Early Hypotheses of Development in Diverse Organisms
From the earliest times, people have noted that a particular organism produced offspring very much like itself in structure and function, and the fully formed adult consisted of numerous cell types and other highly specialized organs and structures, yet it came from one simple egg cell. How could such simplicity, observed in the egg cell, give rise to such complexity in the adult and always reproduce the same structures?
In the seventeenth century, the preformationism hypothesis of embryonic development was advanced to answer these questions by asserting that a miniature organism existed in the sperm or eggs. After fertilization, this miniature creature simply grew into the fully formed adult. Some microscopists of the time claimed to see a homunculus, or little man, inside each sperm cell. That the preformationism hypothesis was ill-conceived became apparent when others noted that developmental abnormalities could not be explained satisfactorily, and it became clear that another, more explanatory hypothesis was needed to account for these inconsistencies.
In 1767, Kaspar Friedrich Wolff published his epigenesis hypothesis, in which he stated that the complex structures of chickens developed from initially homogeneous, structureless areas of the embryo. Many questions remained before this new hypothesis could be validated, and it became clear that the chick embryo was not the best experimental system for answering them. Other investigators focused their efforts on the sea squirt, a simpler organism with fewer differentiated tissues.
Work with the sea squirt, a tiny sessile marine animal often seen stuck to submerged rocks, led to the notion that development followed a mosaic pattern. The key property of mosaic development was that any cell of the early embryo, once removed from its surroundings, grew only into the structure for which it was destined or determined. Thus the early embryo consisted of a mosaic of cell types, each determined to become a particular body part. The determinants for each embryonic cell were found in the cell’s cytoplasm, the membrane-bound fluid surrounding the nucleus. Other scientists, most notably Hans Driesch
in 1892 and Theodor Boveri (working with sea urchin embryos) in 1907, noted that a two-cell-stage embryo could be teased apart into separate cells, each of which grew into a fully formed sea urchin. These results appeared to disagree with the mosaic developmental mechanism. Working from an earlier theory, the germ-plasm theory of August Weismann
(1883), Driesch and Boveri proposed a new mechanism called regulative development.
The key property of regulative development was that any cell separated from its embryo could regulate its own development into a complete organism. In contrast to mosaic development, the determinants for regulative development were found in the nuclei of embryonic cells, and Boveri hypothesized that gradients of these determinants, or morphogens, controlled the expression of certain genes. Chromosomes were assumed to play a major role in controlling development; however, how they accomplished this was not known, and Weismann mistakenly implied that genes were lost from differentiated cells as more and more specific structures formed.
In spite of the inconsistencies among the several hypotheses, a grand synthesis was soon formed. Working with roundworm, mollusk, sea urchin, and frog embryos, investigators realized that both mosaic and regulative mechanisms operate during development, with some organisms favoring one mechanism over the other. The most important conclusion coming from these early experiments suggested that certain genes on the chromosomes interacted with both the cytoplasmic and nuclear morphogenetic determinants to control the proliferation and differentiation of embryonic cells. What exactly were these morphogens, where did they originate, and how did they form gradients in the embryo? How did they interact with genes?
The Morphology of Development
Before the “how and why” mechanistic questions of morphogens could be answered, more answers to the “what happens when” questions were needed. Using new, powerful microscopes in conjunction with cell-specific stains, many biologists were able to precisely map the movements of cells during embryogenesis and to create fate maps of such cell migrations. Fate maps were constructed for sea squirt, roundworm, mollusk, sea urchin, and frog embryos, which showed that specific, undifferentiated cells in the early embryo gave rise to complex body structures in the adult.
In addition, biologists observed an entire stepwise progression of intervening cell types and structures that could be grouped into various stages and that were more or less consistent from one organism to another. Soon after fertilization, during the very start of embryogenesis, specific zones with defining, yet structureless, characteristics were observed. These zones consisted of gradients of different biochemical compounds, some of which were morphogens, and they seemed to function by an induction process. Some of these morphogen gradients existed in the egg before fertilization; thus it became evident that the egg was not an entirely amorphous, homogeneous cell, but one with some amount of preformation. This preformation took the form of specific morphogen gradients.
