PHARYNGEAL ARCHES and FACE

by Michael Rindler, Ph.D.

Objectives
I. PHARYNGEAL ARCHES, CLEFTS AND POUCHES
A. Derivatives of the Arches
B. Derivatives of the Surface Ectoderm
C. The Tongue Mucosa
D. Pharyngeal Endoderm
II. Abnormalities Associated with Arch Development
III. DEVELOPMENT OF THE FACE
A. Palate
B. Developmental Biology of Palate and Face Formation
IV. Cleft Lip and Palate
V. DEVELOPMENT OF THE CRANIAL VAULT
VI. Abnormalities of the Face and Head
VII. DEVELOPMENT OF THE EAR
STUDY QUESTIONS

Objectives:

1. To learn about the contributions of the pharyngeal arches, pouches, and clefts to head and neck structures with particular emphasis on innervation patterns and gland development.

2. To study the development of the face and the palate.

3. To understand some of the molecular mechanisms involved normal and abnormal face and pharyngeal arch development.

4. To examine basic elements of the formation of the inner, middle, and external ear.

I. PHARYNGEAL ARCHES, CLEFTS AND POUCHES

During the fourth week of embryonic development, a series of paired protrusions, or pharyngeal arches, appear sequentially on the ventrolateral surface of the head and neck region (Figs. 15.2/16.2, 15.3/16.3). The arches are also known as branchial arches because they are related phylogenetically to the gill arches (branchia) of fish. In fact, the numbering of the arches ≠ 1, 2, 3, 4, and 6 ≠ reflects their correspondence to the equivalent arches in fish. There is no well-developed fifth arch in human embryos just as there is no fifth aortic arch (see Cardiovascular System chapter). The exterior surface of the arches is covered by ectoderm (Fig. 15.5/16.5). The ectoderm between the arches forms the pharyngeal clefts or grooves (Fig. 15.6/16.6). The inner surface of this pharyngeal region is lined by endoderm. Between each of the arches, where the endoderm approaches the clefts on the outside, are the pharyngeal pouches. The approximation of the ectoderm of the cleft with the endoderm of the pouch forms the pharyngeal membrane, which in fish ruptures to form the gill slits. The four paired clefts and pouches on each side are numbered the same as the preceding arch (Fig. 15.10/16.10).

Each arch contains within it a cartilaginous bar, an aortic arch, and a cranial nerve (Fig. 15.6/16.6). Each cranial nerve supplies the structures that develop from the arch. The mesenchyme of the arches is derived from paraxial (=somitic) mesoderm in the hindbrain region, lateral mesoderm, and neural crest cells from nearby midbrain and hindbrain regions. The cartilages and bones that develop in the arches mainly come from neural crest cells (Table I), while blood vessels, some connective tissue and laryngeal cartilages are from lateral mesoderm. Neural crest cells also contribute to the dermis of the skin in this region and to other connective tissue. Muscle develops from the paraxial mesoderm. Paraxial mesoderm in the cranial region is not organized into recognizable somites, as it is in the rest of the body. However, mesodermal cells adjacent to the developing hindbrain soon migrate into the nearby developing arches. Paraxial mesoderm from occipital somites also contributes to the laryngeal muscles of arches 4 and 6. However, these muscles are innervated by CN X and are considered branchiomeric muscles.

Neural crest cells are also responsible for the specification of the arches and the different structures they make. These cells express homeobox (HOX) genes and other homeodomain transcription factors even before they migrate from the developing midbrain and hindbrain (Fig. 15.12/16.12). Neural crest cell differentiation into the skeletal structures in the different arches is determined by HOX genes and other transcription factors as well as secreted growth factors such as FGF (Fig.15.13/16.13). This is borne out in experiments where neural crest cells from different regions are exchanged before they migrate. Under these circumstances, the neural crest cells will give rise to ectopic skeletal structures appropriate for their natural arch of origin. Targeted disruption of HOX genes causes anteriorization of arch derivatives (transformation of the 2^nd arch into a first arch, for example, yielding a duplicated first arch). Thus, HOX genes determine the fate of arch structures.

Cranial nerve V innervates the first arch, and CN VII supplies the second arch. CN IX is the nerve of the third arch, and the vagus nerve (CN X) supplies arches 4 and 6. The first pharyngeal arch is subdivided into maxillary and mandibular processes, which will contribute in the face to the upper jaw/cheek and lower jaw, respectively. Each has its own cartilaginous bar and branch of CN V as well. The sensory portions of the cranial nerves themselves are derived from neural crest cells and from ectodermal placodes. There are eight ectodermal placodes in all ≠ the lens placode, nasal placode, and otic placode contribute to the formation of the eye, the olfactory epithelium, and the inner ear, respectively. The other five are smaller and not readily visible at the surface of the embryo but they do undergo invagination and contribute cells to CN V, VII, IX, and X.

A. Derivatives of the Arches

Recognizable muscle masses are distinguishable in each of the arches as they form. The masses will then divide into dorsal and ventral halves and migrate before forming their characteristic muscles. The innervation of each muscle mass is established early and hence corresponds to the arch of origin. In general the muscles of mastication derive from the mandibular portion of the first arch and are innervated by the trigeminal nerve (CN V). The muscles of facial expression are second arch derivatives even though their position in the face does not always correspond to their origin. The second arch muscle mass largely grows cranially and ventrally to lie in the center of the face and form the muscles of facial expression. Only one muscle, the stylopharyngeus, is derived from the third arch, and the muscles of the fourth and sixth arches are in the pharynx and larynx. The muscles derived from each of the arches are listed in Table I below.

