Ablation of specific expression domains reveals discrete functions of ectoderm- and endoderm-derived FGF8 during cardiovascular and pharyngeal development

Fibroblast growth factor 8 (Fgf8) is expressed in many domains of the developing embryo. Globally decreased FGF8 signaling during murine embryogenesis results in a hypomorphic phenotype with a constellation of heart, outflow tract, great vessel and pharyngeal gland defects that phenocopies human deletion 22q11 syndromes, such as DiGeorge. We postulate that these Fgf8 hypomorphic phenotypes result from disruption of local FGF8 signaling from pharyngeal arch epithelia to mesenchymal cells populating and migrating through the third and fourth pharyngeal arches. To test our hypothesis, and to determine whether the pharyngeal ectoderm and endoderm Fgf8 expression domains have discrete functional roles, we performed conditional mutagenesis of Fgf8 using novel Crerecombinase drivers to achieve domain-specific ablation of Fgf8 gene function in the pharyngeal arch ectoderm and endoderm. Remarkably, ablating FGF8 protein in the pharyngeal arch ectoderm causes failure of formation of the fourth pharyngeal arch artery that results in aortic arch and subclavian artery anomalies in 95% of mutants; these defects recapitulate the spectrum and frequency of vascular defects reported in Fgf8 hypomorphs. Surprisingly, no cardiac, outflow tract or glandular defects were found in ectodermal-domain mutants, indicating that ectodermally derived FGF8 has essential roles during pharyngeal arch vascular development distinct from those in cardiac, outflow tract and pharyngeal gland morphogenesis. By contrast, ablation of FGF8 in the third and fourth pharyngeal endoderm and ectoderm caused glandular defects and bicuspid aortic valve, which indicates that the FGF8 endodermal domain has discrete roles in pharyngeal and valvar development. These results support our hypotheses that local FGF8 signaling from the pharyngeal epithelia is required for pharyngeal vascular and glandular development, and that the pharyngeal ectodermal and endodermal domains of FGF8 have separate functions.     

An Fgf8 mouse mutant phenocopies human 22q11 deletion syndrome

Deletion of chromosome 22q11, the most common microdeletion detected in humans, is associated with a life-threatening array of birth defects. Although 90% of affected individuals share the same three megabase deletion, their phenotype is highly variable and includes craniofacial and cardiovascular anomalies, hypoplasia or aplasia of the thymus with associated deficiency of T cells, hypocalcemia with hypoplasia or aplasia of the parathyroids, and a variety of central nervous system abnormalities. Because ablation of neural crest in chicks produces many features of the deletion 22q11 syndrome, it has been proposed that haploinsufficiency in this region impacts neural crest function during cardiac and pharyngeal arch development. Few factors required for migration, survival, proliferation and subsequent differentiation of pharyngeal arch neural crest and mesoderm-derived mesenchyme into their respective cardiovascular, musculoskeletal, and glandular derivatives have been identified. However, the importance of epithelial-mesenchymal interactions and pharyngeal endoderm function is becoming increasingly clear. Fibroblast growth factor 8 is a signaling molecule expressed in the ectoderm and endoderm of the developing pharyngeal arches and known to play an important role in survival and patterning of first arch tissues. We demonstrate a dosage-sensitive requirement for FGF8 during development of pharyngeal arch, pharyngeal pouch and neural crest-derived tissues. We show that FGF8 deficient embryos have lethal malformations of the cardiac outflow tract, great vessels and heart due, at least in part, to failure to form the fourth pharyngeal arch arteries, altered expression of Fgf10 in the pharyngeal mesenchyme, and abnormal apoptosis in pharyngeal and cardiac neural crest. The Fgf8 mutants described herein display the complete array of cardiovascular, glandular and craniofacial phenotypes seen in human deletion 22q11 syndromes. This represents the first single gene disruption outside the typically deleted region of human chromosome 22 to fully recapitulate the deletion 22q11 phenotype. FGF8 may operate directly in molecular pathways affected by deletions in 22q11 or function in parallel pathways required for normal development of pharyngeal arch and neural crest-derived tissues. In either case, Fgf8 may function as a modifier of the 22q11 deletion and contribute to the phenotypic variability of this syndrome.     

