2 Prenatal development of the craniofacial region

During evolution, modern humans split from their last common ancestor with the apes around seven million years ago. Since that time, Homo sapiens have acquired features that make them unique as a species and separate them from other primates:

• Expansion of the cerebral hemispheres, with an accompanying increase in brain size and development of higher intelligence;

• Modification of the hand to allow apposition between thumb and fingers; and

• Descent of the larynx relative to the cranial base and the concomitant development of speech and language.

Many of these evolutionary changes are reflected in the form and function of the craniofacial region (Fig. 2.1). The essential morphology of the head and face is fundamental to being human and the development of this region is complicated, beginning early in life and requiring the coordinated growth and interaction of all primary cell populations within the embryo.

Figure 2.1 Comparison of a human (left) and orangutan (right) skull.

In the human skull, the cranium is enlarged and the forehead is upright above the eyes, reflecting the expansion of the frontal lobes. The human face is therefore positioned downwards and backwards relative to the forebrain.

Cranial neural crest

Sensory ganglia

Sympathetic ganglia (V, VII, IX, X)

Parasympathetic ganglia of neck

Pharyngeal arch cartilages

Dermal skull bones

Connective tissue of:

Vascular and dermal smooth muscles

Odontoblasts and pulp of the teeth

Corneal endothelium and stroma

Melanocytes and melanopores

Epidermal pigment cells

Carotid body type I cells

C cells of ultimopharyngeal body

Endoderm

Pharyngotympanic tube

II Tonsillar recess

III Thymus

Inferior parathyroid

IV Superior parathyroid

Ultimopharyngeal body

Mesoderm Head mesoderm

Craniofacial musculature

Paraxial mesoderm

Axial neck skeleton and basal occipital bone

There is also a further contribution from the neural crest (or fourth germ layer). Ectoderm and endoderm are derived from the epiblast and hypoblast, two fundamental cell populations within the bilaminar disc of the early embryo (Fig. 2.2). Mesoderm is produced during the process of gastrulation and the conversion of a bilaminar embryo into one with three primary tissue layers (Fig. 2.3).

Figure 2.2 Early development of the embryo.

(A) Following fertilization, the zygote undergoes a series of mitotic cell divisions to produce a sixteen cell morula. (B) The cells within the morula are quickly organised into outer and inner cell masses and the early embryo is known as a blastocyst. Cells of the outer cell mass form the trophoblast, which mediates implantation of the blastocyst into the uterine wall and contributes to the placenta. The inner cell mass forms the embryo itself. (C) During implantation, the inner cell mass differentiates into two layers; the epiblast (future ectoderm) and hypoblast (future endoderm), which together form the bilaminar disc of the early embryo.

Redrawn from Sandler TW (ed.) (2003), Langman’s Medical Embryology, 9th edn (Baltimore: Lippincott Williams and Wilkins).

Figure 2.3 Gastrulation.

During the third week of embryonic development, the third germ layer or mesoderm is formed by the process of gastrulation. Cells of the epiblast migrate through the primitive streak, a raised structure on the surface of the epiblast, and detach to position themselves between epiblast and hypoblast and form the mesodermal cell layer.

Redrawn from Sandler TW (ed.) (2003), Langman’s Medical Embryology, 9th edn (Baltimore: Lippincott Williams and Wilkins).

In mammals, neural crest cells arise during formation of the neural tube and migrate extensively throughout the embryo in four overlapping domains:

Cranial neural crest cells provide the building blocks for generating much of the skeleton and connective tissue of the head, in addition to the cranial ganglia and peripheral nerves innervating these skeletal structures. In the developing head, neural crest cells arising from the forebrain and midbrain regions populate the upper face, whilst those from the posterior midbrain and hindbrain migrate into the pharyngeal arch system (see Fig. 2.14). Cranial neural crest cells interact with ectodermal and mesodermal cell populations present within these regions, leading to the formation of craniofacial bones, cartilages and connective tissue (Fig. 2.4).

Figure 2.4 Derivatives of neural crest cells.

