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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Mech Dev. Author manuscript; available in PMC Dec 7, 2011.
Published in final edited form as:
PMCID: PMC3233353
NIHMSID: NIHMS340096

Fibronectin and integrin alpha 5 play essential roles in the development of the cardiac neural crest

Abstract

Cardiac neural crest (CNC) plays a requisite role during cardiovascular development and defects in the formation of CNC-derived structures underlie several common forms of human congenital birth defects. Migration of the CNC cells to their destinations as well as expansion and maintenance of these cells are important for the normal development of the cardiac outflow tract and aortic arch arteries; however, molecular mechanisms regulating these processes are not well-understood. Fibronectin (FN) protein is present along neural crest migration paths and neural crest cells migrate when plated on FN in vitro; therefore, we tested the role of FN during the development of the CNC in vivo. Our analysis of the fate of the neural crest shows that CNC cells reach their destinations in the branchial arches and the cardiac outflow tract in the absence of FN or its cellular receptor integrin α5β1. However, we found that FN and integrin α5 modulate CNC proliferation and survival, and are required for the presence of normal numbers of CNC cells at their destinations.

Keywords: cardiac neural crest, fibronectin, integrin alpha5, proliferation, survival

1. Introduction

Neural crest cells (NCCs) are a population of multipotent progenitor cells that originate in the dorsal neural tube during embryogenesis (Crane and Trainor, 2006). In the dorsal neural tube, NCC progenitors undergo the epithelial-to-mesenchymal transition (EMT), detach from the neural tube and migrate to their diverse destinations. Depending on the axial position, NCCs give rise to a wide array of cell types in the embryo, including neurons, glia, vascular smooth muscle cells (VSMCs), cranial bones and cartilage (Le Douarin, 1982).

In chick, a subpopulation of the cranial NC originating from the region of the dorsal neural tube between the mid-otic placode and the posterior boundary of the third somite is termed the cardiac neural crest (CNC). CNC gives rise to VSMCs around aortic arch arteries and the distal portions of aorta and pulmonary artery (Hutson and Kirby, 2007). The importance of the CNC in cardiovascular development was first recognized in seminal studies performed in chick, showing that the ablation of a portion of the dorsal neural tube containing CNC progenitors resulted in aberrant septation and positioning of the cardiac outflow tract, absence of CNC-derived VSMCs and defective remodeling of pharyngeal arch arteries into their canonical mature asymmetric arrangement. These defects resemble some of the most common and severe forms of human congenital cardiovascular disorders, including those found in patients with DiGeorge syndrome (Hutson and Kirby, 2007). Similar to the chick, mouse NC progenitors that give rise to the CNC originate between the pro-rhombomere c and the fourth somite (Chan et al., 2004).

In vitro experiments performed with NCCs isolated from different axial levels of the neural tube showed that depending on their site of origin, NCCs exhibit different migratory responses to extracellular matrix (ECM). These experiments also showed that components of the ECM and levels and repertoire of integrins expressed by NCCs modulate NCC migratory behavior (Delannet et al., 1994; Strachan and Condic, 2003; Testaz et al., 1999; Xu et al., 2006). While there are examples indicating that some ECM components (e.g. laminin alpha 5 and fibulin-1) modulate NC migratory paths in vivo (Coles et al., 2006; Cooley et al., 2008), the functions of many other components of the ECM during the development of the NC are not completely understood.

Fibronectin (FN) is an essential component of the ECM present along the paths of NCC transit (Duband and Thiery, 1982; George et al., 1997; Mayer et al., 1981; Rovasio et al., 1983). The appreciation that both the FN gene and the neural crest are unique to vertebrates led to the hypothesis that FN could have evolved to play an important role during the development of the neural crest and its derived lineages (Hynes and Zhao, 2000; Whittaker et al., 2006). While experiments in vitro indicate that FN serves as a permissive substratum for NCC migration (Rovasio et al., 1983) the function of FN during NC development in vivo is not known.

In order to determine the role of FN in the development of the neural crest, we performed lineage-tracing experiments to follow the fate of CNC precursors in embryos that lack FN or its cellular receptor, integrin α5. While we observed extensive migration of cranial NCCs, including CNCCs in the absence of FN or integrin α5, our experiments also indicate that there is a significant deficiency in the number of CNCCs in the mutant embryos. Our experiments show that this deficiency is not due to defective formation of the CNC or defective exit of CNC precursors from the neural tube, but rather to the depletion of the CNC progenitor pool within the neural tube and decreased proliferation and survival of CNCCs. Our studies are the first to demonstrate the requisite role of FN and integrin α5 during the ontogeny of the CNC.

