Development of the Pharyngeal Apparatus and Face

Origin of Vertebrate Head

The “new head hypothesis” proposed that the vertebrate head evolved by adding structures, derived from neural crest cells, cranial to the notochord. Cell lineage tracing, for example, using genetically modified mice to label neural crest cell–derived and mesoderm-derived structures, has confirmed this hypothesis. In the cranial base, the structures that develop around and caudal to the cranial tip of the notochord (i.e., the occipital bone) are derived from the mesoderm (yellow; see Fig. 17.4A ). Structures that form rostral to the notochord (i.e., the sphenoid and ethmoid bones) are derived from the neural crest (tan; see Fig. 17.4A ). Therefore, the neural crest–mesoderm boundary lies at the sphenoid-occipital interface, with one exception—the hypochiasmatic cartilage , which provides the attachment for the extraocular muscles, contributes to the sphenoid bone, and is derived from mesoderm, even though it lies rostral to the notochord (yellow islands; see Fig. 17.4A ).

These lineage-tracing studies have revealed the contributions of neural crest and mesoderm to the mammalian cranial vault and pharyngeal arches. Neural crest–derived cells contribute to the frontal bone and to a portion of the interparietal bones in the cranial vault, whereas the mesoderm forms the parietal bones and the lateral part of the interparietal bones (see Fig. 17.4B,C ). Neural crest cells are also found between paired parietal bones. Therefore, two sutures ( coronal and sagittal sutures ) are formed at a neural crest–mesoderm interface. This is significant, as mutations affecting formation of these boundaries result in craniosynostosis (see the following “In the Clinic” entitled “Craniosynostosis”). Neural crest cells also give rise to cartilages formed within pharyngeal arches 1 to 3.

The chondrocranium is induced to form in response to signals from surrounding tissues. In the trunk, the notochord, which expresses sonic hedgehog (Shh), is essential for formation of the axial skeleton (see Chapter 8 ). However, the structures of the anterior cranial base develop cranial to the notochord. Therefore, they must form in response to signaling from different tissues. Shh signaling from the developing brain and/or facial ectoderm is required for development of the midline structures of the cranial base, whereas FGF signals from the otic and nasal epithelium induce the formation of the otic and nasal capsules, respectively. Consequently, treatment of zebrafish embryos with cyclopamine (a chemical inhibitor of hedgehog signaling) results in loss of the trabecular bones. Similarly, ablation of the hedgehog receptor, smoothened, in the cranial neural crest in mice results in loss of the structures of the anterior cranial base. Therefore, it seems that the brain and ectoderm induce formation of the cranial base, and that similar signaling networks control development of the trunk and cranial axial structures.

Craniosynostosis

Craniosynostosis , the premature closure of sutures, affects approximately 1 in 2500 children. Sutures, which occur where two membrane bones meet, contain the progenitor cells that will give rise to new bone cells, the osteoblasts. These are the sites where growth of membrane bone occurs. Craniosynostosis can be caused by gene mutations or chromosomal abnormalities and occurs in many syndromes, including Crouzon , Apert , Pfeiffer , Muenke , and Saethre-Chotzen syndromes . Craniosynostosis can also be a consequence of environmental factors such as a restricted intrauterine environment or exposure to teratogens.

The sagittal suture is the most commonly affected. Closure at one suture causes increased growth at other sutures, thereby deforming the brain and skull, which develop a characteristic deformation depending on the suture(s) that is closed. This is shown in Fig. 17.6A . Here, the coronal suture has closed, so that the head cannot grow in length, and now the developing brain forces growth along the other sutures. The result is a skull deformity, with possible changes in neurologic function, increased intracranial pressure, and inability to feed and close the eyes, together with respiratory and hearing abnormalities. Dolichocephaly and scaphocephaly are the terms used to describe long and narrow skulls. Acrocephaly describes a towered skull; trigonocephaly describes a skull that is triangular at the front; brachycephaly describes a skull that is broad and flattened; and plagiocephaly describes a skewed skull (see Fig. 17.6B ).