The muscles developing from pharyngeal arch mesoderm are really skeletal-type (striated) muscles. Because they do not arise from somites per se, the innervation of the branchiomeric muscles has been designated functionally as special visceral efferent (see Table I). Special visceral efferents also have a separate tract in the brain distinct from the tracts of other skeletal muscles (GSE) and from smooth muscle (GVE) hence the distinction does have an important anatomical correlation.

Cartilaginous bars develop in the pharyngeal arches from mesenchyme of neural crest origin (except in the laryngeal region where they are derived from lateral mesoderm). While the cartilages in the laryngeal region, derived from 4^th and 6^th arch mesoderm develop into the cricoid and thyroid cartilages of the larynx, the cartilages in the rest of the arches differentiate into ligaments, become sites of bone deposition, or disappear. In the mandibular process the cartilaginous bar is called Meckelπs Cartilage. Portions of this cartilage are converted into the malleus ear ossicle bone and the sphenomandibular ligament. The mandibular bone itself develops by direct ossification (see Cranial Vault section below for a description of bone formation) from neural crest cells of the mandibular process at a later stage of fetal development,

TABLE I: PHARYNGEAL ARCH DERIVATIVES
ARCH	CRANIAL NERVE	SKELETAL ELEMENTS	MUSCLES
1	Maxillary and mandibular division of trigeminal nerve (V)	Derived from arch cartilages: From maxillary cartilage: alisphenoid (part of sphenoid), incus From mandibular cartilage: malleus Derived by direct ossification from arch dermal mesenchyme: maxilla, zygomatic, squamous portion of temporal bone, mandible	Muscles of mastication (temporalis, masseter, pterygoids), myelohyoid, anterior belly of the digestric, tensor tympani, tensor veli palatini
2	Facial nerve (VII)	Stapes, styloid process, stylohoid ligament, Lesser horns of hyoid	Muscles of facial expression (orbicularis oris, risorius, fronto-occipitalis, auricularis, platysma, buccinator), posterior belly of the digastric, stylohyoid, stapedius
3	Glossopharyngeal (IX)	Greater horns of hyoid	Stylopharyngeus
4	Superior laryngeal branch of vagus (X)	Laryngeal cartilages (derived from fourth arch cartilage lateral plate mesoderm)	Constrictors of pharynx, cricothyroid, levator veli palatini
6	Recurrent laryngeal branch of vagus (X)	Laryngeal cartilages (derived from sixth-arch cartilage lateral plate mesoderm)	Intrinsic muscles of larynx

and therefore it is not considered to be derived from the cartilage per se. The maxillary process cartilage ultimately forms part of the sphenoid bone (alisphenoid) and the incus. However, neural crest cells in the maxillary portion of the first arch subsequently give rise to the maxilla, zygomatic and squamous portion of the temporal bone through direct ossification (see Table I above). Ossification of the second arch cartilage gives rise to the stapes of the ear, the styloid process, the stylohyoid ligament, and the lesser cornu and part of the body of the hyoid bone. The third arch cartilage ossifies to form the greater cornu and rest of the body of the hyoid bone.

The first and second aortic arches coursing through the first two arches degenerate. The third aortic arch passes through the 3^rd pharyngeal arch and persists as the stem of the internal carotid artery (and part of the common carotid artery). The fourth aortic arch persists on the right as the proximal subclavian artery. On the left it develops into a portion of the arch of the aorta. The sixth aortic arch survives as the origin of the pulmonary arteries and the ductus arteriosus (later the ligamentum arteriosum).

B. Derivatives of the Surface Ectoderm

The first pharyngeal cleft enlarges to form the external auditory meatus, which is the external opening of the ear (Figs. 15.10/16.10, 15.11/16.11). The ectoderm from the cleft, which is in close proximity to the first pharyngeal pouch, will contribute to the tympanic membrane (eardrum) as well. The mesenchyme of first and second arches on either side of the cleft will give rise to the external ear, the auricle. The second (hyoid) arch enlarges and grows inferiorly so that by the 6th week it overlaps the 3^rd, 4^th and 6^th arches exteriorly and covers them (Fig. 15.10/16.10). As a consequence, the second arch also covers the openings of the 2^nd, 3^rd, and 4^th pharyngeal clefts, which together form a remnant called the cervical sinus. The cervical sinus is gradually obliterated, but occasionally it will persist and can enlarge and form fluid-filled lateral cervical cysts ( Figs.15.14/16.14, 15.15/16.15). Sometimes the cervical sinus can fail to close externally, leaving a fistula open to the external surface. More rarely, the pharyngeal membrane can rupture internally, leaving the cervical remnant connected to the pharynx by a sinus. A fistula that runs entirely from the external to the internal surface is called a complete branchial fistula.

The ectodermal covering of the 2^nd arch contributes to the epithelium of the auricle and external auditory canal (meatus), and some of the epithelium behind the ear. Since the second arch is innervated by CN VII, its derivatives are innervated by the facial nerve. As noted above, the ectoderm of the 3^rd and 4^th arches is mostly covered by the 2^nd arch ectoderm. What remains can be found around the external ear (innervated by CN IX), and the external auditory meatus, external tympanic membrane and back of the ear (CN X).

