TBX1 regulates epithelial polarity and dynamic basal filopodia in the second heart field

Morphogenesis of the embryonic heart occurs by addition of cardiac progenitor cells to the elongating arterial and venous poles of the heart tube (Cai et al., 2003; Kelly et al., 2001; Mjaatvedt et al., 2001; Waldo et al., 2001). These progenitor cells, termed the second heart field (SHF), are located in splanchnic mesoderm in the dorsal wall of the coelomic or pericardial cavity (dorsal pericardial wall, DPW) and are characterized by elevated proliferation and delayed differentiation (Kelly, 2012; Vincent and Buckingham, 2010). Anterior and posterior SHF populations have been identified that contribute to the right ventricle and outflow tract (OFT) at the arterial pole and atrial myocardium at the venous pole, respectively (Vincent and Buckingham, 2010). Perturbation of SHF development results in a shorter heart tube that fails to remodel correctly during cardiac septation, leading to a spectrum of morphological anomalies (Dyer and Kirby, 2009). Understanding how progressive deployment of SHF cells is regulated is therefore an important step towards deciphering the origins of congenital heart defects. Previous research has focused on the signaling pathways and transcription factors operating in the SHF, including the LIM homeodomain transcription factor Isl1, required for heart tube elongation, and the T-box-containing transcription factor TBX1, required for development of the distal OFT (Cai et al., 2003; Dyer and Kirby, 2009; Xu et al., 2004). TBX1 regulates proliferation and differentiation in the SHF and is the major gene implicated in 22q11.2 deletion (or DiGeorge) syndrome, a primary cause of congenital heart defects affecting the cardiac OFT (Chen et al., 2009; Liao et al., 2008; Pane et al., 2012; Papangeli and Scambler, 2013). However, despite progress in dissecting SHF regulatory networks, basic cellular features of the SHF, including cell polarity, cell shape and cell dynamics, remain to be investigated.

Polarized epithelia play central roles in embryonic development. In particular, the formation and remodeling of epithelia drive diverse morphogenetic processes, and reiterative rounds of mesenchymal-to-epithelial and epithelial-to-mesenchymal transitions accompany organogenesis, including heart development (Thiery et al., 2009). In the early embryo, cardiac progenitor cells, giving rise to the cardiac crescent and linear heart tube, are located in anterior splanchnic mesoderm and form an epithelial sheet in the ventral midline. The epithelial properties of these early cardiac cells have been studied by Linask and colleagues in the avian embryo, where epithelialization and the regulation of N-Cadherin accumulation has been shown to play an important role in the onset of differentiation (Linask, 1992; Linask et al., 2005). The epithelial organization of myocardial cells has also been studied in zebrafish, where basal accumulation of fibronectin, regulated by Hand2, and the polarized epithelial organization of myocardial cells downstream of Has/PKCι are required for normal morphogenesis of the early fish heart (Rohr et al., 2006; Trinh and Stainier, 2004; Trinh et al., 2005). By contrast, the epithelial properties of SHF cells located in the DPW during late stages of vertebrate heart tube elongation have received less attention. In a pioneering study, Virágh and Challice showed, using light and electron microscopy, that cells adjacent to the distal border of the heart tube of the mid-gestation mouse embryo form a cuboidal epithelium of condensed mesenchymal cells (Virágh and Challice, 1973). However, molecular characterization of the epithelial properties of SHF cells has not been performed and the regulators of such properties are unknown.

Here, we characterize apicobasal polarity, cell shape and cell dynamics in the murine SHF. We demonstrate that SHF cells constitute an atypical polarized epithelium with apical monocilia and dynamic basal filopodia, visualized by time-lapse microscopy of living SHF cells. Furthermore, we show that TBX1 is a regulator of epithelial cell morphology in the DPW. Cell shape changes in mutant embryos include increased circularity, a reduced basolateral membrane domain and impaired filopodial activity, and are associated with elevated aPKCζ levels. Using embryo culture, we show that activation of aPKCζ similarly impairs filopodia activity and phenocopies proliferative defects and ectopic differentiation observed in the DPW of Tbx1 null embryos. Our results identify control of the epithelial properties of SHF progenitor cells as a regulatory step in progressive heart tube elongation and OFT morphogenesis.