The most common genetic causes of craniosynostosis syndromes are mutations in genes coding for (1) Fgf receptors 2 ( Apert , Crouzon , and Pfeiffer syndromes ) and 3 ( Muenke and Crouzon syndromes with acanthosis nigricans ); (2) the transcription factor, Twist ( Saethre - Chotzen syndrome ); and (3) a membrane-bound ligand, Ephrin B1 (EfnB1) ( craniofrontonasal syndrome ; Fig. 17.7A ). Pfeiffer syndrome is highly variable, but the most severe form is characterized by multiple synostoses (union of separate bones to form a single bone) that are associated with high mortality. Distinguishing characteristics of the hand and face can be used to help with an initial diagnosis of the syndrome. For example, Pfeiffer syndrome is defined by a broad thumb and a big toe, Apert syndrome by syndactyly, and craniofrontonasal syndrome by split nails.

Molecular Mechanisms of Craniosynostosis

As was just covered, mutations in genes coding for Fgf receptors (Fgfr1, 2, and 3) result in craniosynostosis. All of these mutations cause a gain-of-function in Fgf signaling owing to the constitutive activation of receptors (i.e., these mutations result in an increase in Fgf signaling). Fgfs are expressed in the osteoblastic front of membrane bones (see Fig. 17.6C ). Therefore, the highest levels of signaling are found in the cells adjacent to the bone, whereas lower levels are found in the sutural mesenchyme (see Fig. 17.6C ). The cognate receptors are expressed in the sutural mesenchyme (Fgfr2) and in differentiating osteoblasts (Fgfr1 and 3; see Fig. 17.6C ).

Fgf signaling has two roles in suture development. Low levels of Fgfs in the sutural mesenchyme promote cell proliferation whereas high levels promote osteoblast differentiation. Therefore, the rate of osteogenesis is carefully controlled by balancing the level of Fgf signaling (see Fig. 17.6C ). Increased Fgf signaling through constitutive activation of the receptors decreases cell proliferation and accelerates osteoblast differentiation, resulting in synostosis across the sutures (i.e., premature closure of the fontanelles). In mouse models of craniosynostosis, administration of pharmacologic inhibitors against the Fgf pathway during pregnancy and postnatally prevents premature suture closure. Such approaches may eventually allow non-surgical or reduced surgical intervention for the treatment of craniosynostosis in humans. Curiously, FGFR2 and 3 mutations are usually inherited as spontaneous new mutations through the paternal lineage, increasing in frequency with paternal age. One such mutation in FGFR2 (a mutation that causes Apert syndrome ) increases the clonal expansion of mutant spermatogonia, thereby increasing the number of sperm carrying the mutation.

The EFNB1 and TWIST mutations specifically affect the coronal suture. Long-term lineage tracing studies in mice have shown that the coronal suture arises at a neural crest cell–mesoderm interface (see Fig. 17.4B,C ). The frontal bone is derived from neural crest, whereas the adjacent parietal bone and the coronal sutural mesenchyme are derived from mesoderm. The neural crest–derived mesenchyme and the mesoderm have different cell adhesion properties and do not mix, thereby creating a sharp boundary. These distinct properties are controlled by the differential expression of the membrane-bound ephrin ligands and their Eph receptors in the neural crest– and mesoderm-derived tissues (also see Fig. 8.5 in Chapter 8 and Fig. 20.29 in Chapter 20 , as well as Chapter 13, Chapter 14, Chapter 9 , for other examples of the differential expression of ephrin ligands and Eph receptors inhibiting cell mixing). Mutations in the ephrin signaling pathway and in twist, a transcription factor that regulates ephrin expression, will affect the formation of this boundary. Twist also directly inhibits Runx2, a master transcriptional regulator of bone differentiation (covered further in Chapter 8 ). Therefore, reduction in twist expression will result in bone formation across the suture.

Craniofrontonasal dysplasia , caused by an EfnB1 mutation, is an unusual X-linked syndrome in that heterozygote females show more severe phenotypes than hemizygous males. The ephrin ligand EfnB1 is specifically expressed in the neural crest and prevents mixing of neural crest–derived cells with mesodermal cells. Due to random X-inactivation, in heterozygote females, the frontal bone will consist of a mixture of cells that either express one copy of the functional EFNB1 gene or express the mutated non-functional EfnB1 protein. Cells that express the normal EFNB1 gene have different cell adhesion and signaling properties than cells expressing the non-functional EFNB1 gene, such that the two populations will clump and segregate: the result is that some of these cells cross and abolish the sutural boundary. Additional features of craniofrontonasal dysplasia include severe hypertelorism (widely spaced eyes) and a bifid nasal tip in females (see Fig. 17.7A ).

