Generation of mice with ILK deletion specifically in the neural crest cells
We analyzed expression of ILK in the OFT during development. RNA in situ hybridization (ISH) with a probe to ILK revealed ubiquitous expression of ILK in mouse embryos at E8.5 and E9.5 (Additional file 1: Figure S1A,B,B’). Immunostaining with ILK antibody and β-galactosidase (β-gal) staining of adjacent sections from Wnt1-Cre; Rosa-LacZ embryos at E10.5 showed that ILK was expressed in the OFT, including the OFT mesenchyme that was colocalized with Wnt1-Cre lineage (β-gal+) (Additional file 1: Figure S1C-E). Furthermore, immunostaining of cultured NCCs with antibodies to ILK and Sox10, a neural crest marker, revealed expression of ILK in the cytoplasm and focal adhesions of NCCs (Additional file 1: Figure S1F-H).
To investigate the role of ILK in NCC and OFT morphogenesis, we ablated ILK specifically in NCCs using Wnt1-Cre. Floxed ILK (ILKf/f) [27] mice were crossed with Wnt1-Cre mice. Resulting ILK heterozygous mice (Wnt1-Cre;ILKf/+) were viable, fertile and did not present any phenotypes, and thus were used as a littermate control in this study. Wnt1-Cre;ILKf/+ mice were backcrossed with ILKf/f mice to generate NCC-specific ILK mutant mice (NKO). Ablation of ILK expression in neural crest derivatives of the OFT was confirmed by ILK immunostaining (Additional file 1: Figure S1I-L).
Deletion of ILK in NCCs resulted in embryonic lethality and OFT malformation
To visualize the morphology of the OFT and pharyngeal arch arteries, we did ISH analysis of NKO and control littermates at E10.5 with a probe to connexin 40 (C×40) that is expressed in vascular endothelial cells, thus outlining the blood vessels [30]. At E10.5, the pharyngeal arteries of NKO mutant embryos displayed a pattern similar to that of control littermates (Figure 1A,B). However, a notable dilation of NKO mutant OFT was observed at this early stage (Figure 1B, arrow). From E11.5 onwards, NKO mutant embryos exhibited progressive OFT dilation, hypoplastic thymus and frequent cranial hemorrhage (Figure 1C-F). All NKO mutant embryos died around E13.5. At E13, control littermates exhibited well-defined aorta and pulmonary arteries, but NKO mutant OFT embryos displayed an enlarged common trunk that was stiff and difficult to separate from surrounding tissues (Figure 1E,F, common arterial trunk (CAT)). Histological analysis of control (Figure 1G,I,K,M,O) and NKO mutant (Figure 1H,J,L,N,P) embryos at E11.5 and E13 revealed that NKO mutant OFTs were markedly dilated and protruded in between the tongue and pharynx, pushing the tongue ventrally, and the pharynx of NKO mutants became deformed at E13 (Figure 1L, arrow). Furthermore, we observed ventricular septal defect (VSD), a thinner ventricular myocardium compact zone (Figure 1O,P) and shortened mandibles (Figure 1K,L) in NKO mutants at E13. Furthermore, MRI and 3D reconstruction of control and NKO mutant hearts at E13 revealed severe cardiovascular defects in NKO mutant embryos, including VSD and right ventricular outflow tract (RVOT) and a markedly dilated CAT that appeared to be formed by inclusion of all segments of the OFT and proximal parts of the pharyngeal arch arteries (Figure 1Q-T).
