Homeotic Genes

Kelly Kaminski

Evolution Paper

April 22, 2003

I. Introduction

One of biologyπs and manπs greatest mysteries is how a single fertilized cell develops into a complex organism, like a fly, a mouse, or a human. In the early 19^th century, Von Baer observed that all vertebrates, from salamanders to humans, look very similar in the early stages of embryonic development. At about the same time, French zoologist Geoffroy Saint-Hilaire declared, ≥all animals have the same body plan (Zihlman 2001).≤ From very early on, scientists have realized that there is some fundamental linkage between vertebrates. Today, better technology has enabled biologists to discover the genetic connections between very diverse animal species. The homeotic genes were one of these important connections. Homeotic genes are surprisingly very similar in structure and function across the Phylum Vertebrata and have proved important in scientific research.

II. Early Experiments and History

A tremendous source of knowledge about how homeotic genes function came from three men, who won the Noble Prize in Medicine in 1995. These Noble Prize winners were Dr. Edward B. Lewis, Christiane Nusslein-Volhard, and Eric F. Wieschaus. The work of these three scientists had an enormous impact on our understanding of how invertebrate and vertebrate embryos develop. Dr. Lewis was the first to study how genes could control the further development of individual body segments into specialized organs. Dr. Lewis was interested in certain developmental changes that occurred in the Drosophila fly. He was also interested in how homeotic genes control body segmentation during development (Homeotic Genesä 1995).

The experimental model for many of the early experiments was Drosophila melanogaster. The Drosophila experiments centered on the homeotic genes controlling wings, legs, and eye development. Drosophila's body plan consists of three parts: head, thorax, and abdomen. The thorax is composed of three segments T1, T2 and T3. Drosophila belongs to the insect order Diptera; they only have one pair of wings. The wings are located on T2 segment. The thoracic segment, T3, carries a pair of balancing organs called halteres (Figure 1). In Drosophila, a gene called Ultrbithorax (Ubx) acts within the cells of T3 to suppress wing formation (Kimball 1994).

Figure1: The halteres of a normal Drosophila. (Kimball 1994)

Dr. Lewis carried out a series of experiments on the Ultrabithorax (Ubx) gene. The experiments manipulated the Ubx gene in order to create a double mutation. Consequently, the mutated fly grew a second pair of wings where the halteres normally develop (Figure 2) (Kimball 1994). This puzzled Lewis, because he knew that wings are a very complex structure. A single mutation in one gene resulted in the reprogramming of the T3 body segment. Similar experiments have taken place with the Antennapedia gene (Antp). Normally, Antp is active in the thorax and inactive in the head. Mutations in Antp result in Antp expression in the head; a pair of legs develops where the Antennae would normally develop (Brook 1990).

Figure 2: A mutated Drosophila with two sets of wings. (Kimball 1994)

Lewisπ work with the bithorax genes lead to his co-linearity principle. According to this principle, there is a co-linearity in time and space between the order of genes in the bithorax complex and their affected regions in the body segments. This theory was later expanded to cover all homeotic genes. Therefore, the linear order of the homeotic genes on the chromosome corresponds to the order they will appear when expressed (Burke and Nowicki 1997). For example, the 3π gene in a cluster is expressed earlier and more anteriorly in the embryo than its 5π neighbor (Reichert and Simeone 1999). The end-product of Dr. Lewisπ experiments laid the foundation for one of the most surprising discoveries in developmental biology ≠ ≥the same type of genes which controls the early embryonic development of Drosophila also controls the early embryogenesis of numerous other higher organisms, including man (Homeotic Genesä 1995).≤

These homeotic genes are conserved throughout evolution (Busturia and Sankonju 1999). Over the course of evolution, these genes have been tinkered with by nature to create an enormously diverse range of animal forms. Various changes in the control of homeotic genes have been shown to under lie an evolutionary trend leading to novel body structures. A European lab did a series of experiments on Crustaceans. The researchers found a linkage between changes in activity pattern of two Hox genes and the sudden evolutionary development of useful feeding limbs. These limbs are called maxillipeds (literally jaw-feet), and the maxillipeds develop where the swimming or walking legs once were (Easton 1997). This evidence supports the claim that mutations in homeotic genes may play a part in the formation of new unique organisms.

III. Homeotic Genes

Ubx and Antp are examples of homeotic genes. Homeotic genes are master regulatory genes and are conserved from flies to humans (Basturia and Sakonjo 2003). Homeotic genes are genetic switches that turn on or off different programs of cellular differentiation. Homeotic genes specify the identity of body segments. By studying a particular organism, scientists can discover what role certain homeotic genes play in development. This information is used to look for genes in other organisms that have similar homology, with possible similarities in function as well (Dola and Iredale 2003).

Each homeotic gene is expressed in its appropriate domain along the axis of the body. Each gene directs the appropriate cells on a developmental pathway for its position. If the gene is misexpressed outside of its normal domain, it transforms the misexpressed body segment to look like another segment (Zihlman 2001).

In the 1970s, Christiane Nusslein-Volhard and Eric F. Wieschaus sequenced the homeotic genes controlling the development of Drosophilaπs body plan. They observed a conserved DNA motif of about 180 base pairs in each of the genes (Zihlman 2001). This conserved DNA motif was later coined, the homeobox. The homeobox encodes a protein domain, the homeodomain, which is 60 amino acids long (Burglin 2002). The homeodomain proteins regulate gene expression at the level of RNA synthesis (Purves et al. 2001). These proteins have many important developmental roles in multicellular organisms (Burglin 2002). The expression of the homeobox genes is important in the regulation of other genes and gene products also (Dola and Iredale 2003). The homeobox is found in all homeotic genes, but the sequence differs slightly from individual to individual (Futuyma 1998). The first genes found to contain homeodomain proteins were found in Drosophila melanogaster (Dola and Iredale 2003).

