nerve fibers in dorsal root ganglia aorta between the third and fourth PA arteries in control embryos (arrowheads in Fig. 4A,B). The roots of the vagus (X) and (see Fig. S4A in the supplementary material). However, no apoptotic hypoglossal (XII) nerves were hypoplastic (arrow in Fig. 4B) and cells were detected in Ctnnb1Dermo1-Cre embryos at this stage (see the glossopharyngeal (IX) nerve was missing entirely (dashed arrow Fig. S4B in the supplementary material). in Fig. 4B). Failure to form a normal PNS indicated that Ctnnb1Dermo1-Cre embryos have neural crest cell defects. These Isl1-positive anterior heart field progenitor cells defects could be due to intrinsic defects in neuronal cells or to cell- are not involved in DGS-like phenotypes caused autonomous or cell-non-autonomous defects after neural crest cell by mesenchymal deletion of b-catenin migration. To determine whether early neural crest cells migrated Recent studies indicate that Wnt–b-catenin signaling plays an normally, Ap2a in situ hybridization was used to identify all neural important role in anterior heart field formation and proliferation crest-derived cells at E10.5 (Mitchell et al., 1991). Despite abnormal by modulating Isl1-positive cells and/or directly regulating Isl1 development of the neural crest-derived PNS, there was no expression (Ai et al., 2007; Cohen et al., 2007; Kwon et al., 2007; difference in Ap2a staining at E10.5, suggesting that neural crest cell Lin et al., 2007; Qyang et al., 2007). Isl1 in situ hybridization and migration was intact (Fig. 4C,D). Importantly, Dermo1-Cre real-time qRT-PCR showed that there was no change in recombinase activity in neural crest-derived mesenchyme occurs at expression at E10.5 (Fig. 5A,B; data not shown) and that the relatively late stages of development (starting at E10.5) (Fig. 1C). length of the outflow tract and right ventricle was not changed To determine whether Dermo1-Cre activity was present in neuronal (data not shown). These data suggest that Isl1-positive cells are cells derived from neural crest cells, immunostaining for b- intact and that there is no anterior heart field defect in the Dermo1-Cre

galactosidase and neurofilament was performed at E11.5 on Ctnnb1 line. DEVELOPMENT b-catenin regulation of pharyngeal arch development RESEARCH ARTICLE 1141

Fig. 2. Mesenchymal deletion of b-catenin causes DGS-like phenotypes. (A-D)Vascular structure of the aortic arch branches in E16.5 embryos visualized with India ink injection into the left ventricle. (A,C)Control embryo showing a normal branching pattern. (B,D)Ctnnb1Dermo1-Cre mutants showing increased separation between the innominate and left common carotid artery (arrow), abnormal origin of the left subclavian artery (arrowhead), and pulmonary artery hypoplasia. (E-H)Hematoxylin and Eosin (H&E)-stained sections of E16.5 control (E,G) and Ctnnb1Dermo1-Cre (F,H) embryos. Ctnnb1Dermo1-Cre embryos showed ventricular and atrial septal defects (F) and overriding aorta (H) anomalies. (I-L)Micrognathia (small mandible, arrowhead) and small outer ear (arrow) in E16.5 Ctnnb1Dermo1-Cre (J) compared with control (I) embryos. (K,L)Detached and decreased thymus size in Ctnnb1Dermo1-Cre embryos. (M,N)H&E-stained section of the neck region showing normal location of parathyroid gland (pth) next to the thyroid gland (thy) in control (M). Ctnnb1Dermo1-Cre embryos (N) showing the parathyroid gland located next to the thymus (th). (O,P)Skeleton preparation stained with Alizarine Red and Alcian Blue showing decreased calcification of cranial bones, missing tympanic ring (arrow) and shortened mandible (arrowhead) in Ctnnb1Dermo1-Cre (P) compared with control (O) embryos. aa, aortic arch; asd, atrial septal defect; e, ear; ia, innominate artery; lca, left common carotid artery; lpa, left pulmonary artery; lsa, left subclavian artery; m, mandible; oa, overriding aorta; pt, pulmonary trunk; pth, parathyroid gland; rca, right common carotid artery; rpa, right pulmonary artery; rsa, right subclavian artery; th, thymus; thy, thyroid gland; tr, tympanic ring; vsd, ventricular septal defect. Scale bars: 200m in E-H; 50m in M,N.