Premature emigration and altered migration pattern of cardiac NCC in NKO mutant embryos
Wnt1-Cre lineage analysis was performed to examine the migration and colonization of NKO mutant cardiac NCCs into the pharyngeal arches and OFT. Wnt1-Cre; ILKf/+ mice were crossed with ILKf/f mice on a Rosa-LacZ [31] or Rosa-tdTomato [32] reporter background that allows us to visualize NCCs by β-gal staining and tdTomato Red. At E8.5 (5 to 6 somites, S5, S6), a slightly increased number of β-gal+/tdTomato + cells were observed in the cranial mesenchyme and the forming first pharyngeal arch (trigeminal neural crest-tg) of NKO mutants (Figure 2A,B,G,H, tg). Few, if any, control postotic NCCs were found in the paraxial mesoderm underneath the neural plates (Figure 2A-A’, arrow, inset; 2G’). In contrast, a substantial number of postotic NCCs of somite-matched NKO mutant embryos at E8.5 (S5) were found to migrate to the paraxial mesenchyme (Figure 2B-B’, arrow, inset; 2H’), suggesting ablation of ILK resulted in premature NCC emigration. Control cardiac NCCs at E9 (16 to 18 somites) migrated as distinct streams en route to pharyngeal arch 3, 4, 6 and the OFT, a few of which migrated into the aortic sac (Figure 2C,C’, arrow). In contrast, a significant increase in the number of NKO mutant NCCs at E9 (somite-matched) was observed in cranial regions and the first and second pharyngeal arches (Figure 2D). Migration of postotic NCCs of NKO mutants appeared to be diffuse and the caudal stream seemed slightly wider in the NKO mice (Figure 2D, arrow). Histological analysis revealed an increased number of NCCs accumulated in the pharyngeal arches and aortic sac in NKO mutant embryos at E9.0 and some NCCs had prematurely migrated into the OFT (Figure 2D’). At E10.5, cardiac NCCs of control embryos started to migrate into the OFT up to the conotruncal junction and colonized the subendocardial mesenchyme of the outflow cushion (Figure 2E,E’,E”, red arrowhead). In somite-matched NKO mutants, the OFT and the third pharyngeal arch were significantly enlarged and a marked increase in the number of NCCs was found in the pharyngeal mesoderm. Within the OFT, NKO mutant NCCs migrated beyond the conotruncal junction and invaded entire layers of the OFT (Figure 2F,F’,F”, red arrowhead). At E13, histological analysis of the aortic arch and proximal OFT revealed that the OFT of control littermates were separated into aorta and pulmonary arteries, and control NCCs contributed to a subpopulation of mesenchymal cells (tdTomato Red+) of aortic arch, aorta, pulmonary artery and OFT cushion (Figure 2K,K’). However, the OFT of NKO mutants at E13 exhibited an enlarged common trunk with a significantly increased number of NCCs (tdTomato Red+) (Figure 2L,L’).
An increased number of NCCs in the pharyngeal arches and OFT of NKO mutant embryos may be due to increased NCC proliferation and reduced cell death. We examined NCC proliferation by phosphohistone H3 (Ph3) antibody staining at E8.5 (Figure 2G,H, cranial level; G’,H’, postotic level), E11 (Figure 2I,J) and E13 (Figure 2K-L’). Mitotic indices were determined by counting the number of cells per section that were doubly positive for tdTomato Red (NCCs) and Ph3 staining, normalized to the total number of NCCs and derivatives (tdTomato+) within the pharyngeal arteries and OFTs of NKO mutant and control embryos (Figure 2M). We did not observe significant change in proliferation in the pharyngeal and OFT regions of NKO and control littermates. We also performed terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining to measure neural crest cell death and no significant change in cell death in the pharyngeal and OFT regions of NKO and control littermates was observed at different developmental stages (data not shown).
We further examined the rate and pattern of NCC migration in neural crest explant culture from E8.5 NKO mutant and control embryos. NKO mutant NCCs on fibronectin coated plates were able to migrate out of neural tube explants. However, after 24 h, most NKO mutant NCCs rounded up and detached, suggesting defective adhesion. We cultured neural crest explants embedded in mitrogel. After 24 h, some control NCCs (tdTomato Red+) started to migrate out as distinct streams (Figure 2N,N’). However, NKO mutant NCCs migrated earlier (12 to 24 h after incubation) and lost their stream-like migration pattern as individual cells (Figure 2O,O’). Quantitative analysis of NCC migration [33] revealed that NKO mutant NCCs migrated significantly longer distances and to a bigger outgrowth area (Figure 2P) compared to control NCCs.
