Chris Carter

Dr. Garbutt

22 April 2003

Humpty-Dumpty needed homeotic genes

At this point in your education, you have assimilated a great horde of knowledge about genetics. You probably know all about base pairs, how genes are copied, how cells duplicate, and even how mistakes are made and new genes appear. Yet, if you have ever looked at an insect and wondered why they have so many legs and why they grow where they do, then this paper is for you. Because if you have caught yourself asking yourself that question or nearly any other question concerning how living creatures grow from a cell to a highly-ordered whole, then you have been considering homeotic genes. Perhaps the easiest way to understand homeotic genes is to consider them as ≥genetic switches that turn different programs of cellular differentiation on or off≤ (Brook 2003). However, by relying on just this simple definition, we ignore the vital characteristics that must be in place before homeotic genes can work.

Research History and Conclusions

In 1980, the journal Nature published an article by Christiane N¸sslein-Volhard and Eric F. Wieschaus. N¸sslein-Volhard and Wieschaus had been researching how the segmented body plan of Drosophila melanogaster, a species of fruit fly, develops following fertilization. Their plan was to treat Drosophila females with chemicals that damaged their genes and then to observe how these genetic defects were manifested in their offspring. By relating which offspring suffered segmentation defects to the chemicals the mother was treated with they could determine which genes were responsible for segmentation. At the time, the research strategy was unique, and its reasoning was explained in this way: ≥Studying genes intentionally damaged by mutation was the only accessible way to obtain knowledge about genes controlling development. By disturbing the system and looking for what happens you may be able to learn something about it≤ (≥Hunting≤ 2001). In all, N¸sslein-Volhard and Wieschaus identified roughly 40,000 mutations and settled on 15 different genes responsible for early embryonic development. Later studies have increased that number to 25 genes. Of these genes responsible for segmentation, they all fell into three classes that operated in the following chronological order:

• Gap-genes lay the foundation of a rough body plan along the head-to-tail axis,
• Pair rule-genes govern formation of every second body segment,
• Segment polarity-genes refine the head-to-tail polarity of each individual segment, meaning that the head-end and the tail-end of a segment look different. (≥Hunting≤ 2001)

Mutations of any one of the genes in the first two classes leads to loss of body segments (Fig 1). Mutations in the third class of genes cause the fly to have head and tail ends that are similar (Fig 1) (≥N¸sslein-Volhard≤ 2001).

Figure 1

Loss of gap gene results in a reduced number of segments as shown in the embryo to the right.	Loss of a pair rule-gene, e.g. even-skipped, allows only odd-numbered segments to develop.	Loss of a segment polarity-gene leads to segments with similar head and tail ends.

Once this segmentation takes place, homeotic genes begin to act. Homeotic genes in the Drosophila genome are grouped into complexes and further specialize the segments. The genes are clustered in two major groups which are called the Antennapedia complex (ANT-C) and the bithorax complex (BX-C) (Brook 2003). In general, the homeotic gene complex is referred to as HOM-C in Drosophila. The mammalian homologue is referred to as HOX. Edward B. Lewis studied the bithorax and found that the genes in the DNA are arranged in the same general order as their expression pattern along the head-to-tail axis (≥Lewis≤ 2001). This became the basis for his co-linearity principle, which states that ≥the genetic expression domains overlap and that the first gene in the complex becomes active a little earlier than the second and so on≤ (≥Lewis≤ 2001). According to Brook, Lewis originally thought ≥there was one gene for each segment; however molecular analysis of the BX-C indicates that it is composed of three regions encoding homeodomain genes called Ultrabithorax (Ubx), abdominal-A (abdA), and Abdominal-B (AbdB) (2003). The Antennapedia complex is composed of five more regions (Fig. 2) (Hallick 2002).

The most famous of Lewisπ experiments dealt with the mutant form of Drosophila with four wings instead of two (Fig. 3). Lewisπ research in this area led him to the conclusion that ≥(i)n the mutant-case, inactivity of the first gene in a complex of homeotic genes (the bithorax complex) caused other homeotic genes to duplicate the segment with two wings≤ (≥Lewis≤ 2001). This was the major breakthrough spurring research to the discovery that ≥the more posterior genes will repress the transcription of the more anterior genes≤ (Brook 2003).

Figure 2

Segmentation of Drosophila

Figure 3

As you can see from Figure 2, the first homeotic gene of the bithorax complex lies directly posterior to the last gene of the Antennapedia complex, which encodes for the winged segment of the body. In the case of Lewisπ mutant fly in Figure 3, the first gene of the bithorax complex is inactive and is unable to repress the activity of the neighboring anterior gene. So how do the posterior genes become activated? The answer lies in Lewisπ co-linearity principle. Because the gene anterior to the next coincides with the anterior segment of the body, activation of the anterior ≥genes of the homeotic complex (HOX) encode DNA binding homeodomain proteins that control developmental fates by differentially regulating the transcription of downstream target genes≤ (Chan 1996).