The mouse and Drosophilia homeotic genes show similarities in organization (Figure 3). The amino acid sequences of these genes are conserved in the homeobox domain. The arrangement of gene order on the chromosomes is also conserved. It is believed that gene complexes arose form a common ancestor before the arthropod/vertebrate evolutionary divergence (Purves et al 2001).

Figure 3: The similarities between Drosophila and a mouse embryo. (Purves et al. 2001)

In Drosophila, the homeotic genes are called ≥Hom≤ genes; in vertebrates, the homeotic genes are called ≥Hox≤ genes. The duplication of Hox genes occurred twice in the evolution from invertebrates to vertebrates. Drosophila (invertebrate) has one cluster of approximately 10 genes on one chromosome, compared to the mouse (vertebrate), which has four clusters of about 10 genes each, on four different chromosomes (Zihlman 2001). The fly and mouse Hox genes are very similar in number and chromosomal arrangement. They are so similar that it is possible to transfer a Hox gene from a human to a Drosophila embryo. The inserted Hox gene can perform many of the same functions the corresponding Drosophila Hom gene normally carries out (Homeotic Genesä 1995). In mammals, only 40 genes out of 100,000 control most of development, architecture and appearance of the body plan (Zihlman 2001). It is striking how only a few genes can control the fate of development.

Humans have four Hox clusters, compared to Drosophilaπs one Hox cluster. This indicates that the mammalian genomes were duplicated over evolutionary time too. In Figure 4, Hox A, Hox B, Hox C and Hox D have been lined up. The conservation of the geneπs organization can be observed. Also in each Hox cluster, the genes between each cluster (for example, A4, B4, C4, and D4) are more closely related than the genes within a given cluster (for example, A4, A5, and A6). Through evolutionary time, some clusters have lost genes and other genes have been retained in all four clusters. A4, B4, C4, and D4 are examples of paralogs. Paralogs are genes that correspond to eachother in each duplicated cluster (Purves et al. 2001). Experimental studies have shown that paralogs have similar and overlapping functions in animals, which has made it difficult to study the function of homeotic genes. It is difficult to create a mutation in a gene and study it effects, because the effects are often hidden by the normal functions of the other genes in the same paralogous group (Mutationsä 2003).

Figure 4: The conserved organization of mammalian homeotic genes (Mutationsä 2003)

IV. Research

Experiments have shown that minor changes in genes have an immense effect on the overall body plan (Purves et al. 2001). This has laid the foundation for a variety of research on homeotic genes. Using mice and Drosophila as specimens, scientists can observe the effects mutated homeotic genes have on the organism. The relationship Drosophila and mice have with humans has enabled researchers to correlate the experimental results to humans.

A series of experiments investigated the Hox-A11 and the Hox-D11 genes in mice. The composition of the mouse and human foreleg contains the same structures. In mice and humans, the humerus, radius and ulna make up the bones of the foreleg. There is reason to believe that Hox cluster genes control the development of the entire arm, including the carpals and phalanges. Figure 5 shows the mice forelimb bones with mutations in two genes from the Hox-11 group. A normal forelimb bone with no mutations can be seen in a. It is important to note that b and c carried mutations in only one Hox cluster (A or D). In b and c, bone structure and arrangement were approximately normal. This illustrates that a mutation in a single gene from this paralogous group will have a minimal effect on the animal. A different outcome occurs in a cross between two homozygous mutants in both Hox-A11 and Hox-D11 (d). These mice were born with neither radius nor ulna in the forelimbs. This illustrates that mutations of two genes of a paralogous group have a debilitating effect on the body shape of the animal (Mutationsä 2003).

Figure 5: Mutations in mice forelimbs (Mutationsä 2003)

In humans, a mutation in the homeotic gene PAX 6 may cause Aniridia. Aniridia is a debilitating disease. In Aniridia there is a loss of the iris of the eye, and occurs because the eye stops developing too early. The human Pax 6 gene is homologous to the small eyes and eyeless genes of mice and Drosophila, respectively. The mouse gene, small eyes, is similar in sequence to the Drosophila eyeless gene. These genes are so similar that when the small eyes gene is substituted for the eyeless gene in Drosophila, no loss of function occurs (Homeotic Genesä 1995). Mice and Drosophila have proven to be extremely useful models. Applying knowledge gained from mice and Drosophila experiments, researchers hope to gain more insight into the causes and maybe a possible cure for Aniridia.

Other research studies carried out with mice have shown that a disturbance in homeotic genes may cause a condition known as megacolon, a malformation of the colon. In young children, doctors sometimes diagnose the cause of megacolon as an unknown origin (Homeotic Genesä 1995). It is hopeful that future research may be able to find if defective genes cause megacolon. Also, ongoing research hopes to find the relationship between cleft palate, lips and jaw and defective genes (Homeotic Genesä 1995).

V. Conclusion

Homeotic genes have been of great importance to evolutionary and research biologists. Gene analysis and comparison has provided a picture for evolutionary biologist as to how new organisms develop and original organisms diverge. These genes have also impacted disease research by opening the door for experimentation on diseases that may stem from mutations in homeotic genes. Discoveries have inferred that homeotic genes are important in many aspects of development and play a fundamental role in a vast array of organisms. Homeotic gene research is of great importance. Researchers hope to develop gene therapies for inflicted patients or discover possible cures for many diseases. New techniques may provide this chance. The years to come may bring a whole new understanding about the roles of homeotic genes.

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