Thompson & Thompson Genetics in Medicine, 8th Edition

CHAPTER 14. Developmental Genetics and Birth Defects

Knowledge of the principles and concepts of developmental genetics, including the mechanisms and pathways responsible for normal human development in utero, is essential for the practitioner who seeks to develop a rational approach to the diagnostic evaluation of a patient with a birth defect. With an accurate diagnostic assessment in hand, the practitioner can make predictions about prognosis, recommend management options, and provide an accurate recurrence risk for the parents and other relatives of the affected child. In this chapter, we provide an overview of the branch of medicine concerned with birth defects and review basic mechanisms of embryological development, with examples of some of these mechanisms and pathways in detail. We present examples of birth defects that result from abnormalities in these processes. And finally, we show how an appreciation of developmental biology is essential for understanding prenatal diagnosis (see Chapter 17) and stem cell therapy as applied to regenerative medicine (see Chapter 13).

Developmental Biology in Medicine

The Public Health Impact of Birth Defects

The medical impact of birth defects is considerable. In 2013, the most recent year for which final statistics are available, the infant mortality rate in the United States was 5.96 infant deaths per 1000 live births; more than 20% of infant deaths were attributed to birth defects, that is, abnormalities (often referred to as anomalies) that are present at birth in the development of organs or other structures. Another 20% of infant deaths may be attributed to complications of prematurity, which can be considered a failure of maintenance of the maternal-fetal developmental environment. Therefore nearly half of the deaths of infants are caused by derangements of normal development. In addition to mortality, congenital anomalies are a major cause of long-term morbidity, intellectual disability, and other dysfunctions that limit the productivity of affected individuals.

Developmental anomalies certainly have a major impact on public health. Genetic counseling and prenatal diagnosis, with the option to continue or to terminate a pregnancy, are important for helping individuals faced with a risk for serious birth defects in their offspring improve their chances of having healthy children (see Chapter 17). Physicians and other health care professionals must be careful, however, not to limit the public health goal of reducing disease solely to preventing the birth of children with anomalies through voluntary pregnancy termination. Primary prevention of birth defects can be accomplished. For example, recommendations to supplement prenatal folic acid intake, which markedly reduces the incidence of neural tube defects, and public health campaigns that focus on preventing teratogenic effects of alcohol during pregnancy, are successful public health approaches to the prevention of birth defects that do not depend on prenatal diagnosis and elective abortion. In the future, it is hoped that our continued understanding of the developmental processes and pathways that regulate them will lead to therapies that may improve the morbidity and mortality associated with birth defects.

Dysmorphology and Mechanisms That Cause Birth Defects

Dysmorphology is the study of congenital birth defects that alter the shape or form of one or more parts of the body of a newborn child. Researchers attempt to understand the contribution of both abnormal genes and nongenetic, environmental influences to birth defects, as well as how those genes participate in conserved developmental pathways. The objectives of the medical geneticist who sees a child with birth defects are:

• to diagnose a child with a birth defect,

• to suggest further diagnostic evaluations,

• to give prognostic information about the range of outcomes that could be expected,

• to develop a plan to manage the expected complications,

• to provide the family with an understanding of the causation of the malformation, and

• to give recurrence risks to the parents and other relatives.

To accomplish these diverse and demanding objectives, the clinician must acquire and organize data from the patient, the family history, and published clinical and basic science literature. Medical geneticists work closely with specialists in pediatric surgery, neurology, rehabilitation medicine, and the allied health professions to provide ongoing care for children with serious birth defects.

Malformations, Deformations, and Disruptions

Medical geneticists divide birth defects into three major categories: malformations, deformations, and disruptions. We will illustrate the difference between these three categories with examples of three distinct birth defects, all involving the limbs.

Malformations result from intrinsic abnormalities in one or more genetic programs operating in development. An example of a malformation is the extra fingers in the disorder known as Greig cephalopolysyndactyly (Fig. 14-1). This syndrome, discussed later in the chapter, results from loss-of-function mutations in a gene for a transcription factor, GLI3, which is one component of a complex network of transcription factors and signaling molecules that interact to cause the distal end of the human upper limb bud to develop into a hand with five digits. Because malformations arise from intrinsic defects in genes that specify a series of developmental steps or programs, and because such programs are often used more than once in different parts of the embryo or fetus at different stages of development, a malformation in one part of the body is often but not always associated with malformations elsewhere as well.


FIGURE 14-1 Polydactyly and syndactyly malformations. A, Insertional polydactyly. This patient has heptadactyly with insertion of a digit in the central ray of the hand and a supernumerary postaxial digit. This malformation is typically associated with metacarpal fusion of the third and fourth digits. Insertional polydactyly is common in patients with Pallister-Hall syndrome. B, Postaxial polydactyly with severe cutaneous syndactyly of digits two through five. This type of malformation is seen in patients with Greig cephalopolysyndactyly syndrome. SeeSources & Acknowledgments.

In contrast to malformations, deformations are caused by extrinsic factors impinging physically on the fetus during development. They are especially common during the second trimester of development when the fetus is constrained within the amniotic sac and uterus. For example, contractions of the joints of the extremities, known as arthrogryposes, in combination with deformation of the developing skull, occasionally accompany constraint of the fetus due to twin or triplet gestations or prolonged leakage of amniotic fluid (Fig. 14-2). Most deformations apparent at birth either resolve spontaneously or can be treated by external fixation devices to reverse the effects of the instigating cause.


FIGURE 14-2 Deformation known as congenital arthrogryposis seen with a condition referred to as amyoplasia. There are multiple, symmetrical joint contractures due to abnormal muscle development caused by severe fetal constraint in a pregnancy complicated by oligohydramnios. Intelligence is generally normal, and orthopedic rehabilitation is often successful. SeeSources & Acknowledgments.

Disruptions, the third category of birth defect, result from destruction of irreplaceable normal fetal tissue. Disruptions are more difficult to treat than deformations because they involve actual loss of normal tissue. Disruptions may be the result of vascular insufficiency, trauma, or teratogens. One example is amnion disruption, the partial amputation of a fetal limb associated with strands of amniotic tissue. Amnion disruption is often recognized clinically by the presence of partial and irregular digit amputations in conjunction with constriction rings (Fig. 14-3).


FIGURE 14-3 Disruption of limb development associated with amniotic bands. This 26-week fetus shows nearly complete disruption of the thumb with only a nubbin remaining. The third and fifth fingers have constriction rings of the middle and distal phalanges, respectively. The fourth digit is amputated distally with a small fragment of amnion attached to the tip. SeeSources & Acknowledgments.

The pathophysiological concepts of malformations, deformations, and disruptions are useful clinical guides to the recognition, diagnosis, and treatment of birth defects, but they sometimes overlap. For example, vascular malformations may lead to disruption of distal structures, and urogenital malformations that cause oligohydramnios can cause fetal deformations. Thus a given constellation of birth defects in an individual may represent combinations of malformations, deformations, and disruptions.

Genetic, Genomic, and Environmental Causes of Malformations

Malformations have many causes (Fig. 14-4). Chromosome imbalance accounts for approximately 25%, of which autosomal trisomies for chromosomes 21, 18, and 13 (see Chapter 6) are some of the most common. The recent clinical application of genome-wide arrays in comparative genomic hybridization (CGH or array-CGH; see Chapter 5) has revealed small, de novo submicroscopic deletions and/or duplications, also known as copy number variants (CNVs), in as many as 10% of individuals with birth defects. An additional 20% are caused by mutations in single genes. Some malformations, such as achondroplasia or Waardenburg syndrome, are inherited as autosomal dominant traits. Many heterozygotes with birth defects, however, represent new mutations that are so severe that they are genetic lethals and are therefore often found to be isolated cases within families (see Chapter 7). Other malformation syndromes are inherited in an autosomal or X-linked recessive pattern, such as the Smith-Lemli-Opitz syndrome or the Lowe syndrome, respectively.


FIGURE 14-4 The relative contribution of single-gene defects, chromosome abnormalities, copy number variants, multifactorial traits, and teratogens to birth defects.

Another approximately 40% of major birth defects have no identifiable cause but recur in families of affected children with a greater frequency than would be expected on the basis of the population frequency and are thus considered to be multifactorial diseases (see Chapter 8). This category includes well-recognized birth defects such as cleft lip with or without cleft palate, and congenital heart defects.

The remaining 5% of birth defects are thought to result from exposure to certain environmental agents—drugs, infections, alcohol, chemicals, or radiation—or from maternal metabolic disorders such as poorly controlled maternal diabetes mellitus or maternal phenylketonuria (see Chapter 12). Such agents are called teratogens (derived, inelegantly, from the Greek word for monster plus -gen, meaning cause) because of their ability to cause malformations (discussed later in this chapter).

