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Genes Dev. Mar 1, 2005; 19(5): 530–535.
PMCID: PMC551573

Inactivation of TGFβ signaling in neural crest stem cells leads to multiple defects reminiscent of DiGeorge syndrome

Abstract

Specific inactivation of TGFβ signaling in neural crest stem cells (NCSCs) results in cardiovascular defects and thymic, parathyroid, and craniofacial anomalies. All these malformations characterize DiGeorge syndrome, the most common microdeletion syndrome in humans. Consistent with a role of TGFβ in promoting non-neural lineages in NCSCs, mutant neural crest cells migrate into the pharyngeal apparatus but are unable to acquire non-neural cell fates. Moreover, in neural crest cells, TGFβ signaling is both sufficient and required for phosphorylation of CrkL, a signal adaptor protein implicated in the development of DiGeorge syndrome. Thus, TGFβ signal modulation in neural crest differentiation might play a crucial role in the etiology of DiGeorge syndrome.

Keywords: Neural crest, TGFβ, CrkL, Src kinase, fate decision, pharyngeal apparatus, DiGeorge syndrome

During development, neural crest cells emerge from the dorsal part of the neural tube and emigrate to various locations within the embryo to generate most of the peripheral nervous system and a variety of other structures (Le Douarin and Dupin 2003). In particular, neural crest cells localized in the pharyngeal apparatus contribute to non-neural tissues, such as craniofacial bone and cartilage, thymus, parathyroid glands, and cardiac outflow tract and septum (Kirby and Waldo 1995; Jiang et al. 2000; Graham 2003). The function of neural crest cells in the generation of these tissues, however, has been debated (Graham 2003).

Formation of the pharyngeal apparatus involves complex interactions of neural crest, ectoderm, endoderm, and mesoderm whose development must be coordinated (Graham 2003). Significantly, alterations to the development of this region are often associated with congenital human birth defects such as DiGeorge or Velocardiofacial syndrome. DiGeorge syndrome is the most common microdeletion syndrome in humans, characterized by cardiovascular defects plus thymic, parathyroid, and craniofacial anomalies (Lindsay 2001; Vitelli and Baldini 2003). Approximately 80% of the patients carry a variably sized deletion on chromosome 22 (del22q11). Ablation of genes affected by the microdeletion indicated two pathophysiological mechanisms causing DiGeorge syndrome: Mutations of the transcription factor Tbx1 lead to disturbed pharyngeal arch patterning. Consequently, neural crest cells are unable to populate the pharyngeal apparatus (Jerome and Papaioannou 2001; Lindsay et al. 2001; Merscher et al. 2001). However, upon deletion of the gene encoding the signal adaptor protein CrkL, neural crest cells localize to the pharyngeal apparatus, but they do not form non-neural derivatives from this structure (Guris et al. 2001). In both cases, aberrant neural crest cell migration, survival, or differentiation might contribute to DiGeorge syndrome (Lindsay 2001; Vitelli and Baldini 2003). A direct role of neural crest cells in the development of the pharyngeal apparatus, however, was not demonstrated by the analysis of these mutants. Furthermore, the signals specifying non-neural fates from neural crest cells in the pharyngeal apparatus have not yet been identified.

Previously, cell culture experiments allowed the identification of several growth factors able to instruct migratory and post-migratory neural crest cells to adopt specific lineages (Le Douarin and Dupin 2003; Lee et al. 2004). One of these factors is transforming growth factor (TGF) β, which elicits multiple responses in cultured neural crest stem cells (NCSCs). Depending on the cellular context, it can promote the generation of non-neural smooth-muscle-like cells or autonomic neurons, or induce apoptosis (Shah et al. 1996; Hagedorn et al. 1999, 2000). Here we investigated the role of TGFβ signaling in NCSCs and their derivatives in vivo, with an emphasis on migration, non-neural fate decision, and differentiation processes of neural crest cells in the pharyngeal apparatus.