Regionalisation of early head ectoderm is regulated by endoderm and prepatterns the orofacial epithelium

The oral epithelium becomes regionalised proximodistally early in development, and this is reflected by the spatial expression of signalling molecules such as Fgf8 and Bmp4. This regionalisation is responsible for regulating the spatial expression of genes in the underlying mesenchyme. These genes are required for the spatial patterning of bone, cartilage orofacial development and, in mammals, teeth. The mechanism and timing of this important regionalisation during head epithelium development are not known. Using lipophilic dyes to fate map the oral epithelium in chick embryos, we show that the cells that will occupy the epithelium of the distal and the proximal mandible primordium already occupy different spatial locations in the developing head ectoderm prior to the formation of the first pharyngeal arch and neural crest migration. Moreover, the ectoderm cells fated to become proximal oral epithelium express Fgf8 and this expression requires the presence of endoderm. Thus, the first fundamental patterning process in jaw morphogenesis is controlled by the early separation of specific areas of ectoderm that are regulated by ectoderm-endoderm interactions, and does not involve neural crest cells.     

Sonic hedgehog in the pharyngeal endoderm controls arch pattern via regulation of Fgf8 in head ectoderm

Fgf8 signalling is known to play an important role during patterning of the first pharyngeal arch, setting up the oral region of the head and then defining the rostral and proximal domains of the arch. The mechanisms that regulate the restricted expression of Fgf8 in the ectoderm of the developing first arch, however, are not well understood. It has become apparent that pharyngeal endoderm plays an important role in regulating craniofacial morphogenesis. Endoderm ablation in the developing chick embryo results in a loss of Fgf8 expression in presumptive first pharyngeal arch ectoderm. Shh is locally expressed in pharyngeal endoderm, adjacent to the Fgf8-expressing ectoderm, and is thus a candidate signal regulating ectodermal Fgf8 expression. We show that in cultured explants of presumptive first pharyngeal arch, loss of Shh signalling results in loss of Fgf8 expression, both at early stages before formation of the first arch, and during arch formation. Moreover, following removal of the endoderm, Shh protein can replace this tissue and restore Fgf8 expression. Overexpression of Shh in the non-oral ectoderm leads to an expansion of Fgf8, affecting the rostral-caudal axis of the developing first arch, and resulting in the formation of ectopic cartilage. Shh from the pharyngeal endoderm thus regulates Fgf8 in the ectoderm and the role of the endoderm in pharyngeal arch patterning may thus be indirectly mediated by the ectoderm.     

FGF8 dose-dependent regulation of embryonic submandibular salivary gland morphogenesis