Pleiotropy: Syndromes and Sequences

A birth defect resulting from a single underlying causative agent may result in abnormalities of more than one organ system in different parts of the embryo or in multiple structures that arise at different times during intrauterine life, a phenomenon referred to as pleiotropy. The agent responsible for the malformation could be either a mutant gene or a teratogen. Pleiotropic birth defects come about in two different ways, depending on the mechanism by which the causative agent produces its effect. When the causative agent causes multiple abnormalities in parallel, the collection of abnormalities is referred to as a syndrome. If, however, a mutant gene or teratogen affects only a single organ system at one point in time, and it is the perturbation of that organ system that causes the rest of the constellation of pleiotropic defects to occur as secondary effects, the malformation is referred to as a sequence.

Pleiotropic Syndromes.

The autosomal dominant branchio-oto-renal dysplasia syndrome exemplifies a pleiotropic syndrome. It has long been recognized that patients with branchial arch anomalies affecting development of the ear and neck structures are at high risk for having renal anomalies. The branchio-oto-renal dysplasia syndrome, for example, consists of abnormal cochlear and external ear development, cysts and fistulas in the neck, renal dysplasia, and renal collecting duct malformations. The mechanism of this association is that a conserved set of genes and proteins are used by mammals to form both the ear and the kidney. The syndrome is caused by mutations in one such gene, EYA1, which encodes a protein phosphatase that functions in both ear and kidney development. Similarly, the Rubinstein-Taybi syndrome, caused by loss of function in a transcriptional coactivator, results in abnormalities in the transcription of many genes that depend on this coactivator being present in a transcription complex for normal expression (Fig. 14-5).


FIGURE 14-5 Physical characteristics of patients with Rubinstein-Taybi syndrome, a highly variable and pleiotropic syndrome of developmental delay, distinctive facial appearance, broad thumbs and large toes, and congenital heart defects. The syndrome is caused by loss-of-function mutations in one of two different but closely related transcriptional coactivators, CBP or EP300. A, Distinctive facial features. B, Appearance of hands and feet. SeeSources & Acknowledgments.


In contrast, an example of a sequence is the U-shaped cleft palate and small mandible referred to as the Robin sequence (Fig. 14-6). This sequence comes about because a restriction of mandibular growth before the ninth week of gestation causes the tongue to lie more posteriorly than is normal, interfering with normal closure of the palatal shelves, thereby causing a cleft palate. The Robin sequence can be an isolated birth defect of unknown cause or can be due to extrinsic impingement on the developing mandible by a twin in utero. This phenotype can also be one of several features of a condition known as Stickler syndrome, in which mutations in the gene encoding a subunit of type II collagen result in an abnormally small mandible as well as other defects in stature, joints, and eyes. The Robin sequence in the Stickler syndrome is a sequence because the mutant collagen gene itself is not responsible for the failure of palatal closure; the cleft palate is secondary to the primary defect in jaw growth. Whatever the cause, a cleft palate due to the Robin sequence must be distinguished from a true primary cleft palate, which has other causes with differing prognoses and implications for the child and family. Knowledge of dysmorphology and developmental genetic principles is thus necessary to properly diagnose each condition and to recognize that different prognoses are associated with the different primary causes.


FIGURE 14-6 A, Hypoplasia of the mandible and resulting posterior displacement of the tongue lead to the Robin sequence, in which the tongue obstructs palatal closure. B, Posterior placement of the tongue in the Robin sequence causes a deformationof the palate during development, leading to the constellation of a small chin and a U-shaped cleft palate involving the soft palate and extending into the hard palate. C, In contrast, primary cleft palate resulting from failure of closure of maxillary ridges is a malformation that begins in the anterior region of the maxilla and extends posteriorly to involve first the hard palate and then the soft palate, and it is often V-shaped. D, The delay in jaw development can be observed by serial three-dimensional fetal scans, from as early as 17 weeks (left) to 20 weeks (middle) and 29 weeks (right). SeeSources & Acknowledgments.

Introduction to Developmental Biology

The examples introduced briefly in the previous section serve to illustrate the principle that the clinical practice of medical genetics rests on a foundation of the basic science of developmental biology. For this reason, it behooves practitioners to have a working knowledge of some of the basic principles of developmental biology and to be familiar with the ways that abnormal function of genes and pathways affect development and, ultimately, their patients.

Developmental biology is concerned with a single, unifying question: How can a single cell transform itself into a mature animal? In humans, this transformation occurs each time a single fertilized egg develops into a human being with more than 1013 to 1014 cells, several hundred recognizably distinct cell types, and dozens of tissues. This process must occur in a reliable and predictable pattern and time frame.

Developmental biology has its roots in embryology, which was based on observing and surgically manipulating developing organisms. Early embryological studies, carried out in the 19th and early 20th centuries with readily accessible amphibian and avian embryos, determined that embryos developed from single cells and defined many of the fundamental processes of development. Much more recently, the application of molecular biology, genetics, and genomics to embryology has transformed the field by allowing scientists to study and manipulate development by a broad range of powerful biochemical and molecular techniques.

Development and Evolution

A critically important theme in developmental biology is its relationship to the study of evolution. Early in development, the embryos of many species look similar. As development progresses, the features shared between species are successively transformed into more specialized features that are, in turn, shared by successively fewer but more closely related species. A comparison of embryological characteristics among and within evolutionarily related organisms shows that developmental attributes (e.g., fingers) specific to certain groups of animals (e.g., primates) are built on a foundation of less specific attributes common to a larger group of animals (e.g., mammals), which are in turn related to structures seen in an even larger group of animals (e.g., the vertebrates). Structures in different organisms are termed homologous if they evolved from a structure present in a common ancestor (Fig. 14-7). In the case of the forelimb, the various ancestral lineages of the three species shown in Figure 14-7, tracing all the way back to their common predecessor, share a common attribute: a functional forelimb. The molecular developmental mechanism that created those limb structures is shared across all three of the contemporary species.


FIGURE 14-7 Diagram of the upper limb of three species: human, bird, and bat. Despite the superficially dissimilar appearance of the human arm and hand, the avian wing, and the bat wing, the similarity in their underlying bone structure and functionality reveals the homology of the forelimbs of all three species. In contrast, the two superficially similar wings in the bird and bat are analogous, not homologous structures. Although both the bird and bat wings are used for flying, they are constructed quite differently and did not evolve from a winglike structure in a common ancestor. SeeSources & Acknowledgments.

Not all similarity is due to homology, however. Evolutionary studies also recognize the existence of analogous structures, those that appear similar but arose independently of one another, through different lineages that cannot be traced back to a common ancestor with that structure. The molecular pathways that generate analogous structures are unlikely to be evolutionarily conserved. In the example shown in Figure 14-7, the wing structures of the bat and the birds arose independently in evolution to facilitate the task of aerial movement. The evolutionary lineages of these two animals do not share a common ancestor with a primitive winglike structure from which both bats and birds inherited wings. On the contrary, one can readily see that the birds developed posterior extensions from the limb to form a wing, whereas bats evolved wings through spreading the digits of their forelimbs and connecting them with syndactylous tissue. This situation is termed convergent evolution.

The evolutionary conservation of developmental processes is critically important to studies of human development because the vast majority of such research cannot (for important ethical reasons) be performed in humans (see Chapter 19). Thus, to understand a developmental observation, scientists use animal models to investigate normal and abnormal developmental processes. The ability to extend the results to humans is completely dependent on the evolutionary conservation of mechanisms of development and homologous structures.

Genes and Environment in Development

Developmental Genetics

Development results from the action of genes interacting with cellular and environmental cues. The gene products involved include transcriptional regulators, diffusible factors that interact with cells and direct them toward specific developmental pathways, the receptors for such factors, structural proteins, intracellular signaling molecules, and many others. It is therefore not surprising that most of the numerous developmental disorders that occur in humans are caused by chromosomal, subchromosomal or gene mutations.

Even though the genome is clearly the primary source of information that controls and specifies human development, the role of genes in development is often mistakenly described as a “master blueprint.” In reality, however, the genome does not resemble an architect's blueprint that specifies precisely how the materials are to be used, how they are to be assembled, and their final dimensions; it is not a literal description of the final form that all embryological and fetal structures will take. Rather, the genome specifies a set of interacting proteins and noncoding RNAs (see Chapter 3) that set in motion the processes of growth, migration, differentiation, and apoptosis that ultimately result, with a high degree of probability, in the correct mature structures. Thus, for example, there are no genetic instructions directing that the phalanx of a digit adopt an hourglass shape or that the eye be spherical. These shapes arise as an implicit consequence of developmental processes, thereby generating correctly structured cells, tissues, and organs.


Although genes are the primary regulators of development, other processes must also play a role. That development is regulated but not determined by the genome is underscored by the important role that probability plays in normal development. For example, in the mouse, a mutation in the formin gene produces renal aplasia in only approximately 20% of mice who carry the mutation, even when such carriers are genetically identical. Given that inbred strains of mice are genetically identical throughout their genomes, the 20% penetrance of the formin mutation cannot be explained by different modifying gene variants in the mice affected with renal agenesis versus the mice who are unaffected. Instead, it appears likely that the formin mutation shifts the balance of some developmental process by increasing the probability that a threshold for causing renal aplasia is exceeded, much as we explored in Chapter 8 when discussing complex patterns of inheritance in humans. Thus carrying a formin mutation will not always lead to renal aplasia, but it sometimes will, and neither the rest of the genome nor nongenetic factors are responsible for development of the defect in only a minority of animals. Probabilistic processes provide a rich source of interindividual variation that can lead to a range of developmental outcomes, some normal and some not. Thus it is not the case in development that “nothing is left to chance.”

