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. Author manuscript; available in PMC: 2009 Apr 15.
Published in final edited form as: Dev Biol. 2008 Feb 15;316(2):417–430. doi: 10.1016/j.ydbio.2008.02.006

Disruption of Smad4 in neural crest cells leads to mid-gestation death with pharyngeal arch, craniofacial and cardiac defects

Xuguang Nie 1, Chu-xia Deng 2, Qin Wang 3, Kai Jiao 1,4
PMCID: PMC2362382  NIHMSID: NIHMS46602  PMID: 18334251

Abstract

TGFβ/BMP signaling pathways are essential for normal development of neural crest cells (NCCs). Smad4 encodes the only common Smad protein in mammals, which is a critical nuclear mediator of TGFβ/BMP signaling. In this work, we sought to investigate the roles of Smad4 for development of NCCs. To overcome the early embryonic lethality of Smad4 null mice, we specifically disrupted Smad4 in NCCs using a Cre/loxP system. The mutant mice died at mid-gestation with defects in facial primordia, pharyngeal arches, outflow tract and cardiac ventricles. Further examination revealed that mutant embryos displayed severe molecular defects starting from E9.5. Expression of multiple genes, including Msx1, 2, Ap-2α, Pax3, and Sox9, which play critical roles for NCC development, was downregulated by NCC disruption of Smad4. Moreover, increased cell death was observed in pharyngeal arches from E10.5. However, the cell proliferation rate in these areas was not substantially altered. Taken together, these findings provide compelling genetic evidence that Smad4-mediated activities of TGFβ/BMP signals are essential for appropriate NCC development.

Keywords: Smad4, Pharyngeal arch, Pharyngeal arch artery, Outflow tract, facial primordial, Neural crest cells

Introduction

Neural crest cells (NCCs) are a group multi-potent, migratory cells that are generated from the interface of dorsal ectoderm and neural tube along the neuraxis. After generation and specification, NCCs delaminate from the neural fold and migrate ventral-laterally to participate in organogenesis and constitute essential elements of a number of tissues. Their migration is orchestrated into separately aggregated streams. NCCs in the anterior neural tube (forebrain, midbrain, and hindbrain), also known as cranial NCCs, mainly migrate to the regions of presumptive facial primordia, drive the outgrowth of the facial primordia and later form NCC derivatives in the craniofacial region (Creuzet et al., 2005; Graham et al., 2004; Helms et al., 2005; Osumi-Yamashita et al., 1994). Some NCCs from the caudal hindbrain also participate in the formation of the enteric nervous system in the gut (Burns, 2005; Young et al., 2004). In addition, a group of NCCs located caudal to the otic vesicle (otic vesicle to the third somite) migrates to the pharyngeal region and the outflow tract (OFT) of embryonic hearts through pharyngeal arches (PAs) 3, 4 and 6, where they contribute to cardiovascular development and are thus known as cardiac NCCs (Jiang et al., 2000). These cells are essential participants in the formation of the OFT septum, and hence, the division of pulmonary and aortic arteries (Jiang et al., 2000; Kirby and Waldo, 1995). At the trunk level, NCCs give rise to structures such as the dorsal root ganglia and sympathetic ganglia (Graham et al., 2004).

NCCs play essential roles during craniofacial and cardiovascular development in vertebrates. Experimental ablation of NCCs in animal models inevitably leads to a spectrum of craniofacial and cardiovascular anomalies (Bockman et al., 1990; Kirby, 1990; Kirby et al., 1985). The embryologic development of craniofacial elements and some segments of large vessels are closely related, and all are derived from a set of temporary embryonic apparatus, the PAs. In mammals, five pairs of bilaterally symmetric PAs (PA1, 2, 3, 4 and 6) develop initially in a segmental fashion along the anterior-posterior axis. Each arch has an external covering of ectoderm and an inner covering of endoderm. Between these epithelial layers are mesenchymal cells surrounding a pharyngeal arch artery (PAA) (Graham, 2003; Noden and Trainor, 2005). The mesenchymal cells in PAs are derived from both mesodermal cells and NCCs that have migrated from the dorsal neural tube. In PAs, NCCs actively interact with their neighbor cells, and are differentiated into distinct cell types such as vascular smooth muscle cells, chondrogenic cells, osteogenic cells, neurons, and glial cells (Noden and Trainor, 2005; Trainor and Krumlauf, 2001).

Accumulated evidence has indicated that TGFβ/BMP signaling in NCCs is critical for their normal development. Neural crest (NC) disruption of genes encoding receptors of TGFβ/BMP signaling pathways, including Alk2, 3, 5 and Tgfbr2, inevitably leads to a spectrum of defects in the craniofacial, pharyngeal and cardiac regions (Choudhary. et al., 2006; Dudas et al., 2004; Ito et al., 2003; Kaartinen et al., 2004; Stottmann et al., 2004; Wang et al., 2006) (summarized in Table 1). Smad proteins are direct mediators of the TGFβ/BMP signaling from the cell surface to the nucleus. Upon ligand stimulation, TGFβ/BMP receptor kinase phosphorylates (activates) specific members of the receptor activated Smads (R-Smads), which then oligomerize with the common Smad (co-Smad). The Smad complex then translocates into the nucleus to regulate expression of target genes in coordination with other transcription factors.