2. Results

2.1. Fibronectin synthesis is upregulated in CNC progenitors at the time corresponding with their expansion and the onset of migration

Prior studies in chick and frog showed that NCC progenitors within the dorsal neural tube as well as NCCs exiting the neural tube are surrounded by FN protein (Alfandari et al., 2003; Duband and Thiery, 1982; Le Douarin, 1982; Mayer et al., 1981). However, the cellular source(s) of FN during NC ontogeny are not known. Therefore, we used in situ hybridization to determine the spatio-temporal localization of mouse FN mRNA and immunofluorescence (IF) to examine FN protein expression at different embryonic stages corresponding with induction, expansion and migration of CNCCs.

We first assayed the presence of FN mRNA and protein in embryos with five somites, a time-point when the first known markers (e.g. Pax3) are already expressed by the CNC progenitors in the dorsal neural folds (Goulding et al., 1991). At this stage, we did not detect FN mRNA within the neural folds in wild-type embryos (n=6 embryos), while FN was abundantly transcribed in other embryonic locations such as the foregut endoderm, splanchnic mesoderm and endocardium (Fig. 1A, B). Since FN is a secreted protein, we used immunofluorescence (IF) microscopy to assay its spatio-temporal distribution. Concordant with the FN mRNA expression data, we did not detect FN protein in the dorsal neural tube in embryos with 5 somites, even though other embryonic locations were abundant with FN protein (Fig. 1C). Taken together, these experiments show that FN mRNA and protein are undetectable in the dorsal neural tube prior to CNC formation.

Figure 1
Dynamic expression of fibronectin mRNA during CNC development

By the 10th somite stage, CNCCs have begun exiting from the dorsal neural tube (Chan et al., 2004; Serbedzija et al., 1992; Stottmann et al., 2004). Examination of FN synthesis in 10–11 somite embryos showed that FN mRNA is up-regulated in the top few cell layers of the dorsal neural tube as well as in the cells of apposing surface ectoderm (Figure 1D, E). Concomitantly, FN protein is localized between the surface ectoderm and the neural epithelium containing NC progenitors (Fig. 1F). This localization pattern of FN protein is similar with that observed during NC development in chick and frog (Alfandari et al., 2003; Mayer et al., 1981). FN protein is also localized within the embryonic mesenchyme where it could interact with migrating CNCCs (Supplementary Fig. S1) (Duband and Thiery, 1982; Mayer et al., 1981; Peters and Hynes, 1996).

FN synthesis by NC progenitors is maintained in embryos at E9.5 (23 somites) (Fig. 1G, black arrow) and NCCs exiting the neural tube also synthesize FN (Supplementary Fig. S2A). FN synthesis is downregulated within the arch mesenchyme while within the cardiac outflow tract, mesenchymal cells (presumptive CNCCs) and endocardial cells express FN mRNA (Sup. Fig. S2B, B′). Taken together, our studies indicate that FN synthesis is induced in CNC precursors within the dorsal neural tube at the time corresponding with their expansion and exit (Conway et al., 2000; Serbedzija et al., 1992) and suggest a potential role for FN during these processes.

In mice, CNCCs destined for the cardiac outflow tract, migrate mainly through the fourth pharyngeal arch (Chan et al., 2004) and CNCCs remaining within the third, fourth and sixth pharyngeal arches give rise to VSMCs of aortic arch arteries (Jiang et al., 2000). In embryos with 6–23 somites, FN mRNA is highly enriched in the pharyngeal pocket endoderm and pharyngeal ectoderm, the areas of future pharyngeal arches 3, 4 and 6 (Figs. 1E, G and Supplementary Fig. S2B). In order to determine whether enrichment of FN mRNA within this region corresponds with enrichment in FN protein, we performed IF microscopy and quantified fluorescent pixel intensity within the pharyngeal arches of E9.5 embryos (Fig. 1H). This analysis showed that similar to FN mRNA, FN protein is enriched in the mesenchyme of pharyngeal arches 3 and 4 compared with arches 1 and 2 (Fig. 1H, I). Taken together our studies show that FN protein is expressed by and is localized to tissues important for the development of the CNC.