Impaired Tbx1 signaling in Ctnnb1Dermo1-Cre tissue showed a significant (P<0.04 at E9.5 and P<0.005 at E10.5) embryos increase in Tbx1 mRNA in Ctnnb1Dermo1-Cre embryos at E9.5 and Tbx1 has been identified as the most probable disease-causing E10.5 (Fig. 5E,F). Furthermore, the expression of Gcm2, a Tbx1 gene for DGS (Baldini, 2002; Baldini, 2003). Tbx1 is expressed target gene and early marker for the parathyroid gland, was also in PA endoderm and mesoderm and gain-of-function, as well as increased in Ctnnb1Dermo1-Cre embryos compared with controls loss-of-function, mouse mutants of Tbx1 recapitulate defects (Fig. 5G,H). Recent studies identified Fgf8 as a genetic modifier associated with DGS. To test whether Tbx1 is regulated by of Tbx1 in PA development (Hu et al., 2004). Hypomorphic alleles Wnt–b-catenin signaling, Tbx1 expression was examined in or conditional knockout of the Fgf8 gene recapitulates DGS Ctnnb1Dermo1-Cre embryos. At E9.5, Ctnnb1Dermo1-Cre embryos phenotypes, similar to what was observed in Tbx1 hypomorph or showed increased Tbx1 expression in PA mesenchyme compared knockout mice. Consistent with an established link between Tbx1 with controls (Fig. 5C,D). Consistent with the observed increase and Fgf8, at E9.5, Fgf8 expression was increased in PAs in Dermo1-Cre

of Tbx1 expression, quantitative RT-PCR analysis of PA-region Ctnnb1 embryos (Fig. 5I,J). Additionally, the expression DEVELOPMENT 1142 RESEARCH ARTICLE Development 137 (7) of Fgfr1, one of the receptors for Fgf8, was increased in the PAs Activation of Wnt–b-catenin signaling in in Ctnnb1Dermo1-Cre embryos (Fig. 5K,L), consistent with previous mesenchyme suppresses Tbx1 expression and data (Park et al., 2006). Also consistent with increased Fgf8 signaling signaling, Pea3, a target of Fgf8/Fgfr signaling, was increased in The results shown above indicate that Wnt–b-catenin signaling is pharyngeal mesenchyme in Ctnnb1Dermo1-Cre embryos (Fig. necessary to inhibit Tbx1 and downstream signaling molecules. To 5M,N). determine if Wnt–b-catenin signaling is sufficient to inhibit Tbx1 expression in vivo, a Cre-activatable constitutively active allele of Ctnnb1 (Ctnnb1F(DEx3)/+) was mated with Dermo1-Cre mice to generate embryos with the genotype Ctnnb1F(DEx3)/+; Dermo1-Cre [referred to as Ctnnb1(DEX3)Dermo1-Cre]. At E10.5, embryos expressing Ctnnb1(DEX3)Dermo1-Cre showed decreased expression of

Fig. 4. Neural crest cell defects in Ctnnb1Dermo1-Cre embryos. (A,B)Wholemount immunostaining for neurofilament showing defective peripheral neuronal development in Ctnnb1Dermo1-Cre embryos Fig. 3. Mesenchymal b-catenin is required for normal PA artery (B) compared with control (A) at E10.5. Arrowheads, dorsal root remodeling. (A,B)At E10.5, both control (A) and Ctnnb1Dermo1-Cre (B) ganglia; black arrow, root of the vagus and hypoglossal nerve; white embryos showed similar aortic arch artery structures. (C,D)At E11.5, arrow, glossopharyngeal nerve. (C,D)Wholemount ISH for Ap2a there is a normal atrophy of the dorsal aorta between PA artery 3 and showing no difference between control (C) and Ctnnb1Dermo1-Cre 4 (C, arrow). Ctnnb1Dermo1-Cre mutant embryos (D) do not show embryos (D) at E10.5. Arrows indicate neural crest cell migratory paths. regression of the PA artery 3 and 4 channel (arrow). (E,F)By E13.5, the (E-G)Immunofluorescence staining of an E11.5 embryo for dorsal aorta between PA artery 3 and 4 is normally lost (E, red