Table.

Targeted genes NCC generation and migration Major phenotypes
Alk3 (Stottmann et al., 2004) Not apparent defect Mid-gestation death (E11.5), shortened OFT with defective septation, thin-walled ventricle, reduced proliferation of ventricle myocardium
Alk2 (Dudas et al., 2004; Kaartinen et al., 2004) Migration impaired Cardiac defects: Deficient of smooth muscle differentiation, persistent truncus arteriosus (PTA).
Craniofacial defects: cleft palate and hypotrophic mandible
Alk5 (Wang et al., 2006) Not affected Inappropriate remodeling of pharyngeal arch arteries, abnormal aortic sac development, failure in pharyngeal organ migration and persistent truncus arteriosus, increased migrated NCC death
Tgf RII(Choudhary. et al., 2006; Ito et al., 2003) Not affected Persistent truncus arteriosus PTA, interrupted aortic arch (IAA-B), cleft secondary palate, calvaria agenesis
Smad4 (Jia et al., 2007; Ko et al., 2007, and this study) No apparent defect Mid-gestation death (E12.5), thin wall ventricle, hypoplastic ventricular septum, normal proliferation rate but increased apoptosis in PAs, defective craniofacial and OFT morphogenesis
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Smad4 is unique among the Smad genes because it encodes the only co-Smad capable of interacting with both BMP and TGFβ R-Smads in mammals. Additionally, a germline mutation in SMAD4 was recently identified in a human patient with congenital heart disease (Waite and Eng, 2003). It has been well documented that in addition to the “canonical” pathway of Smad mediated transcription, TGFβ/BMP signaling may also transduce signals through non-canonical kinase pathways, including activation of JNK, p38 MAPK and Erk (Massague and Gomis, 2006; Moustakas and Heldin, 2005; ten Dijke and Hill, 2004). Thus, functions of Smad4 in NCCs may not be simply inferred from “adding up” defects caused by inactivation of individual TGFβ/BMP receptors in NCCs. Furthermore, even in certain Smad-mediated responses, Smad4 is not an essential component for activating transcription of target genes (Chu et al., 2004; He et al., 2006). In this study, we investigated the specific roles of Smad4 in both NCC development and morphogenesis of NCC derivatives, with a focus on craniofacial and OFT development. To overcome the early embryonic lethality of Smad4 null mice (Chu et al., 2004; Sirard et al., 2000), we applied a conditional gene inactivation approach to specifically disrupt Smad4 in NCCs. We show here that NCC inactivation of Smad4 causes severe abnormalities during craniofacial, PA, OFT and cardiac development, suggesting that Smad4 plays central roles in mediating TGFβ/BMP signaling during NCC development.

Materials and Methods

Mouse maintenance, genotyping and histological analysis

All procedures are approved by the Institutional Animal Care and Use Committee at the University of Alabama at Birmingham. Wnt1-Cre mice (Danielian et al., 1998) (purchased from The Jackson Laboratory) were crossed with Smad4loxp/loxp mice (Yang et al., 2002) to generate Wnt1-Cre;Smad4loxp/+ male mice, which were crossed with Smad4loxp/loxp female mice to produce Wnt1-Cre;Smad4loxp/loxp embryos. Mouse genotypes were determined with PCR analysis using Cre and Smad4 primers as described previously (Yang et al., 2002). For morphological analysis, all samples were fixed with 4% PFA and processed into paraffin-embedded sections using routine procedures. For whole mount staining, the embryos were stored in 100% methanol at −20°C after PFA fixation before further processing.

Cardiac ink injection

India ink was injected into the embryo ventricles using a pulled capillary tube. Injected embryos were subsequently fixed in 4% PFA overnight, dehydrated and cleared in benzyl benzoate: benzyl alcohol (1:1).

TUNEL, immunostaining and in situ hybridization analysis

TUNEL staining was performed using DeadEnd Colorimetric TUNEL System (Promega) following the manufacturer’s protocol. For cell proliferation analysis, we used an anti-phosphorylated Histone H3 polyclonal antibody (Upstate) to detect the cells in M phase following procedures described previously (Song et al., 2007a; Song et al., 2007b). Whole mount immunostaining for neurofilament was performed using a 2H3 anti-neurofilament monoclonal antibody (Hybridoma Bank at the Univ. of Iowa). Whole mount and section in situ hybridization was performed as previously described (Barnes et al., 1994; Song et al., 2007b).