The mouse heart tube undergoes rapid elongation by addition of cardiac progenitor cells from the SHF between embryonic days (E) 8.5 and 10.5. At E9.5, SHF cells expressing an Fgf10 enhancer trap transgene (Fgf10-nlacZ) and Isl1 are located in splanchnic mesoderm in the DPW (Fig. 1A-D). We investigated the epithelial properties of SHF cells in the DPW using immunofluorescence microscopy (Fig. 1E-N). aPKCζ and Crumbs3 [crumbs homolog 3 (Drosophila), Crb3 – Mouse Genome Informatics] are localized at the apical membrane and are enriched at cell-cell junctions in the DPW (Fig. 1E,G,H); by contrast, Scribble is localized at the basolateral membrane (Fig. 1F,G). The tight junction protein ZO-1 (Tjp1 – Mouse Genome Informatics) accumulates at apical cell-cell junctions in the DPW (Fig. 1I) together with Par3 and PATJ (Inadl – Mouse Genome Informatics) (supplementary material Fig. S1). Adherens junctions are present at the boundary between apical (aPKCζ positive) and basolateral (Scribble positive) membrane domains, labeled by N-cadherin and, at lower levels, E-cadherin (Fig. 1J-L; supplementary material Fig. S1). Basally, SHF cells are characterized by an irregular basal lamina and a fibronectin-rich extra-cellular matrix (Fig. 1M,N). Together, these results demonstrate that Fgf10-nlacZ- and Isl1-expressing cells in the DPW constitute an apicobasally polarized epithelium, with an apical surface facing the pericardial cavity and a basal surface facing underlying mesenchyme and pharyngeal endoderm. These epithelial properties are shared with the columnar epithelium of the overlying pharyngeal endoderm, although the latter is distinguished by higher levels of E-cadherin accumulation and a more continuous basal lamina (Fig. 1E-N).

SHF cells contribute to the growth of the OFT at the arterial pole of the heart and to atrial myocardium at the venous pole, corresponding to anterior and posterior domains of the SHF (Vincent and Buckingham, 2010). Whereas Isl1 is expressed in both domains, Fgf10-nlacZ expression is restricted to the anterior region (Fig. 2A,B; Cai et al., 2003; Kelly et al., 2001). We investigated whether cells differ in their epithelial properties in the anterior (aDPW, Fgf10-nlacZ expressing) and posterior (pDPW, Fgf10-nlacZ non-expressing) DPW (Fig. 2). ZO-1 and aPKCζ/Scribble are expressed in Isl1-positive cells throughout the DPW (Fig. 2B; supplementary material Fig. S2). N-cadherin is also expressed in the entire DPW, although with a gradient increasing towards the venous pole, and is strongly expressed in differentiated cardiomyocytes (Fig. 2C,G; supplementary material Fig. S2). By contrast, E-cadherin accumulates only in the aDPW and the most lateral regions of the pDPW and is maintained in differentiated cardiomyocytes in the OFT (Fig. 2D,G; supplementary material Fig. S2). A basal lamina is not apparent in the pDPW, in contrast to the aDPW (Fig. 2E-E″; supplementary material Fig. S2), and a low level of fibronectin labeling between cells was observed in the pDPW (Fig. 2F-F″; supplementary material Fig. S2). The pDPW is thus a distinct epithelial domain within the DPW, with elevated N-cadherin levels, lack of E-cadherin and a reduced basal lamina. A day earlier in development, at E8.5, the distribution of ZO-1, aPKCζ, Scribble and N-cadherin accumulation in the DPW is similar to that at E9.5 (supplementary material Fig. S3). However, the DPW is entirely E-cadherin negative and the basal lamina is not well formed, as indicated by punctuated laminin staining (supplementary material Fig. S3). Cells in the coelomic wall are apicobasally polarized as early as E7.5, although N-cadherin expression is less apically enriched and a basal lamina is absent (supplementary material Fig. S3). This spatiotemporal heterogeneity suggests that the epithelial properties of the DPW progressively mature and diversify as SHF cells contribute to the cardiac poles, with the appearance of E-cadherin-containing junctions and a basal lamina in the aDPW by E9.5. Such differential epithelial properties might underlie segregation of anterior and posterior progenitor cell populations during SHF deployment.