The stomodeum (primitive mouth) forms primarily through the longitudinal folding of the early embryo as the buccopharyngeal membrane, and some of the ectoderm adjacent to it, comes to lie inside the oral cavity. The membrane, which will rupture, is located at the boundary of the first and second arches. Thus, the epithelium of the first arch inside the oral cavity is derived from ectoderm. This epithelium will also give rise to the parotid gland, enamel of the teeth, epithelium of the body of the tongue. Structures that develop from the first arch ectoderm are innervated by CN V.

Finally, a portion of the pituitary gland also comes from ectoderm in the oral cavity. The pituitary gland consists of two parts, the anterior (adenohypophysis, which synthesizes LH, FSH, prolactin, growth hormone, ACTH, and thyroid stimulating hormone) and posterior (neurohypophysis, which makes antidiuretic hormone and oxytocin). During the fourth week, ectoderm in the roof of the oral cavity forms a diverticulum, Rathke's pouch, which will eventually become the anterior portion of the gland. The pouch continues to migrate toward the developing brain. As it does so, it makes contact with a diverticulum growing down from the floor of the forebrain called the infundibulum, which is destined to become the posterior pituitary. As the pouch moves up toward the brain, the thin stalk of tissue that connected it to the oral cavity involutes.

In summary, the first cleft ectoderm gives rise to the external auditory meatus and contributes to the tympanic membrane. The ectoderm of the first arch lines the oral cavity and gives rise to the anterior pituitary gland.

C. The Tongue Mucosa

The tongue arises in the region where the stomodeum and primitive pharynx meet. It is seen initially as a proliferation of mesenchyme (Fig. 15.17/16.17). Paired lateral lingual swellings grow superiorly into the oral cavity and then flatten anteriorly, forming most of the body of the tongue. They also largely overgrow another elevation seen in the midline, the tuberculum impar (median tongue bud). The stomodeum is lined by ectoderm and therefore the mucosa of all of these swellings is derived from ectoderm. The root of the tongue develops from a primitive swelling caudal to the tuberculum impar -- the copula (hypobranchial eminence) -- at the levels of the second, third and fourth pharyngeal arches ≠ as well as nearby arch tissue. Further posterior to the copula, an epiglottic swelling arises cranial to the laryngeotracheal groove (see Development of the Respiratory System chapter). Tongue muscles develop primarily from myoblasts derived from occipital somites that migrate into the lower jaw and are innervated by the hypoglossal nerve (CN XII). These tongue muscles are therefore NOT considered branchiomeric muscles and have general somatic efferent innervation.

The general innervation of the tongue mucosa reflects its origin. General afferent innervation is somatic for the anterior two-thirds of the tongue up to the sulcus terminalis because the mucosa is derived from first arch ectoderm. Caudal to this point, the endodermally-derived mucosa gets general visceral afferent innervation. During the formation of the tongue, the second arch migrates out of the oral cavity completely, and therefore all of the sensory innervation in the posterior third of the tongue is from CN IX and CN X, which also innervates the area around the epiglottis.

Taste buds form in the epithelium (mucosa) of the oral cavity as a result of interaction with special visceral afferent nerve fibers beginning in the seventh week. SVA fibers from the chorda tympanic nerve innervate most of the taste buds in the anterior 2/3 of the tongue. This nerve is a branch of the facial nerve (CN VII) that runs with the mandibular division of CN V. A series of large round taste buds called vallate (circumvallate) papillae are found just anterior to the sulcus terminalis, in other words, just inside the margin of the anterior 2/3 of the tongue. These vallate papillae are innervated by CN IX. CN IX also innervates the taste buds in the posterior 1/3 of the tongue in the regions where the mucosa is derived from the 3^rd arch endoderm. A small number of taste buds also develop around the epiglottis that are innervated by the superior laryngeal nerve (CN X), but these buds are designed to help the newborn taste milk and are gradually lost with age.

D. Pharyngeal Endoderm

The thyroid gland, which produces the metabolic regulating hormone thyroxine, originates as a diverticulum in the floor of the stomodeal endoderm at the boundary of the first and second arches adjacent to the rupturing buccopharyngeal membrane (Figs. 15.4/16.4, 15.11/16.11). It is therefore NOT a pouch derivative since the pouches are on the lateral sides of the oral cavity. The thyroid primordium grows down in the midline from the floor of the pharynx and migrates caudally to a position ventral and inferior to the larynx (Fig. 15.18/16.18). This diverticulum forms a right and left lobe with an isthmus of thyroid tissue between. As it migrates inferiorly, the thyroid continues to retain a connection with the pharyngeal lumen known as the thyroglossal duct. Ordinarily, the thyroglossal duct closes off and involutes, leaving only a pit on the tongue (foramen cecum) to mark its point of origin. Retention of all or part of the duct, which is common, may give rise to cysts known as thyroglossal cysts (Figs. 15.19/16.19, 15.20/16.20). Enlargement of that a portion of the thyroglossal duct in contact with the thyroid results in the formation of a pyramidal lobe, which occurs in some individuals. Failure of the thyroid to migrate inferiorly can lead, in rare instances, to a lingual thyroid, where the gland develops at the base of the tongue. Lingual thyroid may produce difficulty in breathing and swallowing.

The first pharyngeal pouch endoderm enlarges adjacent to the first cleft to become the tubotympanic recess, which later will give rise to the tympanic cavity of the middle ear, the internal lining of the tympanic membrane, the auditory (Eustachian) tube, and the lining of the mastoid air cells (Figs. 15-10/16.10, 15.11/16.11). The second pouch endoderm evaginates from the walls of the pharynx on both sides to form the stroma, or supporting structure, of the palatine tonsils (see Table II). The lymphatic cells of the tonsils migrate in from mesodermal precursors later on.