Reduced focal adhesion and Akt phosphorylation, and impaired cytoskeleton organization in NKO mutant NCCs
To examine the formation of focal adhesion complexes of mutant NCCs in culture, we examined expression of paxillin and parvin, two key components of the focal adhesion complex. In control NCCs, expression of paxillin and parvin displayed a well organized pattern associated with focal adhesions (Figure 3A,C). In NKO mutant NCCs, expression of paxillin in focal adhesions appeared to be diffuse. However, expression of parvin and PINCH1 were relatively normal revealed by immunostaining and western blot (Figure 3B,D, and data not shown).
NCCs express multiple integrin subtypes with differential binding affinity to distinct ECM ligands. To test whether deletion of ILK selectively affected the adhesive function mediated by particular integrins, we assayed adhesion of NKO mutant and control NCCs to different integrin ligands. Because of difficulty of isolating enough NCCs from NKO mutant embryos, we established an ILKf/f NCC culture and ablated ILK expression in vitro by infection of Cre recombinase adenoviruses [28]. For this experiment, ILKf/f NCCs were infected with Cre or control adenoviruses, and cultured for additional 48 h and then used to examine adhesion and spreading of ILK mutant (ILKf/f;Cre+) and control NCCs (ILKf/f;Cre-) on different substrates. ILK mutant NCCs on all substrates tested exhibited progressive cell shrinkage (Figure 3E,K) and detachment during the course of culture. Adhesions of ILK mutant NCCs to fibronectin, laminin, collagen and vitronectin were markedly reduced (Figure 3F) suggesting that in NCCs, ILK is required for adhesion to multiple classes of integrins.
Activation of integrins leads to Akt phosphorylation and cytoskeleton reorganization required for cell growth, survival and migration. Coimmunostaining with antibodies to β-gal and phosphorylated Akt revealed a significant reduction in Akt phosphorylation in NKO mutant NCCs compared to the control (Figure 3G-I). In culture, control NCCs were polarized with well-aligned actin filaments. However, in ILK mutant NCCs 48-h post-viral infection, there was a large accumulation of cortical actin in the cell cortex and a significant increase in short, randomized filapodia-like protrusions (Figure 3J,K).
Reduced ECM deposition and cell-cell adhesion in NKO mutant embryos
We examined whether impaired integrin-ILK signaling resulted in changes in ECM expression in NKO mutant OFTs by immunostaining. We observed significantly reduced expression of laminin and fibronectin in NKO mutant OFT at E9.5 relative to control (Figure 4A-D). At E13, elastin, a key component of the elastic matrix of the great vessels, was expressed in the aortic arch of control embryos. However, elastin expression in NKO mutant OFT was significantly reduced (Figure 4E,F). In addition, microarray analysis of RNAs from NKO mutant and control NCCs revealed significantly reduced expression of several ECM components (Additional file 2: Table S1), selected targets were confirmed by quantitative polymerase chain reaction (qPCR) studies, including lamb1, col9a3, col8a2 and Fndc5 (Figure 4G).
We further investigated whether deletion of ILK resulted in defective cell-cell adhesions by examining the expression of NCAM that is required for NCC migration [34]. In culture, control NCCs were elongated and established proper cell-cell contacts and alignment. Clusters of NCAM were found at the cell-cell contacts of control NCCs (Figure 4H). However, ILK mutant (ILKf/f;Cre+) NCCs in culture tended to be separated with markedly reduced NCAM expression (Figure 4I). Consistent with this, we observed a significant reduction in NCAM expression in the dorsal neural tube of NKO mutant embryos at E8.5 (Figure 4J,K, brackets).
Reduced TGFβ signaling and smooth muscle differentiation in NKO mutant OFT
Differentiation of cardiac NCCs to OFT smooth muscle was assessed by immunostaining with an antibody to α-smooth muscle actin (α-SMA) and NCC lineage marker (tdTomato Red). In contrast to control OFT with well-formed smooth muscle layers (Figure 5A,A’), α-SMA expression in E12.5 NKO mutant OFT that were derived from NCCs (tdTomato Red+) was greatly reduced (Figure 5B,B’, arrow), although smooth muscle differentiation from non-NCCs (tdTomato Red-) appeared to be relatively normal (Figure 5B,B’, arrowhead). It has been reported that TGFβ2 signaling can affect OFT development [35, 36], we examined TGFβ2 expression and its downstream mediator Smad2 activation at E9.5 in ILK mutants. Wholemount ISH revealed markedly reduced TGFβ2 expression in NKO mutant OFT relative to control (Figure 5C,D, arrow). In addition, western blot using ILKf/f;Cre+ mutant and control NCCs revealed significantly reduced TGFβ2 expression and Smad2 phosphorylation, although the total Smad2 level was not changed (Figure 5E, E’).