Results and Discussion

To address the role of TGFβ signaling in neural crest development in vivo, we have used the cre/loxP system to conditionally inactivate the TGFβ receptor type II (TβRII) gene essential for TGFβ signal transduction (Leveen et al. 2002). Wnt1-Cre-mediated recombination of a floxed allele of TβRII resulted in loss of TβRII protein in neural crest cells isolated from mutant embryos and in mutant neural crest derivatives in vivo (Fig. 1), consistent with the previously reported activity of wnt1-Cre in virtually all neural crest cells (in addition to its expression in the mid/hindbrain area) (Brault et al. 2001; Lee et al. 2004). At embryonic day 10.5 (E10.5), mutant embryos did not exhibit obvious malformations, and TβRII-deficient neural crest cells migrated correctly into the pharyngeal apparatus, as assessed by in vivo fate mapping using the ROSA26 Cre reporter mouse line (Fig. 1D; Jiang et al. 2000; Hari et al. 2002). Mutants were recovered at the expected Mendelian frequency until E18.5, but they died perinatally, displaying multiple developmental defects including mid/hindbrain abnormalities not covered in this study (Fig. 1E). Control littermates carrying a nonrecombined TβRII allele showed no anomalies.

Figure 1.
Neural crest-specific conditional ablation of TβRII. (A) Exon 4 (ex4) of the TβRII locus, encoding the transmembrane domain (TMD) and the intracellular phosphorylation sites (P) of the TβRII protein, is flanked by loxP-sites and ...

Malformations of cranial bones and cartilage as well as cleft palate were found in all mutant mice examined at E18 (Fig. 2A,B), as described (Ito et al. 2003). As cleft palate together with craniofacial dysmorphism are typical features of DiGeorge syndrome in humans, we examined mutant mice at different developmental stages for other defects characterizing DiGeorge syndrome. Patients suffering from DiGeorge syndrome have absent or hypoplastic parathyroid and thymus glands (Kirby and Waldo 1995). Similarly, in all TβRII-mutant mice at E18, staining of serial cross-sections of the neck for parathyroid hormone indicated either hypoplastic (three of five) or undetectable (two of five) parathyroid glands (Fig. 2C). Likewise, the size of the thymus glands was reduced in mutant mice at E18 (58% ± 7% of control size; n =8; p < 0.01). In normal mouse development, mesenchymal derivatives of neural crest cells are transiently detectable in the cortical region of the developing thymus and parathyroid glands (Fig. 2D; Jiang et al. 2000). Moreover, elimination of cephalic premigratory neural crest in chicken and absence of neural crest in the pharyngeal apparatus of transgenic mice results in hypoplastic parathyroid and thymic glands (Kirby and Waldo 1995; Vitelli and Baldini 2003). This suggests that development of pharyngeal arch-derived glands might depend on interactions between neural crest-derived cells and pharyngeal endoderm. Consistent with the hypothesis that neural crest cells are important for proper thymus gland formation, neural crest-specific inactivation of TGFβ signaling resulted in a reduction of neural crest cells found by in vivo fate mapping in the cortex of the developing thymus at E13.5 (Fig. 2D).

Figure 2.
Features of DiGeorge syndrome in TβRII mutant mice. (A) Hypoplastic or absent bone (red) and cartilage (blue) structures (open arrowheads) in mutant (mt) mice at E18. (co) Control. (B) Cleft palate ([large star]) in macroscopic view (top) and on ...

The main cause of mortality in DiGeorge patients is congenital heart defects such as a single truncus arteriosus and a ventricular septal defect (VSD) (Kirby and Waldo 1995). Aberrant development of cardiac neural crest that normally contributes to the heart and its outflow tract is thought to cause these defects. This assumption is based on genetic manipulations in mouse models and on cardiac neural crest ablation in chicken, which leads to a truncus arteriosus and a VSD (Kirby and Waldo 1995; Vitelli and Baldini 2003). Recently, neural crest-specific inactivation of the type I bone morphogenic protein (BMP) receptor Alk2 resulted in cardiac outflow tract malformations (but not in other tissue anomalies also associated with DiGeorge syndrome), indicating a role of TGFβ family factors in cardiovascular development (Kaartinen et al. 2004). To determine whether the prenatal lethality in the TβRII-mutant mice might result from heart defects, we examined the hearts of mutant mice at E18 histologically and by intracardial ink injection. All mutant hearts (n = 18) displayed a truncus arteriosus together with a VSD (Fig. 3). In addition, we found abnormal patterning of the arteries arising from the aortic arch, as also found in DiGeorge syndrome patients (Fig. 3D; Kirby and Waldo 1995). Overall, these heart defects led to functional right-sided heart failure with venous congestion, resulting in a vasodilatation of the jugular veins (data not shown), a characteristic clinical feature of patients with severe cardiac insufficiency.