The epithelial structure of DPW cells was further investigated by transmission electron microscopy (TEM) at E8.5 and E9.5. DPW cells are characterized by an elongated triangular shape with a smooth convex apical surface (Fig. 3A). Cell contacts are restricted to the apical region, where tight and adherens junction proteins accumulate (Fig. 3A,B, arrows). Scanning EM and immunofluorescence with an anti-acetylated-α-tubulin antibody revealed that the smooth apical surface is characterized by the presence of monocilia with a 9+0 microtubule structure, extending into the pericardial cavity (Fig. 3D,E). The basolateral membrane of epithelial cells in the DPW was observed to form multiple filopodia-like protrusions, which are more commonly observed in mesenchymal cells (Fig. 3A, arrowhead). The shape and organization of epithelial cells in the DPW, in particular the presence of such basolateral protrusions, thus differs significantly from those of columnar epithelia such as pharyngeal endoderm (Fig. 3C). Visualization of basal protrusions was facilitated by Mesp1-Cre activation of a conditional GFP reporter gene (Fig. 3F,G). Together with TEM and phalloidin staining on sagittal and transverse sections, these data revealed that basal filopodia are present throughout the DPW (supplementary material Fig. S4). Furthermore, these protrusions are enriched in actin filaments and microtubules, confirming their identification as filopodia-like structures (Fig. 3H,I).

In order to investigate the dynamic properties and contacts of these filopodia-like structures, we established a thick-slice culture protocol and performed live-imaging of cells in the DPW. Time-lapse microscopy of transverse slices from E8.5 and E9.5 embryos, using the Mesp1-Cre activated GFP reporter, allowed the first visualization of cell behavior in the SHF. These experiments revealed that the basal filopodia are highly dynamic, extending towards and making contact with overlying mesenchyme and pharyngeal endoderm (Fig. 3J; supplementary material Fig. S5 and Movie 1). Quantification of filopodial parameters revealed that basal DPW filopodia extend from 4 µm to 22 µm (Fig. 3K) and have a lifetime ranging from a single timeframe (5 min or less) to 300 min, with a mean duration of 62 min (Fig. 3L). In order to visualize actin filament dynamics, a plasmid encoding an utrophin-GFP fusion protein, which binds F-actin without disturbing actin assembly or disassembly (Burkel et al., 2007), was electroporated into the DPW (Fig. 4M). Time-lapse microscopy of electroporated cells revealed that actin accumulated apically and in the basal protrusions, where dynamic extension and retraction of actin filaments was observed (Fig. 3M; supplementary material Movie 2, left), suggesting that DPW filopodial movement is driven by active actin filament remodeling. Multiple intercellular signaling pathways have been shown to regulate SHF development, including fibroblast growth factor (FGF) signaling that promotes proliferation in the SHF (reviewed by Rochais et al., 2009). We investigated whether basal filopodia could sense FGF signaling, using an anti-phospho-tyrosine (p-Tyr) antibody to score subcellular localization of receptor activation. In support of a sensing role, p-Tyr staining accumulates in the basal filopodia of DPW cells (Fig. 3N).