After these early embryonic events and more cell divisions, in which loosely structured patterns of morphogen gradients were established to form the embryo’s polar axes, the cells aggregated into a structure called a blastula, a hollow sphere of cells. The next stage involved the migration of cells from the surface of the blastula to its interior, a process called gastrulation. This stage is important because it forms three tissue types: the ectoderm (for skin and nerves), the mesoderm (for muscle and heart), and the endoderm (for other internal organs). Continued morphogenesis generates a neurula, an embryo with a developing nervous system and backbone. During axis formation and cell migrations, the embryonic cells are continually dividing to form more cells that are undergoing differentiation into specialized tissue types such as skin or muscle. Eventually, processes referred to as organogenesis transform a highly differentiated embryo into one with distinct body structures that will grow into a fully formed adult.
Experimental Systems for Studying Developmental Genes
In order to understand the details of development, biologists have traditionally studied organisms with the simplest developmental program, those with the fewest differentiated cell types that will still allow them to answer fundamental questions about the underlying processes. Sea squirts and roundworms have been valuable, but they exhibit a predominantly mosaic form of development and are not the best systems for studying morphogen-dependent induction. Frog embryogenesis, with both mosaic and regulative processes, was well described and contributed greatly to answering the “what and when” questions of sequential events, but, at the time, no effective genetic system existed for examining the role of genes in differentiation necessary for answering the “why and how” questions.
Historically, the issue was resolved by focusing once again on the morphogens. These mediators of cellular differentiation were found only in trace amounts in developing embryos and thus were difficult, if not impossible, to isolate in pure form for experimental investigation. An alternative to direct isolation of morphogens was to isolate the genes that make the morphogens. The organism deemed most suitable for such an approach was the fruit fly
Drosophila melanogaster
, even though its development was more complex than that of the roundworm. Fruit flies could be easily grown in large numbers in the laboratory, and many mutants could be generated quickly; most important, an effective genetic system already existed in Drosophila, making it easier to create and analyze mutants. The person who best used the fruit fly system and greatly contributed to the understanding of developmental genetics was Christiane Nüsslein-Volhard, who shared a 1995 Nobel Prize in Physiology or Medicine with Edward B. Lewis and Eric Wieschaus.
The Genes of Development
The first important developmental genes discovered in Drosophila were the latest acting in morphogenesis, which led to the isolation of the gene for one of the morphogens controlling the anterior-posterior axis of the embryo, the bicoid gene. The study of mutants, such as those with legs in place of antennae, allowed the discovery of many other developmental genes, referred to generally as homeotic genes.
The bicoid gene’s discovery validated the gradient hypothesis originally proposed by Boveri because its gene product functioned as a typical morphogen. It was a protein that existed in the highest concentration at the egg’s anterior pole and diffused to lower concentrations toward the posterior pole, thus forming a gradient. Through the use of more fruit fly mutants, geneticists showed that the bicoid protein stimulated the gene expression of another early gene, called hunchback, which in turn affected the expression of other genes: Krüppel
and knirps. The bicoid protein controls the hunchback gene by binding to the gene’s control region.
Since these initial discoveries, a plethora of new developmental genes have been discovered. It is now clear that some fifty genes are involved in development of a fruit fly larva from an egg, with yet more genes responsible for development of the larva into an adult fly. These genes are grouped into three major categories: maternal effect genes, segmentation genes, and homeotic genes. Maternal effect genes include the bicoid gene. These genes, produced by special “nurse” cells of the mother, make proteins that contribute to the initial morphogen gradients along the egg’s axes before fertilization. Segmentation genes comprise three subgroups: gap, pair-rule, and segment polarity genes. Each of these types of segmentation genes determines a different aspect of the segments that make up a developing fruit fly. The hunchback, Krüppel, and knirps genes are all gap genes. Homeotic genes ultimately determine the segment
identity of previously differentiated cell groups.