Environmental Factors

As indicated earlier, the local environment in which a cell or tissue finds itself plays a central role in providing a normal developmental context. It is therefore not unexpected that drugs or other agents introduced from the environment can be teratogens, often because they interfere with intrinsic molecules that mediate the actions of genes. Identification of the mechanism of teratogenesis has obvious implications not only for clinical medicine and public health but also for basic science; understanding how teratogens cause birth defects can provide insight into the underlying developmental pathways that have been disturbed and result in a defect.

Because the molecular and cellular pathways used during development are often not employed in similar developmental processes after adulthood, teratogens that cause serious birth defects may have few or no side effects in adult patients. One important example of this concept is fetal retinoid syndrome, seen in fetuses of pregnant women who took the drug isotretinoin during pregnancy. Isotretinoin is an oral retinoid that is used systemically for the treatment of severe acne. It causes major birth defects when it is taken by a pregnant woman because it mimics the action of endogenous retinoic acid, a substance that in the developing embryo and fetus diffuses through tissues and interacts with cells, causing them to follow particular developmental pathways.

Different teratogens often cause very specific patterns of birth defects, the risk for which depends critically on the gestational age at the time of exposure, the vulnerability of different tissues to the teratogen, and the level of exposure during pregnancy. One of the best examples is thalidomide syndrome. Thalidomide, a sedative widely used in the 1950s, was later found to cause a high incidence of malformed limbs in fetuses exposed between 4 and 8 weeks of gestation because of its effect on the vasculature of the developing limb. Another example is the fetal alcohol syndrome. Alcohol causes a particular pattern of birth defects involving primarily the central nervous system because it is relatively more toxic to the developing brain and related craniofacial structures than to other tissues.

Some teratogens, such as x-rays, are also mutagens. A fundamental distinction between teratogens and mutagens is that mutagens cause damage by creating heritable alterations in genetic material, whereas teratogens act directly and transiently on developing embryonic tissue. Thus fetal exposure to a mutagen can cause an increased risk for birth defects or other diseases (e.g., cancer) throughout the life of the exposed individual and even in his or her offspring, whereas exposure to a teratogen increases the risk for birth defects for current but not for subsequent pregnancies.

Basic Concepts of Developmental Biology

Overview of Embryological Development

Developmental biology has its own set of core concepts and terminology that may be confusing or foreign to the student of genetics. We therefore provide a brief summary of a number of key concepts and terms used in this chapter (see Box on next page).

Core Concepts and Terminology in Human Developmental Biology

Blastocyst: a stage in embryogenesis after the morula, in which cells on the outer surface of the morula secrete fluid and form a fluid-filled internal cavity within which is a separate group of cells, the inner cell mass, which will become the fetus itself (see Fig. 14-10).

Chimera: an embryo made up of two or more cell lines that differ in their genotype. Contrast with mosaic.

Chorion: membrane that develops from the outer cells of the blastocyst and goes on to form the placenta and the outer layer of the sac in which the fetus develops.

Determination: the stage in development in which cells are irreversibly committed to forming a particular tissue.

Dichorionic twins: monozygotic twins arising from splitting of the embryo into two parts, before formation of the blastocyst, so that two independent blastocysts develop.

Differentiation: the acquisition by a cell of novel characteristics specific for a particular cell type or tissue.

Ectoderm: the primary embryonic germ layer that gives rise to the nervous system and skin.

Embryo: the stage of a developing human organism between fertilization and 9 weeks of gestation, when separation into placental and embryonic tissues occurs.

Embryogenesis: the development of the embryo.

Embryonic stem cells: cells derived from the inner cell mass that under appropriate conditions can differentiate into all of the cell types and tissues of an embryo and form a complete, normal fetus.

Endoderm: the primary embryonic germ layer that gives rise to many of the visceral organs and lining of the gut.

Epiblast: a differentiated portion of the inner cell mass that gives rise to the embryo proper.

Fate: the ultimate destination for a cell that has traveled down a developmental pathway.

Fetus: the stage of the developing human between 9 weeks of gestation and birth.

Gastrulation: the stage of development just after implantation in which the cells of the inner cell mass rearrange themselves into the three germ layersRegulative development ceases at gastrulation.

Germ cell: the cells that are the progenitors of the gametes. These cells are allocated early in development and undergo sex-specific differentiation.

Germ layers: three distinct layers of cells that arise in the inner cell mass, the ectodermmesoderm, and endoderm, which develop into distinctly different tissues in the embryo.

Hypoblast: the differentiated portion of the inner cell mass that contributes to fetal membranes (amnion).

Inner cell mass: a group of cells inside the blastocyst destined to become the fetus.

Mesoderm: the primary embryonic germ layer that gives rise to connective tissue, muscles, bones, vasculature, and the lymphatic and hematopoietic systems.

Monoamniotic twins: monozygotic twins resulting from cleavage of part of the inner cell mass (epiblast) but without cleavage of the part of the inner cell mass that forms the amniotic membrane (hypoblast).

Monochorionic twins: monozygotic twins resulting from cleavage of the inner cell mass without cleavage of the cells on the outside of the blastocyst.

Monozygotic twins: twins arising from a single fertilized egg, resulting from cleavage during embryogenesis in the interval between the first cell division of the zygote and gastrulation.

Morphogen: a substance produced by cells in a particular region of an embryo that diffuses from its point of origin through the tissues of the embryo to form a concentration gradient. Cells undergo specificationand then determination to different fates, depending on the concentration of morphogen they experience.

Morphogenesis: the creation of various structures during embryogenesis.

Morula: a compact ball of 16 cells produced after four cell divisions of the zygote.

Mosaic: an individual who develops from a single fertilized egg but in whom mutation after conception results in cells with two or more genotypes. Contrast with chimera.

Mosaic development: a stage in development in which cells have already become committed to the point that removal of a portion of an embryo will not allow normal embryonic development.

Multipotent stem cell: a stem cell capable of self-renewal as well as of developing into many different types of cells in a tissue, but not an entire organism. These are often called adult stem cells or tissue progenitor cells.

Organogenesis: the creation of individual organs during embryogenesis.

Pluripotent cell: an early stem cell capable of self-renewal as well as of becoming any cell in any tissue, including the germ cellsEmbryonic stem cells are pluripotent.

Progenitor cell: a cell that is traversing a developmental pathway on its way to becoming a fully differentiated cell.

Regulative development: a stage in development in which cells have not yet become determined so that the cells that remain after removal of a portion of an embryo can still form a complete organism.

Specification: a step along the path of differentiation in which cells acquire certain specialized attributes characteristic of a particular tissue but can still be influenced by external cues to develop into a different type of cell or tissue.

Stem cell: a cell that is capable both of generating another stem cell (self-renewal) and of differentiating into specialized cells within a tissue or an entire organism.

Zygote: the fertilized egg, the first step in embryogenesis.

Cellular Processes during Development

During development, cells divide (proliferate), acquire novel functions or structures (differentiate), move within the embryo (migrate), and undergo programmed cell death (often through apoptosis). These four basic cellular processes act in various combinations and in different ways to allow growth and morphogenesis (literally, the “creation of form”), thereby creating an embryo of normal size and shape, containing organs of the appropriate size, shape, and location, and consisting of tissues and cells with the correct architecture, structure, and function.

Although growth may seem too obvious to discuss, growth itself is carefully regulated in mammalian development, and unregulated growth is disastrous. The mere doubling (one extra round of cell division) of cell number (hyperplasia) or the doubling of cell size (hypertrophy) of an organism is likely to be fatal. Dysregulation of growth of segments of the body can cause severe deformity and dysfunction, such as in hemihyperplasia and other segmental overgrowth disorders (Fig. 14-8). Furthermore, the exquisite differential regulation of growth can change the shape of a tissue or an organ.


FIGURE 14-8 The clinical consequences of dysregulated growth in a child with Proteus syndrome, a congenital segmental overgrowth disorder affecting his face, abdomen, and right leg. Affected children are usually normal-appearing at birth but then in the first year begin to develop asymmetrical and disproportionate overgrowth of body parts. There are multiple malformations of the vascular system, including veins, capillaries and lymphatics; the osseous skeleton; and the connective tissue. The disorder is caused by somatic mosaicism for de novo activating mutations in AKT1, encoding a cell growth–promoting protein, which explains why the condition is always sporadic and occurs in an irregular pattern throughout the body in different affected individuals. SeeSources & Acknowledgments.

Morphogenesis is accomplished in the developing organism by the coordinated interplay of the mechanisms introduced in this section. In some contexts, morphogenesis is used as a general term to describe all of development, but this is formally incorrect because morphogenesis has to be coupled to the process of growth discussed here to generate a normally shaped and functioning tissue or organ.