Figure 3.
Malformations in the heart of TβRII mutant mice. (A) Ventricular heart at E18 with detailed view of the paired outflow tract in control (co) and of the truncus arteriosus ([large star]) in mutant (mt) mice. (rv) Right ventricle; (lv) left ventricle; ...

Taken together, our findings show that mice with a mutation of TβRII in NCSCs develop all the morphological features of DiGeorge syndrome. The question arises, whether these developmental defects are due to impaired migration, programmed cell death, and/or a failure in fate decision or differentiation of neural crest cells. In vivo fate mapping revealed that, as in the control, mutant neural crest cells were able to populate the pharyngeal apparatus and the forming aorto-pulmonary septum at E10.5 (Figs. (Figs.1,1, ,4).4). Likewise, expression of the NCSC-marker Sox10 (Paratore et al. 2001) and migration were not affected in TβRII-deficient neural crest cells emanating from neural tubes in explant cultures (data not shown). As in the control, neural crest-derived cells in the pharyngeal apparatus of mutant mice lost sox10 expression while expressing the neural crest markers pax3 and ehand (data not shown). In addition, we did not observe differences in the number of apoptotic cells in the pharyngeal apparatus of control and mutant mice at E10.5 (data not shown).

Figure 4.
TβRII-mutant neural crest cells fail to acquire non-neural fates. (A) Equal distribution of βGal-expressing neural crest cells (blue) in the first and second branchial arch of control (co) and TβRII-mutant (mt) mice at E10.5. Presence ...

Our data indicate that neural crest cell differentiation in the pharyngeal apparatus rather than migration or cell survival is defective in the absence of TGFβ signaling. To test this hypothesis, we analyzed the expression of smooth muscle α-actin in neural crest derivatives, since TGFβ is known to induce a smooth-muscle-like cell fate in NCSCs in vitro (Shah et al. 1996). While in the control at E10.5, βGal-expressing neural crest derivatives in the prospective aorto-pulmonary septum stained for smooth muscle α-actin, TβRII-mutant neural crest cells in this region did not contribute to the development of the smooth musculature (Fig. 4B). We therefore suggest that the non-neural, smooth muscle α-actin-positive cells (which have also been termed myofibroblasts and smooth muscle-like cells) (Hagedorn et al. 1999; Morrison et al. 1999) generated from TGFβ-treated NCSCs in culture indeed represent smooth muscle cells. At later developmental stages, neural crest cells that failed to adopt a smooth muscle cell fate underwent apoptotic cell death (data not shown). The absence of neural crest-derived smooth muscle cells in mutants explains the defective separation of the aorta from the pulmonary trunk, leading inevitably to a truncus arteriosus (Fig. 3).

The transcription factor Sox9 is required for the specification of an osteochondroprogenitor cell from neural crest cells, and conditional inactivation of sox9 in NCSCs results in craniofacial anomalies (Mori-Akiyama et al. 2003). During normal development sox9 is expressed in neural crest cells in the first and second pharyngeal arches, which give rise to the craniofacial skeleton. In contrast, sox9 expression was markedly reduced in TβRII-mutant neural crest cells populating the first and second pharyngeal arches (Fig. 4A). These data indicate that neural crest cells are unable to acquire an osteochondroprogenitor cell fate in the absence of TGFβ signaling, which leads to the malformations of cranial bones and cartilage observed in TβRII-mutant mice (Fig. 2A).

The microdeletion on chromosome 22 (del22q11) found in most of the DiGeorge syndrome patients encompasses ~30 genes but does not include genes encoding either TGFβ receptors or its ligands (Lindsay 2001; Vitelli and Baldini 2003). Thus, gene products eliminated by del22q11 may functionally interact with TGFβ signaling in neural crest cells. For instance, orthologs of PCQAP, a gene within the microdeleted region (Berti et al. 2001), have been shown to modulate TGFβ signaling (Kato et al. 2002), although the role of PCQAP in the development of the pharyngeal apparatus is unknown. Inactivation of two of the genes deleted in del22q11, Tbx1 and Crkol, phenocopies symptoms of DiGeorge syndrome (Lindsay 2001; Vitelli and Baldini 2003). In Tbx1 mutants, early steps in pharyngeal arch patterning are disrupted, which, as a secondary effect, interferes with neural crest migration into the pharyngeal arch complex (Vitelli et al. 2002; Vitelli and Baldini 2003). Similarly, manipulation of genes that genetically interact with Tbx1, such as Fgf8 and Vegf, affect migration and/or survival of neural crest cells into the pharyngeal apparatus (Vitelli and Baldini 2003). Inactivation of any one of Tbx1, Fgf8, or Vegf results in abnormal patterning of the aortic arch arteries, leading to conotruncal heart defects (Vitelli and Baldini 2003). In contrast, we found that Tbx1 expression was not altered in mice carrying a neural crest-specific mutation of TβRII (data not shown) and that the development of DiGeorge-like syndrome in these mice is due to a failure in non-neural fate acquisition and differentiation rather than in migration or survival of mutant neural crest cells. Moreover, intracardial ink injection at E10.5 revealed normal formation of the aortic arch arteries in TβRII-mutant mice (data not shown). Thus, although we cannot exclude it at present, the differences in phenotypes do not support an association between a Tbx1 genetic pathway and TGFβ signaling in neural crest cells.