Holoprosencephaly

Holoprosencephaly (HPE) is the most common developmental defect of the forebrain, affecting 1 in 16,000 births and an estimated 1 in 250 fetuses. It results from a disturbance in early patterning of the forebrain (see Fig. 17.7B ). The spectrum of phenotypes is wide and there is variable expressivity, which means that the same mutation can produce different phenotypic outcomes. This indicates the possibility of modifier genes and/or environmental effects, and the fact that HPE should be viewed as a multigenic or multifactorial disorder . It is estimated that approximately 30% of people with HPE mutations have no phenotype.

HPE can be classified into five forms: alobar , lobar , semilobar , microform , which are classic forms of HPE, and middle interhemispheric form ( MIH ; also known as syntelencephaly ). In its most severe form, alobar HPE, only a single cerebral lobe forms (hence the name of the condition) rather than paired right and left hemispheres. Defects of the olfactory nerves, olfactory bulbs, olfactory tracts, basal olfactory cortex, and associated structures including the limbic lobe, hippocampus, and mammillary bodies can also occur. The corpus callosum is sometimes affected; the hindbrain is usually normal. Patients with alobar HPE typically die within the first year. In the milder forms of HPE, semilobar and lobar HPE, partial separation of the single cerebral lobe is noted. In MIH, the anterior forebrain develops normally, but the hemispheres fail to separate in the posterior parietal and frontal regions.

Except for MIH, forebrain defects are accompanied by a spectrum of facial abnormalities that often, but not always, reflect the severity of the forebrain defect. Facial anomalies typical of HPE include a flat nose, ocular hypotelorism (closely spaced eyes), deficient philtrum or cleft lip, high arched or cleft palate, and microcephaly (small skull) (see Fig. 17.7B ). Particularly severe cases involve dramatic defects of the facial structures arising from the frontonasal prominence, most notably the nasal placodes (development of the face is covered in detail later in this chapter). Failure of the medial nasal processes to form results in agenesis of the intermaxillary process and reduction or absence of other midfacial structures such as the nasal bones, nasal septum, and ethmoid. The consequence may be cebocephaly (a single nostril; see Fig. 17.7B ) and, at the most extreme, cyclopia (a single, midline eye). Mild cases of HPE are characterized by relatively minimal midface anomalies and by trigonocephaly , a triangular skull shape that develops as a result of premature closure (synostosis) of the suture between the frontal bones and causes compression of the growing cerebral hemispheres (see Fig. 17.6B ). Occasionally, the presence of a single, central incisor and the loss of the maxillary midline frenulum are the only indications of a holoprosencephalic phenotype. In microform HPE , mild facial abnormalities occur in the absence of forebrain defects.

Mutations in at least 17 genes have been implicated in HPE in humans, and mutations in nine genes in these loci have been identified. Of these, four are components of the sonic hedgehog signaling pathway (SHH, PTC1, GLI2, and DISP1: DISPATCHED HOMOLOG 1). Mutations in SHH and in ZIC2, SIX3, and TRANSFORMING GROWTH FACTOR INTERACTING FACTOR (TGIF) all of three of which encode transcription factors, are the leading genetic causes of non-syndromic HPE.

HPE is also a feature of more than 25 syndromes and is seen in 5% of Smith-Lemli-Opitz syndrome patients. Smith-Lemli-Opitz syndrome affects about 1 in 9000 births (live and stillborn) and is the result of a mutation in the DHCR7 gene, which encodes 7-dehydrocholesterol reductase, an enzyme involved in the penultimate step of cholesterol synthesis. A cholesterol modification of Shh is necessary for full Shh activity (see Chapter 5 ); hence, it is believed that some Smith-Lemli-Opitz phenotypes are due to loss of hedgehog signaling.

Environmental agents associated with HPE include maternal diabetes. Infants of diabetic mothers have a risk of HPE that may be as high as 1%. Maternal exposure to alcohol , excess vitamin A , or statins , which disrupt Shh signaling, have also been implicated.

Conversely, some syndromes have hypertelorism (widely spaced eyes) as a component. This is seen in craniofrontonasal dysplasia , resulting from mutations in EFNB1 (EPHRIN-B1; see Fig. 17.7A ); Gorlin syndrome resulting from mutations in PTC1; and Greig cephalopolysyndactyly , resulting from mutations in GLI3. As Gli3 and Ptc1 are components of the Shh pathway and can repress Shh signaling, it is believed that the hypertelorism observed in Gorlin and Greig cephalopolysyndactyly is due to a gain in Shh signaling (see the following “In the Research Lab” entitled “Cranial Midline and HPE”).

