Principles and Mechanisms of Morphogenesis and Dysmorphogenesis

Summary

Formation of the embryo and its parts involves morphogenesis , a form-shaping process controlled by fundamental cell behaviors that result in differential growth . Perturbation of differential growth due to a genetic mutation , teratogen exposure, or a combination of the two processes results in dysmorphogenesis and the formation of structural birth defects . Structural birth defects consist of both malformations —involving perturbation of developmental events directly involved in forming a particular structure—and deformations —involving indirect perturbation of a developing structure due to mechanical forces. Malformation can involve single organs or body parts or a constellation of organs or body parts. In the latter case, if a single cause is involved, the condition constitutes a syndrome . Understanding how development occurs requires the use of animal models in which experiments can be conducted. Because developmental mechanisms are conserved across species, the use of animal models provides insight into how normal development of the human embryo occurs and how development can be perturbed by genetic mutation or environmental insult, resulting in birth defects. The tool kit for the developmental biologist’s experiments is vast, including techniques derived from the fields of cell biology, molecular biology , and genetics , combined with the classical approaches of cut-and-paste experimental embryology . Manipulation of the mouse genome has been a particularly fruitful approach for understanding how genes function during development and for developing models for human disease and birth defects. Through experimental approaches, a small number of highly conserved signaling pathways have been identified. These pathways are used repeatedly and in various combinations throughout embryonic development. Tools originally used to study mouse embryos have been adapted for use in human embryos. This has resulted in advances in reproductive technologies, such as in vitro fertilization, and recently in the development of stem cells and organoids, which can be used potentially to regenerate diseased or damaged organs.

Clinical Taster

A first-year medical genetics fellow is on full-time service for the month of May. Early in the month, she is asked to consult on an infant with cleft lip and palate and possible brain abnormalities that were identified on prenatal ultrasound. Review of the prenatal and postnatal history is significant only for an abnormal ultrasound during the 24th week of gestation that showed dilation and possible fusion of the lateral ventricles of the brain, and for premature birth at 32 weeks after the onset of preterm labor. The family history seems negative at first, but on further discussion, the fellow elicits a history of a single central incisor in the patient’s father. The physical examination shows microcephaly (small head), ocular hypotelorism (closely spaced eyes), a flat nasal bridge, and bilateral cleft lip and cleft palate ( Fig. 5.1A ). Magnetic resonance imaging (MRI) of the brain shows fusion of the left and right frontal lobes and partial fusion of the parietal lobes characteristic of semilobar holoprosencephaly . Genetic testing discovers a deleterious mutation in one allele of the SONIC HEDGEHOG (SHH) gene in both the patient and her father. As the father and daughter carried the same mutation and both are heterozygous for this mutation, she is curious about why the child is more severely affected. It is explained that the syndrome is highly variable. Phenotypic variability may be due to differences in other genes that affect the expression or function of SHH from the wildtype allele. Alternatively, the daughter may have been exposed to a teratogen that decreased SHH expression or function from the wildtype SHH allele (see Chapter 17 for further coverage).

Near the end of the month, the fellow is called to the nursery to examine a newborn with limb anomalies. She finds an otherwise healthy, full-term girl with polydactyly (extra digits) of both hands and feet occurring on the thumb and great toe side (preaxial; see Fig. 5.1B ). Chromosome analysis shows a translocation involving chromosomes 5 and 7, with the chromosome 7 breakpoint occurring distant from the SHH gene, but in a region known to affect SHH expression in the limb. Disruption of these regulatory elements results in ectopic SHH expression and is known to cause preaxial polydactyly.

The fellow is impressed by the variability of manifestations caused by different defects in the same gene (SHH), with one mutation causing brain and face abnormalities and another causing limb defects.

Principles of Morphogenesis and Dysmorphogenesis

Having described the initial steps in embryogenesis in Chapter 1, Chapter 2, Chapter 3, Chapter 4 , it is appropriate to pause to lay down the basic groundwork for understanding the concepts of normal and abnormal embryology covered in later chapters. Moreover, because these concepts have been formulated using animal models for experimental studies, it is important to understand the attributes each of these models provides for understanding human development. Finally, experimental techniques are described to explain how experiments are conducted in the field of developmental biology, and signaling pathways are covered to place molecules that control developmental events into context.

As covered in preceding chapters, the initially flat three-layered embryonic disc undergoes morphogenesis to form a three-dimensional embryo with a tube-within-a-tube body plan and the beginnings of rudiments that will form all of the adult organs and systems. In this chapter, we consider how morphogenesis occurs and how it goes awry during the formation of birth defects . Morphogenesis results from differential growth . Differential growth is driven by a small number of fundamental cellular behaviors , such as changes in cell shape, size, position, number, and adhesivity. If these behaviors are perturbed during embryogenesis by a genetic mutation, environmental insult (i.e., a teratogen ), or a combination of the two, differential growth is abnormal and dysmorphogenesis results with the formation of a structural birth defect.