dashed neurofilament (E, red) and b-galactosidase (F, green) showing no line, arrow in inset). Ctnnb1Dermo1-Cre mutant embryos (F) retained the overlap (G) between b-galactosidase-positive cells and neurofilament- dorsal aorta between PA artery 3 and 4 (arrow in inset). (G-J)In positive cells in dorsal root ganglia. (H,I)Wholemount immunostaining Ctnnb1Wnt1-Cre mutant embryos (H,J), the PA artery remodeling for neurofilament showing defective peripheral neuron development in occurred normally with no apparent difference from controls (G,I) at Ctnnb1Wnt1-Cre (I), compared with control (H), embryos. Arrowheads, E11.5 and E12.5, respectively. Arrows indicate the dorsal aorta dorsal root ganglia; arrow, root of the vagus and hypoglossal nerve. spanning PA artery 3 to 4. 3, third pharyngeal arch; 4, fourth V, trigeminal nerve; VII and VIII, acousticofacial nerve; IX, pharyngeal arch; 6, sixth pharyngeal arch; ia, innominate artery; lca, glossopharyngeal nerve; X, vagus nerve; XII, hypoglossal nerve; left common carotid artery; lva, left vertebral artery; rca, right common h, heart; ov, otic vesicle; pa1, first pharyngeal arch; pa2, second carotid artery. pharyngeal arch. Scale bar: 100m in E-G. DEVELOPMENT b-catenin regulation of pharyngeal arch development RESEARCH ARTICLE 1143

Fig. 5. Upregulation of the Tbx1 signaling pathway in Ctnnb1Dermo1-Cre embryos. (A,B)Wholemount ISH of lsl1 at E9.5 showing no difference in Isl1 expression between control and Ctnnb1Dermo1-Cre embryos. (C,D)Wholemount ISH of Tbx1 at E9.5 showing increased expression in the pharyngeal arches (arrows) in Ctnnb1Dermo1-Cre (D) compared with control (C) embryos. (E,F)Quantitative RT-PCR analysis showing increased Tbx1 expression in Ctnnb1Dermo1-Cre embryos (white bar) compared with control (black bar) at E9.5 (E) and E10.5 (F). (G,H) Wholemount ISH of Gcm2 (arrows) at E10.5 showing increased expression in Ctnnb1Dermo1-Cre (H) compared with control (G) embryos. (I,J)Wholemount ISH of Fgf8 at E9.5 showing increased expression in pharyngeal arches (arrow) in Ctnnb1Dermo1-Cre (J) compared with control (I) embryos. (K,L)Wholemount ISH of Fgfr1 at E10.5 showing increased expression in pharyngeal arches (arrows) in Ctnnb1Dermo1-Cre (L) compared with control (K) embryos. (M,N)Wholemount ISH of Pea3 at E9.5 showing increased expression in pharyngeal arches (arrows) in Ctnnb1Dermo1-Cre (N) compared with control (M) embryos. *, P<0.04 in E; *, P<0.005 in F.

Tbx1 (Fig. 6A,B). Consistent with the observed decreased Tbx1 catenin signaling could enhance PA artery phenotypes on a expression, qRT-PCR analysis of PA-region tissue showed a sensitized genetic background (Fgf8lacZ/+ or Tbx1–/+), pregnant mice significant (40%-50%, P<0.002) decrease in Tbx1 mRNA in carrying Fgf8LacZ/+ and Tbx1–/+ embryos were treated with NaCl or Ctnnb1(DEX3)Dermo1-Cre embryos at E9.5 and E10.5 (Fig. 6C,D). The LiCl from E8.5 to E9.5 and examined at E10.5 by dye injection into expression of Gcm2 was also decreased in Ctnnb1(DEX3)Dermo1-Cre the heart. No abnormalities were seen in control embryos treated embryos (Fig. 6E,F). Interestingly, at E9.5 and E10.5, we observed with NaCl or LiCl (Fig. 7A-BЈ; Table 2). NaCl-treated Fgf8lacZ/+ no changes in the expression of Fgf8 using in situ hybridization or embryos also formed normal PA arteries, whereas 7 out of 22 (32%) qRT-PCR (data not shown). However, Fgf8 is normally expressed LiCl-treated Fgf8LacZ/+ embryos