Human Embryogenesis

This description of human development begins where Chapter 2 ends, with fertilization. After fertilization, the embryo undergoes a series of cell divisions without overall growth, termed cleavage. The single fertilized egg undergoes four divisions to yield the 16-cell morula by day 4 (Fig. 14-9). At day 5, the embryo transitions to become a blastocyst, in which cells that give rise to the placenta form a wall, inside of which the cells that will make the embryo itself aggregate to one side into what is referred to as the inner cell mass. This is the point at which the embryo acquires its first obvious manifestation of polarity, an axis of asymmetry that divides the inner cell mass (most of which goes on to form the mature organism) from the embryonic tissues that will go on to form the chorion, an extraembryonic tissue (e.g., placenta) (Fig. 14-10). The inner cell mass then separates again into the epiblast, which will make the embryo proper, and the hypoblast, which will form the amniotic membrane.


FIGURE 14-9 Human development begins with cleavage of the fertilized egg. A, The fertilized egg at day 0 with two pronuclei and the polar bodies. B, A two-cell embryo at day 1 after fertilization. C, A four-cell embryo at day 2. D, The eight-cell embryo at day 3. E, The 16-cell stage later in day 3, followed by the phenomenon of compaction, whereby the embryo is now termed a morula (F, day 4). G, T formation of the blastocyst at day 5, with the inner cell mass indicated by the arrow. Finally, the embryo (arrow) hatches from the zona pellucida (H). SeeSources & Acknowledgments.


FIGURE 14-10 Cell lineage and fate during preimplantation development. Embryonic age is given in time after fertilization in humans: A, 6 days. B, 7 days. C, 8 days post fertilization. SeeSources & Acknowledgments.

The embryo implants in the endometrial wall of the uterus in the interval between days 7 and 12 after fertilization. After implantation, gastrulation occurs, in which cells rearrange themselves into a structure consisting of three cellular compartments, termed the germ layers, comprising the ectoderm, mesoderm, and endoderm. The three germ layers give rise to different structures. The endodermal lineage forms the central visceral core of the organism. This includes the cells lining the main gut cavity, the airways of the respiratory system, and other similar structures. The mesodermal lineage gives rise to kidneys, heart, vasculature, and structural or supportive functions in the organism. Bone and muscle are nearly exclusively mesodermal and have the two main functions of structure (physical support) and providing the necessary physical and nutritive support of the hematopoietic system. The ectoderm gives rise to the central and peripheral nervous systems and the skin. During the complicated movements that occur in gastrulation, the embryo also establishes the major axes of the final body plan: anterior-posterior (cranial-caudal), dorsal-ventral (back-front), and left-right axes, which are discussed later.

The next major stages of development involve the initiation of the nervous system, establishment of the basic body plan, and then organogenesis, which occupies weeks 4 to 8. The position and basic structures of all of the organs are now established, and the cellular components necessary for their full development are now in place. It is during this phase of embryonic development that neural tube defects occur, as we explore next.

Neural Tube Defects

Neural tube defects (NTDs) are among the most common and devastating birth defects. Anencephaly and spina bifida are NTDs that frequently occur together in families and are considered to have a common pathogenesis. In anencephaly, the forebrain, overlying meninges, vault of the skull, and skin are all absent. Many infants with anencephaly are stillborn, and those born alive survive a few hours at most. Approximately two thirds of affected infants are female. In spina bifida, there is failure of fusion of the arches of the vertebrae, typically in the lumbar region. There are varying degrees of severity, ranging from spina bifida occulta, in which the defect is in the bony arch only, to spina bifida aperta, in which a bone defect is also associated with meningocele (protrusion of meninges) or meningomyelocele (protrusion of neural elements as well as meninges through the defect; see Fig. 17-3).

As a group, NTDs are a leading cause of stillbirth, death in early infancy, and handicap in surviving children. Their incidence at birth is variable, ranging from almost 1% in Ireland to 0.2% or less in the United States. The frequency also appears to vary with social factors and season of birth and oscillates widely over time (with a marked decrease in recent years; see later discussion).

A small proportion of NTDs have known specific causes, for example, amniotic bands (see Fig. 14-3), some single-gene defects with pleiotropic expression, some chromosomal disorders, and some teratogens. Most NTDs, however, are isolated defects of unknown cause.

Maternal Folic Acid Deficiency and Neural Tube Defects.

NTDs were long believed to follow a multifactorial inheritance pattern determined by multiple genetic and environmental factors, as introduced generally in Chapter 8. It was therefore a stunning discovery to find that the single greatest factor in causing NTDs is a vitamin deficiency. The risk for NTDs was found to be inversely correlated with maternal serum folic acid levels during pregnancy, with a threshold of 200 µg/L, below which the risk for NTD becomes significant. Along with reduced blood folate levels, elevated homocysteine levels were also seen in the mothers of children with NTDs, suggesting that a biochemical abnormality was present at the step of recycling of tetrahydrofolate to methylate homocysteine to methionine (see Fig. 12-8). Folic acid levels are strongly influenced by dietary intake and can become depressed during pregnancy even with a typical intake of approximately 230 µg/day. The impact of folic acid deficiency is exacerbated by a genetic variant of the enzyme 5,10-methylenetetrahydrofolate reductase (MTHFR), caused by a common missense mutation that makes the enzyme less stable than normal. Instability of this enzyme hinders the recycling of tetrahydrofolate and interferes with the methylation of homocysteine to methionine.

The mutant allele is so common in many populations that between 5% and 15% of the population is homozygous for the variant. In studies of infants with NTDs and their mothers, it was found that mothers of infants with NTDs were twice as likely as controls to be homozygous for the mutant allele encoding the unstable enzyme. How this enzyme defect contributes to NTDs and whether the abnormality is a direct result of elevated homocysteine levels, depressed methionine levels, or some other metabolic derangement remain undefined.

Prevention of Neural Tube Defects.

There are two approaches to preventing NTDs. The first is to educate women to supplement their diets with folic acid 1 month before conception and continuing for 2 months after conception during the period when the neural tube forms. Dietary supplementation with 400 to 800 µg of folic acid per day for women who plan their pregnancies has been shown to reduce the incidence of NTDs by more than 75%. Much active discussion is ongoing as to whether the entire food supply should be supplemented with folic acid as a public health measure to avoid the problem of women failing to supplement their diets individually during pregnancy.

The second approach is to apply prenatal screening for all pregnancies and offer prenatal diagnosis to high-risk pregnancies. Prenatal diagnosis of anencephaly and most cases of open spina bifida relies on detecting excessive levels of alpha-fetoprotein (AFP) and other fetal substances in the amniotic fluid and by ultrasonographic scanning, as we shall discuss further in Chapter 17. However, less than 5% of all patients with NTDs are born to women with previous affected children. For this reason, screening of all pregnant women for NTDs by measurements of AFP and other fetal substances in maternal serum is now widespread. Thus we can anticipate that a combination of preventive folic acid therapy and maternal AFP screening will provide major public health benefits by drastically reducing the incidence of NTDs.

Human Fetal Development

The embryonic phase of development occupies the first 2 months of pregnancy and is followed by the fetal phase of development, which is concerned primarily with the maturation and further differentiation of the components of the organs. For some organ systems, development does not cease at birth. For example, the brain undergoes substantial postnatal development, and limbs undergo epiphyseal growth and ultimately closure after puberty.

The Germ Cell: Transmitting Genetic Information

In addition to growth and differentiation of somatic tissues, the organism must also specify which cells will go on to become the gametes of the mature adult. The germ cell compartment serves this purpose. As described in Chapter 2, cells in the germ cell compartment become committed to undergoing gametogenesis and meiosis in order that the species can pass on its genetic complement and facilitate the recombination and random assortment of chromosomes. In addition, the sex-specific epigenetic imprint that certain genes require must be reset within the germ cell compartment (see Chapters 36, and 7).

The Stem Cell: Maintaining Regenerative Capacity in Tissues

In addition to specifying the program of differentiation that is necessary for development, the organism must also set aside tissue-specific stem cells that can regenerate differentiated cells during adult life. The best-characterized example of these cells is in the hematopoietic system. Among the 1011 to 1012 nucleated hematopoietic cells in the adult organism are approximately 104 to 105 cells that have the potential to generate any of the more specialized blood cells on a continuous basis during a lifetime. Hematopoietic stem cells can be transplanted to other humans and completely reconstitute the hematopoietic system (see Chapter 13). A system of interacting gene products maintains a properly sized pool of hematopoietic stem cells. These regulators permit a balance between the maintenance of stem cells through self-replication and the generation of committed precursor cells that can go on to develop into the various mature cells of the hematopoietic system (Fig. 14-11) (see Box).