The 22q11.2 deletion syndrome candidate gene Tbx1 encodes a key regulator of pharyngeal morphogenesis and SHF development (Chen et al., 2009; Liao et al., 2008). We investigated whether altered epithelial properties are associated with abnormal SHF development in Tbx1^−/− embryos. Although SHF cells in Tbx1^−/− embryos are apicobasally polarized, they appear rounder than cells in control embryos (Fig. 4). Quantification of the circularity index, using aPKCζ and Scribble to visualize cell membranes, confirmed that SHF cells are significantly rounder in the aDPW of Tbx1^−/− than in wild-type embryos (Fig. 4A-G). Further analysis revealed that cells in the Tbx1^−/− aDPW have expanded apical (aPKCζ positive) and reduced basolateral (Scribble positive) membrane domains and that cells are partially extruded from the epithelium (Fig. 4A,D; supplementary material Movie 3). Extruded cells with enlarged apical domains were also observed using TEM (Fig. 5F). E-cadherin labeling revealed that the interface between apical and basolateral domains is shifted basally (Fig. 4D-F). Quantification revealed that total circumferential membrane length is reduced, whereas the apical/basolateral membrane ratio is increased in Tbx1^−/− DPW cells (Fig. 4H,I). E- and N-cadherin, ZO-1 and Crumbs3 accumulate in the aDPW of Tbx1^−/− mutant embryos with levels indistinguishable from those in wild-type embryos (Fig. 4J,L; data not shown). However, extruded cell nuclei can be observed on the pericardial side of these apical and junctional markers (Fig. 4J,L, arrowheads; supplementary material Movie 4), a situation not observed in wild-type embryos (Fig. 4M, arrowheads; supplementary material Movie 4), and consistent with a shifted interface between apical and basolateral domains. These phenotypes are restricted to anterior SHF progenitor cells in the aDPW, which is hypoplastic in Tbx1^−/− embryos (Kelly and Papaioannou, 2007; Liao et al., 2008); close to the venous pole, epithelial organization is similar to that in wild-type embryos (Fig. 4). In addition to the changes described above, laminin staining is more punctuated and diffuse in the aDPW of Tbx1^−/− embryos compared with wild-type embryos (Fig. 4N,O), suggesting defects in basal lamina establishment. Finally, increased localization of fibronectin between epithelial cells was observed throughout the Tbx1^−/− DPW (Fig. 4P-S; supplementary material Movie 5). Together, these findings reveal that TBX1 regulates apicobasal polar properties and is required for normal cell shape in the aDPW.

A reduction in the basal membrane of cells in the aDPW of Tbx1^−/− embryos could impact on basal filopodia formation. We therefore investigated the dynamic properties of epithelial cells in Tbx1^−/− embryos, using time-lapse imaging of slice cultures. Individual or small groups of cells were labeled by electroporation of a CMV-GFP plasmid into the DPW. Strong cytoplasmic GFP expression allowed visualization of long dynamic basal filopodia in control embryos at E8.5 (Fig. 5A; supplementary material Movie 6). At this stage, prior to overt morphological anomalies, Tbx1^−/− embryos present a similar, although milder, epithelial phenotype to that in the aDPW at E9.5 (supplementary material Fig. S6). Cells in the DPW of Tbx1^−/− embryos are rounder and form fewer and smaller filopodia (Fig. 5B; supplementary material Movie 6). Measurements of electroporated cells revealed that filopodia in control embryos extend from 5 µm to 42 µm, with a mean length of 13.5 µm (Fig. 5C). By contrast, significantly smaller filopodia were observed in Tbx1^−/− embryos, extending from 4 µm to 14 µm, with a mean length of 7 µm (Fig. 5C). The lifetime of individual filopodia was also reduced in the absence of TBX1 (Fig. 5D). Utrophin-GFP was used to investigate actin filament dynamics, and rounder cells were observed in Tbx1^−/− embryos, with fewer and smaller dynamic actin filaments on the basal side (Fig. 5E; supplementary material Movie 2). TEM revealed a similar filopodial phenotype in the Tbx1^−/− aDPW at E9.5 (Fig. 5F). Basal actin filaments are reduced compared with wild type, and the residual filopodia show reduced p-Tyr labeling (Fig. 5G). By contrast, pDPW cells close to the venous pole of Tbx1^−/− embryos maintain an elongated triangular shape with actin-enriched basal filopodia at E9.5 (supplementary material Fig. S6). Investigation of monocilia, using scanning EM and immunofluorescence, revealed that apically localized primary cilia are present throughout the Tbx1^−/− DPW (Fig. 5H,I).