The third pair of pouches gives rise to two diverticula, dorsal and ventral. From the dorsal part of the pouch arises parathyroid III, which becomes the inferior parathyroid gland on either side (Figs. 15.10/16.10, 15.11/16.11). Parathyroid glands, four in all, synthesize parathyroid hormone, which regulates, along with calcitonin secreted by the thyroid gland, calcium homeostasis. From the ventral part of the pouches the thymic primordia arise. They give rise to the thymus, the site for maturation of so-called thymic or T lymphocytes. During the 7th week, both parathyroid III and the developing thymus separate from the pouch and move caudally. After migrating part way down, parathyroid III will ordinarily detach from the thymus, move medially, and attach to the posterior surface of the thyroid. Occasionally parathyroid III or accessory parathyroid tissue formed from either the 3rd or 4th pharyngeal pouches will be carried into the mediastinum by the migrating thymus. The thymic primordia also may leave thymic tissue along the path as they descend. As was the case for the tonsils, the pouch endoderm contributes to the stroma of the gland while the lymphocytic precursors migrate in subsequently. The thymus continues to grow and develop until puberty, when it is quite large. During adulthood it shrinks in size. It is often difficult to recognize in older people because it is atrophied and fatty tissue accumulates in the region.

The 4th pharyngeal pouches form in the wall of the pharynx (Figs.15.10/16.10, 15.11/16.11). Parathyroid IV arises from the dorsal portion (see Table II). It separates from the pouch and migrates medially but ultimately becomes the superior parathyroid since it does not migrate as far caudally as parathyroid III. A small contribution of thymic tissue may also arise from the ventral portion of the 4th pouch.

TABLE II: PHARYNGEAL POUCH AND CLEFT DERIVATIVES
Number	Type	Structures
1	Cleft	External Auditory Meatus
	Pouch	Tubulotympanic Recess
2	Pouch	Palatine Tonsil (stroma)
3	Pouch	Inferior Parathyroids
		Thymus (stroma)
4	Pouch	Superior Parathyroids
		C cells of Thyroid (Ultimobranchial Bodies)

A branch of the ventral portion of the fourth pharyngeal pouch, which some embryologists consider a fifth pouch for evolutionary reasons, gives rise to the ultimobranchial body (Fig. 15.10/16.10). The ultimobranchial bodies lose their attachment to the pharynx and become incorporated into the thyroid gland as it moves inferiorly into the neck (Fig. 15.11/16.11). The ultimobranchial body on each side disperses in the thyroid and give rise to the calcitonin-secreting parafollicular or C cells (Table II). Experimental evidence indicates, however, that the C cells are actually derived from neural crest cells that migrate into the ultimobranchial body.

In addition to these structures, the endoderm of the oral cavity (but not the pouches) is the origin of the epithelium of the sublingual and submandibular salivary glands. As noted above, the third major salivary gland, the parotid, is derived from ectoderm of the oral cavity. Salivary glandular connective tissues and capsules are derived from mesoderm.

II. Abnormalities Associated with Arch Development

1. First arch Syndromes (Fig. 15.16/16.16):

Treacher Collins syndrome (also called mandibulofacial dysostosis) manifests autosomal dominant inheritance due to a defective gene encoding a nucleolar protein. It is characterized by a midline deficit in the face with underdevelopment of the zygomatic bones and down-slanting palpedral fissures, a hypoplastic mandible with small chin, and other first arch defects. There is often conductive hearing loss and deformed external ears, and occasionally cleft palate.

Pierre Robin sequence is a group of about 60 abnormalities involving the first and, in some cases, the second arches. All of them have a characteristic bilateral cleft palate, malpositioned tongue and apparent arrested facial development with hypoplasia of the mandible. Depending on the severity, the defects can include external ear deformities and hearing loss as well. The cause appears to be deficits in the mandibular and possibly maxillary processes. Failure of sufficient neural crest cells to migrate into the region can lead to arrested development. The tongue remains in a posterior position, filling the oral cavity and preventing the palatal shelves from realigning horizontally and fusing. This accounts for much of the observed deformations. The ears can remain lowset, and the ear bones can also be maldeveloped. Mice with disrupted activin signaling pathways have a version of Pierre Robin syndrome. Activin is a growth factor of the TGFfl family.

2. Lateral facial cleft and hemifacial microsomia involve deficiencies in the formation of the cheek (Fig. 15.16D/16.16D). Lateral cleft leaves a large mouth (macrostomia) on one or both sides. Hemifacial microsomia (small cheek) is a more severe version affecting posterior structures and usually having a lateral cleft as well. 1/3 of the time it is on both sides of mouth. Hemifacial microsomia can affect only the proximal portion of the cheek or extend all the way back to the ear, causing major deformities in the external ear in severe cases, such as in Goldenhar syndrome (Fig. 15.16D/16.16D). Lesions of a similar nature can be induced in experimental animals by causing vascular damage to nearby blood vessels. Thus, vascular accidents are a potential cause, leading to the death of head mesenchyme and neural crest cells. The cell deficit prevents proper growth of the maxillary and mandibular processes in the region where the two processes merge to form the cheek.