Embryonic Stem Cell Technology

Inner cell mass cells are believed to be capable of forming any tissue in the body. This is suspected of being true in humans (but has never been tested for obvious ethical reasons) but has been proved to be true in mice. The full developmental potential of inner cell mass cells is the basis of the experimental field of embryonic stem cell technology in mice, a technology that is crucial for generating animal models of human genetic disease (Fig. 14-12). In this technique, mouse inner cell mass cells are grown in culture as embryonic stem cells and undergo genetic manipulation to introduce a given mutation into a specific gene. These cells are then injected into the inner cell mass of another early mouse embryo. The mutated cells are incorporated into the inner cell mass of the recipient embryo and contribute to many tissues of that embryo, forming a chimera (a single embryo made up of cells from two different sources). If the mutated cells contribute to the germline in a chimeric animal, the offspring of that animal can inherit the engineered mutations. The ability of the recipient embryo to tolerate the incorporation of these pluripotent, nonspecified cells, which then undergo specification and can contribute to any tissue in a living mouse, is the converse of regulative development, the ability of an embryo to tolerate removal of some cells.

Human stem cells (HSCs) made from unused fertilized embryos are the subject of intensive research as well as ethical controversy. Although the use of HSCs for cloning an entire human being is considered highly unethical and universally banned, current research is directed toward generating particular cell types from HSCs to provide cellular models of human genetic diseases or to repair damaged tissues and organs, a goal of regenerative medicine (see Chapter 13).

Induced pluripotent stem (iPS) cells are another source of early stem cells that can be cultured and differentiated in vitro into particular cell types. Human iPS cells are derived through reprogramming of readily available and ethically uncontroversial somatic cells, such as fibroblasts, to very early stem cells through the introduction of certain transcription factors into the cells (e.g., the transcription factors Oct4, Sox2, cMyc, and Klf4). This technology makes what were previously inaccessible tissues from patients with genetic disorders, such as cardiac myocytes from patients with cardiomyopathies, or central nervous system neurons from patients with neurodegenerative diseases available for research and, ultimately, perhaps tissue-based therapy using their own gene-corrected iPS cells. Shinya Yamanaka was awarded the 2012 Nobel Prize in Physiology or Medicine for his demonstration of the feasibility of creating iPS cells.


FIGURE 14-11 The development of blood cells is a continuous process that generates a full complement of cells from a single, totipotent hematopoietic stem cell. This hematopoietic stem cell is a committed stem cell that differentiated from a more primitive mesodermal stem cell. RBC, Red blood cell. SeeSources & Acknowledgments.


FIGURE 14-12 Embryonic stem (ES) cells are derived directly from the inner cell mass, are euploid, and can contribute to the germline. Cultured ES cells differentiated in vitro can give rise to a variety of different cell types.

Fate, Specification, and Determination

As an undifferentiated cell undergoes the process of differentiation, it moves through a series of discrete steps in which it manifests various distinct functions or attributes until it reaches its ultimate destination, referred to as its fate (e.g., when a precursor cell becomes an erythrocyte, a keratinocyte, or a cardiac myocyte). In the developing organism, these attributes not only vary across the recognizable cell types but also change over time. Early during differentiation, a cell undergoes specification when it acquires specific characteristics but can still be influenced by environmental cues (signaling molecules, positional information) to change its ultimate fate. These environmental clues are primarily derived from neighboring cells by direct cell-cell contact or by signals received at the cell surface from soluble substances, including positional information derived from where a cell sits in a gradient of various morphogens. Eventually a cell either irreversibly acquires attributes or has irreversibly been committed to acquire those attributes (referred to as determination). With the exception of the germ cell and stem cell compartments just described, all cells undergo specification and determination to their ultimate developmental fate.

Specification and determination involve the stepwise acquisition of a stable cellular phenotype of gene expression specific to the particular fate of each cell—nerve cells make synaptic proteins but do not make hemoglobin, whereas red blood cells do not make synaptic proteins but must make hemoglobin. With the exception of lymphocyte precursor cells undergoing DNA rearrangements in the T-cell receptor or immunoglobulin genes (see Chapter 3), the particular gene expression profile responsible for the differentiated cellular phenotype does not result from permanent changes in DNA sequence. Instead, the regulation of gene expression depends on epigenetic changes, such as stable transcription complexes, modification of histones in chromatin, and methylation of DNA (see Chapter 3). The epigenetic control of gene expression is responsible for the loss of developmental plasticity, as we discuss next.

Regulative and Mosaic Development

Early in development, cells are functionally equivalent and subject to dynamic processes of specification, a phenomenon known as regulative development. In regulative development, removal or ablation of part of an embryo can be compensated for by the remaining similar cells. In contrast, later in development, each of the cells in some parts of the embryo has a distinct fate, and in each of those parts, the embryo only appears to be homogeneous. In this situation, known as mosaic development, loss of a portion of an embryo would lead to the failure of development of the final structures that those cells were fated to become. Thus the developmental plasticity of the embryo generally declines with time.

Regulative Development and Twinning

That early development is primarily regulative has been demonstrated by basic embryological experiments and confirmed by observations in clinical medicine. Identical (monozygotic) twins are the natural experimental evidence that early development is regulative. The most common form of identical twinning occurs in the second half of the first week of development, effectively splitting the inner cell mass into two halves, each of which develops into a normal fetus (Fig. 14-13). Were the embryo even partly regulated by mosaic development at this stage, the twins would develop only partially and consist of complementary parts. This is clearly not the case, because twins are generally completely normally developed and eventually attain normal size through prenatal and postnatal growth.


FIGURE 14-13 The arrangement of placental membranes in monozygotic twins depends on the timing of the twinning event. Dichorionic twins result from a complete splitting of the entire embryo, leading to duplication of all extraembryonic tissues. Monochorionic diamniotic twins are caused by division of the inner cell mass at the blastocyst stage. Monoamniotic twins are caused by division of the epiblast but not the hypoblast.

The various forms of monozygotic twinning demonstrate regulative development at several different stages. Dichorionic twins result from cleavage at the four-cell stage. Monochorionic twins result from a cleaved inner cell mass. Monoamniotic twins result from an even later cleavage, in this case within the bilayered embryo, which then forms two separate embryos but only one extraembryonic compartment that goes on to make the single amnion. All of these twinning events demonstrate that these cell populations can reprogram their development to form complete embryos from cells that, if cleavage had not occurred, would have contributed to only part of an embryo.

The successful application of the technique of preimplantation diagnosis (see Chapter 17) also illustrates that early human development is regulative. In this procedure, male and female gametes are harvested from the presumptive parents and fertilized in vitro (Fig. 14-14; see also Fig. 17-1). When these fertilized embryos have reached the eight-cell stage (at day 3), a biopsy microneedle is used to remove some of the cells of the developing blastocyst. The isolated cell with its clearly visible nucleus can then be examined using a variety of appropriate cytogenetic or genomic tests to ascertain if the embryo is suitable for implantation. Embryos composed of the remaining seven cells that are not affected by the disease can then be selected and implanted in the mother. The capacity of the embryo to recover from the biopsy of one of its eight cells is attributable to regulative development. Were those cells removed by biopsy fated to form a particular part or segment of the body (i.e., governed by mosaic development), one would predict that these parts of the body would be absent or defective in the mature individual. Instead, the embryo has compensatory mechanisms to replace those cells, which then undergo normal development as specified by their neighboring cells.


FIGURE 14-14 Blastomere biopsy of a human cleavage stage embryo. A, Eight-cell embryo, day 3 after fertilization. B, Embryo on holding pipette (left) with biopsy pipette (right) breaching the zona pellucida. C, Blastomere removal by suction. D, Blastomere removed by biopsy with a clearly visible single nucleus (indicated by arrow). SeeSources & Acknowledgments.

Mosaic Development

Embryonic development generally proceeds from more regulative to more mosaic development. Typical identical twinning early in development, as mentioned earlier, is an illustration of regulative development. However, later embryo cleavage events result in the formation of conjoined twins, which are two fetuses that share body structures and organs because the cleavage occurred after the transition from regulative to mosaic development, too late to allow complete embryos.

Interestingly, in some adult nonhuman species, ablation of a specific tissue may not limit development. For example, the mature salamander can regenerate an entire tail when it is cut off, apparently retaining a population of cells that can reestablish the developmental program for the tail after trauma. One of the goals of research in developmental biology is to understand this process in other species and potentially harness it in practice for human regenerative medicine.

Axis Specification and Pattern Formation

A critical function of the developing organism is to specify the spatial relationships of structures within the embryo. In early development, the organism must determine the relative orientation of a number of body segments and organs, involving the establishment of three axes:

• The head-to-tail axis, which is termed the cranial-caudal or anterior-posterior axis, is established very early in embryogenesis and is probably determined by the entry position of the sperm that fertilizes the egg. (It is referred to as the rostral-caudal axis later in development.)

• The dorsal-ventral axis is the second dimension, and here, too, a series of interacting proteins and signaling pathways are responsible for determining dorsal and ventral structures. The morphogen sonic hedgehog (discussed later) participates in setting up the axis of dorsal-ventral polarity along the spinal cord.

• Finally, a left-right axis must be established. The left-right axis is essential for proper heart development and positioning of viscera; for example, an abnormality in the X-linked gene ZIC3, involved in left-right axis determination, is associated with cardiac anomalies and situs inversus, in which some thoracic and abdominal viscera are on the wrong side of the chest and abdomen.

