Homeosis

The body plans of advanced animals and plants can be viewed as a series of segments with unique identities. This is especially obvious in the annelids (segmented worms), but even in vertebrates, the muscular regions and the backbone are segmented. Occasionally, one segment takes on the identity of another segment of the same organism. This is called homeosis, and it was first described in 1984. Numerous examples of homeosis have been cited, including the antennapedia mutant of the fruit fly, which has a leg that develops in the antennal socket of the head. Much has been learned about developmental patterns in organisms and about the evolution of these patterns from the study of homeotic mutants.

The Genetic Control of Body Plans

The role of homeosis in elucidating genetic control of overall body plans is best illustrated in the development of the fruit fly. Early in its embryogenesis, the basic plan for the adult fly form is established, and this information is stored in imaginal disks (derived from imago, which is the adult form of an insect) through three larval stages and associated molts. Imaginal disks are small groups of cells that differentiate adult structures after the last larval molt. The early determination of these imaginal disks for specific developmental fates is controlled by a hierarchy of genes. This hierarchy of gene regulation has been carefully documented for the establishment of the anterior-posterior axis (a line running from the head to the abdomen) of larval and adult fruit flies. Three levels of genetic control—egg-polarity genes, segmentation genes, and homeotic selector genes—result in an adult fly with anterior head segments, three thoracic segments, and eight abdominal segments.

Egg-polarity genes are responsible for establishing the anterior-posterior axis. Mutations of these genes result in bizarre flies that lack head, thoracic, or abdominal structures. Maternal egg-polarity genes are transcribed, and the resulting ribonucleic acid (RNA) is translocated into the egg and localized at one end. This RNA is not translated into protein until after fertilization. Following translation, the proteins are dispersed unequally in the embryo, forming an anterior-posterior gradient that regulates their expression and that of other related genes.

Segmentation genes are the second tier of the genes that establish the body plan of the fly. Within the segmentation genes, there are three levels of control—gap genes, pair-rule genes, and segment-polarity genes—resulting in progressively finer subdivisions of the anterior-posterior axis. While there is hierarchical control within the segmentation genes, genes in a given level also interact with one another. Gap genes of the embryo respond to the positional information of the gradient established by the maternal egg-polarity genes. Gap genes form boundaries that specify regional domains, and several gap genes with distinct regions of influence have been identified.

Pair-rule genes follow next in the sequence and function at the level of two-segment units. Mutant pair-rule genes are responsible for flies with half the normal number of segments. Ultimately, the larval fly body is divided into visible segments, but while the patterns are forming, genes appear to influence parasegments. A parasegment is half a segment that is “out of phase” with the visible adult segments. Parasegments include the posterior of one segment and the anterior of the adjacent segment. Developmental programming of parasegments ultimately gives rise to visibly distinct segments.

The final level of control of the segmentation genes focuses on individual segments and is controlled by the segment-polarity genes. In response to the pair-rule genes, the segment-polarity genes subdivide each segment into anterior and posterior compartments. Thus, the segmentation genes create a series of finely-tuned boundaries.

Homeotic Selector Genes

It is the third tier of genes, the homeotic selector genes, that actually specifies segment identity. Segmentation gene mutations result in missing body parts, whereas mutations of the homeotic selector genes result in a normal number of segments, some of which have abnormal identities. Homeotic selector genes are found in two gene clusters, the antennapedia complex and the bithorax complex. Genes associated with the antennapedia complex determine the fate of segments associated with the anterior body segments, like the development of fruit flies' legs, while the bithorax complex is responsible for the more posterior segments, such as the abdominal segments. The pattern that arises is modulated both by interactions among the homeotic selector genes and by their interactions with the segment-polarity genes.

The genes found within the antennapedia and bithorax complexes have been identified based on mutant phenotypes that have arisen. These genes function within smaller regions of the anterior or posterior axis, much as pair-rule genes subdivide regions established by the gap genes. It is intriguing that the genes within the two complexes appear to be lined up in the same order in which they function spatially. That is, the position of an antennapedia complex gene on the chromosome relative to other antennapedia complex genes correlates with the actual position of the segments controlled by the gene. Analyses of homeotic mutations in beetles indicate that the homeotic genes are also physically organized in a left-to-right sequence corresponding to the location of the segments they control on the anterior-posterior axis. Unlike the fruit fly, however, the beetle has a single homeotic gene complex controlling the entire anterior-posterior axis.