Results

Deficiency of Smad4 in NCCs led to mid-gestational lethality with PA and facial primordium defects

To identify the roles of Smad4 during NCC development, we specifically disrupted Smad4 in NCCs by crossing Wnt1-Cre;Smad4loxp/+ mice with Smad4loxp/loxp mice. The number of mutants (Wnt1-Cre;Smad4loxp/loxp) obtained conformed to the expected Mendelian ratio until E11.5 (93/351). No live mutant embryo was recovered beyond E12.5, suggesting that embryonic lethality occurred between E11.5 and E12.5. Many mutant embryos isolated between E11.5 and E12.0 showed pericardial edema, dilated heart, and blood pooling in large vessels, suggesting that embryonic lethality is caused by cardiovascular insufficiency.

No obvious gross abnormality was found in mutants at E9.5 or E10.5 (data not shown). At E11.5, craniofacial morphogenesis was visibly delayed in mutants, although their general body size appeared normal. The facial prominences (maxillary, mandibular, lateral nasal and medial nasal prominences) in mutant embryos failed to merge, and therefore could not form a complete face at E11.5 (Fig. 1A, B). The underdevelopment was particularly obvious for the medial nasal prominences; they failed to expand to the midline. The delay in fusion of left and right mandibles appeared subtle, but was consistently observed in all mutants examined at this stage (Fig. 1A, B). The union of the first and the second PAs in mutants was also delayed compared to their littermates (Fig. 1C, D).

Figure 1. Craniofacial defects in Wnt1-Cre;Smad4loxp/loxp embryos.

Figure 1

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(A–D) Whole mount examination of control and mutant embryos at E11.5 showed that craniofacial and PA development was visibly retarded in the mutants. Embryos were stained briefly with BM purple and nuclear-fast-red for better visualization. The medial frontonasal prominences failed to expand and join at the midline in the mutant embryo when compared to the control (indicated with white arrow heads. A, B). The tongue, indicated with white arrows, was evident in the control, but was not observed in the mutant embryo. The fusion between the first and second PAs (indicated with black arrows) was delayed in the mutant embryo (C vs. D). (E, F) Control and mutant embryos at E11.5 were cross-sectioned and HE stained. The tongue structure (indicated with an arrow) was absent in the mutant. (G–J) Section in situ hybridization analysis was performed on cross (G, H) and sagittal (I, J) sections of control and mutant embryos (at E11.5) using a MyoD probe. Arrows indicate examples of positively stained cells. Samples were counterstained with nuclear-fast-red. (K, L) Section in situ hybridization analysis was performed on cross- sections of control and mutant embryos (at E11.5) using a Sox9 probe. The arrows indicate chondrogenic precursors for Meckel’s cartilage. Scale bar: 500 μm. fn: frontonasal process; ls: lingual swelling; man: mandibular process; max: maxillary process; pa: pharyngeal arch; ps: palatal shelf; ton: tongue; control: Smad4loxp/+; mutant: Wnt1-Cre;Smad4loxp/loxp.

Embryonic tongue is formed by merging of bilateral lingual swellings from the mandibular prominences and the tuberculum impar and contains myoblasts derived from somite mesenchyme and connective tissues of NCC origin. A complete tongue was evident in the control embryos at E11.5, but was not seen in the mutants (Fig. 1A, B, E, F). The secondary palatal shelves, although budded from the maxillary prominences and normal in size, grew in a horizontal orientation rather than exhibiting an initial vertical outgrowth, as seen in normal development (Fig. 1E, F). This altered growth pattern of palatal shelves was likely secondary to the abnormal development of the tongue, which usually prevents the palate shelves from horizontal outgrowth by occupying the oral cavity. In order to determine whether myogenic and chondrogenic progenitors are present within the mandibular prominences of mutant embryos, we examined the myogenic and cartilaginous differentiation markers, MyoD and Sox9. Section in situ hybridization analysis revealed that expression of both MyoD and Sox9 was detected in the mandibular prominences of mutant embryos (Fig. 1G–L). Therefore, we conclude that although a complete tongue was never formed in the central oral cavity in the mutant embryos, the primordia of lingual swellings were present in the medial side of the mandibular prominences and failed to expand to the midline.

Next, we examined the development of the cranial and dorsal root ganglia in mutant embryos (E10.5) via whole mount immunostaining with a 2H3 antibody. The three branches of the trigeminal ganglion (the V cranial ganglion), the ophthalmic, maxillary and mandibular branches, were hypoplastic in all mutant embryos examined (Fig. 2). We did not observe any other obvious neural structural defects in mutants from the 2H3 staining study. Therefore, we concluded that expression of Smad4 in NCCs is essential for normal development of the trigeminal ganglion.

Figure 2. Hypoplastic branches of the trigeminal ganglia in Wnt1-Cre;Smad4loxp/loxp embryos.

Figure 2

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(A–D) Wholemount immunostaining of neurofilament using a 2H3 anti-neurofilament monoclonal antibody was performed on control (A, C) and mutant (B, D) embryos at E10.5. The three branches of trigeminal (the V cranial) ganglia were hypomorphic in mutant embryos. Scale bar: 500 μm; max.b: maxillary branch; man.b: mandibular branch; op.b: ophthalmic branch; control: Smad4loxp/+; mutant: Wnt1-Cre;Smad4loxp/loxp.

