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