FGF8 has been shown to play important morphoregulatory roles during embryonic development. The observation that craniofacial, cardiovascular, pharyngeal, and neural phenotypes vary with Fgf8 gene dosage suggests that FGF8 signaling induces differences in downstream responses in a dose-dependent manner. In this study, we investigated if FGF8 plays a dose-dependent regulatory role during embryonic submandibular salivary gland (SMG) morphogenesis. We evaluated SMG phenotypes of Fgf8 hypomorphic mice, which have decreased Fgf8 gene function throughout embryogenesis. We also evaluated SMG phenotypes of Fgf8 conditional mutants in which Fgf8 function has been completely ablated in its expression domain in the first pharyngeal arch ectoderm from the time of arch formation. Fgf8 hypomorphs have hypoplastic SMGs, whereas conditional mutant SMGs exhibit ontogenic arrest followed by involution and are absent by E18.5. SMG aplasia in Fgf8 ectoderm conditional mutants indicates that FGF8 signaling is essential for the morphogenesis and survival of Pseudoglandular Stage and older SMGs. Equally important, the presence of an initial SMG bud in Fgf8 conditional mutants indicates that initial bud formation is FGF8 independent. Mice heterozygous for either the Fgf8 null allele (Fgf8(+/N)) or the hypomorphic allele (Fgf8(+/H)) have SMGs that are indistinguishable from wild-type (Fgf8(+/+)) mice which suggest that there is not only an FGF8 dose-dependent phenotypic response, but a nonlinear, threshold-like, epistatic response as well. We also found that enhanced FGF8 signaling induced, and abrogated FGF8 signaling decreased, SMG branching morphogenesis in vitro. Furthermore, since FGF10 and Shh expression is modulated by Fgf8 levels, we postulated that exogenous FGF10, Shh, or FGF10 + Shh peptide supplementation in vitro would largely "rescue" the abnormal SMG phenotype associated with decreased FGF8 signaling. This is as expected, though there is no synergistic effect with FGF10 + Shh peptide supplementation. These in vitro experiments model the principle that mutations have different effects in the context of different epigenotypes.     

Fgf8 is required for pharyngeal arch and cardiovascular development in the mouse

We present here an analysis of cardiovascular and pharyngeal arch development in mouse embryos hypomorphic for Fgf8. Previously, we have described the generation of Fgf8 compound heterozygous (Fgf8(neo/-)) embryos. Although early analysis demonstrated that some of these embryos have abnormal left-right (LR) axis specification and cardiac looping reversals, the number and type of cardiac defects present at term suggested an additional role for Fgf8 in cardiovascular development. Most Fgf8(neo/-) mutant embryos survive to term with abnormal cardiovascular patterning, including outflow tract, arch artery and intracardiac defects. In addition, these mutants have hypoplastic pharyngeal arches, small or absent thymus and abnormal craniofacial development. Neural crest cells (NCCs) populate the pharyngeal arches and contribute to many structures of the face, neck and cardiovascular system, suggesting that Fgf8 may be required for NCC development. Fgf8 is expressed within the developing pharyngeal arch ectoderm and endoderm during NCC migration through the arches. Analysis of NCC development in Fgf8(neo/-) mutant embryos demonstrates that NCCs are specified and migrate, but undergo cell death in areas both adjacent and distal to where Fgf8 is normally expressed. This study defines the cardiovascular defects present in Fgf8 mutants and supports a role for Fgf8 in development of all the pharyngeal arches and in NCC survival.     

An essential role for Fgfs in endodermal pouch formation influences later craniofacial skeletal patterning

Fibroblast growth factor (Fgf) proteins are important regulators of pharyngeal arch development. Analyses of Fgf8 function in chick and mouse and Fgf3 function in zebrafish have demonstrated a role for Fgfs in the differentiation and survival of postmigratory neural crest cells (NCC) that give rise to the pharyngeal skeleton. Here we describe, in zebrafish, an earlier, essential function for Fgf8 and Fgf3 in regulating the segmentation of the pharyngeal endoderm into pouches. Using time-lapse microscopy, we show that pharyngeal pouches form by the directed lateral migration of discrete clusters of endodermal cells. In animals doubly reduced for Fgf8 and Fgf3, the migration of pharyngeal endodermal cells is disorganized and pouches fail to form. Transplantation and pharmacological experiments show that Fgf8 and Fgf3 are required in the neural keel and cranial mesoderm during early somite stages to promote first pouch formation. In addition, we show that animals doubly reduced for Fgf8 and Fgf3 have severe reductions in hyoid cartilages and the more posterior branchial cartilages. By examining early pouch and later cartilage phenotypes in individual animals hypomorphic for Fgf function, we find that alterations in pouch structure correlate with later cartilage defects. We present a model in which Fgf signaling in the mesoderm and segmented hindbrain organizes the segmentation of the pharyngeal endoderm into pouches. Moreover, we argue that the Fgf-dependent morphogenesis of the pharyngeal endoderm into pouches is critical for the later patterning of pharyngeal cartilages.     

