Early Patterning

The emergence of the mesoderm heralds the transition from the stage of bilaminar to trilaminar disc, or gastrulation. Induction of the mesoderm—the initiation, maintenance, and subsequent patterning of this layer—involves several key families of signaling factors. The Nodal family is involved in initiation, FGFs (fibroblast growth factors) and WNTs are involved in maintenance, and BMPs (bone morphogenetic proteins) are involved in patterning the mesoderm. Signaling pathways are activated when a key ligand binds specific membrane-bound protein receptors. This usually leads to the phosphorylation of a cytoplasmic factor, and this in turn leads to binding with other factor(s). These factors translocate to the nucleus where transcriptional activation of specific targets occurs.

In the case of Nodal and BMP pathways, ligand binding of a specific heterotetramer membrane-bound protein initiates the signaling, which is common to all members of the TGF-β family, the cytoplasmic mediators being SMAD factors (see the following section). The embryo appears to have gradients of Nodal activity along the dorsal-ventral axis, although the significance and role of these gradients in mesoderm induction are uncertain.

The WNT pathway has two main branches: one that is β-catenin–dependent (canonical) and the other independent of β-catenin. In the canonical pathway, Wnt ligand binds to a Frizzled/LRP heterodimer membrane-bound protein complex and the downstream intracellular signaling involves a G protein. The effect of this is to disrupt a large cytoplasmic protein complex that includes Axin, the adenomatous polyposis coli (APC; see p. 221) protein, and the glycogen synthase kinase-3β (GSK-3β) protein. This prevents the phosphorylation of β-catenin, but when β-catenin is not degraded, it accumulates and translocates to the nucleus where it activates the transcription of dorsal-specific regulatory genes. Binding of the ligand to the Fgf receptor results in dimerization of the receptor and transphosphorylation of the receptor’s cytoplasmic domain, with activation of Ras and other kinases, one of which enters the nucleus and activates target transcription factors. Mutated WNT10A in man results in a form of ectodermal dysplasia (odonto-onychodermal dysplasia) but apart from the possibility of WNT4 being implicated in a rare condition called Mayer-Rokitansky-Kuster syndrome, no other members of this gene family are yet implicated in human disease phenotypes.

The TGF-β Superfamily in Development and Disease

Thus far it recognized that there are 33 members of this cytokine family. Cytokines are a category of signaling molecules—polypeptide regulators—that enable cells to communicate. They differ from hormones in that they are not produced by discrete glands. These extracellular signaling polypeptides are transduced through a cascade to regulate gene expression within the cell nucleus. This is achieved through binding with cell surface receptors that, in a series of reactions, induces phosphorylation and activation of specific receptor kinases. This leads to the translocation of complexes into the nucleus, which execute transcriptional activation or repression of responsive target genes. The TGF-β family can be divided into two groups: (1) the BMPs and (2) the TGF-βs, activins, nodal, and myostatin, acting through various SMAD proteins. Ultimately, this superfamily is actively involved in a very broad range of cellular and developmental processes (Figure 6.2). This includes regulation of the cell cycle, cell migration, cell size, gastrulation and axis specification, and metabolic processes. In relation to health and disease, there are consequences for immunity, cancer, heart disease, diabetes, and Marfan syndrome (p. 300). Hyperactive signalling (overexpression) of BMP4 has been found in the rare bony condition fibrodysplasia ossificans progressiva, where disabling heterotopic bone deposition occurs, which is due to mutated ACVR1, encoding a BMP type 1 receptor. A mutated BMP receptor 2 has been shown to be a cause of familial primary pulmonary hypertension. BMP signalling is also involved in both dendritogenesis and axonal transport.

FIGURE 6.2 A summary of biological responses to TGF family signaling. The range of processes that come under the influence of this super family is very broad.

(Modified from Wharton K, Derynck R 2009 TGFβ family signaling: novel insights in development and disease. Development 136[22]:3693.)

Somatogenesis and the Axial Skeleton

The vertebrate axis is closely linked to the development of the primary body axis during gastrulation, and during this process the presomitic mesoderm (PSM), where somites arise, is laid down in higher vertebrates. Wnt and FGF signals play vital roles in the specification of the PSM. The somites form as blocks of tissue from the PSM in a rostro-caudal direction (Figure 6.3), each being laid down with a precise periodicity that, in the 1970s, gave rise to the concept of the ‘clock and wavefront’ model. Since then, molecular techniques have given substance to this concept, and the key pathway here is notch-delta signaling and the ‘oscillation clock’—a precise, temporally defined wave of cycling gene expression (c-hairy in the chick, lunatic fringe and hes genes in the mouse) that sweeps from the tail-bud region in a rostral direction and has a key role in the process leading to the defining of somite boundaries. Once again, not all of the components are fully understood, but the notch receptor and its ligands, delta-like-1, and delta-like-3, together with presenilin-1 and mesoderm posterior-2, work in concert to establish rostro-caudal polarity within the PSM such that somite blocks are formed. Human phenotypes from mutated genes in this pathway are now well known and include presenile dementia (presenilin-1), which is dominantly inherited, and spondylocostal dysostosis (delta-like-3, mesoderm posterior-2, lunatic fringe, and hairy enhancer of split-7), which is recessively inherited (Figure 6.4). Another component of the pathway is JAGGED1, which, when mutated, results in the dominantly inherited and very variable condition known as Alagille syndrome (arteriohepatic dysplasia) (Figure 6.5). Rarely, mutations in NOTCH2 have been shown to cause some cases of Alagille syndrome, usually with renal malformations.

FIGURE 6.3 Somatogenesis and Notch-Delta pathway. T-box genes have a role in PSM specification, whereas the segmentation clock depends on oscillation, or cycling, genes that are important in somite boundary formation where genes of the Notch-Delta pathway establish rostro-caudal polarity. HOX genes have a global function in establishing somite identity along the entire rostro-caudal axis.

