The Modern Era: The Impact of Molecular and Cellular Biology
The Molecular Biology Revolution
The molecular biology revolution in the middle of the Twentieth Century provided the means to study the role of genes in development that Wilson and his contemporaries lacked. The key technological advance for the study of gene control of development was the ability to isolate and clone genes. The roles that individual genes play in development could then be assessed directly. The patterns of expression of individual genes could be followed by tracing the products of their expression in the embryo.
Analyses of expression of specific genes during development have revealed regions of those genes that regulate their expression and have identified proteins in the nuclei of embryonic cells that interact with those regions of genes to mediate their regulation. Furthermore, regulatory cascades have been demonstrated in which early-acting genes encode nuclear proteins that regulate genes that are expressed later.
Much of the current research in developmental biology involves attempts to understand the cellular and intercellular events that signal the nucleus to express genes or initiate a sequence of gene expression.
Among the most powerful techniques in the armament of contemporary biologists is the ability to introduce cloned genes into embryos and assess their effects on development. Another major advance has been the development of techniques to eliminate or "knock out" specific genes and determine the effects on development. These two techniques allow investigators to test directly the roles of specific genes during development.
Molecular biology obtained a very powerful tool to facilitate the study of nucleic acids when the polymerase chain reaction (PCR) was developed. This technique allows investigators to amplify specific sequences of DNA many-fold from a minute amount of starting material, using oligonucleotide primers that flank the region of interest in DNA. Not only did PCR facilitate gene cloning, but it provided developmental biologists with a new tool to study RNA from embryos. Because of their small size, embryos are notoriously poor sources of RNA. This is not critical if one is able to collect very large numbers of synchronously-developing embryos. However, that is not always possible. With a modification of the PCR technique, called RT-PCR, RNA is isolated, a cDNA copy of the RNA is made using the enzyme reverse transcriptase and the cDNA is then amplified many-fold using PCR. Thus, a virtually unlimited amount of DNA representative of RNA at a particular stage of development or from a particular region of the embryo can be produced.
Drosophila: The Molecular Rosetta Stone
The molecular biology era has brought with it unprecedented opportunities for understanding how development occurs. Its most powerful applications and much of the explosive progress in contemporary developmental biology come when molecular biology is coupled with genetic analysis. Let's explore an example of this synergism by considering the development of the eye of the fruit fly, Drosophila melanogaster.
The eye of Drosophila is a so-called compound eye, consisting of multiple facets with photoreceptors that detect light and transmit light images to the brain. Although its structure is very different from that of the human eye and from the simple photoreceptors in primitive worms, the eyes of Drosophila serve essentially the same function as they do in these other organisms.
Evidence to answer some of these questions has come from studies of mutations in a gene called eyeless in Drosophila. These mutations can either cause eye deficiencies or eliminate the eyes altogether. Furthermore, ectopic expression of eyeless (i.e., expression of the gene in an abnormal location) can result in the formation of retinal tissue in those locations. Thus, expression of the wild-type allele of eyeless is necessary for eye development. This gene is at least one of the genes that is capable of triggering the events that result in the formation of eyes.
Mutations that reduce or eliminate eyes have also been observed in mammals. These include Small eye in mice and Aniridia in humans. Molecular analyses have shown that these genes have substantial similarities in their nucleotide sequences to the Drosophila eyeless gene. These similarities have apparently arisen because Drosophila, mice and humans were all derived from ancestors with a similar gene. Genes that share substantial similarity are called homologous genes or homologues. These genes that control eye development are members of a family of genes called Pax-6. Pax-6 homologues have been discovered in organisms as diverse as mammals, squids, ascidians, insects, cephalopods, and nemerteans (Halder et al., 1995; Tomarev et al., 1997).
Pax-6 is now recognized as a master control gene for production of eyes in animals of all sorts. The Pax-6 genes in diverse organisms are so similar in their function that expression of the mouse Small eye gene will cause the formation of ectopic eyes in Drosophila . Although the details of eye development differ dramatically from one species to another, their specification is dependent upon expression of the Pax-6 gene. Pax-6 genes are regulators of gene transcription. Thus, they must have target genes that mediate their role as master control genes. In fact, a complex cascade of events that results in eye formation is triggered by Pax-6 gene expression. Differences among these downstream events will result in different eye morphologies. One of those downstream genes in Drosophila is eyes absent, which also has homologues in vertebrates (Xu et al., 1997).
We have described how the analysis of a mutation that controls the formation of eyes in Drosophila has led to the discovery of a family of genes that controls eye formation in other organisms and has facilitated the discovery of the sequence of events that culminate in formation of these crucial organs. This is but one example of the ways in which comparisons between genes in Drosophila and other organisms have helped to untangle the genetic basis for basic developmental processes and lead to unprecedented progress in understanding embryonic development. Drosophila has acquired its role as a reference organism through the discovery of a large number of mutations that affect specific developmental processes and through extensive analyses of the abnormalities that they produce and the roles of their wild-type counterparts in normal development.