Fgf signalling is required for formation of cartilage in the head

Characterisation of human craniofacial syndromes and studies in transgenic mice have demonstrated the requirement for Fgf signalling during morphogenesis of membrane bone of the cranium. Here, we report that Fgf activity is also required for development of the oro-pharyngeal skeleton, which develops first as cartilage with some elements subsequently becoming ossified. We show that inhibition of FGF receptor activity in the zebrafish embryo following neural crest emigration from the neural tube results in complete absence of neurocranial and pharyngeal cartilages. Moreover, this Fgf signal is required during a 6-h period soon after initiation of neural crest migration. The spatial and temporal expression of Fgf3 and Fgf8 in pharyngeal endoderm and ventral forebrain and its correlation with patterns of Fgf signalling activity in migrating neural crest makes them candidate regulators of cartilage development. Inhibition of Fgf3 results in the complete absence of cartilage elements that normally form in the third, fourth, fifth, and sixth pharyngeal arches, while those of the first, second, and seventh arches are largely unaffected. Inhibition of Fgf8 alone has variable, but mild, effects. However, inhibition of both Fgf3 and Fgf8 together causes a complete absence of pharyngeal cartilages and the near-complete loss of the neurocranial cartilage. These data implicate Fgf3 and Fgf8 as key regulators of cartilage formation in the vertebrate head.     

Sprouty genes are essential for the normal development of epibranchial ganglia in the mouse embryo

Fibroblast growth factor (FGF) signalling has important roles in the development of the embryonic pharyngeal (branchial) arches, but its effects on innervation of the arches and associated structures have not been studied extensively. We investigated the consequences of deleting two receptor tyrosine kinase (RTK) antagonists of the Sprouty (Spry) gene family on the early development of the branchial nerves. The morphology of the facial, glossopharyngeal and vagus nerves are abnormal in Spry1−/−;Spry2−/− embryos. We identify specific defects in the epibranchial placodes and neural crest, which contribute sensory neurons and glia to these nerves. A dissection of the tissue-specific roles of these genes in branchial nerve development shows that Sprouty gene deletion in the pharyngeal epithelia can affect both placode formation and neural crest fate. However, epithelial-specific gene deletion only results in defects in the facial nerve and not the glossopharyngeal and vagus nerves, suggesting that the facial nerve is most sensitive to perturbations in RTK signalling. Reducing the Fgf8 gene dosage only partially rescued defects in the glossopharyngeal nerve and was not sufficient to rescue facial nerve defects, suggesting that FGF8 is functionally redundant with other RTK ligands during facial nerve development.     

Hox genes, neural crest cells and branchial arch patterning

Proper craniofacial development requires the orchestrated integration of multiple specialized tissue interactions. Recent analyses suggest that craniofacial development is not dependent upon neural crest pre-programming as previously thought but is regulated by a more complex integration of cell and tissue interactions. In the absence of neural crest cells it is still possible to obtain normal arch patterning indicating that neural crest is not responsible for patterning all of arch development. The mesoderm, endoderm and surface ectoderm tissues play a role in the patterning of the branchial arches, and there is now strong evidence that Hoxa2 acts as a selector gene for the pathways that govern second arch structures.     

Hox genes, neural crest cells and branchial arch patterning.

Proper craniofacial development requires the orchestrated integration of multiple specialized tissue interactions. Recent analyses suggest that craniofacial development is not dependent upon neural crest pre-programming as previously thought but is regulated by a more complex integration of cell and tissue interactions. In the absence of neural crest cells it is still possible to obtain normal arch patterning indicating that neural crest is not responsible for patterning all of arch development. The mesoderm, endoderm and surface ectoderm tissues play a role in the patterning of the branchial arches, and there is now strong evidence that Hoxa2 acts as a selector gene for the pathways that govern second arch structures.     