The three axes that must be specified in the whole embryo must also be specified early in the developing limb. Within the limb, the organism must specify the proximal-distal axis (shoulder to fingertip), the anterior-posterior axis (thumb to fifth finger), and the dorsal-ventral axis (dorsum to palm). On a cellular scale, individual cells also develop an axis of polarity, for example, the basal-apical axis of the proximal renal tubular cells or the axons and dendrites of a neuron. Thus, specifying axes in the whole embryo, in limbs, and in cells is a fundamental process in development.

Once an organismal axis is determined, the embryo then overlays a patterning program onto that axis. Conceptually, if axis formation can be considered as the drawing of a line through an undeveloped mass of cells and specifying which end is to be the head and which end the tail, then patterning is the division of the embryo into segments and the assignment to these segments of an identity, such as head, thorax, or abdomen. The HOX genes (discussed in the next section) have major roles in determining the different structures that develop along the anterior-posterior axis. The end result of these pattern specification programs is that cells or groups of cells are assigned an identity related primarily to their position within the organism. This identity is subsequently used by the cells as an instruction to specify how development should proceed.

Pattern Formation and the HOX Gene System

The homeobox (HOX) gene system, first described in the fruit fly Drosophila melanogaster, constitutes a paradigm in developmental biology. HOX genes are so named because the proteins they encode are transcription factors that contain a conserved DNA-binding motif called the homeodomain. The segment of the gene encoding the homeodomain is called a homeobox, thus giving the gene family its name, HOX.

Many species of animals have HOX genes, and the homeodomains encoded by these genes are similar; however, different species contain different numbers of HOX genes; for example, fruit flies contain 8 and humans nearly 40. The 40 human HOX genes are organized into four clusters on four different chromosomes. Strikingly, the order of the individual genes within the clusters is conserved across species. The human HOX gene clusters (Fig. 14-15) were generated by a series of gene duplication events, conceptually similar to those described in Chapter 11 for the evolution of the globin gene family. Initially, ancient events duplicated the original ancestral HOX gene in tandem along a single chromosome. Subsequent duplications of this single set of HOX genes and relocation of the new gene set to other locations in the genome resulted in four unlinked HOX gene clusters in humans (and other mammals) named HOXA, HOXB, HOXC, and HOXD.


FIGURE 14-15 Action and arrangement of HOX genes. A, An ancestral HOX gene cluster in a common ancestor of vertebrates and invertebrates has been quadruplicated in mammals, and individual members of the ancestral cluster have been lost. B, The combination of HOX genes expressed in adjacent regions along the anteroposterior axis of developing embryos selects a unique developmental fate (as color-coded in the segments of the fly and human embryo). C, In the developing limbs, different combinations of HOXA and HOXD genes are expressed in adjacent zones that help specify developmental fate along the proximal-distal and anterior-posterior axes. SeeSources & Acknowledgments.

Unique combinations of HOX gene expression in small groups of cells, located in particular regions of the embryo, help determine the developmental fate of those regions. Just as specific combinations of HOXgenes from the single HOX gene cluster in the fly are expressed along the anterior-posterior axis of the body and regulate different patterns of gene expression and therefore different body structures (see Fig. 14-15), mammals use a number of HOX genes from different clusters to accomplish similar tasks. Early, in the whole embryo, HOX transcription factors specify the anterior-posterior axis: the HOXA and HOXBclusters, for example, act along the rostral-caudal axis to determine the identity of individual vertebrae and somites. Later in development, the HOXA and HOXD clusters determine regional identity along the axes of the developing limb.

One interesting aspect of HOX gene expression is that the order of the genes in a cluster parallels the position in the embryo in which that gene is expressed and the time in development when it is expressed (see Fig. 14-15). In other words, the position of a HOX gene in a cluster is collinear with both the timing of expression and the location of expression along the anterior-posterior axis in the embryo. For example, in the HOXB cluster, the genes expressed first and in the anterior portion of the embryo are at one end of the cluster; the order of the rest of the genes in the cluster parallels the order in which they are expressed, both by location along the anterior-posterior axis of the embryo and by timing of expression. Although this gene organization is distinctly unusual and is not a general feature of gene organization in the genome (see Chapter 3), a similar phenomenon is seen within another developmentally regulated human gene family, the globin gene clusters (see Chapter 11). In both cases, the association of spatial organization in the genome with temporal expression in development presumably reflects long-range regulatory elements in the genome that govern the epigenetic packaging and accessibility of different genes at different times in the embryo.

The HOX gene family thus illustrates several important principles of developmental biology and evolution:

• First, a group of genes functions together to accomplish similar general tasks at different times and places in the embryo.

• Second, homologous structures are generated by sets of homologous transcription factors derived from common evolutionary predecessors. For example, flies and mammals have a similar basic body plan (head anterior to the trunk, with limbs emanating from the trunk, cardiorespiratory organs anterior to digestive), and that body plan is specified by a set of genes that were passed down through common evolutionary predecessors.

• And third, although it is not usually the case with genes involved in development, the HOX genes show a remarkable genomic organization within a cluster that correlates with their function during development.

Cellular and Molecular Mechanisms in Development

In this section, we review the basic cellular and molecular mechanisms that regulate development (see Box). We illustrate each mechanism with a human birth defect or disease that results from the failure of each of these normal mechanisms.

Fundamental Mechanisms Operating in Development

• Gene regulation by transcription factors

• Cell-cell signaling by direct contact and by morphogens

• Induction of cell shape and polarity

• Cell movement

• Programmed cell death

Gene Regulation by Transcription Factors

Transcription factors control development by controlling the expression of other genes, some of which are also transcription factors. Groups of transcription factors that function together are referred to as transcriptional regulatory modules, and the functional dissection of these modules is an important task of the developmental geneticist and, increasingly, of genome biologists. Some transcription factors activate target genes and others repress them. Still other transcription factors have both activator and repressor functions (so-called bifunctional transcription factors); noncoding RNAs such as microRNAs also interact with target sequences and can activate or repress gene expression. The recruitment of these various activators and repressors within chromatin can be guided by histone modifications such as acetylation, and the regulation of histone modifications is accomplished by histone acetyltransferases and deacetylases (see Chapter 3). These epigenetic changes to histones are marks that indicate whether a particular gene is likely to be active or inactive. Regulatory modules control development by causing different combinations of transcription factors to be expressed at different places and at different times to direct the spatiotemporal regulation of development. By directing differential gene expression across space and time, the binding of various transcriptional regulatory modules to transcriptional complexes is controlled by histone modifications and is a central element of the development of the embryo.

A transcriptional regulatory complex consists of a large number of general transcription factors joined with the specific transcription factors that are responsible for creating the selectivity of a transcriptional complex (Fig. 14-16). Most general transcription factors are found in thousands of transcriptional complexes throughout the genome, and, although each is essential, their roles in development are nonspecific. Specific transcription factors also participate in forming transcription factor complexes, mostly under the control of epigenetic marks of histone modifications, but only in specific cells or at specific times in development, thereby providing the regulation of gene expression that allows developmental processes to be exquisitely controlled.


FIGURE 14-16 General transcription factors, shown in blue, and RNA polymerase bind to cis-acting sequences closely adjacent to the messenger RNA (mRNA) transcriptional start site; these cis-acting sequences are collectively referred to as the promoter. More distal enhancer or silencer elements bind specialized and tissue-specific transcription factors. Coactivator proteins facilitate a biochemical interaction between specialized and general transcription factors. SeeSources & Acknowledgments.

The importance of transcription factors in normal development is illustrated by an unusual mutation of HOXD13 that causes synpolydactyly, an incompletely dominant condition in which heterozygotes have interphalangeal webbing and extra digits in their hands and feet. Rare homozygotes have similar but more severe abnormalities and also have bone malformations of the hands, wrists, feet, and ankles (Fig. 14-17). The HOXD13 mutation responsible for synpolydactyly is caused by expansion of a polyalanine tract in the amino-terminal domain of the protein; the normal protein contains 15 alanines, whereas the mutant protein contains 22 to 24 alanines. The polyalanine expansion that causes synpolydactyly is likely to act by a gain-of-function mechanism (see Chapter 11), as heterozygosity for a HOXD13 loss-of-function mutation has only a mild effect on limb development, characterized by a rudimentary extra digit between the first and second metatarsals and between the fourth and fifth metatarsals of the feet. Regardless of the exact mechanism, this condition demonstrates that a general function for HOX genes is to determine regional identity along specific body axes during development.


FIGURE 14-17 An unusual gain-of-function mutation in HOXD13 creates an abnormal protein with a dominant negative effect. Photographs and radiographs show the synpolydactyly phenotype. A and B, Hand and radiograph of an individual heterozygous for a HOXD13 mutation. Note the branching metacarpal III and the resulting extra digit IIIa. The syndactyly between digits has been partially corrected by surgical separation of III and IIIa-IV. C and D, Hand and radiograph of an individual homozygous for a HOXD13 mutation. Note syndactyly of digits III, IV, and V and their single knuckle; the transformation of metacarpals I, II, III, and V to short carpal-like bones (stars); two additional carpal bones (asterisks); and short second phalanges. The radius, ulna, and proximal carpal bones appear normal. E and F, Foot and radiograph of the same homozygous individual. Note the relatively normal size of metatarsal I, the small size of metatarsal II, and the replacement of metatarsals III, IV, and V with a single tarsal-like bone (stars). SeeSources & Acknowledgments.