showed hypoplastic fourth PA in the PA region at earlier stages of development and, by E9.5, Fgf8 arteries (Fig. 7C-DЈ; Table 2). In the Tbx1–/+ genetic background, expression normally declines to nearly undetectable levels (Ilagan the incidence of fourth PA artery defects also became more severe et al., 2006). To examine the effects of increased Wnt–b-catenin in LiCl-treated embryos compared with NaCl-treated embryos, signaling at earlier stages (before Dermo1-Cre becomes active), we increasing from 78% in NaCl-treated to 100% in LiCl-treated injected pregnant female mice with LiCl to inhibit Gsk3b and to Tbx1–/+ embryos (Fig. 7E-FЈ; Table 2). Increased severity of the activate b-catenin in the embryos. To monitor Fgf8 expression, we phenotype was also evident as bilateral cases of fourth PA defects used an allele of Fgf8 (Fgf8lacZ/+) in which the b-galactosidase gene increased from 52% in NaCl-treated to 96% in LiCl-treated Tbx1–/+ replaced the Fgf8 coding sequence. The b-galactosidase reporter has embryos (Table 2). Additionally, the incidence of unilateral and the advantage of a relatively long half-life, allowing a cumulative bilateral aplasia of the fourth PA artery was also increased when assessment of changes in gene expression (Ilagan et al., 2006). Tbx1–/+ embryos were exposed to LiCl (Table 2). These data Pregnant mice carrying Fgf8lacZ/+ embryos were injected indicated that administration of LiCl enhances the severity of intraperitoneally with 50 l of 1 M NaCl or 1 M LiCl at E8.5. DGS phenotypes in Fgf8lacZ/+ and Tbx1–/+ genetic backgrounds, Embryos stained for b-galactosidase activity at E9.5 showed supporting the hypothesis that Wnt–b-catenin signaling is important decreased Fgf8 expression in pharyngeal mesenchyme in LiCl- for maintaining the proper level of Tbx1 signaling. treated Fgf8lacZ/+ embryos compared with NaCl-treated embryos (Fig. 6G-HЈ). Rescue of PA artery defects in Ctnnb1Dermo1-Cre; Fgf8lacZ/+ compound embryos Enhancement of DGS pathology with activation of If gene dosage of Tbx1 and/or Fgf8 is crucial for DGS-like Wnt–b-catenin signaling phenotypes, we hypothesize that in Ctnnb1Dermo1-Cre embryos, where Because constitutive activation of Wnt–b-catenin signaling in both Tbx1 and Fgf8 expression is increased, reducing the gene mesenchyme decreased Tbx1 and downstream signaling cascades in dosage of Tbx1 or Fgf8 should reduce the severity of the DGS-like PA mesenchyme, we hypothesized that constitutive activation of phenotype. To test this hypothesis, Ctnnb1Dermo1-Cre; Fgf8lacZ/+ Wnt–b-catenin signaling might also enhance DGS phenotypes. compound embryos were injected with India ink at E12.5 and PA Because Ctnnb1(DEX3)Dermo1-Cre embryos die between E11.5 and arteries were visualized (Fig. 8). Comparing Ctnnb1Dermo1-Cre, which E12.5 (data not shown), we could not assess the consequences of shows a failure of regression of the channel between the third and activation of Wnt–b-catenin signaling at later developmental stages fourth PA arteries (100% penetrance), Ctnnb1Dermo1-Cre;Fgf8lacZ/+ using this genetic system. However, one of the early DGS-like embryos showed normalization of regression of this aortic arch phenotypes is hypoplasia of the fourth PA artery. This phenotype channel (8 of 10 channels regressed). Pea3 expression was also occurs in embryos hypomorphic for both Tbx1 and Fgf8 (Lindsay et restored to normal in Ctnnb1Dermo1-Cre; Fgf8lacZ/+ embryos (see Fig.