Thus, developmental biologists can identify a gene that affects development in one organism like Drosophila and use it to search for genes in other organisms. This approach is similar to the process that led to the decoding of ancient Egyptian writing using the Rosetta Stone. The stone, which was written in 196 BC and discovered in 1799 AD near the seaside town of Rosetta in lower Egypt, bears the same inscription written in three ways: Greek, hieroglyphs and demotic, which is a cursive script derived from hieroglyphs. By comparing the hieroglyphic and demotic texts to the Greek text, the basis for ancient Egyptian writing was uncovered. The comparison of nucleotide sequences of genes between distantly-related organisms has had the same sort of profound impact in developmental biology as the Rosetta Stone has had for understanding ancient Egyptian writing.
Among the most striking mutations in Drosophila are those that affect the basic body plan of the fly. Geneticists realized that these mutations were valuable for understanding how genes regulate the process of development. The Drosophila body consists of three main regions: the head, thorax and abdomen. Mutations were identified that altered the developmental processes that formed these regions. Such alterations imply that the wild-type alleles of these mutations are responsible for controlling formation of the embryo at a very basic level.
For example, a group of genes were discovered that are responsible for specifying the defining characteristic of flies (dipterans): a single pair of wings. The insect thorax has three segments: the prothorax, the mesothorax and the metathorax. Each segment has a pair of legs on it, and the mesothorax also bears a pair of wings. Most insects also have a pair of wings on the metathorax, but in dipterans the metathorax has instead a pair of balancing organs called halteres, which look like rudimentary wings. Mutations of certain genes of the bithorax complex of Drosophila can produce a four-winged creature in which the halteres are replaced by a second pair of wings. Thus, cells in one region of the fly behave as if they are in another. This is a so-called homeotic transformation. The mutations responsible for such a conversion are called homeotic mutations. Edward B. Lewis at the California Institute of Technology studied extensively the genetic basis for homeotic transformations.
(See Browder et al., 1991, Fig. 1.6; Gilbert, Fig. 14.29; Kalthoff, Fig. 21.32; Shostak, Fig. 22.8; Wolpert et al., Fig. 5.36)
Lewis discovered that the bithorax complex consists of a family of genes that control segmentation of the posterior thorax and abdomen along the anterior-posterior body axis. Another complex that is linked to the bithorax complex, the antennapedia complex, controls segment identity in the head and anterior thorax. One of the most important discoveries that Lewis made was that the genes at the beginning of these complexes control the most anterior body segments, whereas genes further down the genetic map control progressively more posterior segments. This is the colinearity principle.
Lewis also showed that these genes have overlapping domains and that interactions among them are necessary to specify the development of individual body segments (Lewis, 1978). The Drosophila homeotic genes have subsequently been cloned and used to identify their homologues (the HOX genes) in higher organisms. The single homeotic gene cluster in Drosophila has been duplicated during evolution, resulting in four complexes of HOX genes in mammals. Conservation of the gene complexes has been so complete that the genes within the HOX gene clusters of mammals occur in the same order as they do in Drosophila and exhibit overlapping domains of expression along the anterior-posterior body axis, in agreement with the colinearity principle.
Clearly, the basic body plan of Drosophila is genetically controlled. Thus, a systematic and comprehensive search for genes that establish the body plan (the so-called pattern formation genes) should provide the means for discovering the blueprint for forming the fly. In 1980, two German scientists, Christiane Nüsslein-Volhard and Eric Wieschaus, published the first results of such a search, which identified and characterized many of the genes that produce the basic body plan of Drosophila. These genes control the anterior-posterior body plan and establish the segments from which the major body regions are produced. These pattern formation genes were cloned, which has allowed investigators to compare them to their counterparts in other organisms. Those studies have shown that most of the pattern formation genes of Drosophila have homologues in both higher and lower organisms. They may not play exactly the same role in humans as they do in flies, but they are critical components of the genetic toolkit that is needed to construct a complex organism.
Like the Pax-6 gene, many of the pattern formation genes have been shown to encode proteins that regulate the expression of other genes. Ultimately, complex genetic pathways were unravelled. For their pioneering work, Lewis, Nusslein-Volhard and Wieschaus were awarded The Nobel Prize in Physiology or Medicine for 1995.