The observation that altered epithelial properties of the DPW in Tbx1^−/− embryos are restricted to the aDPW is consistent with the expression of TBX1 in the aDPW and only the most lateral regions of the pDPW at E9.5 (Fig. 6A,B). Microarray analysis of Tbx1^−/− embryos has identified a number of genes encoding epithelial proteins as potential TBX1 targets, including E-cadherin and Crumbs3 (Liao et al., 2008; van Bueren et al., 2010). However, the level of these two proteins appears to be unchanged in the aDPW of Tbx1^−/− embryos (Fig. 5, and data not shown). By contrast, we observed increased intensity of aPKCζ immunolabeling in the aDPW of Tbx1^−/− compared with wild-type embryos (Fig. 6C-F; supplementary material Fig. S7). This increase is particularly marked in lateral regions of the aDPW (Fig. 6C, arrowheads). aPKCζ is an important regulator of apicobasal polarity (Xiao and Liu, 2013). Indeed, aPKCζ promotes the apical membrane domain of epithelial cells, and elevated aPKCζ levels might contribute to the reduction of the basolateral domain and impairment of basal filopodia in the Tbx1^−/− DPW. We quantified the intensity of aPKCζ immunostaining, using high-magnification immunofluorescence in both the cytoplasm and apical cell membrane. The relative intensity of aPKCζ is significantly increased in both the cytoplasmic pool and at the apical membrane of Tbx1^−/− aDPW cells (Fig. 6G,H), consistent with an increase in aPKCζ protein levels. By contrast, aPKCζ levels are comparable to wild type in adjacent mesenchymal cells, pharyngeal endoderm and the pDPW (Fig. 6, data not shown). Elevated aPKCζ expression is also observed in the most anterior region of the DPW in Tbx1^−/− embryos at E8.5 (supplementary material Fig. S6). Increased aPKCζ levels in the DPW of Tbx1^−/− embryos were confirmed by western blot; furthermore, these experiments revealed that the level of the active phosphorylated form of aPKCζ was increased in the pharyngeal region of Tbx1^−/− embryos (Fig. 6I).

In order to investigate the potential role of elevated aPKCζ levels in the etiology of the Tbx1 null epithelial phenotype, we cultured E8.5 embryos for 24 h in the presence of a physiological activator of aPKCζ (El-Hashash et al., 2012; Limatola et al., 1994). Western blot analysis confirmed that the level of phosphorylated aPKCζ was increased after embryo culture (supplementary material Fig. S8). Investigation of the DPW in treated embryos revealed that apicobasal polarity is perturbed, associated with a significant reduction of the basal membrane (Fig. 7A-D). Strikingly, DPW cells in treated embryos form fewer basal filopodia compared with control embryos (Fig. 7E,F), similar to the situation in Tbx1^−/− embryos. Given the potential role for DPW filopodia in environmental sensing and intercellular signal transmission, as suggested by localized p-Tyr expression, we investigated whether downstream signaling events in the FGF pathway were affected in treated embryos. Indeed, phospho-ERK staining is reduced in the DPW of treated, but not of control embryos, consistent with decreased FGF signaling and observations in Tbx1^−/− embryos (Fig. 7G,H; supplementary material Fig. S8). FGF signaling plays an important pro-proliferative role in the SHF. We evaluated proliferation in the DPW of treated embryos and observed a significant reduction in the number of KI67 (Mki67 – Mouse Genome Informatics) and phosphorylated Histone H3 (PH3)-positive cells (Fig. 8A-D). TBX1 regulates both proliferation and differentiation in the SHF, and ectopic myocardial differentiation has been observed in the aDPW of Tbx1^−/− embryos (Chen et al., 2009; Liao et al., 2008). We investigated whether altered epithelial properties in the DPW of embryos treated with the aPKCζ activator are associated with ectopic cell differentiation. In control embryos, sarcomeric myosin heavy chain (Myhc) is expressed in the distal OFT (Fig. 8E; supplementary material Fig. S8). In both Tbx1^−/− and aPKCζ activator-treated embryos, differentiation is observed in the aDPW contiguous with the OFT (Fig. 8F-I; supplementary material Fig. S8). Quantification revealed elevated numbers of MF20-positive cells in the aDPW of aPKCζ activator-treated embryos compared with control embryos (Fig. 8F,H,I). Abnormal differentiation in the aDPW was confirmed with α-actinin, α-SMA and Troponin I (supplementary material Fig. S8), although differentiated cells in the aDPW continued to express the progenitor cell markers Isl1 and TBX1 (supplementary material Fig. S8). These results suggest that aPKCζ upregulation in Tbx1^−/− embryos is unlikely to be a consequence of increased differentiation; furthermore, aPKCζ levels do not increase in differentiated OFT myocardium of wild-type embryos (supplementary material Fig. S7). Finally, we investigated whether the reduced proliferation and premature differentiation in the SHF of aPKCζ activator-treated embryos has an impact on OFT growth. Quantification of OFT morphology revealed that treated embryos have a significantly shorter and straighter OFT, with a larger angle between the distal and the proximal OFT compared with control embryos (Fig. 8J-L). Together, these results show that directly manipulating the epithelial properties of SHF progenitor cells by increasing activated aPKCζ levels leads to decreased proliferation and premature differentiation in the aDPW, and consequently to reduced extension of the myocardial OFT.