The interactions between homeotic selector genes and other genes in the hierarchy may, in part, be controlled by the homeodomain protein that is coded for by the homeobox associated with many of these genes. The homeobox is a 180-nucleotide-pair sequence that is included in many homeotic selector genes and some segmentation genes, including the pair-rule gene. The homeodomain protein has a unique structure that may bind deoxyribonucleic acid (DNA) and affect its transcription. This may allow genes to regulate the expression of themselves and other related genes. For example, a pair-rule gene known as fushi tarazu (Japanese for “not enough segments”) is found in the antennapedia complex and has a homeobox, although fushi tarazu is not a homeotic selector gene. Its homeodomain can bind to the antennapedia gene and thus can regulate when this gene is turned on and off. Antennapedia, a homeotic selector gene, was first identified when a mutation of it resulted in the replacement of an antenna with a leg. Thus, the ability of the homeodomain to bind to DNA provides a way for the hierarchical control of homeotic selector genes by segmentation genes to occur.

The homeotic selector genes are ultimately linked to gene expression that leads to the development of specific structures associated with different segments. While it is not completely understood exactly how homeotic selector genes regulate segment differentiation, scientists have identified their interaction with other gene regulatory proteins and encoding homeodomain proteins. Although the homeotic selector genes are active early in development, they are also involved in programming cells for fates that are not expressed until much later.

Studying Homeosis through Mutations

Most of what is known about homeosis has been learned from studying mutations. Sometimes, these mutations have arisen spontaneously; sometimes, they have been induced by exposing organisms to mutagenic substances, such as chemicals, X-ray, or ultraviolet radiation. The large number of mutations identified in fruit flies accounts for the wealth of information on homeosis in this organism, in sharp contrast to the limited information on humans, for whom ethical considerations prohibit mutagenesis. Mutations affecting segmentation and determination of segment identity represent defective developmental switches and provide insight into the normal developmental sequence.

Classical genetic approaches have been used with homeotic mutants to map genes to chromosomes and identify gene interactions. For example, it can be determined whether two genes are on the same chromosome by making a series of specific matings between flies with mutations in these genes and wild-type (“normal”) flies. If the genes are not on the same chromosome, offspring with one mutation will not necessarily have the other mutation. If the mutations are both on the same chromosome, they will be inherited together, except in rare situations where there is recombination between chromosomes. Geneticists use the frequency of recombination to assess how close together two genes are on a chromosome. This approach helped geneticists determine the order of genes within the antennapedia and bithorax complexes. Matings between different mutants have also established the hierarchy of genetic control among egg-polarity, segmentation, and homeotic selector genes. For example, a pair-rule mutant will have no effect on gap genes, but a gap gene mutant will affect pair-rule genes.

To visualize the results of these crosses of hierarchical mutants, researchers employed a second technique: in situ hybridization. To investigate the effect of gap genes on pair-rule genes, a wild-type fly embryo and one with a gap mutation affecting the middle section were exposed to radioactively labeled DNA that was a copy of the pair-rule gene fushi tarazu. The DNA hybridized (bound) to fushi tarazu RNA, thus labeling tissues where the fushi tarazu gene was turned on and making RNA copies of itself. Excess radioactive DNA was washed away, and a photographic emulsion that was then placed over the tissue was exposed by the bound radioactive DNA. This permitted researchers to see that the fushi tarazu gene was being expressed in the middle of the wild-type embryo but not in the gap mutant embryo. When this experiment was repeated using radioactive gap-gene DNA in a pair-rule mutant, no effect on gap-gene expression was observed.

In situ hybridization has also provided information on how egg-polarity genes provide segmentation genes with positional information. The egg-polarity gene bicoid was identified by mutations resulting in a fly with abdominal structures but no head or thoracic structures. When radioactive DNA copies of the bicoid gene were hybridized eggs, it was found that all the RNA was located at the anterior tip after being transferred from the mother. When this RNA was translated into protein, the protein was tagged with an antibody that identifies the bicoid protein. Tagging the protein with an antibody is similar to tagging RNA with a radioactive DNA segment; both techniques allow researchers to see how the RNA or protein is distributed in tissues. In this case, the bicoid protein formed an anterior-to-posterior gradient after the egg was fertilized, with more protein being found at the anterior end.

Homeotic genes have been isolated and used for in situ hybridization studies. In addition, the sequence of nucleotides in the DNA in these genes has been established. A variety of techniques is available for DNA sequencing. Generally, the DNA is broken into smaller segments that can be more readily identified. Cutting the DNA yields overlapping segments, and the overall sequence can be established by piecing these overlapping fragments back together. The presence of the homeobox was established by comparing DNA sequences from different genes in flies and other organisms. It was evident, based on these types of data, that the homeobox sequence differs by only a small number of nucleotides, even between very distantly related organisms.