Redrawn from Larsen WJ (1998). Essentials of Human Embryology (Edinburgh: Churchill Livingstone).

The prechordal plate—a molecular organizer for the brain and face

A region of fundamental importance for early development of the brain and face is the prechordal plate, an area of thickened endoderm that lies beneath the future forebrain of the early embryo (Fig. 2.5).

Figure 2.5 Signalling from the prechordal plate is important for patterning ventral regions of the early forebrain and producing bilateral subdivision of the eyefield.

Sonic hedgehog (SHH) signalling from the prechordal plate plays an important role in this process.

The prechordal plate acts as a true head organizer, producing molecular signals that pattern the forebrain and subdivide the eyefield into two. In the absence of normal signalling from this region, holoprosencephaly (HPE) or cyclopia can occur. In the most severe cases, the lower forebrain does not form and the upper part remains as a single undivided vesicle, rather than developing into paired cerebral hemispheres (Muenke & Beachy, 2000). This lack of midline development within the central nervous system has important consequences for the face, with a spectrum of facial deformity reflecting the severity of the brain malformation. Failure of forebrain division leads to a lack of separation of the optic primordia and the formation of a single cyclopic eye, which becomes situated below a rudimentary midline facial proboscis or nose-like structure (see Fig. 13.21).

Early organization of the craniofacial region

Primary architecture of the craniofacial region is established during early development and is based upon segmentation (Fig. 2.6). At the future head end of the human embryo, the neural tube is segmented into three vesicles, which will form:

• Forebrain;

• Midbrain; and

• Hindbrain.

Figure 2.6 Segmentation of the craniofacial region in the early embryo.

The neural tube is segmented into forebrain, midbrain and hindbrain vesicles; the frontonasal process is situated over the developing forebrain and the segmented pharyngeal arches are situated ventrally.

On the lateral side of the head are the pharyngeal arches, which form:

• Neck;

• Pharynx; and

• Jaws.

In the upper region of the head is the frontonasal process, which surrounds the early forebrain and will form:

• The upper face.

In the lower region of the head is the first pharyngeal arch, which will form:

• The midface; and

• The lower face.

The pharyngeal arches

In humans there are six pharyngeal arches, which appear progressively during the fourth week of embryonic development. Each arch is covered externally by ectoderm and internally by endoderm, whilst a core of mesodermal tissue exists within. As development proceeds, this central core becomes infiltrated by cranial neural crest cells that migrate into the arches from their site of origin adjacent to the roof of the neural tube. The junction of each arch is in close proximity with its neighbour, producing a pharyngeal cleft of ectoderm externally and a pouch of endoderm internally (Fig. 2.7).

Figure 2.7 The pharyngeal arches.

Each arch is covered externally by ectoderm and internally by endoderm. Within each arch is a core of mesoderm, which becomes progressively infiltrated with migrating neural crest cells. In addition, a characteristic nerve, cartilage and artery are situated within each arch.

The pharyngeal arches give rise to a number of structures within the head and neck (Fig. 2.8):

• The first pharyngeal arch produces the jaws and dentition;

• The second pharyngeal arch forms the suprahyoid apparatus of the neck;

• The third pharyngeal arch contributes to the infrahyoid region;

• The fourth and sixth pharyngeal arches form the laryngeal structures; and

• The fifth pharyngeal arch is the exception, rapidly degenerating after formation and making no contribution towards any permanent structures in the human.

Figure 2.8 Skeletal derivatives of the pharyngeal arches.

The first arch gives rise to the upper and lower jaws, the dentition, the malleus and incus (middle ear ossicles) and sphenomandibular ligament.

The second arch gives rise to the styloid process, stylohyoid ligament, stapes (middle ear ossicle) and the lesser cornu and upper part of the body of the hyoid bone. The third arch gives rise to the greater cornu and lower part of the body of the hyoid bone. The fourth arch gives rise to the laryngeal cartilages. Those structures formed by the pharyngeal arch cartilages are indicated in colour.