Cardiac progenitor cells giving rise to the linear heart tube differentiate within an N-cadherin-expressing epithelium in anterior lateral mesoderm (Linask, 1992; Linask et al., 2005). Our characterization of apicobasal polarity and cell morphology in the SHF reveals that these late differentiating progenitor cells retain epithelial status within an atypical polarized epithelium in the DPW. Furthermore, analysis of altered apicobasal polarity in the DPW of Tbx1^−/− and aPKCζ activator-treated embryos implicates the control of epithelial properties of SHF cells as a regulatory step in progressive heart tube elongation and OFT morphogenesis.

SHF cells in the DPW have a triangular shape characterized by apical monocilia, and basal actin filament- and microtubule-enriched filopodia-like projections. Our investigation of the micro-behavior of living SHF cells demonstrates that the basal filopodia are highly dynamic. Cell protrusions have recently been reported in the caudal splanchnic mesoderm of mouse embryos, using phalloidin staining (Sinha et al., 2012). Although the dynamic properties of these protrusions were not examined, Sinha and colleagues suggested a potential involvement in cell intercalation, as mesenchymal SHF progenitors incorporate into a cohesive epithelial sheet prior to directional deployment towards the arterial pole. This model is consistent with our finding that the posterior SHF has a less developed basal lamina compared with the anterior SHF. Indeed, the epithelial properties of cardiac progenitor cells in the DPW progressively mature between E7.5 and E9.5 days of development as an E-cadherin- and laminin-positive aDPW domain is established. Such spatiotemporal heterogeneity might facilitate segregation of progenitor cells to the cardiac poles. Filopodia have been implicated in condensation and adhesion of colonization-competent cancer cells after extravasation (Shibue et al., 2012). However, we observed basal filopodia throughout the DPW epithelium, suggesting that their function is not limited to cell intercalation. Filopodia at the leading edge of migrating cells have been implicated in cell motility (Martín-Blanco and Knust, 2001); however, the basal localization of filopodia in DPW cells suggests that they are not involved in epithelial spreading. Filopodia have been recently implicated in environmental sensing, mediating, for example, Shh (Sanders et al., 2013), Notch (Cohen et al., 2010) or FGF signaling (Cooley et al., 2011). Proliferation and progressive differentiation of SHF cells is regulated by the concerted activity of multiple signaling pathways, including Hedgehog, Notch, FGF, BMP and canonical and non-canonical WNT signaling (Dyer and Kirby, 2009; Rochais et al., 2009). Filopodial extensions from the DPW directed to the overlying endoderm and adjacent mesenchymal cells might play a role in mediating signaling events required to maintain cardiac progenitor cell status in the DPW during heart tube extension. In support of a sensing role we observed elevated anti-phospho-tyrosine antibody staining in filopodia, consistent with FGF receptor activation. Furthermore, loss of filopodia in Tbx1^−/− and aPKCζ activator-treated embryos is associated with decreased FGF signaling, reduced proliferation and ectopic differentiation in the SHF (Vitelli et al., 2002; Fig. 8).