OFT, ventricle and PAA defects in Wnt1-Cre;Smad4loxp/loxp embryos

Previous studies have demonstrated that TGFβ/BMP signaling in NCCs is critical for normal development of the OFT and ventricular myocardial wall (summarized in Table 1). As shown in Fig. 3A–D, the distal part of the OFT, which had started to separate into two vessels in control embryos, failed to separate in mutants (Wnt1-Cre;Smad4loxp/loxp). This phenotype was more obvious from the side view (Fig. 3C, D). The defect was further confirmed with section studies (Fig. 3E, F). The aortic sac was separated into two orifices in control embryos, while it remained unseparated in mutants. In contrast to the well-formed OFT cushions in controls, the distal OFT cushions of mutants were hypoplastic. In addition to the OFT defects, Wnt1-Cre;Smad4loxp/loxp embryos displayed evident thin-walled ventricles and hypoplastic ventricular septa (Fig. 3G, H), suggesting that NCC Smad4 is required for normal development of myocardial wall in mouse embryos.

Figure 3. Cardiac and OFT defects in Wnt1-Cre;Smad4loxp/loxp embryos.

Figure 3

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(A–D) Embryonic hearts (at E11.5) were dissected for whole mount examination. Panels A, B show the front view; panels C, D show the side view. The OFT of the control embryo began to separate at the distal end, while no septation was observed in the mutant OFT. This phenotype was more obvious in the side view. Arrows in panels A–D indicate the OFT. (E–H) Embryos at E11.5 were cross-sectioned and HE stained for histological examination. In the control embryo, the OFT cushions were well formed and the opposing ridges of OFT cushions were beginning to fuse, separating the OFT into aorta and pulmonary trunk (E). In the mutant embryo, however, the OFT cushions were hypoplastic and the OFT remained a single channel (F). Arrows in panels E and F indicate OFT cushions. The ventricle wall and ventricular septum of the mutant embryo were hypoplastic compared to those of the control (G vs. H). Boxed areas were magnified and shown in the upper-right corners. Scale bar: 200 μm. as: aortic sac; la: left atria; lv: left ventricle; ra: right atrial; rv; right ventricle; vs: ventricular septum; control: Smad4loxp/+; mutant: Wnt1-Cre; Smad4loxp/loxp.

To determine whether the PAAs were properly patterned in Wnt1-Cre;Smad4loxp/loxp mice, we performed cardiac India ink injection experiments. The PAAs were well formed and patent by E10.5 (Fig. 4A, B), suggesting that disruption of Smad4 in NCCs does not disrupt initial PAA formation. We further examined whether the PAAs were correctly remodeled at E11.5. Results in Fig. 4C, D confirmed our previous observation that the distal OFT septum was well formed in littermate controls but not in mutant embryos. In control embryos at this stage, PAAs were in the process of active remodeling. On the left side of control embryos, the distal ends of the fourth and sixth arteries were adjoined (Fig. 4E). However, no obvious remodeling was observed in mutant PAAs (Fig. 4F). A similar retarded remodeling defect was also observed on PAAs of the right side in mutant embryos (data not shown). We did not observe abnormal regression of PAAs at this stage, a defect which was reported in Wnt1-Cre;Alk2loxp/loxp and Wnt1-Cre;Alk5loxp/loxp embryos (Kaartinen et al., 2004; Wang et al., 2006).

Figure 4. Cardiac ink injection analysis.

Figure 4

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(A, B) India ink injection was performed in control (A) and mutant (B) embryos at E10.5. No obvious defect was observed in PAA formation in the mutant embryo. (C–F) Control and mutant embryos at E11.5 were injected with India ink. At this stage, the OFT was dividing in the control OFT (C), but not in the mutant (D). The fourth and sixth PAAs on the left side of a control embryo (E) were joined at their distal ends; however, this remodeling process was not observed in the mutant embryo (F). Scale bar: 200 μm. as: aortic sac; lv: left ventricle; oft: outflow tract; pa: pharyngeal arch; paa: pharyngeal arch artery; rv; right ventricle; control: Smad4loxp/+; mutant: Wnt1-Cre;Smad4loxp/loxp.

Early NCC generation and migration was largely intact, but expression of some NCC markers was decreased from the postmigratory NCCs

Motivated by the discovery that NCC disruption of Smad4 caused severe morphological abnormalities in craniofacial, PA, and cardiac morphogenesis, we further explored the molecular pathological defects in mutant embryos. We first examined expression of different NC markers including Ap-2α, Msx1, Sox9, and Pax3 in E8.5–9.0 embryos by whole mount in situ hybridization analysis and did not observe any obvious abnormal expression of these genes (Fig. 5), suggesting that the initial formation of NC was intact in mutant embryos. This conclusion is supported by our cell lineage analysis (see below).

Figure 5. Normal expression of neural crest markers in Wnt1-Cre;Smad4loxp/loxp embryos at E8.5.