(Adapted from Tickle C, ed 2003 Patterning in vertebrate development. Oxford: Oxford University Press.)

FIGURE 6.4 Disrupted development of the vertebrae in patient with spondylocostal dysostosis type 1 resulting from mutations in the delta-like-3 gene, part of the notch signaling pathway.

(Courtesy Dr. Meriel McEntagart, Kennedy-Galton Centre, London.)

FIGURE 6.5 A, Boy with Alagille syndrome and confirmed mutation in JAGGED1 who presented with congenital heart disease. B, The same boy a few years earlier with his parents. His mother has a pigmentary retinopathy and was positive for the same gene mutation.

The Sonic Hedgehog–Patched GLI Pathway

The Sonic hedgehog gene (SHH) is as well known for its quirky name as for its function. SHH induces cell proliferation in a tissue-specific distribution and is expressed in the notochord, the brain, and the zone of polarizing activity of developing limbs. After cleavage and modification by the addition of a cholesterol moiety, the SHH protein binds with its receptor, Patched (Ptch), a transmembrane protein. The normal action of Ptch is to inhibit another transmembrane protein called Smoothened (Smo), but when bound by Shh this inhibition is released and a signaling cascade within the cell is activated. The key intracellular targets are the GLI family of transcription factors (Figure 6.6).

FIGURE 6.6 The Sonic hedgehog (Shh)-Patched (Ptch)-Gli pathway and connection with disease. Different elements in the pathway act as activators (arrows) or inhibitors (bars). The Shh protein is initially cleaved to an active N-terminal form, which is then modified by the addition of cholesterol. The normal action of Ptch is to inhibit Smo, but when Ptch is bound by Shh this inhibition is removed and the downstream signaling proceeds. CREBBP, cAMP response element-binding binding protein.

Molecular defects in any part of this pathway lead to a number of apparently diverse malformation syndromes (see Figure 6.6). Mutations in, or deletions of, SHH (chromosome 7q36) cause holoprosencephaly (Figure 6.7), in which the primary defect is incomplete cleavage of the developing brain into separate hemispheres and ventricles. The most severe form of this malformation is cyclopia—the presence of a single central eye. (The complexity of early development can be appreciated by the fact that a dozen or so chromosomal regions have so far been implicated in the pathogenesis of holoprosencephaly [p. 257].) Mutations in PTCH (9q22) result in Gorlin syndrome (nevoid basal cell carcinoma syndrome; Figure 6.8), which comprises multiple basal cell carcinomas, odontogenic keratocysts, bifid ribs, calcification of the falx cerebri, and ovarian fibromata. Mutations in SMO (7q31) are found in some basal cell carcinomas and medulloblastomas. Mutations in GLI3 (7p13) cause Pallister-Hall and Grieg syndromes, which are distinct entities with more or less the same body systems affected. However, there are also links to other conditions, in particular the very variable Smith-Lemli-Opitz syndrome (SLOS), which may include holoprosencephaly as well as some characteristic facial features, genital anomalies and syndactyly. This condition is due to a defect in the final step of cholesterol biosynthesis, which in turn may disrupt the binding of SHH with its receptor Ptch. Some, or all, of the features of SLOS may therefore be due to loss of integrity in this pathway (p. 288). Furthermore, a cofactor for the Gli proteins, CREBBP (16p13) is mutated in Rubenstein-Taybi syndrome (Figure 6.9). Disturbance to different components of the SHH is also clearly implicated in many types of tumor formation.

FIGURE 6.7 Facial features in holoprosencephaly. The eyes are close together and there is a midline cleft lip because of a failure of normal prolabia development.

FIGURE 6.8 Gorlin (nevoid basal cell carcinoma) syndrome. A, This 6-year-old girl from a large family with Gorlin syndrome has macrocephaly and a cherubic appearance. B, Her affected sister developed a rapidly enlarging odontogenic keratocyst (arrows) in the mandible at the age of 9 years, displacing the roots of her teeth.

FIGURE 6.9 A baby with characteristic facial features (A) of Rubenstein-Taybi syndrome, angulated thumbs (B), and postaxial polydactyly of the feet (C). A young adult (D) with same condition, though more mildly affected.

Homeobox (HOX) Genes

In Drosophila a class of genes known as the homeotic genes has been shown to determine segment identity. Incorrect expression of these genes results in major structural abnormalities; the Antp gene, for example, which is normally expressed in the second thoracic segment, will transform the adult fly’s antennae into legs if incorrectly expressed in the head. Homeotic genes contain a conserved 180-base pair (bp) sequence known as the homeobox, which is believed to be characteristic of genes involved in spatial pattern control and development. This encodes a 60-amino-acid domain that binds to DNA in Hox-response enhancers. Proteins from homeobox-containing (or HOX) genes are therefore important transcription factors that activate and repress batteries of downstream genes. At least 35 downstream targets are known. The Hox proteins regulate other ‘executive’ genes that encode transcription factors or morphogen signals, as well as operating at many other levels, on genes that mediate cell adhesion, cell division rates, cell death, and cell movement. They specify cell fate and help to establish the embryonic pattern along the primary (rostro-caudal) axis as well as the secondary (genital and limb bud) axis. They therefore play a major part in the development of the central nervous system, axial skeleton and limbs, the gastrointestinal and urogenital tracts, and external genitalia.

Drosophila has eight Hox genes arranged in a single cluster, but in humans, as in most vertebrates, there are four homeobox gene clusters containing a total of 39 HOX genes (Figure 6.10). Each cluster contains a series of closely linked genes. In vertebrates such as mice, it has been shown that these genes are expressed in segmental units in the hindbrain and in global patterning of the somites formed from axial presomitic mesoderm. In each HOX cluster, there is a direct linear correlation between the position of the gene and its temporal and spatial expression. These observations indicate that these genes play a crucial role in early morphogenesis. Thus, in the developing limb bud (p. 99) HOXA9 is expressed both anterior to, and before, HOX10, and so on.