2.2. CNCCs arrive at their destinations in the branchial arches and the cardiac outflow tract in the absence of FN

To study the function of FN in CNC development we performed lineage-tracing experiments to determine the fate of the CNC in the absence of FN. For these experiments, we utilized Wnt1-Cre transgenic and R26R reporter strains of mice to mark the NC (Jiang et al., 2000; Soriano, 1999). Analysis of β-gal staining of E9.5 FN-null; Wnt1-Cre/+; R26R/+ embryos showed that the overall distribution of cranial NCCs (which include the CNC) was not severely perturbed by the absence of FN (Fig. 2A, B). However, we observed an interruption in the NC distribution at the site of putative trigeminal ganglia (Fig. 2B, asterisk) in each of the FN-null; Wnt1-Cre/+;R26R/+ embryos (n=4) compared with controls. This could be due to the absence or truncation of the hindbrain in FN-null mutants (data not shown) or, alternatively, this interruption may indicate that FN modulates migration of a subset of cranial NCCs. Notably, by E9.5, NCCs in FN-null mutants filled pharyngeal arches to their furthest and ventral-most extent (Fig. 2A′, B′) and entered into the cardiac outflow tract (Fig. 2A″, B″). Since CNCCs begin entering the cardiac outflow tract at about E9.5 in wild-type embryos (Jiang et al., 2000; Waldo et al., 1999), our observations indicate that CNCCs in FN-null mutants have reached their distal-most destinations within the pharyngeal arches and the heart at the expected embryonic stages of development. Taken together, these fate-mapping studies show that NCCs migrate into the pharyngeal arches and the cardiac outflow tract in the absence of FN.

Figure 2
Distribution of NCCs in control, FN and integrin α5-null embryos

2.3. Absence of fibronectin leads to depletion of the CNC

Sparse β-gal staining in whole FN-null; Wnt1-Cre/+; R26R/+ embryos compared with controls, suggested that there were fewer NCCs in these mutants (Fig. 2A, B). To test the idea that FN was required for the presence of normal numbers of NCCs, we focused on the CNC population of the cranial NC and quantified the numbers of CNCCs per section in serial transverse sections in the pharyngeal region located between the otic pit and the atria. Analysis of control and FN-null littermates indicated that the average number of CNCCs per section was three to six-fold lower in FN-null embryos than in controls (Fig. 3A).

Figure 3
FN and integrin α5 are required for the presence of normal numbers of CNCCs in branchial arches at E9.5

We confirmed this decrease with an independent method by detecting NCCs utilizing antibodies recognizing the transcription factor AP2α (TFAP2α). At E8.5, TFAP2α is expressed by NCC progenitors in the dorsal neural tube and by NCCs. At E9.5, TFAP2α is expressed by NCCs and some NC derivatives at E9.5 (Conway et al., 2000). In order to quantify the number of NCCs outside the neural tube in E9.5 FN-null and control embryos, we counted the number of TFAP2α-positive mesenchymal cells per section excluding the cells of the surface ectoderm. Using this method, we also observed approximately a three-fold decrease in the number of NCCs at each branchial arch level (Fig. 3B). Taken together, these studies indicate that FN is required for the presence of normal numbers of CNCCs.

2.4. Fibronectin is not required for specification of the NC and pharyngeal lineages

The presence of LacZ-positive cells in FN-null embryos following lineage tracing analyses indicated that FN is not required to induce the NC lineage. This conclusion is also supported by the observation that transcription factors Foxd3 and Pax3, which are the earliest genes known to be expressed by the NC progenitors as well as Cx43 transcripts are expressed in the dorsal neural tube of FN-null embryos (Fig. 4).

Figure 4
Expression of neural crest markers at E8.5 does not depend on FN

It has been established that the pharyngeal endoderm, mesoderm and ectoderm play essential roles during the development of CNC-derived lineages (Calmont et al., 2009; Goddeeris et al., 2007; High et al., 2009; Macatee et al., 2003; Park et al., 2006; Park et al., 2008; Zhang et al., 2005). Therefore, in this study, we addressed tissue-specific expression of genes normally present within lineages of the pharynx. Our IF studies demonstrated that similar to controls, pharyngeal endoderm and mesoderm express the transcription factor Isl1, and pharyngeal ectoderm expresses both Isl1 and TFAP2α (Figs. 6, ,77 and Fig. S3). Taken together, these studies and work prior to ours (George et al., 1997; George et al., 1993; Georges-Labouesse et al., 1996) indicate that decreased abundance of CNCCs in FN-null embryos is not due to misspecification of pharyngeal, NC or other embryonic lineages.