Figure 5

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(A–H) Whole mount in situ hybridization analysis was performed on embryos isolated between E8.5 and E9.0 using probes against Ap-2α (A, B), Msx1 (C, D), Sox9 (E, F) and Pax3 (G, H). No obvious differences were observed between the control and mutant embryos. The arrows indicate positively stained areas. Scale bar: 200μm. control: Smad4loxp/+; mutant: Wnt1-Cre;Smad4loxp/loxp.

We proceeded to evaluate expression of NCC markers in E9.5 embryos including Msx1, 2, CrabPI, Sox9 and Ap-2α, and our results were consistent with previously published studies describing their expression patterns (Fig. 6). We observed that Msx1 and Msx2 were highly expressed in the first and second PAs, and Sox9 was moderately expressed within the first PAs. AP-2α, was widely expressed in all PAs and NCC migration streams, while CrabPI was found in the migration streams adjacent to rhombomere 2, 4, and 6. Therefore, these genes were expressed with non-identical, yet partially overlapping, patterns and represented distinct subpopulations of NCCs that were destined to different developmental fates. Significantly, the expression of Msx1, −2, Sox9, and Ap-2α were visibly reduced (but not eliminated) from their expression domains by NCC knockout of Smad4, whereas expression of CrabPI was not noticeably affected. To determine whether reduced expression of some of the NCC markers is due to a reduced number of NCCs, we performed cell lineage analysis. We crossed Rosa26-reporter (R26R) mice with Smad4loxp/loxp mice to acquire Smad4loxp/loxp;R26R+/− mice (female), which were then crossed with Wnt1-Cre;Smad4loxp/+ mice (male) to produce Wnt1-Cre;Smad4loxp/loxp;R26R+/− embryos. Results from both wholemount and section studies showed that comparable amounts of NCCs are present on their migration paths and in their destinations in control and mutant embryos at E9.5 (Fig. 6K–N), indicating that generation and migration of NCCs were grossly normal in mutants.

Figure 6. Expression of NCC markers in Wnt1-Cre;Smad4loxp/loxp embryos at E9.5.

Figure 6

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(A–J) Whole mount in situ hybridization analysis was performed on E9.5 embryos using probes against Msx1 (A, B), Msx2(C, D), Sox9 (E, F), CrabPI (G, H), and Ap-2α (I, J). The arrows indicate positively stained areas. control: Smad4loxp/+; mutant: Wnt1-Cre;Smad4loxp/loxp. (K, L) A control (Wnt1-Cre;Smad4loxp/+;R26R+/−) and a mutant (Wnt1-Cre;Smad4loxp/loxp;R26R+/−) embryo at E9.5 were stained with X-gal for whole mount examination. (M, N) X-gal stained control and mutant embryos were cross-sectioned. Scale bar: 500μm. pa: pharyngeal arch.

Altered gene expression in developing PAs and their derivatives in mutant embryos

To further determine the molecular defects in PAs and their derivatives caused by NCC disruption of Smad4 at later stages, we examined expression of multiple genes with in situ hybridization assays in embryos from E10.0 to E11.5. In addition to their early roles in neural crest specification, Msx1 and Msx2 are critical for the development of PAs and their derivatives via mediating epithelial-mesenchymal interactions. Expression of both Msx1 and Msx2 (Fig. 7A–H) was markedly reduced from the facial primordia of mutant embryos at E10.5. By E11.5, expression of both genes was absent from the mandibular and maxillary prominences of the mutants, though their transcripts were still present in the frontonasal prominences. By contrast, expression of the two genes in these areas was robust in controls. Pax3 failed to be expressed in the maxillary and mandibular prominences of the mutants at both E10.5 and E11.5 stages (Fig. 7I–L).

Figure 7. Altered gene expression in facial primordia and PAs between E10.0 and E11.5 in Wnt1-Cre;Smad4loxp/loxp embryos.

Figure 7

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(A–R) Whole amount in situ hybridization analysis was performed on embryos between E10.0 and E11.5. Expression of both Msx1 (A–D) and Msx2 (E–H) were markedly reduced from developing facial primordia and PAs of mutant embryos. Pax3 was not expressed in the maxillary and mandibular prominences in E10.5 or E11.5 mutant embryos (I–L). Expression of Fgf8 appeared to be normal in mutant embryos at E10.0 and E11.0 (M–P). Expression of Tbx1 in the mutant embryo was down-regulated within the mesenchyme of PAs. (Q, R). Arrows indicate sites of expression. Scale bar: 500μm. control: Smad4loxp/+; mutant: Wnt1-Cre;Smad4loxp/loxp.