Cranial Midline and HPE

Establishment of the cranial midline involves three major steps during development ( Fig. 17.8A ): (1) formation of the prechordal plate during gastrulation; (2) signaling from the prechordal plate to the overlying forebrain, which initially consists of a single cerebral lobe (covered in Chapter 9 ), to divide the forebrain into two cerebral lobes; surgical ablation of the prechordal plate results in cyclopia, owing to loss of this signaling; and (3) signaling by the forebrain to the facial ectoderm in post-neurulation stages to induce and/or maintain gene expression in this area. Signaling by the facial ectoderm regulates the outgrowth of the frontonasal prominence during subsequent formation of the midline of the face (development of the face is covered later in this chapter).

In humans, SHH mutations are the leading genetic cause of non-syndromic HPE. In animal models, Shh is expressed, and is required, in the prechordal plate, forebrain, and facial ectoderm to establish the midline (see Fig. 17.8A ). Clearly emphasizing the importance of the Shh pathway, Shh−/− mutant mice exhibit cyclopia and develop a proboscis (see Fig. 17.8B ). The developing upper face normally consists of the medial nasal and lateral nasal processes (see Fig. 17.8C ; also see Fig. 17.18 ). In Shh mutants, a total collapse of the midline is shown by loss of the medial nasal processes (see Fig. 17.8C ). With loss of these processes, the lateral nasal processes become positioned at the midline of the upper face (see Fig. 17.8C ).

During the establishment of the midline, Shh signaling is first required in the prechordal plate region to divide the single midline eye primordium in the forebrain into eye (Pax6 expressing) and non-eye (non-Pax6 expressing) territories (see Fig. 17.8A ). Shh expression also maintains cell survival of the prechordal plate and activates Shh expression in the developing diencephalon (see Fig. 17.8A ). Subsequently, Shh signaling from the diencephalon activates the expression of Shh in the ectoderm of the frontonasal prominence and creates a unique zone called the frontonasal ectodermal zone (FEZ; see later “In the Research Lab” entitled “Patterning of Facial Prominences”), which is required for proximodistal outgrowth and dorsoventral patterning of the upper face (see Fig. 17.8A ). Application of Shh-blocking antibodies in the diencephalon or frontonasal prominence of developing chick embryos results in hypotelorism, showing that both domains of Shh expression are needed for appropriate midline development.

HPE is due to haploinsufficiency (i.e., the presence of one mutated gene and one wild-type allele) and two aspects of the syndrome should be emphasized. First, mice with heterozygous deletion of Shh do not show a phenotype. Second, there is extreme phenotypic variability from cyclopia to normality between individuals carrying the same gene mutation. Treatment of developing chick embryos with cyclopamine, an inhibitor of hedgehog signaling, has shown that loss of Shh function earlier in development (e.g., in the prechordal plate) has a more severe effect than loss of Shh function later in development (e.g., in the facial ectoderm). This could, in part, explain the spectrum of phenotypes observed as a result of Shh mutations and why phenotypes are observed in humans with one copy of mutated Shh. An environmental insult, such as in utero exposure to alcohol or retinoic acid, would downregulate the expression of the wild-type allele of Shh, further decreasing the levels of Shh. An early environmental insult would have the most severe consequences, as this would affect both the forebrain and subsequently the facial prominence, whereas later insults would affect only the facial structures, thereby resulting in milder forms of HPE.

Mutations in silent modifier genes may also determine the phenotypic spectrum of particular Shh mutations. The severity of the HPE defect would be dependent on when and where these silent modifier genes are expressed. Modifier genes would include components or regulators of the Shh pathway, such as the cell-surface molecules Gas1 and Cdo, which are expressed at sites distant to the source of Shh and are thought to increase levels of Shh signaling.

Mutations in TGIF, ZIC2, and SIX3 also result in loss of Shh signaling, explaining why their loss of function causes HPE. Tgif and Zic2 are required for development of the prechordal plate. Therefore, mutations in these factors results in early loss of Shh signaling. Six3 directly regulates Shh expression in the forebrain and, hence, affects later stages of midline patterning. Conversely, gain of function of Shh signaling results in expansion of the midline. Misexpression of Shh in the diencephalon or the ectoderm of the frontonasal prominence in chick embryos results in an expansion of the midline. Hence, it is believed that the levels of Shh signaling may define a spectrum of facial disorders from cyclopia to diprosopus—the duplication of facial structures (see Fig. 17.8D ).