NK-2 class homeobox genes and pharyngeal/oral patterning: Nkx2-3 is required for salivary gland and tooth morphogenesis

Head development in vertebrates requires reciprocal patterning interactions between cranial neural crest and the ectodermal, mesodermal and endodermal components of the branchial arches. Patterning elements within the pharyngeal endoderm and oral ectoderm appear to play defining roles in this process. Several homeobox genes of the NK-2 class (Nkx2-1, Nkx2-3, Nkx2-5 and Nkx2-6) are expressed regionally in the developing pharynx, and Nkx2-1 mutants and Nkx2-5/Nkx2-6 double mutants show loss of thyroid and distal lung progenitors, and pharyngeal cell viability, respectively. Here we examined the expression and genetic role of Nkx2-3 in pharyngeal development. Nkx2-3 was expressed in the pharyngeal floor and pouches, as well as in oral and branchial arch ectoderm. Expression persisted in the developing thyroid until birth, in mucous-forming cells of the lingual and sublingual salivary glands, and in odontogenic epithelium of the mandible. Examination of Nkx2-3 null mice revealed defects in maturation and cellular organisation of the sublingual glands. Furthermore, cusps were absent from mandibular molars and the third molar was occasionally missing. These data suggest roles for Nkx2-3 during pharyngeal organogenesis, although the considerable potential for genetic redundancy within and outside of this gene family may mask earlier functions in organ specification.     

Fgf20b is required for the ectomesenchymal fate establishment of cranial neural crest cells in zebrafish

In cranial skeletal development, the establishment of the ectomesenchymal lineage within the cranial neural crest is of great significance. Fgfs are polypeptide growth factors with diverse functions in development and metabolism. Fgf20b knockdown zebrafish embryos showed dysplastic neurocranial and pharyngeal cartilages. Ectomesenchymal cells from cranial neural crest cells were significantly decreased in Fgf20b knockdown embryos, but cranial neural crest cells with a non-ectomesnchymal fate were increased. However, the proliferation and apoptosis of cranial neural crest cells were essentially unchanged. Fgfr1 knockdown embryos also showed dysplastic neurocranial and pharyngeal cartilages. The present findings indicate that Fgf20b is required for ectomesenchymal fate establishment via the activation of Fgfr1 in zebrafish.     

FGF8 signaling is chemotactic for cardiac neural crest cells

Cardiac neural crest cells migrate into the pharyngeal arches where they support development of the pharyngeal arch arteries. The pharyngeal endoderm and ectoderm both express high levels of FGF8. We hypothesized that FGF8 is chemotactic for cardiac crest cells. To begin testing this hypothesis, cardiac crest was explanted for migration assays under various conditions. Cardiac neural crest cells migrated more in response to FGF8. Single cell tracing indicated that this was not due to proliferation and subsequent transwell assays showed that the cells migrate toward an FGF8 source. The migratory response was mediated by FGF receptors (FGFR) 1 and 3 and MAPK/ERK intracellular signaling. To test whether FGF8 is chemokinetic and/or chemotactic in vivo, dominant negative FGFR1 was electroporated into the premigratory cardiac neural crest. Cells expressing the dominant negative receptor migrated slower than normal cardiac neural crest cells and were prone to remain in the vicinity of the neural tube and die. Treating with the FGFR1 inhibitor, SU5402 or an FGFR3 function-blocking antibody also slowed neural crest migration. FGF8 over-signaling enhanced neural crest migration. Neural crest cells migrated to an FGF8-soaked bead placed dorsal to the pharynx. Finally, an FGF8 producing plasmid was electroporated into an ectopic site in the ventral pharyngeal endoderm. The FGF8 producing cells attracted a thick layer of mesenchymal cells. DiI labeling of the neural crest as well as quail-to-chick neural crest chimeras showed that neural crest cells migrated to and around the ectopic site of FGF8 expression. These results showing that FGF8 is chemotactic and chemokinetic for cardiac neural crest adds another dimension to understanding the relationship of FGF8 and cardiac neural crest in cardiovascular defects.     