Morphogens and Cell to Cell Signaling

One of the hallmarks of developmental processes is that cells must communicate with each other to develop proper spatial arrangements of tissues and cellular subtypes. This communication occurs through cell signaling mechanisms. These cell-cell communication systems are commonly composed of a cell surface receptor and the molecule, called a ligand, that binds to it. On ligand binding, receptors transmit their signals through intracellular signaling pathways. One of the common ligand-receptor pairs is the fibroblast growth factors and their receptors. There are 23 recognized members of the fibroblast growth factor gene family in the human, and many of them are important in development. The fibroblast growth factors serve as ligands for tyrosine kinase receptors. Abnormalities in fibroblast growth factor receptors cause diseases such as achondroplasia (Case 2) (see Chapter 7) and certain syndromes that involve abnormalities of craniofacial development, referred to as craniosynostoses because they demonstrate premature fusion of cranial sutures in the skull.

One of the best examples of a developmental morphogen is hedgehog, originally discovered in Drosophila and named for its ability to alter the orientation of epidermal bristles. Diffusion of the hedgehog protein creates a gradient in which different concentrations of the protein cause surrounding cells to assume different fates. In humans, several genes closely related to Drosophila hedgehog also encode developmental morphogens; one example is the gene sonic hedgehog (SHH). Although the specific programs controlled by hedgehog in Drosophila are very different from those controlled by its mammalian counterparts, the underlying themes and molecular mechanisms are similar. For example, secretion of the SHH protein by the notochord and the floor plate of the developing neural tube generates a gradient that induces and organizes the different types of cells and tissues in the developing brain and spinal cord (Fig. 14-18A). SHH is also produced by a small group of cells in the limb bud to create what is known as the zone of polarizing activity, which is responsible for establishing the posterior side of the developing limb bud and the asymmetrical pattern of digits within individual limbs (see Fig. 14-18B).


FIGURE 14-18 A, Transverse section of the developing neural tube. Sonic hedgehog protein released from the notochord diffuses upward to the ventral portion of the developing neural tube (brown); high concentrations immediately above the notochord induce the floor plate, whereas lower concentrations more laterally induce motor neurons. Ectoderm above (dorsal to) the neural tube releases bone morphogenetic proteins that help induce neural crest development at the dorsal edge of the closing neural tube (dark purple). B, Morphogenetic action of the sonic hedgehog (SHH) protein during limb bud formation. SHH is released from the zone of polarizing activity (labeled polarizing region in B) in the posterior limb bud to produce a gradient (shown with its highest levels as 4, declining to 2). Mutations or transplantation experiments that create an ectopic polarizing region in the anterior limb bud cause a duplication of posterior limb elements. SeeSources & Acknowledgments.

Mutations that inactivate the SHH gene in humans cause birth defects that may be inherited as autosomal dominant traits, which demonstrates that a 50% reduction in gene expression is sufficient to produce an abnormal phenotype, presumably by altering the magnitude of the hedgehog protein gradient. Affected individuals usually exhibit holoprosencephaly (failure of the midface and forebrain to develop), leading to cleft lip and palate, hypotelorism (eyes that are closely spaced together), and absence of forebrain structures. On occasion, however, the clinical findings are mild or subtle such as, for example, a single central incisor or partial absence of the corpus callosum (Fig. 14-19). Because variable expressivity has been observed in members of the same family, it cannot be due to different mutations and instead must reflect the action of modifier genes at other loci, chance, environment, or some combination of all three.


FIGURE 14-19 Variable expressivity of an SHH mutation. The mother and her daughter carry the same missense mutation in SHH, but the daughter is severely affected with microcephaly, abnormal brain development, hypotelorism, and a cleft palate, whereas the only manifestation in the mother is a single central upper incisor. SeeSources & Acknowledgments.

Cell Shape and Organization

Cells must organize themselves with respect to their position and polarity in their microenvironment. For example, kidney epithelial cells must undergo differential development of the apical and basal aspects of their organelles to effect reabsorption of solutes. The acquisition of polarity by a cell can be viewed as the cellular version of axis determination (as discussed in a previous section) with respect to the development of the overall embryo. Under normal circumstances, each renal tubular cell elaborates on its cell surface a filamentous structure, known as a primary cilium. One hypothesis is that the primary cilium is designed to sense fluid flow in the developing kidney tubule and signal the cell to stop proliferating and to polarize. Another hypothesis is that the primary cilium is a sort of cellular antenna that concentrates signal transduction components to facilitate activation or repression of developmental pathways.

There is substantial evidence that the sonic hedgehog signal transduction pathway acts in this fashion. Adult polycystic kidney disease (Case 37) is caused by loss of function of one of two protein components of primary cilia, polycystin 1 or polycystin 2, so that the cells fail to sense fluid flow or to activate or repress signal transduction pathways properly. As a result, they continue to proliferate and do not undergo the appropriate developmental program of polarization, in which they stop dividing and display polarized expression of certain proteins on either the apical or basal aspect of the tubular epithelial cells (Fig. 14-20). The continued cell division leads to the formation of cysts, fluid-filled spaces lined by renal tubular cells.


FIGURE 14-20 Polarization of epidermal growth factor receptor (EGFR) in epithelium from a normal fetus, a normal adult, and a patient with polycystic kidney disease. Fetal cells and epithelial cells from patients with polycystic kidney disease express a heterodimer of EGFR and erb-b2 at apical cell membranes. In normal adults, tubular epithelia express homodimeric complexes of EGFR at the basolateral membrane. SeeSources & Acknowledgments.

Cell Migration

Programmed cell movement is critical in development, and nowhere is it more important than in the central nervous system. The central nervous system is developed from the neural tube, a cylinder of cells created during weeks 4 to 5 of embryogenesis. Initially, the neural tube is only a single cell layer thick, a pseudostratified columnar epithelium. Once sufficient neuroepithelial cells are produced by symmetrical division, these cells divide asymmetrically as neural stem cells. These neural stem cells stretch from the apical surface adjacent to the ventricle to the basal surface. The nucleus of these neural stem cells is adjacent to the apical surface in the ventricular cell layer situated adjacent to the ventricle, and the fiber of these cells stretches to the basal or pial surface as the so-called radial glial cells. These radial glia are one type of neural stem cells, which divide asymmetrically to generate new neural stem cells as well as committed neuronal precursors and secondary neural stem cells. These set up more basally located neural stem cells that can amplify the number of cells produced from a given radial glial progenitor. Postmitotic neuronal precursors then migrate outwards toward the pial surface along the radial glia. The central nervous system is built by waves of migration of these neuronal precursors. The neurons that populate the inner layers of the cortex migrate earlier in development, and each successive wave of neurons passes through the previously deposited, inner layers to form the next outer layer (Fig. 14-21).


FIGURE 14-21 The role of neuronal migration in normal cortical development and the defective migration in individuals heterozygous for an LIS1 mutation causing lissencephaly. Top, A radial slice is taken from a normal developing neural tube of the mouse, showing the progenitor cells at the ventricular zone (VZ). These cells divide, differentiate into postmitotic cells, and migrate radially along a scaffold made up of glia. The different shapes and colors represent the cells that migrate and form the various cortical layers: IZ, intermediate zone; SP, subplate; CP, cortical plate; MZ, marginal zone; PS, pial surface. The six distinguishable layers of the normal cortex (molecular, external granular, external pyramidal, internal granular, internal pyramidal, multiform) that occupy the region of the cortical plate are labeled I through VI. Bottom, Aberrant migration and failure of normal cortical development seen in lissencephaly. SeeSources & Acknowledgments.

Lissencephaly (literally, “smooth brain”) is a severe abnormality of brain development causing profound intellectual disability. This developmental defect is one component of the Miller-Dieker syndrome (Case 32), which is caused by a contiguous gene deletion syndrome that involves one copy of the LIS1 gene on chromosome 17. When there is loss of LIS1 function, the progressive waves of migration of cortical neurons do not occur in an organized fashion because of reduced speeds of migration. The result is a thickened, hypercellular cerebral cortex with undefined cellular layers and poorly developed gyri, thereby making the surface of the brain appear smooth.

In addition to the neuronal migrations described, another remarkable example of cell migration involves the neural crest, a population of cells that arises from the dorsolateral aspect of the developing neural tube (see Fig. 14-18A). Neural crest cells must migrate from their original location at the dorsal and lateral surface of the neural tube to remarkably distant sites, such as the ventral aspect of the face, the ear, the heart, the gut, and many other tissues, including the skin, where they differentiate into pigmented melanocytes.