Figure 6
Defective survival of the cranial NC in the absence of FN at E9.5
Figure 7
Proliferation and survival of the CNC is regulated by FN and integrin α5 at E8.5

2.5. Exit of CNC progenitors from the neural tube does not depend on FN

The decrease in the number of CNCCs observed at E9.5 could be due to defective emigration of CNC progenitors from the neural tube earlier in embryonic development. If this were the case, we would have observed accumulation of CNCCs in the dorsal neural tube of FN-null embryos compared with controls. In mice, emigration of CNCCs from the neural tube is already evident by the 10th somite stage, corresponding with approximately E8.5 of development (Chan et al., 2004; Serbedzija et al., 1992). At this stage, we identified FN-null embryos that were morphogenetically similar to controls (Fig. 5A, B). FN-null embryos lack somites (George et al., 1993; Georges-Labouesse et al., 1996) and therefore, we used an alternative approach (Downs and Davies, 1993) and staged the null and control embryos by their overall morphology. In order to determine whether the absence of FN resulted in accumulation of CNC progenitors within the neural tube during early stages of CNC development and before morphogenetic abnormalities in FN-nulls became grossly evident, we quantified the number of TFAP2α+ cells per section within the dorsal neural tube in FN-null embryos that looked similar to controls containing 10–11 somites. Our quantifications showed that the average number of CNC progenitors was 20% lower in FN-null embryos compared with controls. We think that this decrease is due to the already ongoing depletion of the CNC progenitor pool because of defective proliferation and survival of these cells in FN-null mutants (see sections 2.6 and 2.7 below). Nonetheless, these quantifications demonstrate the absence of accumulation of CNC progenitors within the dorsal neural tube in FN-null embryos.

Figure 5
FN is not essential for the emigration of CNC progenitors from the neural tube

To complement the above analysis, we quantified distribution of CNCCs in the mesenchyme of FN-null and control embryos analyzed above. If there were defective exit of CNC progenitors from the neural tube, we would expect altered distribution of CNCCs outside of the neural tube. The onset of NC exit from the neural tube proceeds in rostral to caudal order, thus rostral sections from an embryo are expected to contain more NCCs within their mesenchyme compared with caudal sections. We counted the number of CNCCs per section in each of the six control and six FN-null embryos isolated at E8.5. The results were binned according to the number of observed CNCCs per section and plotted against the y-axis designating the number of sections contained within each bin. The resultant distributions of CNCCs were similar between FN-null and control embryos (Fig. 5D). Taken together, these complementary analyses indicate that FN is not required for the exit of CNC progenitors from the neural tube and suggest that the process of epithelial-to-mesenchymal transition does not depend on FN.

2.6. Fibronectin is essential for the survival of CNC progenitors

Cell adhesion to FN is required for growth factor-mediated cell survival of normal non-transformed cells in vitro (Matter and Ruoslahti, 2001; Zhang et al., 1995). Our initial experiments demonstrated extensive cells death primarily in the neural tube and the NC of FN-null embryos at E9.5 (Figs. 6A–C). Since apoptosis within the Isl1+ mesodermal cells in the pharyngeal arches did not yield statistically significant differences between the null and control embryos (Figs. 6D), our data suggest a selective role for FN in the survival of cells of the neural tube and the NC at E9.5.

Since we observed enrichment in NC apoptosis at E9.5, we reasoned that the deficiency in the CNC in FN-null embryos at this stage could be a result of decreased survival of CNC progenitors earlier in embryonic development, e.g. at E8.5. To test this hypothesis, we isolated FN-null embryos at E8.5 that were similar in size and shape to controls having 10 -11 somites. CNC progenitors reside between the otic pit and the fourth somite in mice (Chan et al., 2004), and therefore, we obtained serial sections through this region of control embryos and through a comparable region of the nulls. Sections were then co-stained to detect both the NC and apoptotic cells (Fig. 7). FN-null mutants contained four-fold more apoptotic CNC progenitors than controls within the dorsal neural tube, p<0.001 (Fig. 8A). We also detected a ten-fold increase in the apoptosis of TFAP2α+ cells outside the neural tube, p<0.0001 (Fig. 8A). Concordant with the above findings, detection of apoptosis using the TUNEL assay indicated increased apoptosis in CNC progenitors and CNCCs in E8.5 FN-null embryos relative to controls (data not shown).