3. Di George sequence (Fig. 15.16C/16.16C). This group of abnormalities (referred to as CATCH), which includes parathyroid hypoplasia and thymic insufficiency, can be accounted for by defects in neural crest cell proliferation and/or migration. It is often associated with persistent truncus arteriosus as well, since neural crest cells normally migrate into the aortic arch and form the truncoconal septa. Neural crest ablation studies in animals can induce many of the same effects. Generally the third and fourth arches and nearby pouches are affected, but often the syndrome extends to the first arch as well causing deformations in the neck and a facial dysmorphism known as fish mouth deformity. In humans, a deletion in chromosome 22 has been linked to a large percentage of cases. Alcohol ingestion is also known to cause DiGeorge as well, presumably as a result of toxicity to neural crest cells. One of the major structures affected is the thymus gland, whose stroma has a major contribution from third pharygeal pouch. Failure of the gland to develop properly prevents lymphocytic infiltration and differentiation of T lymphocytes, resulting in immunocompromised children.

III. DEVELOPMENT OF THE FACE

The development of the face is dependent on the development of the nearby forebrain and the prechordal plate mesoderm. The prechordal plate, which originated from axial mesoderm migrating through the primitive node, acts as the organizer of face development. Growth factors, including sonic hedgehog (shh) secreted by the prechordal plate (Fig. 19.33/17.33), induce forebrain (prosencephalon) development and the eventual development of the right and left lobes (a.k.a., cerebral hemispheres, lateral ventricles). The forebrain, in turn, sends signals (also including shh) back to the mesoderm to induce the growth of a prominence in the midline, the frontonasal prominence, which overhangs the cranial end of the oral cavity and develops at the end of the sixth week (Fig. 15.21/16.21). The homeobox-containing transcription factor msx-1 is also important for face development and is expressed in the mesenchyme at the tips of the face primordia. Mice with altered msx genes have severe facial abnormalities. In addition, retinoic acid (RA) is a secreted molecule related structurally to vitamin A that is heavily involved in the development of the lower part of the face and first arch structures. RA binds to specific receptors within cells that regulate the transcription of a number of genes, including HOX genes. Mice with altered RA signaling pathways, both those with increased sensitivity and those with diminished response, have striking facial abnormalities. RA derivatives are used to treat acne (Accutane, Retin A). However, Accutane, if taken during pregnancy, increases the incidence of abnormalities in the head and neck region of the fetus, presumably by premature activation of RA signaling pathways (see Abnormalities below).

Paired depressions, or nasal placodes, which appear on either side in the ectoderm of the frontonasal prominence, are induced by the adjacent forebrain. As these ectodermal placodes invaginate to form nasal pits, the tissue surrounding them enlarges into a horseshoe-shaped protrusion, which on the medial side is called the medial nasal process and on the lateral side, the lateral nasal process (Fig. 15.22/16.22). The lateral nasal process is separated from the maxillary process (the cranial portion of the first branchial arch) by a furrow that reaches the inner aspect of the developing eye, the nasolacrimal groove (naso-optic furrow). The oral cavity is bounded inferiorly by the mandible, which has formed by the merging of the right and left mandibular processes of the first pharyngeal arch as cells migrate into the midline. The maxillary processes also expand, and as they do they crowd the medial nasal processes toward the midline (Fig. 15.23/16.32). The medial nasal processes merge with one another to form the intermaxillary segment, which will ultimately become the philtrum of the upper lip (Table III). As this occurs, the frontal prominence in the midline forms the bridge of the nose, and the medial nasal processes fuse laterally with the maxillary processes to complete the formation of the upper lip. Later, the lateral nasal processes fuse with the maxillary processes, obliterating the nasolacrimal groove.

A. Palate

Internally, the nasal pits grow and approach the primitive oral cavity (stomodeum). As they do so, the tissue in the midline separating the pits becomes the nasal septum, an extension of the frontonasal process (Fig. 15.30/16.32). Soon, a thin oronasal membrane is all that separates the pits from the oral cavity. This membrane then ruptures and primitive choanae, or openings, now connect the oral and nasal cavities. The ectodermal placode tissue differentiates into the olfactory epithelium, which is the sensory organ for smell. It has the unusual characteristic that the sensory cell neurons in the mucosa itself send their axons into the nearby olfactory lobe of the brain, whereas normally axons extend from nerve cells of the brain into peripheral tissues (Fig. 19.29/17.29). In addition, these olfactory neurons can be regenerated and are replaced on a regular basis whereas most neurons are seldom if ever replaced.

At the end of the second month, a partition forms to separate the primitive nasal cavities from the oral cavity. The anterior aspect of this partition is derived from the intermaxillary segment, the product of the merged medial nasal processes (Fig. 15.24/16.24). In addition, the intermaxillary segment extends posteriorly into the oral cavity. This extension is called the primary palate. Most of the palatine partition, however, is derived from the growth of shelf-like processes called palatine shelves (lateral palatine processes), which form the secondary palate (Fig. 15.25/16.25). These processes extend from the maxillary processes as neural crest cells migrate in. Initially the shelves grow inferiorly on either side of the developing tongue. However, as the lingual swellings develop, the tongue is flattened and displaced anteriorly allowing the palatine shelves to assume a horizontal orientation within the oral cavity (Fig. 15.26/16.26). As the secondary palate is formed, it fuses with the primary palate just as the medial nasal processes are fusing with the maxillary processes (Fig. 15.27/16.27). In addition, the nasal septum grows inferiorly toward it. The nasal septum and the two palatine shelves unite in the midline to form separate right and left nasal chambers, the oral cavity, and the definitive choanae, now narrow posterior openings on either side connecting the oral and nasal cavities (Fig. 15.30/16.32). Bone forms from neural crest cells of the palate except in the posterior segment where the soft palate and uvula will develop.