FIGURE 6.10 A, Drosophila has eight Hox genes in a single cluster whereas there are 39 HOX genes in humans, arranged in four clusters located on chromosomes 7p, 17q, 12q, and 2q for the A, B, C, and D clusters, respectively. B, Expression patterns of Hox and HOX genes along the rostro-caudal axis in invertebrates and vertebrates, respectively. In vertebrates the clusters are paralogous and appear to compensate for one another.

(Redrawn from Veraksa A, Del Campo M, McGinnis W: Developmental patterning genes and their conserved functions: from model organisms to humans. Mol Genet Metab 2000;69:85–100, with permission.)

Mutations in HOXA13 cause a rare condition known as the hand-foot-genital syndrome. This shows autosomal dominant inheritance and is characterized by shortening of the first and fifth digits, with hypospadias in males and bicornuate uterus in females. Experiments with mouse Hoxa13 mutants have shown that expression of another gene, EphA7, is severely reduced. Therefore, if this gene is not activated by Hoxa13, there is failure to form the normal chondrogenic condensations in the distal limb primordial. Mutations in HOXD13 result in an equally rare limb developmental abnormality known as synpolydactyly. This also shows autosomal dominant inheritance and is characterized by insertion of an additional digit between the third and fourth fingers and the fourth and fifth toes, which are webbed (Figure 6.11). The phenotype in homozygotes is more severe and reported mutations take the form of an increase in the number of residues in a polyalanine tract. This triplet-repeat expansion probably alters the structure and function of the protein, thereby constituting a gain-of-function mutation (p. 26). Mutated HOXA1 has been found in the rare, recessively inherited Bosley-Saleh-Alorainy syndrome, consisting of central nervous system abnormalities, deafness, and cardiac and laryngotracheal anomalies. A mutation in HOXD10 was found in isolated congenital vertical talus in a large family demonstrating autosomal dominant inheritance, and duplications of HOXD have recently been found in mesomelic limb abnomality syndromes.

FIGURE 6.11 Clinical (A) and (B) radiographic views of the hands in synpolydactyly.

Given that there are 39 HOX genes in mammals, it is surprising that so few syndromes or malformations have been attributed to HOX gene mutations. One possible explanation is that most HOX mutations are so devastating that the embryo cannot survive. Alternatively, the high degree of homology between HOX genes in the different clusters could lead to functional redundancy so that one HOX gene could compensate for a loss-of-function mutation in another. In this context HOX genes are said to be paralogous because family members from different clusters, such as HOXA13 and HOXD13, are more similar than adjacent genes in the same cluster.

Several other developmental genes also contain a homeobox-like domain. These include MSX2 and EMX2. Mutations in MSX2 can cause craniosynostosis—premature fusion of the cranial sutures. Mutations in EMX2 have been implicated in some cases of schizencephaly, in which there is a large full-thickness cleft in one or both cerebral hemispheres.

Paired-Box (PAX) Genes

The paired-box is a highly conserved DNA sequence that encodes a 130-amino-acid DNA-binding transcription regulator domain. Nine PAX genes have been identified in mice and humans. In mice these have been shown to play important roles in the developing nervous system and vertebral column. In humans, loss-of-function mutations in five PAX genes have been identified in association with developmental abnormalities (Table 6.2). Waardenburg syndrome type 1 is caused by mutations in PAX3. It shows autosomal dominant inheritance and is characterized by sensorineural hearing loss, areas of depigmentation in hair and skin, abnormal patterns of pigmentation in the iris, and widely spaced inner canthi (Figure 6.12). Waardenburg syndrome shows genetic heterogeneity; the more common type 2 form, in which the inner canthi are not widely separated, is sometimes caused by mutations in the human microphthalmia (MITF) gene on chromosome 3.

Table 6.2 Developmental Abnormalities Associated with PAX Gene Mutations

FIGURE 6.12 Iris heterochromia and marked dystopia canthorum in an infant with Waardenburg syndrome type 1, caused by a mutation in PAX3.

The importance of expression of the PAX gene family in eye development is illustrated by the effects of mutations in PAX2 and PAX6. Mutations in PAX2 cause the renal-coloboma syndrome, in which renal malformations occur in association with structural defects in various parts of the eye, including the retina and optic nerve. Mutations in PAX6 lead to absence of the iris, which is known as aniridia (Figure 6.13). This is a key feature of the WAGR syndrome (p. 282), which results from a contiguous gene deletion involving the PAX6 locus on chromosome 11.

FIGURE 6.13 An eye showing absence of the iris (aniridia). The cornea shows abnormal vascularization.

(Courtesy Mr. R. Gregson, Queen’s Medical Centre, Nottingham, UK.)

SRY-Type HMG Box (SOX) Genes

SRY is the Y-linked gene that plays a major role in male sex determination (p. 102). A family of genes known as the SOX genes shows homology with SRY by sharing a 79-amino-acid domain known as the HMG (high-mobility group) box. This HMG domain activates transcription by bending DNA in such a way that other regulatory factors can bind with the promoter regions of genes that encode for important structural proteins. These SOX genes are thus transcription regulators and are expressed in specific tissues during embryogenesis. For example, SOX1, SOX2, and SOX3 are expressed in the developing mouse nervous system.

In humans it has been shown that loss-of-function mutations in SOX9 on chromosome 17 cause campomelic dysplasia. This very rare disorder is characterized by bowing of the long bones, sex reversal in chromosomal males, and very poor long-term survival. In-situ hybridization studies in mice have shown that SOX9 is expressed in the developing embryo in skeletal primordial tissue, where it regulates type II collagen expression, as well as in the genital ridges and early gonads. SOX9 is now thought to be one of several genes that are expressed downstream of SRY in the process of male sex determination (p. 102). Mutations in SOX10 on chromosome 22 cause a rare form of Waardenburg syndrome in which affected individuals have a high incidence of Hirschsprung disease. Mutations in SOX2 (3q26) have been shown to cause anophthalmia or microphthalmia, but also a wider syndrome of esophageal atresia and genital hypoplasia in males: the anophthalmia-esophageal-genital syndrome.