Despite Lewisπ groundbreaking discovery of the Antennapedia and bithorax complexes, his most important discovery for developmental biology was yet to come. Lewis discovered that the early embryogenesis of many higher organisms, including man, was controlled by the same kind of genes as that of Drosophila (≥Lewis≤ 2001). Meaning that for roughly 650 million years, the mechanisms by which organisms develop embryologically stayed roughly the same. According to Abzhanov and Kaufman ≥the function of HOX gene products as selective transcription factors is apparently highly conserved in animals, and the expression patterns of these genes have been used as stable molecular markers for judging evolutionary relationships, such as homologies, of structures along the anteroposterior axis≤ (1999). But what happens when mistakes are made?

Mutation

The mutations of homeotic genes cause ≥dramatic changes in body appearance≤ (Hallick 2002). An example can be seen in Figure 4 (Hallick 2002) where a mutation in the Antennapedia complex causes the flyπs legs to be expressed on its head. Obviously, the proper transcription of an organismπs homeotic genes are vital for itπs function, and because the embryological basis of growth is so similar in many higher organisms these mutations are not reserved strictly for Drosophila. Both mice and humans are known to express mutant forms of homeotic genes. In one example of homeotic mutation in mice (Fig. 5), the addition of retinoic acid (Vitamin A) during pregnancy causes the expression of homeotic genes 1-4 in cells where it usually is dormant (≥Consequences≤ 2001). The result is a mouse with posterior regions either incompletely developed or practically non-existent. These deformities are not reserved for mice, as retinoic acid is also known to cause defects in humans. Fortunately, mammals appear to have evolved a safety protocol for these mutations, as evidenced by the fact that their ≥homeotic genesä appear to have been duplicated over evolutionary time≤ (≥Mutations≤ 2003). This means that in most cases a mutated homeotic gene can be masked in mammals due to the presence of wild-type paralogous genes. To observe the effects of a mutation in mammals, all the copies of the homeotic gene must be mutated. One example is that of Figure 6. Provided by Mario Capecchi's research group at the University of Utah, the figure shows the mutation of two out of three copies of a homeotic gene responsible for fore limb growth(≥Mutations≤ 2003). As you can see, the results of homeotic gene mutations can be quite drastic when expressed, and can seriously alter or destroy the functionality of the mutated segment of the body.

Figure 4

Mutation in Attenapedia of Drosophila

Figure 5

Mice with mutations from varying retinoic acid levels

Normal mouse embryo

Retinoic acid: loss of many vertebrae

More retinoic acid: no posterior region formed

Figure 6

Mice with mutations in paralogous genes

The capitol letters A and D represent the normal HOX A-11 or HOX D-11 genes. The small letters a and d represent mutated genes. For each picture, the genotype of the animal at these two genes is shown. For example, "aa; DD" represents a mouse carrying two mutated copies of the HOX A-11 gene and two normal copies of the HOX D-11 gene.

• (a) is a normal forelimb. The letters h, r, and u indicate the three main bones of the forelimb: r=radius, u=ulna, h=humerus.
• (b) shows the forelimb from a mouse carrying two mutated copies of HOX A-11 and two normal copies of HOX D-11.
• (c) shows the forelimb from a mouse carrying two mutated copies of HOX D-11 and two normal copies of HOX A-11. Note that the structure and arrangement of the bones in (b) and (c) are both approximately normal. Therefore, mutation of a single gene in this paralogous group has only a mild effect on the animal.

∑ (d) shows the forelimb from a mouse carrying mutations in both copies of both genes. Note that two of the forelimb bones, the radius and ulna, are almost completely missing. The mutations in both genes have produced a striking change in body shape.

In humans, homeotic mutations are suspected to cause some structural deformities involving the jaws, lips, and palate. Known conditions in human beings caused by homeotic mutations include at least two others besides those due to retinoic acid. Waardenburgπs Syndrome and aniridia are two conditions that are definitely caused through homeotic gene mutation. Waardenburgπs Syndrome is ≥a very rare disease, affecting one of 42,000 born children. It involves deafness, defects in the facial skeleton and altered pigmentation of the iris≤ (≥Consequences≤ 2001). Aniridia results when the homeotic gene PAX 6 is mutated. A child born with aniridia has no iris and cannot control the amount of light entering the eye (≥Aniridia≤ 2001). Someone with this condition is as good as blind because their world is too bright for them to even look at. If youπve ever accidently looked at the sun (who hasnπt?), then you have an idea of what it would be like to not have an iris. The only treatment for those suffering from aniridia is the use of colored lenses that reduce the amount of light entering the eye (≥Aniridia≤ 2001).

How do homeotic genes work?