Pattern Formation
Through the use of highly specific stains to track the morphogens in normal and mutant fruit fly embryos, a fascinating picture of the interactions among developmental genes has emerged. Even before fertilization, shallow, poorly defined gradients are established by genes of the mother, such as the bicoid gene and related genes. These morphogen gradients establish the anterior-to-posterior and dorsal-to-ventral axes. After fertilization, these morphogens bind to the control regions of gap genes, whose protein products direct the formation of broadly defined zones that will later develop into several specific segments. The gap proteins then bind to the control regions of pair-rule genes, whose protein products direct further refinements in the segmentation process. The last group of segmentation genes, the segment polarity genes, direct the completion of the segmentation patterns observable in the embryo and adult fly, including definition of the anterior-posterior orientation of each segment. Homeotic genes then define the specific functions of the segments, including what appendages will develop from each one. Mutations in any of these developmental genes cause distinct and easily observed changes in the developing segment patterns. Genes such as hunchback, giant, gooseberry, and hedgehog were all named with reference to the specific phenotypic changes that result from improper control of segmentation.
Homeotic genes are often called the master genes because they control large numbers of other genes required to make a whole wing or leg. Several clusters of homeotic genes have been discovered in Drosophila. Mutations in a certain group of genes of the bithorax complex result in adult fruit flies with two sets of wings. Similarly, mutations in some of the genes in the antennapedia complex can result in adult fruit flies with legs, rather than antennae, on the head.
A general principle applying to developmental processes in all organisms has emerged from the elegant work with Drosophila mutants: finer and finer patterns of differentiated cells are progressively formed in the embryo along its major axes by morphogens acting on genes in a cascading manner, in which one gene set controls the next in the sequence until a highly complex pattern of differentiated cells results. Each cell within its own patterned zone then responds to the homeotic gene products and contributes to the formation of distinct, identifiable body parts.
Another important corollary principle was substantiated by the genetic analysis of development in Drosophila and other organisms: in direct contrast to Weismann’s implication about gene loss during differentiation, convincing evidence showed that genes were not systematically lost as egg cells divided and acquired distinguishing features. Even though a muscle cell was highly differentiated from a skin cell or a blood cell, each cell type retained the same numbers of chromosomes and genes as the original, undifferentiated, but fertilized egg cell. What changed in each cell was the pattern of gene expression, so that some genes were actively transcribed, whereas other genes were turned off. The morphogens, working in complex combinatorial patterns during the course of development, determined which genes would stay “on” and which would be turned “off.”
Modern Tools for Studying Developmental Genetics
Innovations in genetic manipulation technologies have transformed the study of developmental genetics. Creation of specific DNA alterations—ranging from single base-pair changes (called point mutations) to large-scale deletions or rearrangements of chromosome segments—provides a unique forum for researchers to assess gene function during development. Moreover, conditional mutations, wherein altered gene products are only produced within certain tissues or at specific times during development, allow for the study of gene function within the context of one or more systems of interest. The ability to create designer mutations was made possible through the work of Mario R. Capecchi and Oliver Smithies, who conceptualized and studied the use of homologous
recombination for gene modification. Their work, combined with murine embryonic stem cell technologies pioneered by Martin J. Evans, gave birth to the first mice with targeted gene mutations. For these discoveries, Capecchi, Evans, and Smithies were awarded the 2007 Nobel Prize in Physiology or Medicine. With genome sequences complete for a number of model organisms, the possibilities for targeted mutations are limitless.
The zebra fish (Danio rerio) is a model organism uniquely suited to studies of developmental genetics. Genetic manipulations are possible with zebra fish that are not possible with higher model organisms. The effects of gene overexpression can be studied in zebra fish through direct injection of mRNA or DNA constructs into one- to four-cell stage embryos. Underexpression or “knock down” approaches to studying gene function in zebra fish include the use of morpholinos, antisense oligonucleotides that bind to complementary mRNA transcripts and either prevent their translation or inhibit proper splicing. Morpholinos have become a popular technique for assessing developmental gene function in zebra fish.