Redrawn from Wendell-Smith CP, Williams RL, Treadgold S (1984), Basic Human Embryology, 3rd edn (London: Pitman Publishing).

The embryonic tissue components within each arch give rise to specific structures:

• Ectoderm produces the skin and sensory neurons of the lower face and neck;

• Endoderm forms the mucosal lining of the pharynx and associated endocrine organs (thyroid, parathyroid and thymus) of the neck;

• Cranial neural crest cells produce most of the skeletal and connective tissues within the head and neck; and

• Mesoderm produces the associated craniofacial musculature, cardiac outflow tract and endothelial cells of the pharyngeal arch arteries.

Within the pharyngeal arch system are also characteristic sets of bilaterally symmetrical arteries, nerves and cartilages. Those associated with the first two arches make contributions to the maxillary and stapedial arteries in the adult, whilst those within the lower pharyngeal arches are responsible for generating major arteries within the neck and cardiothoracic region. Neural crest cells that migrate into the third and fourth pharyngeal arches are known collectively as the cardiac neural crest, these cells making an important contribution to remodelling of the pharyngeal arch arteries and to the formation of a functional cardiac outflow tract and cardiothoracic vascular system. Any disruption within the embryonic pharyngeal region can have serious implications for normal development, which is exemplified by a group of related disorders known as the 22q11 deletion syndromes (Box 2.1).

Box 2.1 TBX1: a mediator of normal pharyngeal arch development

T-box genes encode a large group of transcription factors and TBX1 is thought to be a key player in the aetiology of the DiGeorge (DGS; OMIM 601362) and Velocardiofacial (VCFS; OMIM 192430) or 22q11 Deletion syndromes (22q11DS). These syndromes form part of a group of related human dysmorphic disorders that result from deletion or rearrangement of a large region of chromosome 22q11 (Baldini, 2005; Scambler, 2000). 22q11DS subjects are characterized by defects in the cardiac outflow tract and aortic arch, thymic and parathyroid aplasia or hypoplasia, and anomalies of the craniofacial region, which include micrognathia, cleft palate and a characteristic facial appearance. The principle clinical phenotypes present within these syndromes are thought to result from an absence of normal pharyngeal pouch signalling and localized disruption of neural crest migration within the pharyngeal arch system.

Three-dimensional facial scans of individuals with 22q11DS (left) and control subjects (right) aged between 2 weeks and 24 years. The 22q11DS face is only subtly different from the average, but careful examination reveals an exaggerated length of the nose, with a narrow nares and nasal base, but fullness above the tip. The eyes are upslanted and the ears cupped or unusual in shape (Hammond et al, 2005).

Reproduced with kind permission of Professor Peter Hammond, Institute of Child Health, University College London.

The candidacy of TBX1 for 22q11DS has been based upon its location within the deleted region of chromosome 22, its expression domain in endoderm and mesoderm of the developing pharyngeal arches and the finding that mice generated with a targeted deletion in Tbx1 exhibit a spectrum of phenotypic effects encompassing most of the common 22q11DS malformations (Lindsay et al, 2001). More recent genetic experiments in mice, designed to ablate the function of Tbx1 in different regions of the early pharynx at different developmental time points, have provided clues as to the role of this transcription factor during patterning and differentiation of the pharyngeal arch derivatives. In particular, Tbx1 activity influences the proliferation and expansion of endoderm lining the embryonic pharynx; a role that facilitates normal segmentation of the pharyngeal arches and the formation of structures derived from these regions.

Development of the face

Development of the face is a dynamic process, relying upon complex tissue interactions that are closely coordinated, both temporally and spatially (Fig. 2.9). Growth and development of this region is driven by neural crest migration and proliferation, which directs the formation and approximation of a series of swellings or processes. Ultimately, these processes fuse with each other to produce seamless regions of ectoderm and the characteristic features of a face. At the molecular level, a host of signalling molecules, transcription factors and extracellular matrix proteins control the cellular activities underlying these processes (Francis-West et al, 2003).