T-Box (TBX) Genes

The T gene in mice plays an important role in specification of the paraxial mesoderm and notochord differentiation. Heterozygotes for loss-of-function mutations have a short tail and malformed sacral vertebrae. This gene, which is also known as Brachyury, encodes a transcription factor that contains both activator and repressor domains. It shows homology with a series of genes through the shared possession of the T domain, which is also referred to as the T-box. These T-box or TBX genes are dispersed throughout the human genome, with some family members existing in small clusters. One of these clusters on chromosome 12 contains TBX3 and TBX5. Loss-of-function mutations in TBX3 cause the ulnar-mammary syndrome in which ulnar ray developmental abnormalities in the upper limbs are associated with hypoplasia of the mammary glands. Loss-of-function mutations in TBX5 cause the Holt-Oram syndrome. This autosomal dominant disorder is characterized by congenital heart abnormalities, most notably atrial septal defects, and upper limb radial ray reduction defects that can vary from mild hypoplasia (sometimes duplication) of the thumbs to almost complete absence of the forearms.

Zinc Finger Genes

The term zinc finger refers to a finger-like loop projection consisting of a series of four amino acids that form a complex with a zinc ion. Genes that contain a zinc finger motif act as transcription factors through binding of the zinc finger to DNA. Consequently they are good candidates for single-gene developmental disorders (Table 6.3).

Table 6.3 Developmental Abnormalities Associated with Genes Containing a Zinc Finger Motif

For example, a zinc finger motif–containing gene known as GLI3 on chromosome 7 (as mentioned, a component of the SHH pathway) has been implicated as the cause of two developmental disorders. Large deletions or translocations involving GLI3 cause Greig cephalopolysyndactyly, which is characterized by head, hand and foot abnormalities such as polydactyly and syndactyly (Figure 6.14, A). In contrast, frameshift mutations in GLI3 have been reported in the Pallister-Hall syndrome (Figure 6.14, B), in which the key features are polydactyly, hypothalamic hamartomata and imperforate anus.

FIGURE 6.14 A, The feet of a child with Greig cephalopolysyndactyly. Note that they show both preaxial polydactyly (extra digits) and syndactyly (fused digits). B, The left hand of a woman with Pallister-Hall syndrome and a proven mutation in GLI3. Note the postaxial polydactyly and the surgical scar, where an extra digit arising from between the normal metacarpal rays (mesoaxial polydactyly) was removed.

Mutations in another zinc finger motif-containing gene known as WT1 on chromosome 11 can cause both Wilms’ tumour and a rare developmental disorder, the Denys-Drash syndrome, in which the external genitalia are ambiguous and there is progressive renal failure as a result of nephritis. Mutations in two other zinc finger motif–containing genes, ZIC2 and ZIC3, have been shown to cause holoprosencephaly and laterality defects, respectively. Just as polarity is a key concept in development, so too is laterality, with implications for the establishment of a normal left-right body axis. In very early development, integrity of many of the same gene families previously mentioned—Nodal, SHH, and Notch—is essential to the establishment of this axis. Clinically, situs solitus is the term given to normal left-right asymmetry and situs inversus to reversal of the normal arrangement. Up to 25% of individuals with situs inversus have an autosomal recessive condition—Kartagener syndrome, or ciliary dyskinesia. Other terms used are isomerism sequence, heterotaxy, asplenia/polyasplenia, and Ivemark syndrome. Laterality defects are characterized by abnormal positioning of unpaired organs such as the heart, liver, and spleen, and more than 20 genes are now implicated from studies in vertebrates, with a number identified in humans by the study of affected families, with all of the main patterns of inheritance represented.

Signal Transduction (’Signaling’) Genes

Signal transduction is the process whereby extracellular growth factors regulate cell division and differentiation by a complex pathway of genetically determined intermediate steps. Mutations in many of the genes involved in signal transduction play a role in causing cancer (p. 213). In some cases they can also cause developmental abnormalities.

FGF Receptors

FGFs play key roles in embryogenesis, including cell division, migration, and differentiation. The transduction of extracellular FGF signals is mediated by a family of four transmembrane tyrosine kinase receptors. These are the fibroblast growth factor receptors (FGFRs), each of which contains three main components: an extracellular region with three immunoglobulin-like domains, a transmembrane segment, and two intracellular tyrosine kinase domains (Figure 6.15).

FIGURE 6.15 Structure of the fibroblast growth factor receptor (FGFR). Arrows indicate the location of mutations in the craniosynostosis syndromes and achondroplasia group of skeletal dysplasias. Ig, immunoglobulin-like domain; TM, transmembrane domain; TK, tyrosine kinase domain; P, Pfeiffer syndrome; A, Apert syndrome; C, Crouzon syndrome; J, Jackson-Weiss syndrome; TD, thanatophoric dysplasia; ACH, achondroplasia; HCH, hypochondroplasia.

