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.     

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.     

The role of Hoxa3 gene in parathyroid gland organogenesis of the mouse.

Mice with a targeted deletion of the Hoxa3 gene have defects of derivatives of the third branchial arch and pouch. To address the role of the Hoxa3 gene in parathyroid organogenesis, we examined the third pharyngeal pouch development by immunohistochemistry (IHC) using the secretory protein (SP)-1/chromogranin A antiserum, which recognizes the parathyroid from its initial formation onward. At embryonic day (E) 11.5, the SP-1/chromogranin A-immunoreactive primary rudiment of the parathyroid appeared in the cranial region of the third pharyngeal pouch of wild-type embryos. In Hoxa3-null mutants, the third pharyngeal pouch was normally formed but failed to differentiate into the parathyroid rudiment, showing no immunoreactivity for SP-1/chromogranin A. Classic studies using chick-quail chimeras have demonstrated that the ectomesenchymal neural crest cells are required for proper development of the pharyngeal pouch-derived organs, including the thymus and parathyroid glands. To visualize the migration and development of mesenchymal neural crest cells in Hoxa3 mutants, the heterozygotes were crossed with connexin43-lacZ transgenic mice in which beta-galactosidase expression was specific to the neural crest cells. In Hoxa3 homozygotes and in wild types, ectomesenchymal neural crest cells densely populated the pharyngeal arches, including the third one, and surrounded the third pouch epithelium. These results indicate that lack of the Hoxa3 gene affects the intrinsic ability of the third pharyngeal pouch to form the parathyroid rudiment and has no detectable effect on the migration of neural crest cells.     

Fgf15 is required for proper morphogenesis of the mouse cardiac outflow tract

Evidence in animal models indicates that signaling networks functioning in the developing pharyngeal arches regulate stereotyped processes critical for proper development of the aortic arch and cardiac outflow tract. Here, we describe the phenotype of mice lacking fibroblast growth factor 15 (Fgf15), which encodes a secreted signaling molecule expressed within the developing pharyngeal arches. Homozygous Fgf15 mutants present heart defects consistent with malalignment of the aorta and pulmonary trunk. These defects correlate with early morphological defects of the outflow tract due to aberrant behavior of the cardiac neural crest. We demonstrate that Fgf15 expression within the pharyngeal arches is unaltered by a loss of Tbx1, a key regulator of pharyngeal arch development implicated in DiGeorge syndrome. In addition, Fgf15 and Tbx1 do not interact genetically, suggesting that Fgf15 operates through a pathway independent of Tbx1. These studies reveal a novel role of Fgf15 during development of the cardiac outflow tract.     

Abnormalities of caudal pharyngeal pouch development in Pbx1 knockout mice mimic loss of Hox3 paralogs

Pbx1 is a TALE-class homeodomain protein that functions in part as a cofactor for Hox class homeodomain proteins. Previous analysis of the in vivo functions of Pbx1 by targeted mutagenesis in mice has revealed roles for this gene in skeletal patterning and development and in the organogenesis of multiple systems. Both RNA expression and protein localization studies have suggested a possible role for Pbx1 in pharyngeal region development. As several Hox mutants have distinct phenotypes in this region, we investigated the potential requirement for Pbx1 in the development of the pharyngeal arches and pouches and their organ derivatives. Pbx1 homozygous mutants exhibited delayed or absent formation of the caudal pharyngeal pouches, and disorganized patterning of the third pharyngeal pouch. Formation of the third pouch-derived thymus/parathyroid primordia was also affected, with absent or hypoplastic primordia, delayed expression of organ-specific differentiation markers, and reduced proliferation of thymic epithelium. The fourth pouch and the fourth pouch-derived ultimobranchial bodies were usually absent. These phenotypes are similar to those previously reported in Hoxa3(-/-) single mutants and Hoxa1(-/-);Hoxb1(-/-) or Hoxa3(+/-);Hoxb3(-/-);Hoxd3(-/-) compound mutants, suggesting that Pbx1 acts together with multiple Hox proteins in the development of the caudal pharyngeal region. However, some aspects of the Pbx1 mutant phenotype included specific defects that were less severe than those found in known Hox mutant mice, suggesting that some functions of Hox proteins in this region are Pbx1-independent.     