Figure 8
Quantitative analysis of CNC apoptosis and proliferation in FN-null and integrin α5-null embryos at E8.5

At E8.5 we observed the presence of some apoptotic cells in the pharyngeal endoderm and ectoderm in addition to the CNC of FN-null embryos. However, whole mount immunohistochemistry performed on embryos using the antibody to cleaved caspase 3 and examination of serial sections stained with this antibody indicated very low overall levels of apoptosis in the mutants and controls (Fig. 7A–D, E, I). These experiments attest to the relative health of the analyzed FN-null mutants. Taken together, our studies indicate that FN is required for the survival of CNC progenitors and their descendants.

2.7. Fibronectin is essential for proliferation of CNC progenitors

Cell binding to FN is required for growth factor-mediated proliferation of normal untransformed cells in vitro. (Assoian and Schwartz, 2001; Roovers et al., 1999; Schwartz and Assoian, 2001). In order to determine whether FN is required for CNC proliferation in vivo, we labeled NCCs with the antibody against TFAP2α and assayed cell proliferation by using the antibody to phosphohistone H3 in FN-null embryos isolated at E8.5 and in controls containing 10 to 11 somites. We found that at this early stage of CNC ontogeny, FN-null embryos contained fewer mitotic CNC progenitors and CNCCs (Fig. 7F, F′, F″, J, J′ and Fig. 8B). Our quantitative studies indicate that FN plays a requisite role in maintenance and expansion of the CNC both within and outside of the dorsal neural tube.

2.8. CNCCs migrate to branchial arches and the cardiac outflow tract in the absence of integrin α5

Integrins comprise a major group of cell surface receptors mediating cellular migration, survival and proliferation upon binding to FN. A number of integrin heterodimers (αvβ3, αvβ1, α8β1, α5β1, α4β1) bind FN in vitro (Humphries et al., 2006; Saoncella et al., 1999). Individual deletion of integrin α4, αv, or α8 permits embryonic development past mid-gestation and/or until birth, while the deletion of integrin α5 leads to early embryonic lethality with similar (albeit a slightly milder) embryonic lethal phenotype as FN-null embryos (Bader et al., 1998; Muller et al., 1997; Yang et al., 1999). Since the phenotypes of integrin α5-null and FN-null embryos are similar and since integrin α5 directly binds FN in vitro and activates intercellular signaling pathways modulating cell migration, survival and proliferation, integrin α5 is thought to function as a major FN receptor during early embryogenesis (Yang et al., 1999).

To uncover the potential downstream players mediating the function of FN during CNC development, we asked whether integrin α5 was similarly required for the formation of quantity of the CNC. To answer this question, we performed lineage-tracing experiments utilizing either Wnt1-Cre or Pax3Cre/+ strains to mark the NC in integrin α5-null mutants. Our experiments indicated that extensive migration of NCCs took place in the absence of integrin α5 (Fig. 2C, D, C′, D′), including migration of CNCCs into the cardiac outflow tract (Fig. 2C″, D″). We also detected CNCCs within the cardiac outflow tracts of integrin α5-null embryos at E9.5 using the antibody to TFAP2α (Fig. 7K). This experiment serves as an independent confirmation that CNCCs successfully reach and enter the cardiac outflow tract in integrin α5-null embryos and indicate that NCCs are capable of long-range migration in the absence of integrin α5.

Whole mount staining of embryos in which the NC was marked with the use of Pax3Cre/+ knock-in or Wnt1-Cre transgenic alleles suggested that similar to FN-null embryos, integrin α5-nulls contained fewer CNCCs in branchial arches (Fig. 2C′, D′ and data not shown). Quantitative analysis of sections indicated that this was indeed the case (Fig. 3C, D). Taken together, our experiments indicate that while both FN and integrin α5 are dispensable for the gross migration of the CNCCs to their target destinations, they are required for the presence of normal numbers of CNCCs.

2.9. Integrin α5 is required for survival and proliferation of the CNC

Prior studies indicated that integrin α5 is required for the survival of cranial NCCs within the second branchial arch (Goh et al., 1997). Therefore, we tested whether integrin α5 was also required for the survival of the CNC, a different population of the cranial NC. We observed statistically significant increase in apoptosis of the CNC outside the neural tube in integrin α5-null embryos (Figs. 7G, K and 8C). However, we did not find an appreciable decrease in survival of CNC progenitors within the neural tube of integrin α5-null embryos. Possibly, other FN-binding integrins in the dorsal neural tube mediated survival of CNC progenitors in the absence of integrin α5. Consistent with our results, isolated integrin α5-null trunk NCCs can survive in vitro during the initial four days of culture and express additional integrins (Haack and Hynes, 2001). Quantification of proliferation in CNC progenitors within the dorsal neural tube and their descendants within the embryonic mesenchyme, demonstrated a significant decrease in the proportion of mitotic CNC progenitors and CNCCs in integrin α5-null embryos (Figs. 7H, L and 8D). Taken together, these data indicate that similar to FN, integrin α5 is required for proliferation of CNC progenitors and for the proliferation and survival of the CNC.