Mutations in the genes that code for FGFRs have been identified in two groups of developmental disorders (Table 6.4). These are the craniosynostosis syndromes and the achondroplasia family of skeletal dysplasias. The craniosynostosis syndromes, of which Apert syndrome (Figure 6.16) is the best known, are characterized by premature fusion of the cranial sutures, often in association with hand and foot abnormalities such as syndactyly (fusion of the digits). Apert syndrome is caused by a mutation in one of the adjacent FGFR2 residues in the peptides that link the second and third immunoglobulin loops (see Figure 6.15). In contrast, mutations in the third immunoglobulin loop can cause either Crouzon syndrome, in which the limbs are normal, or Pfeiffer syndrome, in which the thumbs and big toes are broad. Achondroplasia is the most commonly encountered form of genetic short stature (Figure 6.17). The limbs show proximal (‘rhizomelic’) shortening and the head is enlarged with frontal bossing. Intelligence and life expectancy are entirely normal. Achondroplasia is almost always caused by a mutation in, or close to, the transmembrane domain of FGFR3. The common transmembrane domain mutation leads to the replacement of a glycine amino-acid residue by an arginine—an amino acid that is never normally found in cell membranes. This in turn appears to enhance dimerization of the protein that catalyzes downstream signaling. Hypochondroplasia, a milder form of skeletal dysplasia with similar trunk and limb changes but normal head shape and size, is caused by mutations in the proximal tyrosine kinase domain (intracellular) of FGFR3. Finally, thanatophoric dysplasia, a much more severe and invariably lethal form of skeletal dysplasia, is caused by mutations in either the peptides linking the second and third immunoglobulin domains (extracellular) of FGFR3, or the distal FGFR3 tyrosine kinase domain.

Table 6.4 Developmental Disorders Caused by Mutations in Fibroblast Growth Factor Receptors

Gene	Chromosome	Syndrome
Craniosynostosis syndromes
FGFR1	8p11	Pfeiffer
FGFR2	10q25	Apert Crouzon Jackson-Weiss Pfeiffer
FGFR3	4p16	Crouzon (with acanthosis nigricans)
Skeletal dysplasias
FGFR3	4p16	Achondroplasia Hypochondroplasia Thanatophoric dysplasia

FIGURE 6.16 Views of the face (A), hand (B), and foot (C) of a child with Apert syndrome.

FIGURE 6.17 A young child with achondroplasia.

The mechanism by which these mutations cause skeletal shortening is not understood at present. The mutations cannot have loss-of-function effects as children with the Wolf-Hirschhorn syndrome (pp. 280–281), which is due to chromosome microdeletions that include FGFR3, do not show similar skeletal abnormalities. Instead, the mutations probably involve a gain of function mediated by increased ligand binding or receptor activation.

The Pharyngeal Arches

The pharyngeal (or branchial) arches correspond to the gill system of lower vertebrates and appear in the fourth and fifth weeks of development. Five (segmented) pharyngeal arches in humans arise lateral to the structures of the head (Figure 6.18) and each comprises cells from the three germ layers and the neural crest. The lining of the pharynx, thyroid, and parathyroids arises from the endoderm, and the outer epidermal layer arises from the ectoderm. The musculature arises from the mesoderm, and bony structures from neural crest cells. Separating the arches are the pharyngeal clefts externally and the pharyngeal pouches internally; these have important destinies. Numbered from the rostral end, the first arch forms the jaw and muscles of mastication, the first cleft is destined to be the external auditory meatus, and the first pouch the middle ear apparatus. The second arch forms the hyoid apparatus and muscles of facial expression, whereas the third pouch develops into the thymus, and the third and fourth pouches become the parathyroids. The arteries within the arches have important destinies too and, after remodeling, give rise to the aortic and pulmonary arterial systems. Some of the syndromes, malformations, inheritance patterns and genetic mechanisms associated with the first and second pharyngeal arches are listed in Table 6.5.

FIGURE 6.18 The pharyngeal (or branchial) apparatus. The lateral view (A) shows the five pharyngeal arches close to the embryonic head and the cross-section (B) shows the basic arrangement from which many head and neck structures, as well as the heart, develop. Humans and mice do not have arch no. 5.

(Redrawn from Graham A, Smith A 2001 Patterning the pharyngeal arches. Bioessays 23:54–61, with permission of Wiley-Liss Inc., a subsidiary of John Wiley & Sons, Inc.)

However, the most well known, and probably most common, condition due to disturbed development of pharyngeal structures—the third and fourth pouches—is DiGeorge syndrome (DGS), also known as velocardiofacial syndrome (VCFS), and well described even earlier by Sedláčková of Prague in 1955. This is described in more detail in Chapter 18 (pp. 282–283); it results from a 3Mb submicroscopic chromosome deletion of band 22q11 with the loss of some 30 genes. Studies in mice (the equivalent, or syntenic, region is on mouse chromosome 16) suggest that the most significant gene loss is that of Tbx1, strongly expressed throughout the pharyngeal apparatus. Heterozygous Tbx1 knock-out mice show hypoplastic or absent fourth pharyngeal arch arteries, suggesting that TBX1 in humans is the key. Indeed, mutations in this gene have now been found in some congenital heart abnormalities and it is possible that TBX1 is the key gene for other elements of the phenotype. However, there are still unanswered questions in the whole DGS/VCFS/Sedláčková story.

Initiation and Specification

Limb bud formation is thought to be initiated at around 28 days by a member of the FGF family as illustrated by the development of an extra limb if FGF1, FGF2, or FGF4 is applied to the side of a developing chick embryo. During normal limb initiation FGF8 transcripts have been identified in mesenchyme near the initiation site. FGF8 expression is probably controlled by HOX genes, which determine limb type (forelimb or hindlimb) and number.

Tissue Differentiation and Growth

Once limb formation has been initiated, a localized area of thickened ectoderm at the limb tip, known as the apical ectodermal ridge (AER), produces growth signals such as FGF4 and FGF8, which maintain further growth and establish the proximo-distal axis (Figure 6.21). Expression of the gene TP63 is crucial for sustaining the AER and, when this gene is mutated, split hand-foot (ectrodactyly) malformations result, often together with oral clefting and other anomalies—ectrodactyly-ectodermal dysplasia-clefting syndrome. Signals from another localized area on the posterior margin of the developing bud, known as the zone of polarizing activity, determine the anteroposterior axis. One of these signals is SHH (pp. 86–88), which acts in concert with other FGF genes, GLI3, and another gene family, which produces BMPs. Another morphogen, retinoic acid, is believed to play a major role at this stage in determining development at the anterior margin of the limb bud.