The role of the homeobox was investigated by inserting DNA containing a homeobox from the fushi tarazu gene into bacteria so that the bacteria then produced large amounts of the homeodomain protein. This protein was then tested for its ability to bind the DNA and was found to bind to specific fragments of DNA that were involved in homeosis, such as the antennapedia gene. Homeodomain protein from a mutant fushi tarazu gene was defective in its DNA binding ability. This provided evidence that the homeobox may regulate gene expression via the direct binding of the homeodomain to DNA. This is only one of numerous examples illustrating how researchers have utilized genetic mutants and molecular biology to investigate the role of homeotic genes in the development of segmented organisms.

Implications of Homeotic Research

Research on homeosis and homeoboxes has made significant contributions to the fields of developmental biology and evolution. Since all higher animals and plants exhibit some form of segmented development, the common link of the homeobox has intrigued scientists who are interested in how body plans are established. Questions concerning the evolution of segmentation patterns in animals have also arisen as more is understood about the genes affecting segmentation and how they are regulated.

Significant similarities both among the homeoboxes identified in fruit flies and among the homeoboxes of distantly related species have been found. In fruit flies, homeoboxes have been identified in homeotic selector genes, segmentation genes, and egg-polarity genes. How homeoboxes affect gene regulation and segmentation in other organisms is an exciting and open question. Most, if not all, multicellular organisms, and even some single-celled organisms, have homeoboxes, and the homeobox sequences found in most mammals are not dissimilar to those of fruit flies. Different mouse homeobox genes are expressed in specific regions during embryogenesis. However, the expression of homeotic mutations in mammalian genes is more difficult to observe, as mammals often have more than one copy of homeotic genes, which can mask the effects of any mutations. Also, segmentation may have evolved separately in vertebrates and invertebrates, so the presence of a homeobox may or may not indicate a common developmental process for segmentation among animals.

The addition and modification of homeotic selector genes were likely responsible for the evolution of insects from segmented worms. The presence of two homeotic gene complexes in fruit flies, compared to only one in beetles, suggests that duplication of the gene complex, followed by subsequent specialization, may have allowed for greater fine-tuning of segmental identity. Evolutionary alterations of the initial homeotic gene complex may have been responsible for the addition of legs to a wormlike creature composed of similar segments, giving rise to a millipede-like creature. The reduction of all legs except for the walking legs in the thoracic region and, ultimately, the addition of wings to the thoracic region could also reflect changes in homeotic selector genes. Parts of this evolutionary journey can be reconstructed with homeotic fruit fly mutants that bear similarities to their ancestors. This is exemplified by the deletion of the antennapedia gene, which results in a wingless fly; winged insects presumably arose from non-winged insects. It is impossible, however, to create a millipede from a fly via mutations of the homeotic selector genes, so caution must be taken in speculating on the role of homeotic selector genes in insect evolution.

Fruit flies remain a common and useful tool in the twenty-first century in investigating homeotic selector genes and other genes, many of which offer insight into treating human illness. For example, the homeotic gene Cdx2 also called the homeobox protein CDX-2, controls organ morphogenesis and regulates major intestinal-specific genes during cell differentiation in animals. However, when well expressed, it has applications for preventing the growth of cancerous tumors and helps prevent colorectal cancer brought on by exposure to carcinogens. Additionally, it can be used as a prognostic tool and predictive marker for colorectal cancer.

Principal Terms

Egg-Polarity Genes: genes whose expression in maternal cells results in products being stored in the egg in such a way as to establish polarity, such as the anterior-posterior axis

Gap Genes: genes expressed in the zygote that divide the anterior-posterior axis of fruit flies into several regions

Homeobox: a sequence of about 180 nucleotide pairs that code for a protein called the homeodomain, known to influence body-plan formation in numerous organisms

Homeosis: a process that results in the formation of structures in the wrong place in an organism, such as a leg developing in place of a fly’s antenna

Homeotic Selector Genes: genes that determine the identity and developmental fate of segments established in fruit flies by a hierarchy of genes

Imaginal Disk: a small group of cells that differentiates adult fruit-fly structures after the last larval molt

Pair-Rule Genes: segmentation genes in insects that divide the anterior-posterior axis into two-segment units

Segmentation Genes: genes that regulate segmentation in organisms, including gap genes, pair-rule genes, and segment-polarity genes

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