TBX1 is required in pharyngeal mesoderm to regulate SHF proliferation and differentiation (Chen et al., 2009; Liao et al., 2008; Zhang et al., 2006). Our results show that TBX1 also regulates epithelial properties of SHF cells: in Tbx1^−/− embryos aDPW cells are rounder, have expanded apical and decreased basolateral membrane domains, and form fewer filopodia with a shorter lifetime. Consequently, a fraction of DPW cells are partly extruded into the pericardial cavity. In addition to these changes, we observed that the basal lamina is disorganized in the absence of TBX1, with an elevated incidence of fibronectin mislocalization. Altered basal extracellular matrix properties might also impact on the formation and duration of filopodia. Increased circularity of cardiomyocytes in tbx1 mutant zebrafish hearts has recently been reported (Choudhry and Trede, 2013). This appears to reflect a block in the transition from a rounded morphology in the early heart tube to an anisotropic elongated shape in the outer curvature of the fish heart. Our results provide further evidence of a role for TBX1 in regulating cell shape, and suggest that the regulation of epithelial status and basal filopodia are additional mechanisms by which TBX1 controls SHF development.

We observed that aPKCζ levels are upregulated in the aDPW of Tbx1^−/− embryos. aPKC proteins play important roles in the regulation of epithelial polarity, including the regulation of adherens junction formation in Drosophila and tight junction formation in mammalian epithelial cell lines (Knust and Bossinger, 2002; Suzuki and Ohno, 2006; Suzuki et al., 2001; Xiao and Liu, 2013). Localized Has/PRKCι at the apical junction of myocardial cells is required for heart morphogenesis in zebrafish (Rohr et al., 2006). In addition, aPKC at the apical membrane and junctions has been shown to maintain the apical domain by restricting basal protein localization during epithelial development in Drosophila and Xenopus (Chalmers et al., 2005; Hutterer et al., 2004). We found that elevated aPKCζ levels in the DPW of Tbx1^−/− embryos and on exposure to an activator of aPKCζ are associated with an increased apical/basolateral membrane ratio. Activation of aPKCζ results in filopodial loss as well as differentiation and proliferative defects in the SHF and impaired OFT elongation. This suggests that aPKCζ is a downstream effector of TBX1 function in the SHF. Analysis of aPKCζ transcript distribution by in situ hybridization did not reveal a detectable increase in the DPW of Tbx1^−/− embryos (data not shown). aPKCζ protein levels might be regulated post-transcriptionally, as is the case for TBX1 regulation of SRF (Chen et al., 2009). Alternatively, the regulation of aPKCζ levels and filopodia formation might be indirect. For example, upstream FGF signaling events are required for polarized localization of aPKC in the early mouse embryo (Saiz et al., 2013). Wnt5a has recently been shown to be regulated by TBX1 in the SHF (Chen et al., 2012), and Wnt5a mutant embryos display OFT defects, potentially mediated by impaired planar cell polarity and loss of filopodia (Schleiffarth et al., 2007; Sinha et al., 2012). Finally, although mesoderm has been shown to be the crucial site for TBX1 function in SHF development, Tbx1 is also expressed in pharyngeal endoderm, and a non-cell type autonomous role in cell-shape regulation cannot be discounted.

In conclusion, our analysis of the Tbx1 phenotype and aPKCζ activation in embryo culture suggests that epithelial and progenitor cell states are tightly linked in the DPW and that the control of cell polarity and shape is a regulatory step in SHF development, perturbation of which might contribute to congenital heart defects.

The following mouse lines were used: Mlc1v-nlacZ-24 [an Fgf10 enhancer trap line, also termed Fgf10-nlacZ (Kelly et al., 2001)], Tbx1^+/− (Jerome and Papaioannou, 2001), Mesp1-Cre (Saga et al., 1999), Z/EG (Novak et al., 2000) and CD1 mice. Mice were genotyped as described previously. Animal care was performed in accordance with national and European law.

Embryos were fixed for 1 h in 4% PFA and embedded in paraffin for immunofluorescence or in OCT and were cryosectioned for phalloidin staining. Immunohistochemistry was carried out using standard procedures. Sections were imaged using a Zeiss AxioImager fluorescent microscope with an Apotome module and a Zeiss LSM 780 confocal microscope. Primary antibodies used are listed in supplementary material Table S1. Fluorescent secondary antibodies were obtained from Jackson and Invitrogen and Phalloidin-TRITC was obtained from Sigma (P1951).