FIGURE 6.21 Simplified representation of vertebrate limb development.

Subsequent development involves the activation of genes from the HOXA and HOXD clusters in the undifferentiated proliferating mesenchymal cells beneath the AER. This area is known as the progress zone. Cells in different regions express different combinations of HOX genes that determine local cell proliferation, adhesion, and differentiation. Downstream targets of the HOX gene clusters remain to be identified. Other genes that clearly have a key role are those of the T-box family, already discussed, and SALL4, which is mutated in Okihiro syndrome (radial ray defects with abnormal eye movements resulting from congenital palsy affecting the sixth cranial nerve).

FGFs continue to be important during the later stages of limb development. In this context it becomes easy to understand why limb abnormalities are a feature of disorders such as Apert syndrome (see Figure 6.16), in which mutations have been identified in the extracellular domains of FGFR2.

Gain-of-Function versus Loss-of-Function Mutations

Mention has already been made of the causal role of the RET proto-oncogene in familial Hirschsprung disease, as well as in both inherited and sporadic thyroid cancer (p. 93). The protein product encoded by RET consists of three main domains: an extracellular domain that binds to a glial cell line–derived neurotrophil factor, a transmembrane domain, and an intracellular tyrosine kinase domain that activates signal transduction (Figure 6.22). Mutations causing loss of function result in Hirschsprung disease. These include whole gene deletions, small intragenic deletions, nonsense mutations, and splicing mutations leading to synthesis of a truncated protein.

FIGURE 6.22 The RET proto-oncogene. The most common mutation sites in the different clinical entities associated with RET are indicated. Numbers refer to amino-acid residues. SP, signal peptide; ECD, extracellular domain; TMD, transmembrane domain; TKD, tyrosine kinase domain; MEN, multiple endocrine adenomatosis; FMTC, familial medullary thyroid carcinoma. The arrow above PTC (papillary thyroid carcinoma) indicates the somatic rearrangement site for the formation of new hybrid forms of RET.

(Adapted from Pasini B, Ceccherini I, Romeo G 1996 RET mutations in human disease. Trends Genet 12:138–144.)

In contrast, mutations causing a gain-of-function effect result in either type 2A or type 2B multiple endocrine neoplasia (MEN). These disorders are characterized by a high incidence of medullary thyroid carcinoma and pheochromocytoma. The activating mutations that cause MEN-2A are clustered in five cysteine residues in the extracellular domain. MEN-2B, which differs from MEN-2A, in that affected individuals are tall and thin, is usually caused by a unique mutation in a methionine residue in the tyrosine kinase domain.

Somatic Rearrangements

Activation of the RET proto-oncogene can occur by a different mechanism whereby the genomic region encoding the intracellular domain is juxtaposed to one of several activating genes that are normally preferentially expressed in the thyroid gland. The newly formed hybrid RET gene produces a novel protein whose activity is not ligand dependent. These somatic rearrangements are found in a high proportion of papillary thyroid carcinomas, which show a particularly high incidence in children who were exposed to radiation following the Chernobyl accident in 1986.

PAX3 provides another example of a developmental gene that can cause cancer if it is fused to new DNA sequences. A specific translocation between chromosomes 2 and 13 that results in a new chimeric transcript leads to the development in children of a rare lung tumor called alveolar rhabdomyosarcoma.

Partial Hydatidiform Mole

Chromosome analysis of tissue from partial moles reveals the presence of 69 chromosomes—i.e., triploidy (p. 276). Using DNA polymorphisms, it has been shown that 46 of these chromosomes are always derived from the father, with the remaining 23 being maternal in origin. This doubling of the normal haploid paternal contribution of 23 chromosomes can be due to either fertilization by two sperm, which is known as dispermy, or to duplication of a haploid sperm chromosome set by a process known as endoreduplication.

In these pregnancies the fetus rarely if ever survives to term. Triploid conceptions survive to term only when the additional chromosome complement is maternally derived, in which cases partial hydatidiform changes do not occur. Even in these situations, it is extremely uncommon for a triploid infant to survive for more than a few hours or days after birth.

Complete Hydatidiform Mole

Complete moles have only 46 chromosomes, but these are exclusively paternal in origin. A complete mole is caused by fertilization of an empty ovum either by two sperm or by a single sperm that undergoes endoreduplication. The opposite situation of an egg undergoing development without being fertilized by a sperm, a process known as parthenogenesis, occurs in lower animals such as arthropods but has been reported in a human on only one occasion, this being in the form of chimeric fusion with another cell line that had a normal male-derived complement.

The main importance of complete moles lies in their potential to undergo malignant change into invasive choriocarcinoma. This can usually be treated successfully by chemotherapy, but if untreated the outcome can be fatal. Malignant change is seen only very rarely with partial moles.

Different Parental Expression in Trophoblast and Embryoblast

Studies in mice have shown that when all nuclear genes in a zygote are derived from the father, the embryo fails to develop, whereas trophoblast development proceeds relatively unimpaired. In contrast, if all of the nuclear genes are maternal in origin, the embryo develops normally, but extra-embryonic development is poor. The observations outlined previously on partial and complete moles indicate that a comparable situation exists in humans, with paternally derived genes being essential for trophoblast development and maternally derived genes being necessary for early embryonic development. These phenomena are relevant to the concept of epigenetics (see the following section, p. 103) and genomic imprinting (p. 121).

The Testis-Determining Factor—SRY

In 1990 it was shown that the testis-determining factor or gene is located on the short arm of the Y chromosome close to the pseudoautosomal region (p. 118). This gene is now referred to as being located in the sex-determining region of the Y chromosome (SRY). It consists of a single exon that encodes a protein of 204 amino acids that include a 79-amino-acid HMG box (p. 92), indicating that it is likely to be a transcription regulator.