Prior studies extensively characterized gene expression in integrin α5-null embryos and determined that the spatio-temporal gene expression patterns as well as patterning of the dorsal neural tube caudal to the otic pit were similar between integrin α5-null and control embryos (Goh et al., 1997). Moreover, these studies showed that absence of integrin α5 does not lead to significant differences in proliferation and survival of cells in mesodermal tissues (Goh et al., 1997). Taken together, prior work and our studies indicate that deficiency in CNCC numbers, proliferation and survial is not due to mispartening of embryonic tissues and the neural tube in integrin α5-null embryos, and that integrin α5 plays a selective role in proliferation and survival of the NC.

3. Discussion

The importance of FN in the development of the NC was hypothesized based on observations that FN protein is present along the neural crest transit routes in vivo, on studies implicating FN in NC migration in vitro, and on evolutionary studies implicating FN in the development of vertebrate-specific features such as the neural crest. Our work is the first to specifically examine the function of FN during neural crest development in vivo. Using a genetic fate mapping approach, we found that CNCCs reached the branchial arches and invaded the cardiac outflow tract in the absence of FN, suggesting that FN is not required for the migration of the CNC. Instead, we observed a decrease in the number of CNCCs at their destinations and found that proliferation and survival of CNC progenitors and their descendants were compromised in the absence of FN. These data indicate that in vivo, FN is an important regulator of CNC abundance and suggest that it regulates this process by modulating proliferation and survival of the CNC.

In order to understand how FN functions during CNC development, we examined the role of integrin α5 (a major transducer of FN signals) in CNC migration, proliferation and survival. Our fate mapping analysis showed that similar to FN, integrin α5 is not required for the CNC to reach and fill branchial arches and to invade the cardiac outflow tract. However, integrin α5 is important for regulating CNC numbers and facilitates proliferation and survival of the CNC, suggesting that α5β1 transduces FN proliferation and survival signals into the CNC during embryonic development.

In agreement with our findings, earlier studies showed that mouse integrin α5-null NCCs migrated along ventral paths when injected into the dorsal neural tube of a chick host embryo (Haack and Hynes, 2001). Thus two independent assays, fate mapping (this study) and heterologous transplantation (Haack and Hynes, 2001), indicate that integrin α5 is dispensable for the gross migration of the NC.

Concordant with our studies, earlier studies also indicated compromised survival of a subset of cranial NCCs in integrin α5-null embryos while proliferation and survival of mesodermal tissues were not significantly defective in these mutants, indicating a specific role for integrin α5 during the development of the NC (Goh et al., 1997). Finally, in accord with our conclusion that integrin α5 regulates CNC proliferation in vivo, analysis performed in vitro indicated that trunk NCCs isolated from integrin α5-null embryos failed to proliferate even in the presence of transforming oncogenes, SV40 large and small T antigens (Haack and Hynes, 2001). Taken together these experiments demonstrate an essential role for FN and integrin α5 in maintaining CNC cell number by modulating CNC proliferation and survival.

Extracellular environment is thought to play a major role in NC ontogeny (Duband and Thiery, 1987; Hynes, 1990; Le Douarin, 1982). In order to gain an insight into cellular and molecular roles of FN during NC development, we performed in situ hybridization and IF studies to determine which cell types synthesize FN and to determine the localization of FN protein during NC development. These experiments led to a novel observation that FN mRNA is not produced uniformly by all cells and that FN protein is enriched near the sites of its synthesis in the developing embryo. Analysis of the published literature also indicates that other components of the ECM are not uniformly distributed throughout the embryo. For example, laminin α5 and fibulin-1 transcripts are enriched in distinct embryonic loci; and a number of ECM proteins implicated in NC development are also highly localized within distinct embryonic regions (Coles et al., 2006; Cooley et al., 2008; Duband and Thiery, 1987). These studies indicate that NCCs encounter both different types and different concentrations of ECM proteins during their long-range transit and imply that these differences may be important during the development of the NC and its derivatives. Indeed, absence of laminin α5 chain leads to the widening of NC streams (Coles et al., 2006). Similarly, the absence of fibulin-1 leads to widening and fusion of some NC streams and gives rise to severe defects in the positioning of the cardiac outflow vessels, patterning of aortic arch arteries and NC survival (Cooley et al., 2008). Our studies demonstrated that FN mRNA and protein are upregulated in the dorsal neural tube at the time of expansion and exit of CNC progenitors. FN is also upregulated within the branchial arch regions into which and through which CNCCs migrate. We hypothesize that FN expressed by CNC progenitors and by the tissues comprising branchial arches 3 and 4 play specific roles during CNC development. Future studies using conditional mutagenesis will provide important insights into cell-autonomous and non-cell autonomous functions of FN during the development of the NC.