To recapitulate, the secondary palate is formed as the palatal shelves fuse in the midline with the nasal septum. The secondary palate also fuses with the primary palate, which is derived from the merged medial nasal processes.

Paranasal air sinuses are air-filled extensions of the nasal cavities within the nearby facial bones. Maxillary sinuses (in the maxillae) form as ectodermal diverticula from the wall of the nasal cavity during late fetal life but remain very small at birth (~4 mm). The frontal, and sphenoid sinuses are not present at birth. They develop from outgrowths of the nasal cavities during childhood.

B. Developmental Biology of Palate and Face Formation

Three separate processes contribute to the formation of the palate and the face. Firstly, the migration of sufficient neural crest cells into the face, arches, and palatal shelves is crucial. Cleft palate and other facial abnormalities can be induced in experimental animals by ablation of neural crest cells during their migration into the first arch. Secondly, programmed cell death, or apoptosis, occurs at the edges of the palatal shelves just before fusion. Failure of cells to undergo apoptosis in a timely fashion is known to affect palate formation, and drugs that interfere with this process can prevent fusion. Finally, on a molecular level, growth factors such as TGFfl also play a role in fusion of the palatal shelves. Mice whose TGF-b3 gene is knocked out have cleft palates. In addition, genetic disruption of other growth factor genes can mimic some of the human abnormalities. Knockout of the endothelin-1 gene in mice, for example, leads to malformation of first arch structures, with severe dysgnathia (jaw maldevelopment) and mandibular hypoplasia. The role of growth factors such as activins, retinoic acid, and sonic hedgehog will be discussed in the sections on abnormalities of the face and arches.

IV. Cleft Lip and Palate

1. Cleft lip and cleft palate: By our definition, cleft lip can involve just the lip itself or can extend through the anterior/alveolar portion of the palate (primary palate) to the incisive foramen. Cleft palate means cleft secondary palate, sometimes involving only the uvula. Cleft lip can be accompanied by a cleft secondary palate and vice versa, but the two abnormalities are considered independent. Cleft lip and cleft palate are thought to be caused by failure of neural crest cell migration and proliferation.

In cleft lip, the medial nasal processes fail to fuse with the maxillary processes, oftentimes accompanied by the failure of the primary and secondary palates to fuse (Figs. 15.28/16.28, 15.29/16.30). It can be unilateral or bilateral and is more common in males for unknown reasons.

In cleft palate, the palatine shelves fail to fuse with each other, leading to a cleft secondary palate (Figs. 15.28/16.28, 15.29/16.30). If the nasal septum fuses properly with one of the shelves, then the lesion can be unilateral. Otherwise it is bilateral. Sometimes only the posterior region is affected, and there can be, for example, only a cleft uvula. Cleft palate can be accompanied by cleft lip if the palatal shelves also fail to fuse with the primary palate. Cleft palate is more common in females. The general explanation for this is that the palate forms more slowly in females and is completed one week later (week 9), making the process more susceptible to teratogens and poor migration of neural crest cells.

Cleft lip and palate have what is called multifactorial inheritance. This means that genetic and environmental factors figure in their rate of occurrence. Siblings and twins have a significantly higher risk of having either cleft lip or cleft palate, as do children from parents who have the same congenital defect, however, the increased incidence (2 to 7 percent in different studies) is relatively small. It is important to recognize that if a parent or sibling has isolated cleft lip, there is an increased risk that a subsequent offspring will also have cleft lip but no increased risk for cleft palate and vice versa. Trisomy 13 infants have cleft lip, holoprosencephaly and other facial abnormalities, reinforcing the notion of a genetic component. Equally significant is the evidence that teratogens such as Retin A (Accutane, a retinoic acid derivative) and the anticonvulsant dilantin (phenylhydantoin) cause increased incidence of facial abnormalities including cleft lip. These drugs are widely prescribed to treat acne and epilepsy, respectively.

2. Median cleft of upper lip (Fig. 15.29/16.30). In this case, the two medial nasal processes fail to merge properly. This is very rare and is often accompanied by mental retardation suggesting that there is a defect in forebrain development as well.

3. Median cleft of lower lip: Failure of merger of the mandibular arches on either side. Also very rare.

4. Oblique facial cleft (Fig. 15.29/16.30). The nasolacrimal duct remains patent due to failure of the lateral nasal process to fuse with the maxillary process. If it occurs together with a cleft lip, which is rare, the cleft can extend from the lip to the caruncle of the eye.

V. DEVELOPMENT OF THE CRANIAL VAULT

As mentioned above, the bones and cartilages of the arches are derived from neural crest cells either by direct ossification (intramembranous bone formation) or by ossification of pre-existing cartilage models (endochondral bone formation, see chapter on Limb Development). While a detailed description of these processes will be part of the histology course, at this stage we will define intramembranous bone formation as direct ossification of head mesenchyme. That is, cells differentiate into osteoblasts (bone forming cells) and transform the mesenchyme into bone directly. Membranous bones are evolutionarily related to the dermal bones of fish. Endochondral bone formation involves the initial differentiation of mesenchyme into cartilage with the shape of the bone that will replace it (Fig. 8.15/9.16). Osteoblasts differentiating from the margins of the cartilage then migrate into it and replace the chondrocytes (cartilage forming cells). Thus the bones of the middle ear (malleus, incus, and stapes), for example, are endochondral and form from the first arch cartilages and the hyoid bone is derived from second and third arch cartilages.