The study of developmental genetics has also benefited from improvements in the ability to visualize cell lineage and movements. Embryonic cells can be tagged with constructs that express markers continuously, at a desired time point, or within a target tissue type. Unlike injectable dyes, labeling is noninvasive and is not diluted by successive cell divisions.
The next frontier for the study of developmental genetics is the integration of the vast resources of molecular, cellular, and genetic data through a systems approach. Rather than assess gene function, cell division, and cell movements in isolation, these data can be integrated through computational methodology to generate graphic models. This technology is still in its infancy, but promises to yield a better understanding of the networks that underpin development.
Impact and Applications
The discovery and identification of the developmental genes in Drosophila and other lower organisms led to the discovery of similarly functioning genes in higher organisms, including humans. The base-pair sequences of many of the developmental genes, especially shorter subregions coding for sections of the morphogen that bind to the control regions of target genes, are conserved, or remain the same, across diverse organisms. This conservation of gene sequences has allowed researchers to find similar genes in humans. For example, thirty-nine Hox genes located in four clusters have been found in mice and humans, even though only eight homeobox genes localized in a single cluster were initially discovered in Drosophila. Some of the late-acting human homeobox genes are responsible for such developmental abnormalities as fused fingers and extra digits on the hands and feet. One of the most interesting abnormalities is craniosynostosis, a premature fusion of an infant’s skull bones that can cause intellectual disabilities. In 1993, developmental biologist Robert Maxson and his research group at the University of Southern California’s Norris Cancer Center were the first to demonstrate that a mutation in a human homeobox gene MSX2
was directly responsible for craniosynostosis and other bone/limb abnormalities requiring corrective surgeries. Maxson made extensive use of “knockout” mice, genetically engineered mice lacking particular genes, to test his human gene isolates. He and his research group made great progress in understanding the role of the MSX2 gene as an inducer of surrounding cells in the developing embryo. When this induction process fails because of defective MSX2 genes, the fate of cells destined to participate in skull and bone formation and fusion changes, and craniosynostosis occurs.
A clear indication of the powerful cloning methods developed in the late 1980s was the discovery and isolation in 1990 of an important mouse developmental gene called brachyury
(“short tails”). The gene’s existence in mutant mice had been inferred from classical genetic studies sixty years prior to its isolation. In 1997, Craig Basson, Quan Yi Li, and a team of coworkers isolated a similar gene from humans and named it T-box brachyury (TBX5). Discovered first in mice, the “T-box” is one of those highly conserved subregions of a gene, and it allowed Basson and Li to find the human gene. When mutated or defective in humans, TBX5 causes a variety of heart and upper limb malformations referred to as Holt-Oram syndrome. TBX5 codes for an important morphogen affecting the differentiation of embryonic cells into mesoderm, beginning in the gastrulation phase of embryonic development. These differentiated mesodermal cells are destined to form the heart and upper limbs.
One of the important realizations emerging from the explosive research into developmental genetics in the 1990s was the connection between genes that function normally in the developing embryo but abnormally in an adult, causing cancer. Cancer cells often display properties of embryonic cells, suggesting that cancer cells are reverting to a state of uncontrolled division. Some evidence indicates that mutated developmental genes participate in causing cancer. Taken together, the collected data from many isolated human developmental genes, along with powerful reproductive and cloning technologies, promise to lead to cures and preventions for a variety of human developmental abnormalities and cancers.
Key Terms
differentiation
:
the process in which a cell establishes an identity that is distinct from its parent cell, usually involving alterations in gene expression
epigenesis
:
the formation of differentiated cell types and specialized organs from a single, homogeneous fertilized egg cell without any preexisting structural elements
fate mapping
:
following the movements of a cell and its descendants during development, often through introduction of a temporary or permanent marker into the cell
gene expression
:
the combined biochemical processes, called transcription and translation, that convert the linearly encoded information in the bases of DNA into the three-dimensional structures of proteins
induction
:
the process by which a cell or group of cells signals an adjacent cell to pursue a different developmental pathway and so become differentiated from its neighboring cells
morphogen
:
a chemical compound or protein that influences the developmental fate of surrounding cells by altering their gene expression or their ability to respond to other morphogens
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