Dysmorphogenesis can result from both malformation and deformation . Malformations consist of primary morphologic defects in an organ or body part resulting from abnormal developmental events that are directly involved in the development of that organ or body part. For example, failure of the neural groove to close results in a malformation called a neural tube defect . Similarly, failure of the digits to separate fully results in syndactyly , that is, fusion of the digits. Deformations consist of secondary morphologic defects that are imposed upon an organ or body part owing to mechanical forces, that is, deformations affect the development of an organ or body part indirectly . For example, if insufficient amniotic fluid forms (i.e., oligohydramnios), deformation of the feet can occur as the result of mechanical constraints, leading to club foot. Dysmorphogenesis can occur in an isolated organ or body part or as a pattern of multiple primary malformations with a single cause. In the latter case, the condition is referred to as a syndrome . Common examples, covered elsewhere in the text, include Down syndrome (trisomy 21) and 22q11.2 deletion syndrome—two syndromes that result from chromosomal abnormalities.

Other syndromes can result from teratogen exposure. A common example is fetal alcohol syndrome , also known as fetal alcohol spectrum disorder . This disorder affects 2 in 1000 live-born infants ( Fig. 5.2 ). Fetal alcohol syndrome is most prevalent in alcoholic women, especially those in their third or fourth pregnancies, suggesting that maternal health status interacts with alcohol to produce the syndrome. Nevertheless, consumption of amounts of alcohol as low as 80 g/day (i.e., between two and three shots of a grain liquor such as rum) by a non-alcoholic woman during the first month of pregnancy can cause significant defects, and it has been suggested that even a single binge may be teratogenic. In addition, chronic consumption of even small amounts of alcohol even in later pregnancy can be dangerous, as it may affect development of the fetal brain, resulting in behavioral and cognitive deficiencies that may last a lifetime. Thus, there is no known safe level of alcohol consumption during pregnancy.

Common components of the disorder include defects of brain and face development, namely, microcephaly (small head), short palpebral fissures (eye openings), epicanthal folds (folds over eye lids), a low nasal bridge with a short nose, flat midface, minor external ear anomalies, and jaw anomalies, including a thin upper lip with indistinct philtrum and micrognathia (small jaw) (see Fig. 5.2 ).

Animal Models

The aim of research in developmental biology/embryology is to understand how development occurs at tissue, cellular, and molecular levels. This aim speaks largely to our innate curiosity to understand nature and how it works. An additional aim is to understand how normal development can go awry, resulting in birth defects, particularly in humans. Understanding how both normal development and abnormal development occur could lead to ways to detect (diagnose), prevent, and cure birth defects. Thus, this aim speaks to our desire to prevent and relieve human suffering.

Although the only perfect organism for studying how the human embryo develops is the human embryo, animal models provide useful surrogates because of the principle that developmental mechanisms are highly conserved from organism to organism ( Fig. 5.3 ). Five animal models have been especially useful for deciphering mechanisms and principles of embryogenesis: one invertebrate and four vertebrates. In vertebrates, similarity among the animal models is most pronounced at the phylotypic stage of development when embryos are starting to undergo organogenesis. At this stage of development, the basic body plan has been established and embryos are characterized by the presence of somites, pharyngeal arches, ventral heart tube, head, eyes, and tail ( Fig. 5.4 ). Many conserved genes that establish the patterning of the embryo and organs (e.g., Hox genes) are also expressed during the phylotypic stage. Individual animal models provide complementary information, which, when compared across animal models, provides considerable insight into how the human embryo develops. Amphibians and zebrafish are also used to understand the ability to regenerate organs such as the limbs, tail, lens, and heart. All of these models are practical to obtain, use, and maintain in the laboratory, and all can be acquired and used throughout the year (i.e., they are not seasonal breeders). The unique strengths of each of these organisms for understanding mechanisms of development are covered in the following.

Using Animal Models to Predict Human Risk

Developmental toxicologists usually choose animal models different from those used by developmental biologists because the goal in their studies is different: to predict the risk to humans of exposure to drugs and potential environmental pollutants. Thus, such studies are concerned with similarities between animal models and human placentation (i.e., How similar is placental function?) or pharmacodynamics (i.e., How similar is the metabolism of drugs?). Following the thalidomide-induced birth defects in the 1960s, new guidelines were introduced and now at least two animal models must be used in toxicity studies (see Chapter 20 for further coverage). Rodents or rabbits are typically used for initial studies, and if the results of these studies warrant further testing, non-human primate models are used for later studies.

Experimental Techniques

Understanding how normal development and abnormal development occur requires a detailed understanding of what happens during development—that is, a detailed understanding of descriptive embryology . However, descriptive embryology alone cannot reveal how development occurs. Descriptive embryology provides a catalog of developmental events, which, when carefully studied and reflected upon, can lead to the formulation of hypotheses about how a developmental event occurs. The investigator then designs and conducts tests of the formulated hypotheses. Hypotheses are tested through a series of experiments (specific manipulations that usually perturb a developmental process) as compared with controls (non-specific manipulations used to ensure that results obtained from particular manipulations are specific and not artifactual). Through this approach, hypotheses are refuted, modified, or supported (never truly proven to be correct, but often proven to be incorrect). The cycle continues as new hypotheses are crafted, based on additional data obtained through experiments, leading to new experimental tests of their veracity.

Conducting experiments on developing model embryos constitutes the science of experimental embryology . Classically, experimental embryology has been used to define the tissue and cellular basis of development through a series of microsurgical manipulations. More recently, experimental embryology has merged with cell biology, molecular biology, and genetics, allowing investigators to define the molecular-genetic basis of development.