Microarray analysis of the OFT of NKO mutants and control littermates
To gain insight into genetic programs required for NCC and OFT development, we performed microarray analysis of cardiac NCCs that were fluorescence-activated cell sorting (FACS)-sorted from the OFT of NKO mutant and control littermate embryos at E10.5 (Additional file 2: Table S1). Microarray data were submitted to the Gene Expression Omnibus database under GEO accession numbers GSE41179. Consistent with the observed phenotypes, our data revealed significant changes (fold of change ≥ ±1.5) in the expression of genes associated with migration/cytoskeleton/ECM, muscle/heart development, neurogenesis and osteogenesis (Additional file 2: Table S1).
Among genes implicated in migration/cytoskeleton/ECM, we found increased expression of Plek, Fermt3 and Ly86, but decreased expression of Col9a3, Lamb1-1, St3gal4 and Limk1. We found a significant reduction in the expression of genes of muscle differentiation including Myog, Scx, Zfp874, Xist, Neb and Nppa, suggesting reduced myocardialization of NKO mutant OFT. Sall4, a zinc finger transcription factor associated with heart and OFT development, was upregulated in NKO mutant [37]. Gp1bb, a gene located within the DiGeorge syndrome critical region, was upregulated in NKO mutants. Components of a number of signaling pathways involved in cardiovascular development were upregulated, among which, Sost and Wfikkn1 have been reported to be mediators of TGFβ, BMP and Wnt signaling, and Fabp7 is a direct target of Notch signaling in glial cells. Lbxcor1, downregulated in NKO mutants, is a Smad binding protein and negatively regulates BMP signaling. Selected targets were verified by qPCR (Additional file 2: Table S1 and Figure 6A,B).
Development of the parathyroid from the third pharyngeal pouch is initiated via inductive signals that involve NCCs. Parathyroid hypoplasia is a primary feature of DiGeorge syndrome. We found parathyroid hormone (Pth) expression was greatly reduced in NKO mutants (Additional file 2: Table S1).
Ectopic ossification and neurogenesis in NKO mutant OFT
NCCs have the potential to differentiate into multiple cell types, including osteoblasts and neurons of the peripheral nervous system. Interestingly, in NKO mutant OFT, we found significantly increased expression of genes associated with neural differentiation, including a number of neuronal cytoskeleton and associated proteins (Sncg, Stmn2, 3, Nefm, Nefl, Tubb1) and neural signaling factors (Neurod1, Neurod4, Nhlh2, Fabp7, Elavl4) (Additional file 2: Table S1 and Figure 6B). Neurofilament immunostaining (Figure 6C,D) and β-gal staining (Figure 6C’,D’) of adjacent sections of control embryos at E12.5 revealed the formation of the superior cervical ganglia (scg) and cardiac ganglia (red arrowhead in D) around aorta and pulmonary arteries. However, in NKO mutant embryos at E12.5, we observed ectopic innervation or ganglia in the distal wall of the CAT and within the proximal wall of the OFT (Figure 6E-F’ , red arrowhead).
In addition, a number of genes associated with osteogenesis exhibited altered expression patterns, for example, expression of Spp1 was upregulated, whereas Aspn and Lect1, negative regulators of osteoblast differentiation, were downregulated, suggesting increased ossification (Additional file 2: Table S1). Consistent with this, we found, by Alcian blue (AB) staining, ectopic ossification in NKO mutant OFT at E12.5 (Figure 6G,H). In addition, ISH and qPCR revealed marked upregulation of Col1a2, a marker of osteoblast differentiation [38], in NKO mutant OFT at E12.5 (Figure 6I-K). BMP2 signaling has been implicated in osteoblast differentiation [39]. We observed significantly increased phosphorylation of Smad1 and expression of BMP2K (BMP2-inducible kinase) in the OFT of NKO mutant embryos at E10.5 (Figure 6L).