The bones of the head and neck have three separate origins that reflect our evolution from lower animals (Fig. 8.3/9.3). Occipital structures forming the base of the skull and occipital bones are mainly from occipital sclerotome and form by endochondral mechanisms. Somitic sclerotome is the origin of the entire encasement of the brain in fish, but only the base of the skull forms from somitic sclerotome in humans. The remainder of the cranial vault comes from so-called head mesenchyme, primarily mesoderm and not neural crest, and is intramembranous.

Cranial sutures (fontanelles) are the regions between bones of the cranial vault that fuse late in gestation or in some cases after birth to allow for adequate brain development (Fig. 8.4/9.4). Their development is controlled in part by growth factors of the fibroblast growth factor (FGF) family. Mutations in FGF receptors that affect FGF signal transduction pathways can adversely affect the development of the sutures (see below).

VI. Abnormalities of the Face and Head

1. Craniosynostosis refers to a family of abnormalities characterized by abnormal shape of the head and premature closure of the cranial sutures (Fig. 8.9/9.9). The most familiar of these syndromes are Pfeifferπs, Crouzonπs, and Apertπs. Each syndrome differentially affects the different sutures, and the heads of the children have a characteristic maorphology in each case. Most of these syndromes are associated with skeletal abnormalities in other parts of the body as well. Syndactyly, or malformation of fingers and toes, is found in Apertπs syndrome while certain cervical vertebrae are abnormal in Crouzonπs, for example. Recent molecular genetic analyses have led to the identification of mutations in receptors for FGF in the craniosynostosis syndromes. There are four FGF receptors. Mutations in specific regions of two of them (FGFR2 and FGFR2) are responsible for most of the observed cranial suture anomalies (see Sadler, Table 8.1/9.1). Interestingly, these mutations activate the FGF signaling pathways and are effective when only one allele is mutated. FGF receptors are ligand-regulated tyrosine kinases. In the mutant forms, the kinase activity is activated even in the absence of FGF. This leads to more rapid differentiation of the calvarial cells in the sutures and premature fusion of cranial bones. FGF receptor mutations, albeit in a different FGF receptor (FGFR3), also cause achondroplasia, the principal form of dwarfism. In these individuals, the epiphyseal plates, the areas at the ends of long bones of the body where growth occurs, differentiate and stop growing prematurely. While craniosynostosis can have adverse effects on brain development, leading to mental retardation if not corrected, achondroplasia does not affect the skull in the same way and therefore these ≥little people≤ are of normal intelligence.

2. Holoprosencephaly literally means a single lobed forebrain. Normally the forebrain develops separate right and left lobes, but in individuals with holoprosencephaly there is only a single medial lobe or inadequately separated lobes (Fig. 19.35/17.34). The syndrome varies from mild, where there may be a single incisor tooth, to a severe, lethal form in which only a single eye develops along with a proboscis (instead of a nose). Gene mapping studies have linked specific mutations in the growth factor sonic hedgehog to some forms of holoprosencephaly in humans. Shh is produced by the prechordal plate mesoderm and is required for bilobular forebrain development. Inadequate production or synthesis of mutated, inactive forms leads to inadequate forebrain development which in turn impedes the appropriate formation of midfacial structures.

3. Fetal Alcohol Syndrome (FAS). Epidemiological studies have linked mild forms of holoprosencephaly and mental retardation to alcohol ingestion by pregnant women. Children are born with a characteristic facial appearance including an indistinct philtrum, a thin upper lip and a flattened midface (Fig. 6.8/7.8). As will be discussed in the Congenital Malformations chapter, the drinking of alcoholic beverages is discouraged throughout pregnancy because even moderate alcohol consumption may be harmful to the fetus. FAS is the leading cause of mental retardation.

VII. DEVELOPMENT OF THE EAR

The ear consists of three parts ≠ external, middle, and inner (see Figure 1 below). The external ear consists of the auricle and the external auditory meatus. The tympanic membrane (eardrum) separates the external ear from the middle ear, which includes the tympanic cavity and the three ear ossicles (malleus, incus, and stapes). The inner ear, which is connected to the middle ear by the oval window at the base of the stapes, includes the cochlea, which detects sound, the semicircular canals, which are used for balance, and the endolymphatic duct, which produces the fluid that bathes these structures. Sound vibrates the tympanic membrane, which then vibrates the ossicles. The stapes vibrates the oval window of the inner ear, which in turn transmits the wave to the endolymphatic fluid where it is detected by neurosensory hair cells in the organ of Corti of the cochlea.

The auricle is derived from the first and second arches surrounding the first pharyngeal cleft (Fig. 16.10/18.10). Six elevations called auricular hillocks, three from each arch, arise and grow to form the auricle itself. Variations and abnormalities are very common but most do not affect hearing to a great extent (Fig. 16.11/18.11). The first cleft gives rise to the external auditory meatus.

The tympanic membrane forms from the region of apposition of the first pharyngeal cleft ectoderm and pouch endoderm (Figs. 16.7/18.7, 16.9/18.9). Mesoderm does migrate into the region as well and forms connective tissue. The first pouch endoderm also gives rise to the tubotympanic recess. As the recess enlarges, its endoderm grows to surround the developing ossicle bones of the middle ear in what will be the tympanic cavity (Fig. 16.9/18.9).