Population of the gut by neural crest progenitors gives rise to the autonomic innervation of the gut; failure of that migration leads to the aganglionic colon seen in Hirschsprung disease (Case 22). The genetics of Hirschsprung disease are complex (see Chapter 8), but a number of key signaling molecules have been implicated. One of the best characterized is the RET proto-oncogene. As discussed in Chapter 8, mutations in RET have been identified in approximately 50% of patients with Hirschsprung disease.

Another example of defects in neural crest development is the group of birth defects known as the Waardenburg syndrome, which includes defects in skin and hair pigmentation, coloration of the iris, and colon innervation (Fig. 14-22). This syndrome can be caused by mutations in at least four different transcription factors, each resulting in abnormalities in neural crest development.


FIGURE 14-22 Patients with type I Waardenburg syndrome. A, Mother and daughter with white forelocks. B, A 10-year-old with congenital deafness and white forelock. C, Brothers, one of whom is deaf. There is no white forelock, but the boy on the right has heterochromatic irides. Mutations of PAX3, which encodes a transcription factor involved in neural crest development, cause type I Waardenburg syndrome. SeeSources & Acknowledgments.

Programmed Cell Death

Programmed cell death is a critical function in development and is necessary for the morphological development of many structures. It occurs wherever tissues need to be remodeled during morphogenesis, as during the separation of the individual digits, in perforation of the anal and choanal membranes, or in the establishment of communication between the uterus and vagina.

One major form of programmed cell death is apoptosis. Studies of mice with loss-of-function mutations in the Foxp1 gene indicate that apoptosis is required for the remodeling of the tissues that form portions of the ventricular septum and cardiac outflow tract (endocardial cushions), to ensure the normal positioning of the origins of the aortic and pulmonary vessels. By eliminating certain cells, the relative position of the cushions is shifted into their correct location. It is also suspected that defects of apoptosis underlie some other forms of human congenital heart disease (see Chapter 8), such as the conotruncal heart defects of DiGeorge syndrome caused by deletion of the TBX1 gene located in chromosome 22q11 (see Chapter 6). Apoptosis also occurs during development of the immune system to eliminate lymphocyte lineages that react to self, thereby preventing autoimmune disease.

Interaction of Developmental Mechanisms in Embryogenesis

Embryogenesis requires the coordination of multiple developmental processes in which proliferation, differentiation, migration, and apoptosis all play a part. For example, many processes must occur to convert a mass of mesoderm into a heart or a layer of neuroectoderm into a spinal cord. To understand how these processes interact and work together, developmental biologists typically study embryogenesis in a model organism, such as worms, flies, or mice. The general principles elucidated by these simpler, more easily manipulated systems can then be applied to understanding developmental processes in humans.

The Limb as a Model of Organogenesis

The vertebrate limb is a relatively simple and well-studied product of developmental processes. There is no genomic specification for a human arm to be approximately 1 m long, with one proximal bone, two bones in the forelimb, and 27 bones in the hand. Instead, the limb results from a series of regulated processes that specify development along three axes, the proximal-distal axis, the dorsal-ventral axis, and the anterior-posterior axis (Fig. 14-23).


FIGURE 14-23 This scanning electron micrograph of a 4-week human embryo illustrates the early budding of the forelimb. Overlaid onto the bud are the three axes of limb specification: Do-V, dorsal-ventral (dorsal comes out of the plane of the photo, ventral goes into the plane of the photo); Px-Di, proximal-distal; and A-Po, anterior-posterior. SeeSources & Acknowledgments.

Limbs begin as protrusions of proliferating cells, the limb buds, along the lateral edge of the mesoderm of the human embryo in the fourth week of development. The location of each limb bud along the anterior-posterior axis of the embryo (head-to-tail axis) is associated with the expression of a specific transcription factor at each location, Tbx4 for the hindlimbs and Tbx5 for the forelimbs, whose expression is induced by various combinations of fibroblast growth factor ligands. Thus the primarily proliferative process of limb bud outgrowth is activated by growth factors and transcription factors.

The limb bud grows primarily in an outward, lateral expansion of the proximal-distal axis of the limb (see Fig. 14-18B). Whereas proximal-distal expansion of the limb is the most obvious process, the two other axes are established soon after the onset of limb bud outgrowth. The anterior-posterior axis is set up soon after limb bud outgrowth, with the thumb considered to be an anterior structure, because it is on the edge of the limb facing the upper body. The fifth finger is a posterior structure because it is on the side of the limb bud oriented toward the lower part of the body. During limb formation, the morphogen SHH is expressed in the posterior aspect of the developing limb bud, and its expression level forms a gradient that is primarily responsible for setting up the anterior-posterior axis in the developing limb (see Fig. 14-18B). Defects in anterior-posterior patterning in the limb cause excessive digit patterning, manifested as polydactyly, or failure of complete separation of developing digits, manifested as syndactyly. The dorsal-ventral axis is also established, resulting in a palm or sole on the ventral side of the hand and foot, respectively.

One can now begin to understand the mechanisms underlying birth defect syndromes by applying knowledge from molecular developmental biology to human disorders. For example, mutations in the GLI3transcription factor gene cause two pleiotropic developmental anomaly syndromes, the Greig cephalopolysyndactyly syndrome (GCPS) and the Pallister-Hall syndrome (see Fig. 14-1). These two syndromes comprise distinct combinations of limb, central nervous system, craniofacial, airway, and genitourinary anomalies that are caused by perturbed balance in the production of two variant forms of GLI3, referred to as GLI3 and GLI3R, as shown in Figure 14-24. GLI3 is part of the SHH signaling pathway. SHH signals, in part, through a cell surface receptor encoded by a gene called PTCH1, which is concentrated in the cilium of cells during development. Mutations in PTCH1 cause the nevoid basal cell carcinoma syndrome. Also known as Gorlin syndrome, this syndrome comprises craniofacial anomalies and occasional polydactyly that are similar to those seen in GCPS, but in addition, Gorlin syndrome manifests dental cysts and susceptibility to basal cell carcinoma. By considering Gorlin syndrome and GCPS, one can appreciate that the two disorders share phenotypic manifestations precisely because the genes that are mutated in the two disorders have overlapping effects in the same developmental genetic pathway. A third protein in the SHH signaling pathway, the CREB-binding protein, or CBP, is a transcriptional coactivator of the GLI3 transcription factor. Mutations in CBP cause the Rubinstein-Taybi syndrome (see Fig. 14-5), which also shares phenotypic manifestations with GCPS and Gorlin syndrome.


FIGURE 14-24 Schematic diagram of the anterior-posterior and proximal-distal axes of the limb bud and its molecular components. In this diagram, the anterior aspect is up and the distal aspect is to the right. SHH expression occurs in the zone of polarizing activity of the posterior limb bud, and SHH is activated by the dHand gene. SHH inhibits conversion of the GLI3 transcription factor to GLI3R in the posterior regions of the limb bud. However, SHH activity does not extend to anterior regions of the bud. The absence of SHH allows GLI3 to be converted to GLI3R (a transcriptional repressor) in the anterior limb bud. By this mechanism, the anterior-posterior axis of the limb bud is established with a gradient of GLI3 versus GLI3R. SeeSources & Acknowledgments.

Concluding Comments

Many other examples of this phenomenon could be cited, but the key points to emphasize are that genes are the primary regulators of developmental processes, their protein products function in developmental genetic pathways, and these pathways are employed in related developmental processes in a number of organ systems. Understanding the molecular basis of gene function, how those functions are organized into modules, and how abnormalities in those modules cause and correlate with malformations and pleiotropic syndromes forms the basis of the modern clinical approach to human birth defects. The understanding of these developmental pathways in great detail may also provide an avenue in the future to devise therapies that target appropriate parts of these pathways.

General References

Carlson BM. Human embryology and developmental biology. ed 5. WB Saunders: Philadelphia; 2014.

Dye FJ. Dictionary of developmental biology and embryology. ed 2. Wiley-Blackwell: New York; 2012.

Epstein CJ, Erickson RP, Wynshaw-Boris AJ. Inborn errors of development: the molecular basis of clinical disorders of morphogenesis. ed 2. Oxford University Press: New York; 2008.

Gilbert SF. Developmental biology. ed 10. Sinauer Associates: Sunderland, MA; 2013.

Wolpert L, Tickle C. Principles of development. ed 4. Oxford University Press: New York; 2011.

References Specific to Particular Topics

Acimovic I, Vilotic A, Pesl M, et al. Human pluripotent stem cell-derived cardiomyocytes as research and therapeutic tools. Biomed Res Int. 2014;2014:512831.

Ross CA, Akimov S. Human induced pluripotent stem cells: potential for neurodegenerative diseases. Hum Mol Genet. 2014;23(R1):R17–R26.


1. What is the difference between regulative and mosaic development? What is the significance of these two stages of development for reproductive genetics and prenatal diagnosis?

2. Match the terms in the left-hand column with the terms that best fit in the right-hand column.


3. Match the terms in the left-hand column with the terms that best fit in the right-hand column.


4. What type of diploid cells would not be appropriate nucleus donors in an animal cloning experiment and why?

5. For discussion: Why do some mutations in transcription factors result in developmental defects even when they are present in the heterozygous state?