The anterior pharyngeal regions from wild-type and Tbx1^−/− embryos were dissected and pooled prior to protein extraction. Anti-aPKCζ (1:4000) and anti-phospho-aPKCζ (1:100) antibodies were used, with anti-actin antibody as loading control. Protein levels were quantified using ImageJ software (gel module, NIH). Two biological replicates were performed for each experiment.

A thick transverse slice culture system was adapted from Molyneaux et al. (2001). Embryos were dissected in slice culture medium (Boisset et al., 2011), embedded in medium with low melting-point agarose, and 250 µm transverse slices were cut using a tissue chopper (McIlwain). Slices were transferred to culture medium and imaged using a straight two-photon microscope (Nikon A1R-MP) or an inverted spinning-disk microscope (Roper/Nikon Eclipse TI). Z-stacks were captured every 5-10 min for 6-8 h. During acquisition explants were maintained at 37°C in a heated chamber.

For electroporation experiments, embryos were dissected, keeping the yolk sac intact. DNA was prepared at 3 µg/µl, injected into the pericardial cavity and electroporated into the DPW (Itasaki et al., 1999). After electroporation, embryos were cultured in rolling bottles for 3 h (see section on whole-embryo culture method), embedded in agarose, sliced and imaged as described above.

Mesp1-Cre and Z/EG (TgACTB) mice were used to visualize the entire DPW and the dynamics of basal filopodia. An EGFP expression vector pCX-EGFP-N1 was used to express cytoplasmic GFP in DPW cells. An EGFP-Utrophin plasmid was used to visualize actin filament dynamics (a gift from A. LeBivic, IBDM; see Burkel et al., 2007). Quantification of filopodia length and lifetime was performed using ImageJ software. Data are presented as mean±s.e.m. P values were obtained using a bilateral Mann–Whitney test.

Embryo culture was performed as previously described (Dominguez et al., 2012). Embryos were cultured from E8.5 (7-11 somites) to E9.5 (18-23 somites). Control embryos were cultured with 0.45% chloroform and treated embryos were cultured with 300 μg/ml of phosphatidic acid (Sigma, P9511), a specific physiological aPKCζ activator (Limatola et al., 1994).

Membrane length was measured using segmented lines on ImageJ. Apical and basolateral membrane length were measured using aPKCζ and Scribble, respectively. Total membrane length was used to calculate the circularity of the cells. Data are presented as mean±s.e.m. P values were obtained using a bilateral Mann–Whitney test.

To quantify fibronectin distribution, the number of DPW cells was counted for each transverse section and the incidence of fibronectin labeling on the lateral and/or apical side of cells was scored to obtain the percentage of fibronectin intercalation between epithelial cells.

aPKCζ immunostaining was performed on paraffin transversal sections at different levels on the anteroposterior axis. Images were acquired using a Zeiss LSM 780 confocal microscope with a 63× objective and measurements were obtained using ImageJ. The cytoplasmic intensity was measured in polygonal areas and the apical membrane intensity was measured on segmented lines along aPKCζ-positive apical membranes. Similar quantification in ventral endodermal cells provided an internal control; a mean intensity ratio was obtained by comparing DPW and endodermal values.

Proliferation in the aDPW and endoderm was quantified on sagittal sections by scoring the percentage of Ki67- and PH3-positive cells. Data were based on at least three sections from three or more embryos. Differentiation in the DPW was quantified relative to a reference point in the distal OFT. The number of MF20-positive cells in the DPW and the extent of differentiated tissue were measured. Right lateral views of whole embryos were used to measure OFT length and the angle between distal and proximal regions using ImageJ. Data are presented as mean±s.e.m. P values were obtained using a bilateral Mann–Whitney test.

We are grateful to our colleagues in the Kelly group for helpful discussions, to André Le Bivic for discussions and for the Utrophin-GFP plasmid and anti-Crumbs3 and anti-PatJ antibodies, and to the IBDM Electron Microscopy service. We acknowledge the France-BioImaging/PICsL infrastructure [ANR-10-INSB-04-01].