Ablation of the del22q11 gene Crkol in mice, however, generates a phenotype reminiscent of that obtained by TGFβ signal inactivation in NCSCs, although with a lower penetrance (Guris et al. 2001). While pharyngeal arch patterning is normal, Crkol-mutant mice develop cardiovascular and mild craniofacial anomalies, as well as hypoplastic thymic and parathyroid glands. These deficiencies conceivably result from a post-migratory defect of neural crest cells lacking Crkol (Guris et al. 2001). This is analogous to the deficiencies observed in mice with TβRII-mutant neural crest cells, which also result from post-migratory defects (Figs. (Figs.4,4, ,5).5). The gene product of Crkol, CrkL, belongs to the Crk family of adaptor proteins that are involved in different signaling pathways (Feller 2001). In particular, CrkII—which shares 60% identical residues with CrkL—mediates oncogenic transformation induced by epidermal growth factor and TGFβ (Kizaka-Kondoh et al. 1996), and overexpression of a dominant negative mutant of CrkII prevents TGFβ-induced cell differentiation in vitro (Ota et al. 1998). Therefore, CrkL might be involved in TGFβ-dependent differentiation of neural crest cells. To address this hypothesis, we first assessed the status of CrkL phosphorylation during development of the pharyngeal apparatus. Tyrosine-phosphorylated CrkL was readily detectable in several structures of the wild-type pharyngeal apparatus at E10, including the pharyngeal arches 1 and 2 and the prospective aorto-pulmonary septum (Fig. 5A; Supplementary Fig. 1). However, phosphorylation of CrkL was restricted to a narrow, early time window during development of the pharyngeal apparatus and was no longer observed from E11.5 onward (Supplementary Fig. 1). Intriguingly, CrkL phosphorylation was impaired in TβRII-deficient neural crest cells in the pharyngeal apparatus at E10, concomitant with absence of TGFβ-dependent phosphorylation of Smad2 in the mutant (Fig. 5A; Supplementary Fig. 1). Moreover, TGFβ signaling promoted CrkL phosphorylation in an NCSC line that gives rise to smooth-muscle-like cells in response to TGFβ (Fig. 5B,C; Sommer et al. 1995; Chen and Lechleider 2004). This demonstrates that CrkL phosphorylation is downstream of TGFβ signaling in neural crest cells. Thus, CrkL might mediate TGFβ-dependent non-neural fate acquisition of neural crest cells at early stages of pharyngeal apparatus development. Conceivably, at later developmental stages TGFβ signaling might have CrkL-independent activities in neural crest-derived structures, as suggested by the milder phenotype of Crkol-mutant mice (Guris et al. 2001) as compared to the mutant described here.

Figure 5.
TGFβ-dependent CrkL phosphorylation and neural crest differentiation. (A) Mutant (mt) neural crest cells expressing βGal (blue) in branchial arch 1 (ba1), branchial arch 2 (Supplementary Fig. 1), and the developing aorto-pulmonary septum ...