The Cellular Basis of Development
A fully-developed human consists of approximately 1 x 10 ^14 cells. Coordinating the activities and locations of that many cells is a monumental task of logistics. Each cell has its own location and particular role to play in the body. Without coordination, chaos would result. Production of embryonic form and structure (morphogenesis) depends upon the concerted activities of many cells. Cells must often move relatively large distances within the embryo, and once they have arrived at their final destinations, they must establish stable multicellular structures with specific morphologies and functions. These activities require that cells have the ability to control their shape and to interact effectively with their environment. An understanding of how individual cells acquire their location, form and function during development is also a monumental task. Cell biology has provided the tools to study cellular behavior during development, and developmental biologists have seized upon the opportunities that these technologies provide for understanding how development proceeds.
Effective interactions of cells with both their non-cellular environment and other cells are key to coordinating cellular activities. In order to interact with their surroundings, cells must perceive signals and have the ability to respond appropriately. These signals are often large molecules such as peptides or large proteins that cannot enter their target cell. Hence, cells must have receptors on their surface that enable them to detect these molecules in their environment and transduce those external signals into signals that can be transmitted into cell interior. Signals are often passed within the cell from one molecule to another numerous times before they reach their final destination, which could be either the nucleus or a cytoplasmic organelle. This is referred to as a signal transduction cascade. The most commonly-used cascades involve a sequence of phosphorylations. Phosphorylation can be like a binary switch that alters molecular function. As each consecutive member of the cascade is phosphorylated, it - in turn - phosphorylates the next member. A profound change in cells can be produced when a signal arrives in the nucleus and either activates or represses the expression of a specific gene. Such is the consequence of signaling by growth factors during embryonic induction.
Mutations of the genes encoding proteins of this system can occur later in life and deregulate cellular function, particularly growth control, leading to cancer. It is ironic that the system that is so important in coordinating cell growth during embryonic development can also be so destructive in later life if it malfunctions.
A Relevant Science
Developmental biology explores the continuity of life itself. As we acquire a deeper understanding of the developmental process, we not only satisfy our curiosity about one of the most fascinating and elegant processes in nature, but we acquire additional tools that enable us to intervene as never before in the reproductive process.
The Brave New World of Animal Cloning
Driesch's experiments in separating sea urchin blastomeres led to the concept that nuclei of cleavage-stage blastomeres retain the entire genetic repertoire that is necessary to program the development of the complete organism. The retention of complete developmental potential by cleavage-stage nuclei is called totipotency. Since Driesch's original observation, investigators have attempted to discover whether potency is lost as development proceeds. In the 1950's Robert Briggs and Thomas King developed an elegant procedure to test nuclear potency using the frog Rana pipiens.
By transplantation of nuclei from cells of embryos into enucleated frog eggs, Briggs and King demonstrated that embryonic nuclei remain totipotent during cleavage, but that potency is progressively lost as development proceeds past the cleavage stage. They also demonstrated that multiple totipotent nuclei from a single embryo could be transplanted into a number of enucleated eggs to produce multiple individuals that were genetically identical. Thus, the donor embryo could be cloned. Various investigators have attempted to demonstrate whether cells of later-stage embryos, tadpoles or even adults retain totipotency. Conventional wisdom was that nuclei eventually undergo changes during cell differentiation that are so drastic that they cannot be coaxed into reiterating the complete developmental program.
Investigators have now shown that cells of mammalian adults can be reprogrammed to substitute for the zygote nucleus, allowing for cloning of sheep and mice (Campbell et al., 1996; Wakayama et al., 1998). Thus, nuclear changes during cell differentiation are not necessarily irreversible. The implications of cloning of mammals are far-reaching and have caused considerable anxiety as the possibility of cloning humans has become very real.
As with cloning, recent progress in understanding and manipulating the reproductive process has posed new ethical challenges for society as well as presenting new opportunities for individuals. Manipulation of the human female menstrual cycle with steroid hormones led to the development of the contraceptive pill, which has enabled couples to prevent unplanned pregnancies. The development of in vitro reproduction technology has enabled couples who would otherwise be unable to conceive a child to do so. Embryos produced by in vitro technology can be frozen and later implanted in a uterus. Whereas this is done routinely with domesticated animals with little anguish, the fate of frozen human embryos is subject to considerable debate.
Techniques have been developed for making transgenic animals, in which embryos are produced that incorporate cloned genes. This is a powerful experimental technique for use with flies, mice and frogs. However, should it also be applied to humans? What genes, and for what purposes? Clearly, developmental biology presents us with new opportunities and new challenges - not only for scientists, but for society as a whole.
Molecular Biology Techniques
The Cellular Basis of Development
Drosophila: The Molecular Rosetta Stone
1995 Nobel Prize Announcement summarizes the accomplishments of Lewis, Nusslein-Volhard and Wieschaus.
A Relevant Science
Model Systems in Development
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Gilbert, S.F. 1997. Developmental Biology. Fifth Edition. Sinauer.
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Leon Browder & Laurie Iten (Ed.) Dynamic Development
Last revised Monday, August 17, 1998