Differential expression of Sonic hedgehog along the anterior-posterior axis regulates patterning of pharyngeal pouch endoderm and pharyngeal endoderm-derived organs

Previous studies have implicated Sonic hedgehog (Shh) as an important regulator of pharyngeal region development. Here we show that Shh is differentially expressed within the pharyngeal endoderm along the anterior-posterior axis. In Shh-/- mutants, the pharyngeal pouches and arches formed by E9.5 and marker expression showed that initial patterning was normal. However, by E10.5-E11.0, the first arch had atrophied and the first pouch was missing. Although small, the second, third, and fourth arches and pouches were present. The expression patterns of Fgf8, Pax1, and Bmp4 suggested that pouch identity was abnormal at E10.5 and that Shh is a negative regulator of these genes in the pouches. Despite the loss of pouch identity and an increase in mesenchymal cell death, arch identity markers were expressed normally. Our data show that a Shh-dependent patterning mechanism is required to maintain pouch patterning, independent or downstream of arch identity. Changes in the distribution of Bmp4 and Gcm2 in the third pouch endoderm and subsequent organ phenotypes in Shh-/- mutants suggested that exclusion of Shh from the third pouch is required for dorsal-ventral patterning and for parathyroid specification and organogenesis. Furthermore, this function for Shh may be opposed by Bmp4. Our data suggest that, as in the posterior gut endoderm, exclusion of Shh expression from developing primordia is required for the proper development of pharyngeal-derived organs.     

moz regulates Hox expression and pharyngeal segmental identity in zebrafish

In vertebrate embryos, streams of cranial neural crest (CNC) cells migrate to form segmental pharyngeal arches and differentiate into segment-specific parts of the facial skeleton. To identify genes involved in specifying segmental identity in the vertebrate head, we screened for mutations affecting cartilage patterning in the zebrafish larval pharynx. We present the positional cloning and initial phenotypic characterization of a homeotic locus discovered in this screen. We show that a zebrafish ortholog of the human oncogenic histone acetyltransferase MOZ (monocytic leukemia zinc finger) is required for specifying segmental identity in the second through fourth pharyngeal arches. In moz mutant zebrafish, the second pharyngeal arch is dramatically transformed into a mirror-image duplicated jaw. This phenotype resembles a similar but stronger transformation than that seen in hox2 morpholino oligo (hox2-MO) injected animals. In addition, mild anterior homeotic transformations are seen in the third and fourth pharyngeal arches of moz mutants. moz is required for maintenance of most hox1-4 expression domains and this requirement probably at least partially accounts for the moz mutant homeotic phenotypes. Homeosis and defective Hox gene expression in moz mutants is rescued by inhibiting histone deacetylase activity with Trichostatin A. Although we find early patterning of the moz mutant hindbrain to be normal, we find a late defect in facial motoneuron migration in moz mutants. Pharyngeal musculature is transformed late, but not early, in moz mutants. We detect relatively minor defects in arch epithelia of moz mutants. Vital labeling of arch development reveals no detectable changes in CNC generation in moz mutants, but later prechondrogenic condensations are mispositioned and misshapen. Mirror-image hox2-dependent gene expression changes in postmigratory CNC prefigure the homeotic phenotype in moz mutants. Early second arch ventral expression of goosecoid (gsc) in moz mutants and in animals injected with hox2-MOs shifts from lateral to medial, mirroring the first arch pattern. bapx1, which is normally expressed in first arch postmigratory CNC prefiguring the jaw joint, is ectopically expressed in second arch CNC of moz mutants and hox2-MO injected animals. Reduction of bapx1 function in wild types causes loss of the jaw joint. Reduction of bapx1 function in moz mutants causes loss of both first and second arch joints, providing functional genetic evidence that bapx1 contributes to the moz-deficient homeotic pattern. Together, our results reveal an essential embryonic role and a crucial histone acetyltransferase activity for Moz in regulating Hox expression and segmental identity, and provide two early targets, bapx1 and gsc, of moz and hox2 signaling in the second pharyngeal arch.     