It has been previously demonstrated that NCCs can regulate normal developmental processes in neighboring cell populations in PAs (Noden and Trainor, 2005; Rinon et al., 2007; Stottmann et al., 2004; Tzahor et al., 2003). To examine whether NCC disruption of Smad4 also alters gene expression in other cell populations in PAs, we examined expression of Tbx1, Fgf8 and Shh, which are all known to play critical roles during PA and OFT morphogenesis. Fgf8 encodes a signaling molecule expressed in the epithelia of PAs, and is required for normal growth of PAs and remodeling of OFT (Brown et al., 2004; Frank et al., 2002; Macatee et al., 2003; Vitelli et al., 2006). We did not observe obvious defects in expression of Fgf8 in mutant embryos at E10.5 and E11.0 (Fig. 7M–P). Similar results were observed for Shh (data not shown). Tbx1 is expressed in non-NCC derived mesenchymal cells in developing PAs, and plays critical roles in mediating interactions between NCCs and other cell populations (Baldini, 2004; Epstein, 2001; Grossfeld, 2003; Jerome and Papaioannou, 2001; Lindsay et al., 2001; Merscher et al., 2001; Vitelli et al., 2002a; Yamagishi and Srivastava, 2003; Zhang et al., 2006). Significantly, expression of Tbx1 in the first and second PAs was noticeably reduced in mutants (Fig. 7Q, R).

Disruption of Smad4 in NCCs caused massive cell death within the PAs, but had little effect on their proliferation

To address the potential cellular mechanism underlying the defects in mutant embryos, we examined cell proliferation and apoptosis during the development of PAs and facial primordia. We first examined cell proliferation using an anti-phosphorylated Histone H3 antibody (Fig. 8), but did not observe any apparent reduction in overall proliferation rate in the mutants at E9.5 (data not shown), E10.5 or E11.5 stages (Fig. 8). We therefore concluded that the defects in mutant embryos is unlikely caused by reduced cell proliferation.

Figure 8. Normal proliferation in Wnt1-Cre;Smad4loxp/loxp embryos.

Figure 8

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(A–D) Immunohistochemistry assays were performed on cross-sections of a control and a mutant embryo at E10.5 using an anti-phospho-H3 antibody. No obvious difference was observed between control and mutant samples. Panels C, D correspond to the boxed areas of panels A, B, respectively. (E–F) Sagittal sections of control and mutant embryos at E10.5 were immunostained with an anti-phospho-H3 antibody. (G–J) Sagittal sections of a control and a mutant embryo at E11.5 were immunostained with an anti-phospho-H3 antibody. Panels I, J correspond to the boxed regions of panels G, H, respectively. The size of PA1 in the mutant embryo was clearly reduced. Arrows indicate examples of positively stained cells. Scale bar: 200μm. control: Smad4loxp/+; mutant: Wnt1-Cre;Smad4loxp/loxp.

We next performed TUNEL analysis on embryos between E9.5 and E11.5 (the most advanced survival stage of mutants) with both whole mount and section studies. At E9.5, no increase in apoptosis was observed in mutant PAs compared to control samples (data not shown). At E10.5, the littermate controls exhibited active cell death in the developing PAs (Fig. 9A), suggesting that apoptosis is normally involved in remodeling of PAs. We observed massive and aberrant cell death in the facial primordia and PAs of mutant embryos, most notably in the ventral portion of the mandibular prominences, frontonasal prominences, and the second arches (Fig. 9A–F). Increased apoptosis was also seen in mesenchymal cells located between PA3 and PA6 in mutant embryos (Fig. 9C, D), a region containing cardiac NCCs (Jiang et al., 2000). Increased apoptosis in mutants was further confirmed with section studies at E10.5 (data not shown) and E11.5 (Fig. 9E, F). Aberrant apoptosis was mainly observed in craniofacial and PA regions, but not in other regions of mutants. To test if these apoptotic cells were limited to NCCs, we performed X-gal staining on Wnt1-Cre;Smad4loxp/loxp;R26R+/−embryos (at E11.0) to label NCCs and their derivatives followed by the TUNEL assay. As expected, many NCCs (blue) are positively stained with the TUNEL signal (brown). Significantly, in addition to NCCs, we clearly observed non-NCC derived mesenchymal cells that are also apoptotic (Fig. 9G–I), suggesting that NCC Smad4 is required for normal survival of both NCC-derived ecto-mesenchyme and mesoderm-derived mesenchyme in PAs of E11.0 embryos.

Figure 9. Increased apoptosis in Wnt1-Cre;Smad4loxp/loxp embryos.

Figure 9

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(A–D) Whole mount TUNEL analysis was performed on control and mutant embryos at E10.5. The control embryo exhibited active cell death in developing PAs, but most apoptotic cells were located laterally within the facial primordia and PAs. By contrast, the mutant embryo displayed increased cell death in the facial primordia and PAs, notably in the ventral portion of the mandibular prominences and frontonasal prominences. Massive apoptosis was also seen in cells that were located between PA3 and PA6, where apoptosis was rare in the control. Panels C and D correspond to the boxed areas of panels A and B, respectively. (E, F) TUNEL analysis was also performed on sections of control and mutant embryos at E11.5. Apoptotic cells were widely distributed in the mutant PAs and facial primordia, but few in the littermate control. control: Smad4loxp/+; mutant: Wnt1-Cre;Smad4loxp/loxp. (G–I) A control (Wnt1-Cre;Smad4loxp/+;R26R+/−, G) and a mutant (Wnt1-Cre;Smad4loxp/loxp;R26R+/−, H) embryo at E11.0 was stained with X-gal and cross-sectioned followed by TUNEL analysis. Panel I corresponds to the boxed area of panel H. Black arrows indicate examples of apoptotic cells that were NCCs (blue), while red arrows indicate examples of apoptotic cells that were non NCC-derived. Scale bar: 500μm. ton: tongue.