TGFβ elicits a wide range of cellular processes, involving activation and cross-talk of multiple signaling pathways (Derynck and Zhang 2003). For instance, TGFβ signaling has been reported to induce phosphorylation and activation of Src tyrosine kinase regulating TGFβ-dependent apoptosis, cell migration, or tumor cell invasiveness (Fukuda et al. 1998; Kim and Joo 2002; Park et al. 2004; Tanaka et al. 2004). Interestingly, a recent report identified CrkL as a downstream signaling component of Src kinase in integrin-induced migration (Li et al. 2003). Together, these studies point to a potential association between TGFβ, Src, and CrkL signaling. In agreement with this hypothesis, levels of phosphorylated active Src were increased by TGFβ treatment of an NCSC line, concomitant with increased phosphorylation of CrkL (Fig. 5C). Importantly, the pharmacological Src inhibitors PP1 and PP2 reduced CrkL phosphorylation in TGFβ-treated neural crest cells to levels of untreated cells (Fig. 5D). These data indicate that Src kinase activity is involved in TGFβ-dependent phosphorylation of CrkL, possibly linking TGFβ signaling to the etiology of DiGeorge syndrome in humans.

In conclusion, we propose that TGFβ isoforms expressed in the pharyngeal apparatus (Millan et al. 1991) promote non-neural fate decisions in post-migratory neural crest cells, allowing these cells to contribute to multiple craniofacial, cardiovascular, and glandular structures (Fig. 5E-G). Accordingly, inactivation of TGFβ signaling in neural crest cells prevents development of these structures and recapitulates all morphological features of DiGeorge syndrome.

Materials and methods

Generation of mutant animals

Animals homozygous for the TβRII floxed allele (Leveen et al. 2002) were mated with wnt1-Cre mice heterozygous for the floxed allele (Hari et al. 2002). For in vivo fate mapping of NCSCs, wnt1-Cre mice heterozygous for the TβRII floxed allele were mated with animals homozygous for the TβRII floxed allele and carrying a ROSA26 Cre reporter (R26R) allele, which expresses β-galactosidase (βGal) upon Cre-mediated recombination (Soriano 1999). Genotyping was performed as described (Hari et al. 2002; Leveen et al. 2002).

Staining procedures

In situ hybridization with digoxigenin-labeled riboprobes and detection of LacZ-reporter gene expression was performed as described (Hari et al. 2002). Whole-mount X-Gal-stained embryos were paraffin-embedded and sectioned. Paraffin sections (7 μm) were stained with goat anti-PTH antibodies using the Vectastain method (VectorLab). Specificity of the antibody was tested by saturation with PTH(1-34). Anti-smooth muscle α-actin (Clone 1A4; Sigma) and anti-TβRII antibodies (C-16; Santa Cruz Biotechnology Inc.) were visualized by Alexa546-linked secondary antibodies (Abcam Inc.) or by the Vectastain method using NovaRed as a substrate (VectorLab), and phospho-Smad2 and phospho-CrkL (Cell Signaling Technology, Inc.) by Metal Enhanced DAB (Pierce). Standard protocols were used for bone and cartilage staining with alizarin S red and alcian blue and for staining by van Kossa's method and haematoxylin/eosin. TUNEL assays on paraffin-embedded tissue sections were performed following the producer's guidelines (Roche Diagnostics). Each marker was analyzed on at least three embryos per stage. Intracardial Indian ink injection was carried out as reported (Jerome and Papaioannou 2001).

Cell culture experiments

Neural tube explant cultures were done as described (Lee et al. 2004). Monc1 cells were cultured and treated with TGFβ (5 ng/mL) for 15 min at 37°C (Chen and Lechleider 2004). To inhibit Src kinases, PP1 or PP2 (Calbiochem) was added to the medium 40 min prior to TGFβ treatment. Western blot analysis of Monc1 cell extracts was carried out as described (Koller et al. 2004). Primary antibodies were against Actin (Chemicon Inc.), phospho-Smad2 (S465/467; Cell Signaling Technology, Inc.), phospho-Src (Y416; Cell Signaling Technology, Inc.), and phospho-CrkL (Y207; Cell Signaling Technology, Inc.). Immunocytochemistry of Monc1 cells treated with TGFβ for 3 d was done as described (Lee et al. 2004).

Acknowledgments

We thank B. Langsam, H.R. Sommer, and A. Wiesner for technical assistance; A. McMahon and P. Soriano for providing transgenic animals; and R. Zinkernagel, J.B. Relvas, and N. Mantei for helpful discussions. This work was supported by the Swiss National Science Foundation, by the National Center of Competence in Research “Neural Plasticity and Repair,” by the Swiss Federal Institute of Technology, and by the Roche Research Foundation.

Notes

Supplemental material is available at http://www.genesdev.org.

Article and publication are at http://www.genesdev.org/cgi/doi/10.1101/gad.317405.

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