4. Experimental Procedures

4.1. Mouse strains

All experiments involving vertebrate animals were performed according to the NIH guidelines and were approved by the institutional animal care and use committee. Animals heterozygous for the null alleles of fibronectin (FN+/−) or integrin α5 (Itga5+/) on C57BL6/J genetic background were obtained from Dr. Richard Hynes (MIT) (George et al., 1993; Yang et al., 1993). Wnt1-Cre transgenic strain, Pax3Cre/+ knock-in strain and R26R reporter mice were obtained from the Jackson labs (Engleka et al., 2005; Jiang et al., 2000; Lang et al., 2005; Soriano, 1999).

4.2. Lineage tracing analysis

FN+/−;R26R/R26R females were mated with FN+/−; Wnt1-Cre males and embryos were dissected at E9.5. Similarly, Itga5+/−; R26R/R26R females were mated with either Itga5+/−; Wnt1-Cre or Itga5+/−; Pax3Cre/+ males to obtain embryos at E9.5. Embryos were dissected in ice-cold phosphate buffered saline (PBS) and stained using bluo-gal as the substrate (#B2904, Sigma-Aldrich) and a standard protocol to detect β-gal enzymatic activity. Staining reactions were allowed to proceed for two days at 37°C. Embryos were then photographed using Zeiss Stemi 2000-C stereomicroscope and DFC420 digital camera (Leica). Embryos were dehydrated, embedded into paraffin and serially sectioned in transverse orientation. Serial sections (10–15 microns apart) between the otic pit and the atria were used to quantify the numbers of CNCCs per section. Alternatively, FN-null and control embryos were collected from matings between FN+/− animals and serial sections (10–15 microns apart) were stained using mouse monoclonal antibody recognizing TFAP2α clone 3B5, generated by Trevor Williams and obtained from the Developmental Studies Hybridoma Bank (University of Iowa, Iowa City, IA).

4.3. In situ hybridization

In situ hybridizations were performed according to a standard protocol (Henrique et al., 1995), obtained from the Dr. Janet Rossant’s lab web page, http://www.sickkids.ca/research/rossant/protocols/Conlon/WM2_Henrique.pdf. Plasmids encoding Foxd3 and Pax3 probes were obtained from Drs. Patricia Labosky (Vanderbilt University) and Andrew McMahon (Harvard University), respectively. Plasmid encoding Cx43 probe was generated using primers: 5′-gaattcgtcgacGAGGTGCCCAGACATGGGTG and 5- gatatgcggccgcGACACCACCAGCATGAAGATG. These primers were used to amplify 662 bp of the 2nd exon by PCR utilizing mouse genomic DNA. The resulting fragment was digested with NotI and SalI and subcloned into pBluescript. For anti-sense probe preparation, the plasmid was cut with SalI and transcribed using T7 polymerase (Promega) and DIG RNA labeling mix (Roche).

4.4. Detection of fibronectin and integrin α5 proteins

All stainings were performed on tissues fixed in 4% PBS-buffered paraformaldehyde and embedded into paraffin. FN protein was detected using rabbit polyclonal antibody 297.1 (a gift from Dr. Richard Hynes) and Alexa Fluor 488 secondary anti-rabbit IgG (Invitrogen). Fluorescence intensity was quantified using the rectangle tool in ImageJ, according to the software specifications. Double detection of FN and TFAP2α were performed using paraffin sections, antigen retrieval in 10 mM Citrate buffer, anti-FN antibody obtained from Millipore (cat # AB2033, at 1:200 dilution) and the anti-TFAP2α antibody at the dilution of 1:50. Use of the latter FN antibody on sections from FN-null embryos gave minimal background staining (Supplemental Fig. 1B). Additional control stainings were performed using normal IgG from naïve rabbits.