al., 2001; Vitelli et al., 2002b). To test whether increased Wnt–b- S5 in the supplementary material). Thus, reducing the gene dosage DEVELOPMENT 1144 RESEARCH ARTICLE Development 137 (7)

Fig. 6. Decreased expression of the Tbx1 signaling pathway Fig. 7. Activation of Wnt–b-catenin signaling enhances PA artery following activation of Wnt–b-catenin signaling. developmental anomalies. Pregnant mice were treated with NaCl or (A,B)Wholemount ISH of Tbx1 at E10.5 showing decreased expression LiCl from E8.5 to E9.5. At E10.5, India ink was injected into the left in PAs (arrows) in Ctnnb1(DEX3)Dermo1-Cre (B) compared with control (A) ventricle to visualize PA artery morphology. (A-BЈ) Wild-type embryos embryos. (C,D)Quantitative RT-PCR analysis showing decreased Tbx1 treated with NaCl (A,AЈ) or LiCl (B,BЈ) show normal PA artery formation. lacZ/+ expression in Ctnnb1(DEX3)Dermo1-Cre embryos (white bar) compared (C-DЈ) Fgf8 embryos treated with LiCl (D,DЈ) showing unilateral with control (black bar) at E9.5 (C) and E10.5 (D). (E,F)Wholemount fourth PA artery hypoplasia (arrowhead) compared with normal PA ISH of Gcm2 at E10.5 showing decreased expression in PAs (arrows) in artery formation in NaCl-treated Fgf8lacZ/+ embryos (C,CЈ). (E-FЈ) Tbx1–/+ Ctnnb1(DEX3)Dermo1-Cre (F) compared with control (E) embryos. embryos treated with NaCl show unilateral fourth PA artery hypoplasia –/+ (G,H)Wholemount b-galactosidase staining of Fgf8lacZ/+ at E9.5 (arrowhead, E,EЈ). Tbx1 embryos treated with LiCl show bilateral showing decreased expression in PAs (between arrowheads) in LiCl- fourth PA artery aplasia (F,FЈ). Arrowhead indicates location of the treated Fgf8lacZ/+ embryos (H) compared with NaCl-treated Fgf8lacZ/+ fourth PA arteries. 3, third pharyngeal arch; 4, fourth pharyngeal arch; embryos (G). (GЈ-HЈ) Sections of embryos from G and H showing 6, sixth pharyngeal arch. decreased b-galactosidase activity in pharyngeal mesenchyme (between arrows) in LiCl-treated Fgf8lacZ/+ embryos (HЈ) compared with NaCl treated Fgf8lacZ/+ embryos (GЈ). *, P<0.02 in C; *, P<0.01 in D. Scale bar: 200m in GЈ,HЈ. of Fgf8 can rescue the PA artery defect in Ctnnb1Dermo1-Cre embryos, in pharyngeal apparatus development, we have used the Dermo1- and the Tbx1-Fgf8 signaling axis is at least in part responsible for Cre allele to inactivate Ctnnb1 in the mesenchymal components of the PA artery defects seen in Ctnnb1Dermo1-Cre embryos. the PAs and surrounding mesenchyme. Phenotypes associated with mesenchymal loss of Ctnnb1 are within the spectrum of phenotypes DISCUSSION seen in DGS. Because mutation or changes in gene copy number of Recent studies have identified essential roles for Wnt–b-catenin the Tbx1 transcription factor are closely associated with the etiology signaling in mesenchymal tissues such as bone and lung (Day et al., of DGS, and mesenchymal Tbx1 is necessary and sufficient for 2005; De Langhe et al., 2008; Hu et al., 2005; Yin et al., 2008). To pharyngeal arch development (Zhang et al., 2006), phenotypic

examine developmental requirements for Wnt–b-catenin signaling similarities resulting from loss of mesenchymal Wnt–b-catenin DEVELOPMENT b-catenin regulation of pharyngeal arch development RESEARCH ARTICLE 1145

Table 2. Incidence of fourth PA artery defects in Fgf8lacZ/+ and Tbx1–/+ embryos treated with NaCl or LiCl Right fourth PA artery Left fourth PA artery Abnormal/ Bilateral Fourth PA Genotype Treatment Totala Aplasia Hypoplasia Aplasia Hypoplasia defectsb artery aplasiac Wt NaCl 0/24 0 0 0 0 0 0 Wt LiCl 0/19 0 0 0 0 0 0 Fgf8lacZ/+ NaCl 0/11 0 0 0 0 0 0 Fgf8lacZ/+ LiCl 7/22d 0502 00 Tbx1–/+ NaCl 18/23 7 12 3 12 12 10 Tbx1–/+ LiCl 23/23e 10 11 8 11 22f(4)g 15h aEmbryos were examined by intracardiac dye injection at E10.5-E11.0. Abnormal embryos were scored by the presence of either a hypoplastic or aplastic fourth PA artery. bNumber of embryos with bilaterally abnormal (hypoplastic or aplastic) fourth PA artery. Numbers