Discussion

This study provides compelling mouse genetic evidence demonstrating that expression of Smad4 in NCCs is essential for normal development of NCCs and their derivatives. Depletion of Smad4 from NCCs causes severe abnormalities in craniofacial, pharyngeal and cardiovascular structures. Furthermore, compared to the published mouse models, in which genes encoding TGFβ/BMP receptors were specifically inactivated in NCCs (Choudhary. et al., 2006; Dudas et al., 2004; Ito et al., 2003; Jia et al., 2007; Kaartinen et al., 2004; Ko et al., 2007; Stottmann et al., 2004; Wang et al., 2006) (Table 1), Wnt1-Cre;Smad4loxp/loxp embryos exhibit the most severe embryonic phenotype, namely early lethality at E11.5. These data strongly suggest that Smad4, which encodes the only co-Smad in mammals, plays central roles in mediating TGFβ/BMP signaling during NCC development.

Our molecular examination of E8.5 embryos suggests that initial generation and specification of NC occurs normally in Wnt1-Cre;Smad4loxp/loxp embryos. This conclusion is further supported by our cell lineage analysis on E9.5 embryos. We cannot exclude that the lack of abnormality in mutant embryos at E8.5 is due to incomplete depletion of Smad4 in NCCs. We began to detect molecular defects in E9.5 mutant embryos, though no morphological abnormality is obvious at this stage. Expression of NCC markers including Ap-2α, Sox9, Msx1,-2 was clearly reduced in mutant embryos. Our cell lineage analysis at E9.5 revealed that comparable amounts of NCCs are present in their destinations (Fig. 6), and furthermore, we did not observe any obvious abnormal cell proliferation/apoptosis in mutant embryos at this stage. Therefore, we conclude that the reduction in expression of these genes is unlikely caused by a reduced number of NCCs in these areas, but rather, is due to downregulation of expression of these genes by inactivation of Smad4. Downregulation of Msx1, 2 is anticipated because these two genes are known to be direct downstream targets of BMP R-Smads (Brugger et al., 2004; Gonzalez et al., 1998). Depletion of Smad4 in NCCs is expected to impair transcription mediated by BMP R-Smads. Regulation of Ap-2α and Sox9 expression by TGFβ/BMP signaling pathways has been reported in several developmental processes (Lincoln et al., 2006; Luo et al., 2003). It is currently unclear whether the two genes are also direct downstream targets of Smads. Interestingly, despite the obvious molecular defects in mutants at E9.5, no obvious cellular defect was observed from cell lineage, proliferation or apoptosis analysis at this stage. It has been previously reported that cellular defects in NCC migration and survival can be readily detected in Ap-2α−/− or Msx1−/−;Msx2−/− embryos at E9.5 (Ishii et al., 2005; Schorle et al., 1996; Zhang et al., 1996). The apparent lag between the molecular defects and the observed cellular defects in Wnt1-Cre;Smad4loxp/loxp embryos can be explained by the fact that although expression of the markers (Ap-2α, Msx1, 2) is reduced, it is not fully eliminated in NCCs. The remaining expression of these genes may be sufficient to support normal growth of NCCs up to E9.5. The reduced expression of these genes would contribute to the cellular and morphological defects in mutant embryos at later stages.

Our further molecular characterization of Wnt1-Cre;Smad4loxp/loxp embryos between E10.0 and E11.5 revealed an apparent reduction in expression of various genes (including Msx1, Msx2, Pax3, and Tbx1) from the facial primordia and developing PAs (Fig. 9). Functions of these genes have been implicated in mediating interactions between NCCs and their neighboring cells in facial primordia and PAs, and subsequent organogenesis. Of particular significance, Wnt1-Cre;Smad4loxp/loxp embryos displayed notably decreased expression of Tbx1, which is the leading candidate gene for DiGeorge syndrome and is expressed in non-NCC cells in developing PAs (Brown et al., 2004; Chapman et al., 1996; Vitelli et al., 2002b). What has yet to be determined is how Smad4 expressed in NCCs regulates expression of Tbx1. It has been reported that altered gene expression in NCCs disturbs expression of Tbx1 and other skeletal muscle markers in PAs, suggesting that NCCs may secrete certain cytokines to promote myogenic differentiation of mesoderm-derived mesenchyme (Rinon et al., 2007; Tzahor et al., 2003). Therefore, one possible reason for reduced expression of Tbx1 is that the elevated NCC apoptosis in Wnt1-Cre;Smad4loxp/loxp could reduce the amount of cytokines secreted from NCCs, and thus lead to a reduction in Tbx1 expression. It is also worth noting that, in addition to NCCs, we also observed non-NCC derived mesenchymal cells undergoing apoptosis at E11.0. Therefore, an alternative explanation, which does not necessarily exclude the first, is that the reduced expression of Tbx1 may be merely due to the reduced number of cells expressing this gene. Inactivation of Tbx1 in mouse embryos caused severe pharyngeal phenotypes including the hypoplasia of PA2, absence of PA3, 4, 6, abnormal formation of PAAs, failure in septation of OFT, and others (Arnold et al., 2006; Jerome and Papaioannou, 2001; Lindsay et al., 2001; Merscher et al., 2001; Zhang et al., 2006). Reduced expression of Tbx1 would likely contribute to the pharyngeal arch and heart defects observed in Wnt1-Cre;Smad4loxp/loxp embryos.