The regional pattern of retinoic acid synthesis by RALDH2 is essential for the development of posterior pharyngeal arches and the enteric nervous system

Targeted inactivation of the mouse retinaldehyde dehydrogenase 2 (RALDH2/ALDH1a2), the enzyme responsible for early embryonic retinoic acid synthesis, is embryonic lethal because of defects in early heart morphogenesis. Transient maternal RA supplementation from E7.5 to (at least) E8.5 rescues most of these defects, but the supplemented Raldh2(-/-) mutants die prenatally, from a lack of septation of the heart outflow tract (Niederreither, K., Vermot, J., Messaddeq, N., Schuhbaur, B., Chambon, P. and Dollé, P. (2001). Development 128, 1019-1031). We have investigated the developmental basis for this defect, and found that the RA-supplemented Raldh2(-/-) embryos exhibit impaired development of their posterior (3rd-6th) branchial arch region. While the development of the first and second arches and their derivatives, as well as the formation of the first branchial pouch, appear to proceed normally, more posterior pharyngeal pouches fail to form and the pharyngeal endoderm develops a rudimentary, pouch-like structure. All derivatives of the posterior branchial arches are affected. These include the aortic arches, pouch-derived organs (thymus, parathyroid gland) and post-otic neural crest cells, which fail to establish segmental migratory pathways and are misrouted caudally. Patterning and axonal outgrowth of the posterior (9th-12th) cranial nerves is also altered. Vagal crest deficiency in Raldh2(-/-) mutants leads to agenesis of the enteric ganglia, a condition reminiscent of human Hirschprung's disease. In addition, we provide evidence that: (i) wildtype Raldh2 expression is restricted to the posteriormost pharyngeal mesoderm; (ii) endogenous RA response occurs in both the pharyngeal endoderm and mesoderm, and extends more rostrally than Raldh2 expression up to the 2nd arch; (iii) RA target genes (Hoxa1, Hoxb1) are downregulated in both the pharyngeal endoderm and mesoderm of mutant embryos. Thus, RALDH2 plays a crucial role in producing RA required for pharyngeal development, and RA is one of the diffusible mesodermal signals that pattern the pharyngeal endoderm.     

The zebrafish van gogh mutation disrupts tbx1, which is involved in the DiGeorge deletion syndrome in humans

The van gogh (vgo) mutant in zebrafish is characterized by defects in the ear, pharyngeal arches and associated structures such as the thymus. We show that vgo is caused by a mutation in tbx1, a member of the large family of T-box genes. tbx1 has been recently suggested to be a major contributor to the cardiovascular defects in DiGeorge deletion syndrome (DGS) in humans, a syndrome in which several neural crest derivatives are affected in the pharyngeal arches. Using cell transplantation studies, we demonstrate that vgo/tbx1 acts cell autonomously in the pharyngeal mesendoderm and influences the development of neural crest-derived cartilages secondarily. Furthermore, we provide evidence for regulatory interactions between vgo/tbx1 and edn1 and hand2, genes that are implicated in the control of pharyngeal arch development and in the etiology of DGS.     

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.     

Targeted mutagenesis tools for modelling psychiatric disorders

Hes genes are required to maintain diverse progenitor cell populations during embryonic development. Loss of Hes1 results in a spectrum of malformations of pharyngeal endoderm-derived organs, including the ultimobranchial body (progenitor of C cells), parathyroid, thymus and thyroid glands, together with highly penetrant C-cell aplasia (81%) and parathyroid aplasia (28%). The hypoplastic parathyroid and thymus are mostly located around the pharyngeal cavity, even at embryonic day (E) 15.5 to E18.5, indicating the failure of migration of the organs. To clarify the relationship between these phenotypes and neural crest cells, we examine fate mapping of neural crest cells colonized in pharyngeal arches in Hes1 null mutants by using the Wnt1-Cre/R26R reporter system. In null mutants, the number of neural crest cells labeled by X-gal staining is markedly decreased in the pharyngeal mesenchyme at E12.5 when the primordia of the thymus, parathyroid and ultimobranchial body migrate toward their destinations. Furthermore, phospho-Histone-H3-positive proliferating cells are reduced in number in the pharyngeal mesenchyme at this stage. Our data indicate that the development of pharyngeal organs and survival of neural-crest-derived mesenchyme in pharyngeal arches are critically dependent on Hes1. We propose that the defective survival of neural-crest-derived mesenchymal cells in pharyngeal arches directly or indirectly leads to deficiencies of pharyngeal organs.     

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.     

