In this study, we have examined the phenotypes resulting from targeted deletion of Fak in NCCs. Conditional Fak mutants present craniofacial and cardiovascular malformations that lead to early postnatal lethality and resemble common genetic forms of human congenital heart disease. Mutants display cleft palate together with several cardiovascular defects, including persistent truncus arteriosus, overriding aorta, ventricular septal defect, and type B interruption of the aortic arch.
FAK functions in multiple signaling pathways, including the ones initiated by integrins, FGFs, and TGF-β (8
). We found that TGF-β, FGF2, and FGF8 are able to induce FAK phosphorylation and FAK-mediated phosphorylation of Erk1/2 and Crkl in NCCs in vitro. Interestingly, we found that conditional Fak
mutant mice share strikingly similar phenotypes with murine models of DiGeorge syndrome, such as mice with a 1.5-Mb deletion in the critical 22q11 region (21
); ablation of Crkl
, which maps within this region (5
); or Erk2
deletion, localized to a distal region in chromosome 22q11 (51
). Disruption of Tbx1, FGF8, and TGF-β signaling also generates many features of DiGeorge syndrome (7
). However, inactivation of Tbx1 or FGF8 results in abnormal patterning of the aortic arch arteries and defective migration or survival of NCCs, leading to conotruncal heart defects (7
). In contrast, the development of aortic arch defects in Fak
mutant mice is due to failures in NCC differentiation, not migration or survival, and initial formation of aortic arch arteries seems normal. Thus, phenotypic differences do not support a close association between Tbx1 or FGF8 genetic pathways and FAK signaling in NCCs. Moreover, recent studies have provided evidence that NCCs are not direct targets of secondary heart field–derived FGF signaling (55
In this study, we show that Fak
mutant outflow tracts have reduced Crkl and Erk1/2 phosphorylation, indicating that Crkl and Erk1/2 are FAK effectors in NCCs during outflow tract septation. This is especially interesting, since NCC-specific Erk2
mutant mice recapitulate the major features of DiGeorge syndrome. Crkl is an adaptor that functions downstream of integrin, FGF, and TGF-β receptors to recruit signaling complexes that activate Ras and Rac (13
). Based upon our data, we propose that FAK, Crkl, and Erk1/2 participate in a common pathway, which is involved in the NCC morphogenetic program during outflow tract development, that when perturbed results in DiGeorge syndrome–associated cardiac phenotypes (Figure ). In the future, it will be interesting to determine whether there are genetic interactions between FAK and DiGeorge syndrome–associated genes.
Tests on murine mutants have shown that defects in multiple signaling pathways that affect cardiac NCCs prevent normal development of the aortic arch arteries and cardiac outflow tract, with individual mutants affecting NCC proliferation, survival, migration, or differentiation (57
). Our results indicate that NCCs lacking FAK migrated normally, which is surprising given the importance of FAK in cell motility (8
). NCC migration in vivo and in vitro is primarily mediated by β1
). Integrins promote NCC motility, in part, through protein tyrosine kinase activation (61
). In this regard, a recent study has shown that FAK signaling is required for α5
but not α4
integrin-stimulated neuroblastoma cell motility (62
). Moreover, in Fak
-null fibroblasts, expression of α4
rescued cell motility defects (63
). Thus, our results are consistent with prior studies, documenting a major role for α4
integrin in NCC migration (59
). As noted above, NCC-specific integrin β1
deletion, using Ht-PA-Cre, which is not expressed before NCCs begin migration, does not result in cardiac abnormalities (17
). The same paper cited unpublished observations, indicating that cardiac NCCs are perturbed when integrin β1
is deleted at an earlier time in NCC precursors. Thus, it is not clear if β1
integrins are required for NCC migration. These data indicate, however, that they are not essential for later cardiac NCC differentiation.