Requirement for endoderm and FGF3 in ventral head skeleton formation

The vertebrate head skeleton is derived in part from neural crest cells, which physically interact with head ectoderm, mesoderm and endoderm to shape the pharyngeal arches. The cellular and molecular nature of these interactions is poorly understood, and we explore here the function of endoderm in this process. By genetic ablation and reintroduction of endoderm in zebrafish, we show that it is required for the development of chondrogenic neural crest cells, including their identity, survival and differentiation into arch cartilages. Using a genetic interference approach, we further identify Fgf3 as a critical component of endodermal function that allows the development of posterior arch cartilages. Together, our results reveal for the first time that the endoderm provides differential cues along the anteroposterior axis to control ventral head skeleton development and demonstrate that this function is mediated in part by Fgf3.     

Loss of Gbx2 results in neural crest cell patterning and pharyngeal arch artery defects in the mouse embryo

Several syndromes characterized by defects in cardiovascular and craniofacial development are associated with a hemizygous deletion of chromosome 22q11 in humans and involve defects in pharyngeal arch and neural crest cell development. Recent efforts have focused on identifying 22q11 deletion syndrome modifying loci. In this study, we show that mouse embryos deficient for Gbx2 display aberrant neural crest cell patterning and defects in pharyngeal arch-derived structures. Gbx2(-/-) embryos exhibit cardiovascular defects associated with aberrant development of the fourth pharyngeal arch arteries including interrupted aortic arch type B, right aortic arch, and retroesophageal right subclavian artery. Other developmental abnormalities include overriding aorta, ventricular septal defects, cranial nerve, and craniofacial skeletal patterning defects. Recently, Fgf8 has been proposed as a candidate modifier for 22q11 deletion syndromes. Here, we demonstrate that Fgf8 and Gbx2 expression overlaps in regions of the developing pharyngeal arches and that they interact genetically during pharyngeal arch and cardiovascular development.     

Ectodermally derived FGF8 defines the maxillomandibular region in the early chick embryo: epithelial-mesenchymal interactions in the specification of the craniofacial ectomesenchyme

The most rostral cephalic crest cells in the chick embryo first populate ubiquitously in the rostroventral head. Before the influx of crest cells, the ventral head ectoderm expresses Fgf8 in two domains that correspond to the future mandibular arch. Bmp4 is expressed rostral and caudal to these domains. The rostral part of the Bmp4 domain develops into the rostral end of the maxillary process that corresponds to the transition between the maxillomandibular and premandibular regions. Thus, the distribution patterns of FGF8 and BMP4 appear to foreshadow the maxillomandibular region in the head ectoderm. In the ectomesenchyme of the pharyngula embryo, expression patterns of some homeobox genes overlap the distribution of their upstream growth factors. Dlx1 and Barx1, the targets of FGF8, are expressed in the mandibular ectomesenchyme, and Msx1, the target of BMP4, in its distal regions. Ectopic applications of FGF8 lead to shifted expression of the target genes as well as repatterning of the craniofacial primordia and of the trigeminal nerve branches. Focal injection of a lipophilic dye, DiI, showed that this shift was at least in part due to the posterior transformation of the original premandibular ectomesenchyme into the mandible, caused by the changed distribution of FGF8 that defines the mandibular region. We conclude that FGF8 in the early ectoderm defines the maxillomandibular region of the prepharyngula embryo, through epithelial-mesenchymal interactions and subsequent upregulation of homeobox genes in the local mesenchyme. BMP4 in the ventral ectoderm appears to limit the anterior expression of Fgf8. Ectopic application of BMP4 consistently diminished part of the mandibular arch.