Evidence that the SRY gene is the primary factor that determines maleness comes from several observations:

From a biological viewpoint (i.e., the maintenance of the species), it would clearly be impossible for the SRY gene to be involved in crossing over with the X chromosome during meiosis I. Hence SRY has to lie outside the pseudoautosomal region. However, there has to be pairing of X and Y chromosomes, as otherwise they would segregate together into the same gamete during, on average, 50% of meioses. Nature’s compromise has been to ensure that only a small portion of the X and Y chromosomes are homologous, and therefore pair during meiosis I. Unfortunately, the close proximity of SRY to the pseudoautosomal region means that, occasionally, it can get caught up in a recombination event. This almost certainly accounts for the majority of XX males, in whom molecular and fluorescent in-situ hybridization studies show evidence of Y-chromosome sequences at the distal end of one X-chromosome short arm (see Figure 18.22, p. 288).

Expression of SRY triggers off a series of events that involves other genes such as SOX9 (17q24), leading to the medulla of the undifferentiated gonad developing into a testis, in which the Leydig cells begin to produce testosterone (Figure 6.23). This leads to stimulation of the Wolffian ducts, which form the male internal genitalia, and also to masculinization of the external genitalia. This latter step is mediated by dihydrotestosterone, which is produced from testosterone by the action of 5α-reductase (pp. 174, 287). The Sertoli cells in the testes produce a hormone known as müllerian inhibitory factor, which causes the müllerian duct system to regress. In campomelic dysplasia, resulting from mutated SOX9, ambiguous genitalia is common in cases with a 46,XY karyotype. Ambiguous genitalia, or sex reversal, is also frequent in cases of deletion 9p24.3 syndrome. This is probably due to haploinsufficiency for the DMRT1 gene, a transcriptional regulator expressed in Sertoli cells, spermatogonia, and spermatocytes.

FIGURE 6.23 Summary of the main events involved in sex determination. SRY, Sex determining region of the Y chromosome; MIF, müllerian inhibitory factor.

In the absence of normal SRY expression, the cortex of the undifferentiated gonad develops into an ovary. The müllerian duct forms the internal genitalia. The external genitalia fail to fuse and grow as in the male, and instead evolve into normal female external genitalia. This normal process of female development is sometimes referred to rather chauvinistically as the ‘default’ pathway. Without the stimulating effects of testosterone, the Wolffian duct system regresses.

Normally sexual differentiation is complete by 12 to 14 weeks’ gestation, although the testes do not migrate into the scrotum until late pregnancy. Abnormalities of sexual differentiation are uncommon but they are important causes of infertility and sexual ambiguity. They are considered further in Chapter 18.

X-Chromosome Inactivation

As techniques were developed for studying chromosomes, it was noted that in female mice one of the X chromosomes often differed from all other chromosomes in the extent to which it was condensed. In 1961 Dr Mary Lyon proposed that this heteropyknotic X chromosome was inactivated, citing as evidence her observations on the mosaic pattern of skin coloration seen in mice known to be heterozygous for X-linked genes that influence coat color. Subsequent events have confirmed the validity of Lyon’s hypothesis, and in recognition of her foresight the process of X-chromosome inactivation (XCI) is often referred to as lyonization.

The process of XCI occurs early in development at around 15 to 16 days’ gestation, when the embryo consists of approximately 5000 cells. Normally either of the two X chromosomes can be inactivated in any particular cell. Thereafter the same X chromosome is inactivated in all daughter cells (Figure 6.24). This differs from the case in marsupials, in which the paternally derived X chromosome is consistently inactivated.

FIGURE 6.24 X-chromosome inactivation during development. The maternally and paternally derived X chromosomes are represented as X^m and X^p, respectively.

The inactive X chromosome exists in a condensed form during interphase when it appears as a darkly staining mass of chromatin known as the sex chromatin, or Barr body. During mitosis the inactive X chromosome is late replicating. Laboratory techniques have been developed for distinguishing which of the X chromosomes is late replicating in each cell. This can be useful for confirming that one of the X chromosomes is structurally abnormal, as usually an abnormal X chromosome will be preferentially inactivated; or, more correctly, only those hematopoietic stem cells in which the normal X chromosome is active will have survived. Apparent non-random inactivation also occurs when one of the X chromosomes is involved in a translocation with an autosome (p. 116).

The epigenetic process of XCI is achieved by differential methylation (a form of imprinting; see p. 121) and is initiated by a gene, XIST (‘X inactivation specific transcript’), which maps within the X-inactivation centre at Xq13.3. XIST is expressed only from the inactive X chromosome and produces RNA that spreads an inactivation methylation signal up and down the X chromosome on which it is located. This differential methylation of the X chromosomes has been utilized in carrier detection studies for X-linked immunodeficiency diseases (e.g., Wiskott-Aldrich syndrome) using methylation-sensitive restriction enzymes (p. 204). Not all of the X chromosome is inactivated. Genes in the pseudoautosomal region at the tip of the short arm remain active, as do other loci elsewhere on the short and long arms, such as XIST. There are more genes that escape XCI in Xp compared with Xq. This probably explains why more severe phenotypic effects are seen in women with small Xp chromosome deletions compared with those in women with small deletions in Xq. If all loci on the X chromosome were inactivated, then all women would have the clinical features of Turner syndrome and the presence of more than one X chromosome in a male (e.g., 47,XXY) or two in a female (e.g., 47,XXX) would have no phenotypic effects. There are, in fact, quite characteristic clinical features in these disorders (p. 278).

XCI provides a satisfactory explanation for several observations, described below.

Mosaicism

Mice that are heterozygous for X-linked genes affecting coat color show mosaicism with alternating patches of different color rather than a homogeneous pattern. This is consistent with patches of skin being clonal in origin in that they are derived from a single stem cell in which one or other of the X chromosomes is expressed, but not both. Thus, each patch reflects which of the X chromosomes was active in the original stem cell. Similar effects are seen in tissues of clonal origin in women who are heterozygous for X-linked mutations such as ocular albinism (Figure 6.25).