One particularly significant discovery from this study is that elevated apoptosis is also observed in non-NCC derived mesenchymal cells by NCC disruption of Smad4. It was previously demonstrated that NCCs are not required for early cell survival in PA2. For example, in Hoxa1−/−;Hoxb1−/− 3’RARE−/− embryos (E9.5), which lose NCCs in their second arches, mesoderm-derived mesenchymal cells can still migrate, proliferate and survive (Gavalas et al., 2001). Consistent with this report, we did not observe abnormal proliferation/apoptosis in E9.5 Wnt1-Cre;Smad4loxp/loxp embryos. Our further examination at later stages provides direct evidence suggesting that the activities of NCCs are essential for normal survival of mesoderm-derived mesenchymal cells in PAs at E11.0. Another possible reason for cell death in PAs is poor circulation caused by the myocardial wall defect in mutant mice (see below).

Cardiac NCCs normally migrate to the OFT region via the third, fourth and sixth PAs to form the two-pronged cushions at the distal portion of the OFT where they contribute to the septation of aorta and pulmonary outlets (Gittenberger-de Groot et al., 2005). As expected from NCC survival defects, the distal portion of the OFT septum failed to form in Wnt1-Cre;Smad4loxp/loxp embryos, as shown in Fig. 3. The OFT septation defect appears to be a common feature of all mouse models in which TGFβ/BMP signaling is impaired in NCCs (Table 1), indicating that TGFβ/BMP signaling pathways within NCCs are critical for OFT morphogenesis. However, the defect in OFT septation is unlikely the primary cause of the embryonic lethality at E11.5. Rather, we also observed thin-walled ventricles caused by NCC disruption of Smad4, similar to that seen in Wnt1-Cre;Alk3loxp/loxp embryos. It is likely that this abnormal ventricle development and subsequent inadequate blood supply are the primary causes of embryonic lethality for both Wnt1-Cr;Alk3loxp/loxp (Stottmann et al., 2004) and Wnt1-Cre;Smad4loxp/loxp mice. It was proposed that a small population of NCC-derived epicardial cells signal through Alk3 to promote cardiomyocyte proliferation in ventricles (Stottmann et al., 2004). If this hypothesis is true, our data would suggest that Smad4 encodes an essential component necessary for NCC-derived epicardial cells to stimulate ventricular myocardial growth. In addition to their potential roles in morphogenesis of ventricles, NCCs may also be required for proper physiological functions of cardiomyocytes (Conway et al., 1997; Kirby and Waldo, 1990; Kirby and Waldo, 1995). For example, splotch mutant mice, which are caused by mutation in Pax3 and die by E14.5, display poor myocardial contractility, a defect which is attributed to defective cardiac NCCs (Conway et al., 1997). Therefore, our study provides a valuable mouse model for exploring how NCCs regulate myocardial wall development.

In summary, our current study, together with two other independent studies on the Wnt1-Cre;Smad4loxp/loxp mouse model (Jia et al., 2007; Ko et al., 2007), have provided compelling mouse genetic evidence demonstrating that Smad4 expressed in NCCs play critical roles for NCC development. Specifically, disruption of Smad4 in NCCs results in embryonic death at midgestation with severe morphological defects in the craniofacial, pharyngeal, OFT, and cardiac ventricular regions. Information acquired from these studies will help us to understand the functions of TGFβ/BMP signaling during embryogenesis and its potential contributions to congenital diseases involving craniofacial and cardiovascular defects.

Acknowledgments

We thank Drs. B. Yoder (UAB), C. Haycraft (UAB), B. Zhou (Vanderbilt), S. Baldwin (Vanderbilt), C. Chiang (Vanderbilt) and R. Maxson (U. Southern CA) for providing various in situ probes. We thank Dr. V. Kaartinen (U. Southern CA) for valuable suggestions on India ink injection. We thank members of the Jiao laboratory for assistance on the project. We thank Drs. C. Haycraft and C. Chang (UAB) for critically reading and commenting on the manuscript. This project is supported by the Scientist Development Grant from AHA (National Center), a NIH grant (1R21HL085510-01) and a HSF-GEF Scholar Award to K. J.

Abbreviations

NC

neural crest

NCCs

neural crest cells

OFT

outflow track

PA

pharyngeal arch

PAA

pharyngeal arch artery

Footnotes

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