The inner ear is derived from the otic placode (Fig. 16.2/18.2). The otic placode is induced by the nearby forebrain and invaginates to form the otic pit, which becomes the otic vesicle. The otic vesicle migrates inferiorly to a position near the forebrain and the middle ear ossicles. It then differentiates into the cochlea, semicircular canals and endolymphatic sac (Fig. 16.3/18.3). Soon it becomes encased in a cartilaginous otic capsule, derived from head mesenchyme, that later ossifies to form the bony labyrinth.

There are many causes of congenital deafness. While a few of these are associated with malformations in the middle ear bones and with first arch abnormalities such as Pierre Robin sequence, most affect only the sound detecting neurosensory apparatus of the inner ear and are of unknown causes. Recently, gene mapping of heritable forms has identified at the molecular level several of the defects that affect the ability of hair cells to detect sound waves. One mutation, in a myosin protein, is thought to yield defective actin-rich microvilli in the hair cells. The defective microvilli, extensions of the plasma membrane, cannot deform properly when sound waves approach and consequently do not transmit a signal to the underlying neurons of the vestibulocochlear nerve. Incidence of congenital deafness in this country has declined dramatically since the introduction of rubella vaccinations. Recall that rubella virus could cross the placenta and infect the fetus. It was a major cause of deafness and other facial abnormalities.

1. Schematic illustration of the three divisions of the ear: external ear (auricle and external auditory meatus), middle ear (tympanic cavity, ossicles, tympanic membrane, auditory tube), and inner ear (vestibule, semicircular canals, and cochlea (adapted from Ross, Reith, and Romrell, Histology, 2^nd Ed., Williams & Wilkins, Baltimore, 1989).

STUDY QUESTIONS

Which of major bones are considered to be from first arch mesenchyme (remember to include those not from the cartilage model)? What cartilages and bones are derived from each of the arches?

What muscles are derived from each of the arches? How are they innervated?

The face is derived from five facial swellings. What are they?

What is the prechordal plate? How does it form? What is its role in face formation?

How do midline structures like the nose and the nasal cavities form?

What structures contribute to the formation of the primary palate? The secondary palate? What are the lateral and medial nasal processes, the palatine shelves? What is the nasal septum and what does it do as the palate forms?

What is the nasolacrimal groove and what does it give rise to?

Large craniofacial anomalies can be classified as falling into three categories, those that affect the front of the face and skull primarily (sometimes including the forebrain), those that are primarily deficits are in pharyngeal arches one and/or two, and those that affect primarily the fusion or merging of the facial swellings. What do the following abnormalities represent and which categories would you put them in?

Holoprosencephaly, Apertπs syndrome (craniosynostosis), cleft lip, cleft palate, oblique facial cleft, lateral facial cleft (macrostomia), hemifacial microsomia, Pierre Robin sequence, Treacher-Collins syndrome

What is the difference between cleft lip and cleft palate? Do they ever occur together?

What are the genetic, gender and teratogenic factors that contribute to cleft lip and palate? What two commonly prescribed drugs are contraindicated during pregnancy because of increased teratogenic risk?

How does the nasal cavity form? What are choanae? What separates the nasal from the oral cavity, leaving only the definitive choanae? Where do the olfactory nerves originate from?

In general, what can you say about nasal sinuses of infants and their growth during childhood (you will not be asked to name them or describe the development of each).

The second arch endoderm is largely displaced by third arch endoderm and first arch ectoderm. How does this explain the general afferent innervation of the mucosa of the tongue? How do the taste buds fit in? What nerves innervate the taste buds in each part of the tongue mucosa? What are vallate (=circumvallate) papillae?

What gives rise to the tongue muscles and how are they innervated?

What is Rathke's pouch? What does it give rise to?

What structures are derived from each of the pharyngeal pouches? Which gives rise to the inferior parathyroids and which to the superior?

What is the physiological function of the thymus? The thyroid gland? The parathyroids?

What happens to the ultimobranchial body?

The lymphocytes of the thymus and palatine tonsils are not pharyngeal pouch derivatives. What parts of these structures do come from the pouches?

What happens to the second, third, and fourth pharyngeal grooves/clefts? When this process is not completed, what can result?

Where does the thyroid gland come from? What is the foramen cecum of the tongue?

A number of anomalies are the result of a complete or partial failure of structures derived from the pharyngeal pouches and endoderm to migrate properly. What are these? Are there any physiological or pathological consequences?

What is DiGeorge syndrome? Which arches are primarily affected? What structures? What relationship does persistent truncus arteriosus have to this syndrome?

How might growth factors such as retinoic acid, sonic hedgehog, fibroblast growth factor, and activin play a role in development of the face?

What are some of the known facial syndromes that are linked to defects in the FGF receptors?

What specific defects in FGF receptor signaling lead to craniosynostosis? Why are they dominant mutations? What is the relationship between these syndromes and achondroplasia (dwarfism)?

What is the otic placode and what does it give rise to?

What part of the ear does the first pharyngeal cleft give rise to? The first pouch? What is their relationship to the tympanic membrane (eardrum)?

What does the middle ear consist of?

From which pharyngeal cartilages do the incus, maleus, and stapes derive? Which of the three bones are attached to the eardrum and which to the oval window?

What are auricular hillocks? From which arches do they originate?

Why is congenital deafness less common now in this country than it was 40 years ago?