FIGURE 6.25 The fundus of a carrier of X-linked ocular albinism showing a mosaic pattern of retinal pigmentation.

(Courtesy Mr. S.J. Charles, The Royal Eye Hospital, Manchester, UK.)

Other evidence confirming that X-inactivation leads to mosaicism in females comes from studies of the expression of the enzyme glucose-6-phosphate dehydrogenase (p. 187) in clones of cultured fibroblasts from women heterozygous for variants of this gene. Each clone is derived from a single cell and expresses one of the variants, but never both. The clonal origin of tumors can be confirmed in women who are heterozygous for such variants by demonstration of the expression of only one of the variants in the tumor.

Problems of Carrier Detection

Carrier detection for X-linked recessive disorders based only on examination of clinical features or on indirect assay of gene function is notoriously difficult and unreliable. Cells in which the X chromosome with the normal gene is active can have a selective advantage, or they can correct the defect in closely adjacent cells in which the X chromosome with the mutant gene is active. For example, only a proportion of carriers of Duchenne muscular dystrophy (DMD) show evidence of muscle damage as indicated by measurement of creatine kinase in serum (p. 314). Similarly, distorted ratios of very long chain fatty acids are seen in many, but not all, carriers of X-linked adrenoleukodystrophy.

Fortunately, the development of molecular methods for carrier detection in X-linked disorders can bypass these problems by use of PCR primers that distinguish the products of methylated and unmethylated DNA—if there has been selection against the cell line in which the mutant-bearing X chromosome is active (Figure 6.26).

FIGURE 6.26 A, Normal X-chromosome inactivation resulting in survival of roughly equal numbers of cells with the paternally (P) and maternally (derived X chromosome active). B, In this situation the maternally derived X chromosome has a mutation (*) that results in selection against the cells in which it is active. Thus surviving cells show preferential expression of the paternally derived X chromosome.

Monozygotic Twins

MZ twinning occurs in about 1 in 300 births in all populations that have been studied. MZ twins originate from a single egg that has been fertilized by a single sperm. A very early division, occurring in the zygote before separation of the cells that make the chorion, results in dichorionic twins. Division during the blastocyst stage from days 3 to 7 results in monochorionic diamniotic twins. Division after the first week leads to monoamniotic twins. However, the reason(s) why MZ twinning occurs at all in humans is not clear. As an event, the incidence is increased two- to five-fold in babies born by in-vitro fertilization. There are rare cases of familial MZ twinning that can be transmitted by the father or mother, suggesting a single-gene defect that predisposes to the phenomenon.

We tend to think of MZ twins as being genetically identical, and basically this is of course true. However, they can be discordant for structural birth defects that may be linked to the twinning process itself—especially those anomalies affecting midline structures. There is probably a two- to three-fold increased risk of congenital anomalies in MZ twins (i.e., 5% to 10% of MZ twins overall). Discordance for single-gene traits or chromosome abnormalities may occur because of a post-zygotic somatic mutation or non-disjunction, respectively. One example of the latter is the rare occurrence of MZ twins of different sex: one 46,XY and the other 45,X. Curiously, MZ female twins can show quite striking discrepancy in X-chromosome inactivation. There are several reports of female MZ twin pairs of which only one is affected by an X-linked recessive condition such as DMD or hemophilia A. In these rare examples, both twins have the mutation and both show non-random X-inactivation, but in opposite directions.

MZ twins have traditionally provided ideal research material for the study of genetic versus environmental influences. In a study of 40 pairs of MZ twins, geneticists measured levels of two epigenetic modifications, DNA methylation, and histone acetylation. Two-thirds of the twin pairs had essentially identical profiles, but significant differences were observed in the remaining third. These differences were broadly correlated with the age of the twins, with the amount of time spent apart and the differences in their medical histories, suggesting a cumulative effect on DNA modification over time. It also suggests a possible causal link between epigenetic modification and susceptibility to disease.

Very late division occurring more than 14 days after conception can result in conjoined twins. This occurs in about 1 in 100,000 pregnancies, or approximately 1 in 400 MZ twin births. Conjoined twins are sometimes referred to as Siamese, in memory of Chang and Eng, who were born in 1811 in Thailand, then known as Siam. They were joined at the upper abdomen and made a successful living as celebrities at traveling shows in the United States, where they settled and married. They both managed to have large numbers of children despite remaining conjoined until they died within a few hours of each other at age 61.

The sex ratio for conjoined twins is markedly distorted, with about 75% being female. The later the twinning event, the more distorted the sex ratio in favor of females, and X-inactivation studies suggest that MZ twinning occurs around the time of X-inactivation, a phenomenon limited to female zygotes, of course.

Dizygotic Twins

DZ twins result from the fertilization of two ova by two sperm and are no more closely related genetically than brothers and sisters, as they share, on average, 50% of the same genes from each parent. Hence they are sometimes referred to as fraternal twins. DZ twins are dichorionic and diamniotic, although they can have a single fused placenta if implantation occurs at closely adjacent sites. The incidence varies from approximately 1 in 100 deliveries in black Caribbean populations to 1 in 500 deliveries in Asia and Japan. In western European whites, the incidence is approximately 1 in 120 deliveries and has been observed to fall with both urbanization and starvation, but increases in relation to the amount of seasonal light (e.g., in northern Scandinavia during the summer). Factors that convey an increased risk for DZ twinning are: increased maternal age, a positive family history (from a familial increase in follicle-stimulating hormone levels), and the use of ovulation-inducing drugs such as clomiphene.

Determination of Zygosity

Zygosity used to be established by study of the placenta and membranes and also by analysis of polymorphic systems such as the blood groups, the human leukocyte antigens and other biochemical markers. Now it is determined most reliably by the use of highly polymorphic molecular (DNA) markers (pp. 69–70) and single nucleotide polymorphisms (SNPs).