Combined deficiencies of Msx1 and Msx2 cause impaired patterning and survival of the cranial neural crest

The neural crest is a multipotent, migratory cell population that contributes to a variety of tissues and organs during vertebrate embryogenesis. Here, we focus on the function of Msx1 and Msx2, homeobox genes implicated in several disorders affecting craniofacial development in humans. We show that Msx1/2 mutants exhibit profound deficiencies in the development of structures derived from the cranial and cardiac neural crest. These include hypoplastic and mispatterned cranial ganglia, dysmorphogenesis of pharyngeal arch derivatives and abnormal organization of conotruncal structures in the developing heart. The expression of the neural crest markers Ap-2alpha, Sox10 and cadherin 6 (cdh6) in Msx1/2 mutants revealed an apparent retardation in the migration of subpopulations of preotic and postotic neural crest cells, and a disorganization of neural crest cells paralleling patterning defects in cranial nerves. In addition, normally distinct subpopulations of migrating crest underwent mixing. The expression of the hindbrain markers Krox20 and Epha4 was altered in Msx1/2 mutants, suggesting that defects in neural crest populations may result, in part, from defects in rhombomere identity. Msx1/2 mutants also exhibited increased Bmp4 expression in migratory cranial neural crest and pharyngeal arches. Finally, proliferation of neural crest-derived mesenchyme was unchanged, but the number of apoptotic cells was increased substantially in neural crest-derived cells that contribute to the cranial ganglia and the first pharyngeal arch. This increase in apoptosis may contribute to the mispatterning of the cranial ganglia and the hypoplasia of the first arch.     

sucker encodes a zebrafish Endothelin-1 required for ventral pharyngeal arch development

Mutation of sucker (suc) disrupts development of the lower jaw and other ventral cartilages in pharyngeal segments of the zebrafish head. Our sequencing, cosegregation and rescue results indicate that suc encodes an Endothelin-1 (Et-1). Like mouse and chick Et-1, suc/et-1 is expressed in a central core of arch paraxial mesoderm and in arch epithelia, both surface ectoderm and pharyngeal endoderm, but not in skeletogenic neural crest. Long before chondrogenesis, suc/et-1 mutant embryos have severe defects in ventral arch neural crest expression of dHAND, dlx2, msxE, gsc, dlx3 and EphA3 in the anterior arches. Dorsal expression patterns are unaffected. Later in development, suc/et-1 mutant embryos display defects in mesodermal and endodermal tissues of the pharynx. Ventral premyogenic condensations fail to express myoD, which correlates with a ventral muscle defect. Further, expression of shh in endoderm of the first pharyngeal pouch fails to extend as far laterally as in wild types. We use mosaic analyses to show that suc/et-1 functions nonautonomously in neural crest cells, and is thus required in the environment of postmigratory neural crest cells to specify ventral arch fates. Our mosaic analyses further show that suc/et-1 nonautonomously functions in mesendoderm for ventral arch muscle formation. Collectively our results support a model for dorsoventral patterning of the gnathostome pharyngeal arches in which Et-1 in the environment of the postmigratory cranial neural crest specifies the lower jaw and other ventral arch fates.     

A genetic link between Tbx1 and fibroblast growth factor signaling

Tbx1 haploinsufficiency causes aortic arch abnormalities in mice because of early growth and remodeling defects of the fourth pharyngeal arch arteries. The function of Tbx1 in the development of these arteries is probably cell non-autonomous, as the gene is not expressed in structural components of the artery but in the surrounding pharyngeal endoderm. We hypothesized that Tbx1 may trigger signals from the pharyngeal endoderm directed to the underlying mesenchyme. We show that the expression patterns of Fgf8 and Fgf10, which partially overlap with Tbx1 expression pattern, are altered in Tbx1(-/-) mutants. In particular, Fgf8 expression is abolished in the pharyngeal endoderm. To understand the significance of this finding for the pathogenesis of the mutant Tbx1 phenotype, we crossed Tbx1 and Fgf8 mutants. Double heterozygous Tbx1(+/-);Fgf8(+/-) mutants present with a significantly higher penetrance of aortic arch artery defects than do Tbx1(+/-);Fgf8(+/+) mutants, while Tbx1(+/+);Fgf8(+/-) animals are normal. We found that Fgf8 mutation increases the severity of the primary defect caused by Tbx1 haploinsufficiency, i.e. early hypoplasia of the fourth pharyngeal arch arteries, consistent with the time and location of the shared expression domain of the two genes. Hence, Tbx1 and Fgf8 interact genetically in the development of the aortic arch. Our data provide the first evidence of a genetic link between Tbx1 and FGF signaling, and the first example of a modifier of the Tbx1 haploinsufficiency phenotype. We speculate that the FGF8 locus might affect the penetrance of cardiovascular defects in individuals with chromosome 22q11 deletions involving TBX1.