In the conditional Fak
mutant, NCC differentiation into smooth muscle is impaired in the aortic arch arteries but appears to be comparatively normal in the cardiac outflow tract region. Thus, the essential roles of FAK in NCC derivatives must differ between these regions, probably due to the different environmental signals to which NCCs are being exposed. Alternatively, outflow tract NCCs could be expressing a different morphogenic program from the one in the aortic arch arteries that includes differentiation to smooth muscle cells through FAK-independent pathways. Interestingly, mice hemizygous for the 22q11 homologous region or with disrupted Notch or TGF-β signaling also exhibit impaired cardiac NCC differentiation into smooth muscle in the aortic arch arteries (21
). Our study and others have shown that TGF-β can activate FAK and enhance Crkl phosphorylation (11
). Consequently, our data suggests that the presence of FAK is required for normal TGF-β signaling in this region through control of Crkl and possibly additional effectors.
We found that FAK is required by NCCs for correct cardiac outflow tract rotation during early cardiovascular development. Defective rotation of the outflow tract underlies the overriding aorta and persistent truncus arteriosus phenotypes found in conditional Fak
mutants. It also appears to be related to a more rounded NCC morphology, with deficient cytoskeletal organization and reduced peripheral cell–associated cortactin in the aorticopulmonary septum. In the conotruncal cushions, we found abnormal condensed mesenchyme formation, with reduced NCC expression of perlecan, osteoglycin, and semaphorin 3C. Deficiencies in semaphorin 3C or perlecan result in congenital cardiovascular defects in mice (44
). Interestingly, TGF-β induces perlecan expression, which binds and modulates integrin and growth factor activities (FGF, PDGF) (67
NCC influx is required for outflow tract rotation (39
(also known as Pax3
) mutant mice have reduced NCC colonization of the cardiac outflow tract and developed several outflow tract defects, including persistent truncus arteriosus and double-outlet right ventricle, together with defective rotation of the outflow tract myocardial wall (47
). Conditional Fak
mutants, however, show defective rotation despite the presence of normal NCC numbers. The mechanisms resulting in cardiac outflow tract rotation remain largely unknown, although the cytoskeleton appears essential to induce the rotational forces associated with heart looping (69
). Several mouse models with an incomplete cardiac outflow tract rotation exhibit abnormal cytoskeleton organization of the outflow tract myocardium (70
). Also, mutation of the folate transport gene, Folr1,
affects cytoskeletal organization in conotruncal tissues, preventing normal outflow tract development (72
FAK signaling may modulate the NCC actin cytoskeleton through many mechanisms (8
). FAK promotes focal adhesion turnover through dynamin. Also, FAK regulates Rho family GTPases through Crkl, p190RhoGEF, p190RhoGAP, and other upstream regulators. FAK regulates N-WASP, an Arp2/3 complex activator. In addition, binding of the FAK FERM domain to Arp2/3 controls protrusive lamellipodia formation and cell spreading (73
). Through Rho, FAK controls cytoskeletal contractility and microtubule stability.
Cardiac outflow tract morphogenesis requires crosstalk between NCCs, secondary heart field, myocardium, mesenchymal, and endothelial cells. As part of this study, we performed an RNA profile analysis using control and Fak mutant E11.5 cardiac outflow tracts. Results revealed interesting candidates downregulated in the Fak mutant, including several microtubule regulators (Stmn3, Dcx) and a FAK-associated phospholipid kinase (Pip5k1b). Interestingly, both Stmn3 and Pip5k1b are known to interact with Rac GTPase signaling and may help explain the altered NCC morphology and actin cytoskeleton observed in mutant NCCs.
In conclusion, our results demonstrate that the presence of FAK in NCCs is required for appropriate cardiac outflow tract morphogenesis and aortic arch remodeling. Given the multiple targets of FAK, future studies will be necessary to clarify the roles of various FAK-regulated signaling pathways in cardiac development. In addition, future analyses of NCC-specific knockout mice that survive to adulthood should reveal functions of FAK in other NCC derivatives.