in parentheses indicate cases of bilateral aplasia of fourth PA arteries. cNumber of embryos with bilateral fourth PA artery aplasia. dSignificantly different from that of Fgf8+/–: NaCl group (Fisher’s exact test, P<0.07). eSignificantly different from that of Tbx1+/–: NaCl group (Fisher’s exact test, P<0.005). fSignificantly different from that of Tbx1+/–: NaCl group (Fisher’s exact test, P<0.002). gSignificantly different from that of Tbx1+/–: NaCl group (Fisher’s exact test, P<0.109). hSignificantly different from that of Tbx1+/–: NaCl group (Fisher’s exact test, P<0.067). signaling suggested possible genetic interactions between Tbx1 or Crkl in thymic development (Guris et al., 2006). The mesenchymal Wnt–b-catenin signaling and Tbx1. Here, we showed observation that Wnt–b-catenin signaling negatively regulates Tbx1 that loss of Wnt–b-catenin signaling in pharyngeal mesenchyme expression suggests that the Tbx1 gene functions as an integrative causes DGS-like phenotypes through a mechanism in which Wnt–b- node for multiple signaling pathways that are active in PA catenin inhibits Tbx1 expression. Most cases of DGS are caused by development. deletion of chromosome 22q11.2, which includes the TBX1 gene. PA mesenchyme comprises neural crest-derived mesenchyme and However, overexpression of human TBX1 in mice, or that mesoderm-derived mesenchyme. Dermo1-Cre is first active in PA stabilize TBX1 in some DGS patients, also causes the same core mesenchyme and later becomes active in the remaining neural spectrum of DGS phenotypes (Torres-Juan et al., 2007; Zweier et crest-derived mesenchyme. Defects in neural crest-derived tissues al., 2007). This indicates that the level of expression of Tbx1 is identified in Ctnnb1Dermo1-Cre embryos could result directly from loss crucial for PA formation. Changes in the level of TBX1 expression of Ctnnb1 in neural crest-derived mesenchyme or indirectly from (either increase or decrease) might be responsible for PA loss of Ctnnb1 in pharyngeal mesoderm. Comparison of phenotypes malformations that are consistent with a diagnosis of DGS. from Ctnnb1Dermo1-Cre and Ctnnb1Wnt1-Cre embryos showed normal PA Understanding the mechanisms that regulate the level of TBX1 could artery remodeling in Ctnnb1Wnt1-Cre compared with Ctnnb1Dermo1-Cre be important for understanding the severity of DGS phenotypes. embryos, suggesting that Wnt–b-catenin signaling in pharyngeal Shh signaling has been shown to activate Tbx1 expression in PA mesoderm-derived mesenchymal cells is important to the expression endoderm and mesoderm through regulation of the Foxa and Foxc of DGS-like phenotypes. Also, deletion of Tbx1 causes neural crest transcription factors (Garg et al., 2001; Yamagishi et al., 2003). defects even though Tbx1 is not expressed in neural crest cells Recent data also indicate that retinoic acid signaling might function (Vitelli et al., 2002a). These observations, in addition to defects in to inhibit Tbx1 expression in paraxial mesoderm or tongue muscle PNS development in Ctnnb1Dermo1-Cre embryos, demonstrate that (Abe et al., 2008; Okano et al., 2008). Also, deletion of one copy of mesenchymal cells function cell-non-autonomously through Tbx1 the Raldh2 gene in Crkl–/+; Tbx1–/+ compound heterozygous mice to regulate neural crest development. reduced thymic hypoplasia, consistent with negative regulation of Wnt–b-catenin signaling has been shown to promote Isl1-positive anterior heart field progenitor cell proliferation and inhibit their differentiation by directly regulating Isl1 transcription factor expression (Tzahor, 2007) Anterior heart field progenitor cells comprise the outflow tract and right ventricle in the heart (Laugwitz et al., 2008) As TOPGAL and BATGAL reporter expression indicated, Wnt–b-catenin signaling is first active in the pharyngeal mesenchymal region at E8.5 and remains active at later stages (Fig. 1) (Cohen et al., 2007; Lin et al., 2007). This suggests