4.5. Immunofluorescent analysis of proliferation and apoptosis

FN-null and control embryos were isolated from FN+/− matings at E8.5. FN-null embryos that were similar in size and shape to control embryos having 10–11 somites were chosen for our analyses. Embryos were fixed in 4% paraformaldehyde overnight at 4 °C and stored at 4 °C in 70% ethanol until use. Embryos were dehydrated, embedded into paraffin and serially sectioned in transverse orientation. Serial sections between the otic vesicle and atria (thirty-fourty 5-μm thick sections 5–10 micron apart, per embryo) were analyzed using immunofluorescence microscopy following antigen retrieval in 0.01 M NaCitrate, pH 6.0. NCCs were detected using mouse monoclonal anti-TFAP2α antibody (1:50). Rabbit polyclonal antibodies recognizing cleaved caspase 3 (#9661) or anti-phosphohistone H3 antibody (#9701) were purchased from Cell Signaling Technology (Danvers, MA). Fluorescent anti-rabbit IgG conjugated to Alexa-Fluor 488 and anti-mouse IgG conjugated to AlexaFluor 555 (Invitrogen) were used as secondary antibodies. TUNEL staining was performed using In situ cell death detection kit (Roche, #11684795910). Slides were mounted using ProLong® Gold anti-fade reagent with DAPI (Invitrogen) and imaged using Zeiss Axiophot epifluorescence microscope equipped with Hamamatsu digital camera. Alternatively, slides were imaged using Zeiss LSM510 confocal microscope. Integrin α5-null and control embryos of similar size and shape were isolated at E9.0 (the number of somites in control embryos ranged from 17 to 19). These embryos were processed in the manner similar to the FN-null and control samples described above. Serial sections 10–15 microns apart were used for quantification to avoid double-counting of cells. Statistical analyses were performed using Z-test for two proportions http://dimensionresearch.com/resources/calculators/ztest.html and/or chi-square test. Data in Fig. 5C was analyzed using Student’s t test, and the box plot was generated using a web-based program (http://www.physics.csbsju.edu/stats/t-test_bulk_form.html). The box represents the interquartile range (IQR, central 50% of the data points), horizontal lines inside the boxes represent the median values, and vertical bars represent a spread of 1.5× IQR, while dots represent outliers, which were included into the calculation of significance.

Supplementary Material

Supplementary Figures

Figure S1. A. Confocal IF microscopy shows co-localization of CNCCs (detected using anto-TFAP2α antibody, red nuclei) and FN (green) in the same plane. Magnified box 1 shows localization of FN (green) between non-neural surface ectoderm (arrowhead) and CNC progenitors (red, arrow). Magnified box 2 shows mesenchymal CNCCs (red, arrow) surrounded by FN (green). op-otic pit. B. Control. Staining of a section from FN-null embryos using equivalent concentration of anti-TFAP2α antibody (red), anti-FN antibody (green) and DAPI. Arrow points at the branchial arch mesenchyme. Arrowhead points to the non-neural surface ectoderm. Note that anti-FN antibody produces minimal staining in the absence of FN.

Figure S2. Expression of FN mRNA at E9.5. A. CNC progenitors and CNCCs express FN mRNA (arrows). B. FN mRNA is enriched in pharyngeal ectoderm (black arrow) and pharyngeal pocket endoderm, asterisks. Presumptive CNCCs inside the outflow tract (open arrows) also express FN mRNA, while mesenchymal cells lateral to dorsal aorta and in pharyngeal arches (magnified in panel B′) express low levels of FN mRNA. dAo – dorsal aorta. Endothelial cells in panel B are marked by the black arrowheads.

Figure S3. Pharyngeal ectoderm (ec, small arrow), endoderm (en, arrowhead) and mesoderm (notched arrowhead) are marked by the expression of Isl1 in control and FN-null embryos at E9.5. Long arrows point at the outflow tract myocardium expressing Isl1 in FN-null and controls.

Acknowledgments

We thank Dr. Richard Hynes for providing FN and integrin α5 mutant mouse strains and rabbit polyclonal antibody to FN. Drs. Patricia Labosky and Andrew McMahon for plasmids encoding Foxd3 and Pax3 probes. We are grateful to Drs. Heidi Stuhlmann and Yutaka Nibu for the use of their microscopes and imaging equipment. We thank Shivaprasad Bhuvanendran in the Bio-Imaging resource center at the Rockefeller University for help with confocal imaging, and thank Drs. Bruce Gelb, Ann Foley, Nathan Astrof, and Monn Monn Myat for critical reading of the manuscript. This work was supported in part by the American Heart Association Scientist Development grant # 0835556D to S.A. and by the start-up funds to S.A. from the Division of Cardiology, Weill Cornell Medical College.

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