that Wnt–b- catenin signaling might be important to maintain pharyngeal mesenchyme, including mesenchyme in the anterior heart field. Reporter gene analysis to identify the expression of Dermo1-Cre indicates that Cre recombinase was first activated at E9.5 in the pharyngeal mesenchyme, including the anterior heart field. Therefore, the defects that result from inactivation of Ctnnb1 using Fig. 8. Reduced gene dosage of Fgf8 rescues PA artery defects in the Dermo1-Cre driver might be due to disruption of anterior heart Dermo1-Cre Ctnnb1 embryos. Embryos were injected with India ink at field progenitor cells. However, as Isl1-positive cells are not affected E12.5. (A)Control embryo showing normal regression of the dorsal in Ctnnb1Dermo1-Cre embryos, the phenotype in Ctnnb1Dermo1-Cre aorta between the third and fourth PA arteries (arrow). embryos is probably independent of Wnt– -catenin signaling in (B)Ctnnb1Dermo1-Cre embryo showing a sustained dorsal aorta between b the third and fourth PA arteries (arrow). (C)Ctnnb1Dermo1-Cre; Fgf8lacZ/+ anterior heart field progenitor cells. We posit that Wnt–b-catenin compound embryo showing restored regression of the dorsal aorta signaling has dual functions in anterior heart field development. between the third and fourth PA arteries (arrow). Boxed regions are When the anterior heart field progenitor cells need to proliferate at ϫ magnified 2 and diagramed below each panel. early developmental stages, Wnt–b-catenin signaling promotes Isl1- DEVELOPMENT 1146 RESEARCH ARTICLE Development 137 (7) positive cell proliferation and inhibits its differentiation. Later, DasGupta, R. and Fuchs, E. (1999). Multiple roles for activated LEF/TCF Wnt–b-catenin signaling functions to inhibit Tbx1 expression and transcription complexes during hair follicle development and differentiation. Development 126, 4557-4568. downstream signaling pathways to maintain proper levels of Tbx1 Day, T. F., Guo, X., Garrett-Beal, L. and Yang, Y. (2005). Wnt/beta-catenin signaling. Sustained expression of Tbx1 causes defects in PA artery signaling in mesenchymal progenitors controls osteoblast and chondrocyte remodeling at E12.5. differentiation during vertebrate skeletogenesis. Dev. Cell 8, 739-750. The mechanism by which Wnt–b-catenin signaling regulates De Langhe, S. P., Carraro, G., Tefft, D., Li, C., Xu, X., Chai, Y., Minoo, P., Hajihosseini, M. K., Drouin, J., Kaartinen, V. et al. (2008). Formation and Tbx1 expression is not known. One possibility is that Tbx1 is differentiation of multiple mesenchymal lineages during lung development is regulated directly by Wnt–b-catenin signaling. However, no regulated by beta-catenin signaling. PLoS ONE 3, e1516. conserved Tcf/Lef binding sites are found in the regions flanking the Garg, V., Yamagishi, C., Hu, T., Kathiriya, I. S., Yamagishi, H. and Srivastava, Tbx1 gene (data not shown). Other possible mechanisms could D. (2001). Tbx1, a DiGeorge syndrome candidate gene, is regulated by sonic hedgehog during pharyngeal arch development. Dev. Biol. 235, 62-73. include direct regulation of Tbx1 through interaction with another Goddeeris, M. M., Schwartz, R., Klingensmith, J. and Meyers, E. N. (2007). transcription factor or regulation of Tbx1 indirectly through Independent requirements for Hedgehog signaling by both the anterior heart modulation of other signaling pathways. However, Shh signaling field and neural crest cells for outflow tract development. Development 134, was not changed in Ctnnb1Dermo1-Cre embryos (data not shown). 1593-1604. Graham, A. (2003). Development of the pharyngeal arches. Am. J. Med. Genet. Whether retinoic acid signaling, the only other known inhibitor of 119A, 251-256. Tbx1, is involved remains to be explored. Grieshammer, U., Cebrian, C., Ilagan, R., Meyers, E., Herzlinger, D. and DGS is a disease that has variable penetrance, ranging from life Martin, G. R. (2005). 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