The honest answer to the question is that no one knows. Despite all the research that has been completed and all the hours spent studying homeotic genes, the scientific community is still not certain of their mechanism of function. Researchers do know that ≥(a)ll the members of the BX-C and ANT-C encode homeodomain proteins and several have been proven to act as transcription factors in vitro≤ (Brook 2003). However, this information yields little in the way of explaining their function. This uncertainty has led to disagreement as researchers struggle to elucidate how the genes work. Still, some clues have become apparent, as stated by Chan:

Despite their unique in vivo functions, disparate HOX proteins often bind to very similar DNA sequences in vitro. Thus, a critical question is how HOX proteins select the correct sets of target genes in vivo. The homeodomain proteins encoded by the Drosophila extradenticle gene and its mammalian homologues, the pbx genes, contribute to HOX specificity by cooperatively binding to DNA with HOX proteins. (1996)

In addition, Li and McGinnis add, ≥HOX proteins each contain a highly homologous DNA-binding homeodomain and recognize in vitro similar DNA sequences as monomers≤ (1996). So, it seems that HOX proteins could have a mediator of some kind that facilitates their binding to their specific gene sequences. Also, considering HOX proteins may have disparate functions but bind to very similar DNA sequences, it can be inferred that HOX proteins do not bind different DNA sequences based on structure alone. This point of view may be supported by Lohman and McGinnis, who state: ≥Recent studies provide compelling new evidence that HOX gene effects depend on fine-structure spatial and temporal information. Further, in a specific cell type, only one or a few downstream genes may mediate HOX morphogenetic functions≤ (2002). Spatial and temporal orientation in the nucleus may depend on other proteins similar in function to those active during RNA synthesis. Also, because the function of HOX is specific for cell type due to downstream genes but the genome is replicated in each cell, there must be some other device at work which interacts with these downstream genes in varying cell types.

Though part of the mystery of homeotic genes blooms from the ignorance of how their targets are selected, this is further complicated by ≥the lack of genes known to be directly regulated by the HOM-C proteins≤ (Pederson et al 2000). Most troubling is that there doesnπt appear to have been much progress from when Reuter et al. stated ten years earlier that ≥the genes probably coordinate the expression of as yet unidentified target genes that carry out cell differentiation processes≤ (1990). Without even the basic information of what genes the homeotic genes are targeting, it is easily understood why there has been such slow progress in understanding the mechanisms of homeotic genes. As McGinnis states in Genetics:

Though the amount of research that has been done on HOM/Hox-type homeodomain proteins is enormous, it is still unknown how many genetic or cofactor inputs are required for a homeotic switch to be thrown that changes cells (or even a single gene for that matter) from being assigned to a head, thoracic, or abdominal fate. (1994)

Unfortunately, until there are significant advances in the research of homeotic genes and their proteins, there will be no definitive answers on how they work.

What can homeotic genes tell us about evolution?

As mentioned previously, the homeotic regions of the genome are highly conserved and serve to judge evolutionary relationships. So homeotic genes may offer the possibility of helping us judge when divergence events occurred. The truly exciting possibility is that homeotic genes may have supplied the mutations through which structural change and speciation occurred. As explained by the Genetic Science Learning Center at the University of Utah:

Organisms can survive and reproduce even with homeotic gene mutations that produce differences in body shape. This means that homeotic mutations can be an effective means for evolutionary change. For example, in a mammal, a single homeotic mutation might produce an arm that is shorter, or longer, or broader. Regardless, it will probably still look and work like an arm. A change in body shape might lead to an advantage for an organism. For example, the mutation may allow it to capture food more effectively or be more attractive in some way. If this is the case, then the mutant organism may have greater reproductive fitness. Its genes may be preferentially passed along to the next generation, thus influencing the course of evolution. (2003)

We may consider one argument against evolution that is made in reference to its speed. The argument is sometimes made that the changes conferred by mutation are too small to effectively confer an advantage to an organism. The point follows logically then that these new genes would not be transferred to succeeding generations at the increased rate necessary to alter the genotypic frequencies of the population. However, in the case of homeotic gene mutation, the expressed mutant phenotypes deviate drastically from the previous generation. The mutations are easily large enough to confer an advantage to an organism, and if they did then they would be passed on to the progeny at a rate necessary to ensure a change in genotypic frequency. These changes could lead to permanent structural changes and eventually new species as successive generations deviated from their ancestors. Because the homeotic gene complex is so highly conserved, any mutation passed on the next generation would stick around in the gene pool for some time.

Conclusion

There is much work left if we are to unravel the mystery of homeotic genes. The future is ripe with potential, but the nagging questions about the basics of how homeotic genes work appear to be a stumbling block for the time being. Until the seemingly obvious questions about what the basic targets of HOX and HOM-C are can be answered, research will be a tedious process. Once the specific mechanisms of homeotic genes are discovered, it shouldnπt be long before these new contributions yield data on how speciation occurs and new body structures develop. Considering the conditions in humans known and suspected to be caused by mutations, the medical implications are tremendous. If we know how the genes work and affect transcription downstream, we may be able to stop mutations before they occur or correct them. Research has come a long way since N¸sslein-Volhard and Wieschaus discovered gap, pair-rule, and segment polarity genes, but in the end their mantra still stands: ≥By disturbing the system and looking for what happens you may be able to learn something about it.≤

Works Cited

Abzhanov, A., and T.C. Kaufman. ≥Homeotic genes and the arthropod head: Expression patterns of labial, proboscipedia, and Deformed gens in crustaceans and insects.≤ Proceedings of the National Academy of Sciences 96.18 (1999): 10224-10229.

≥Aniridia.≤ Nobel e-Museum. 2002. Nobel Foundation. 10 May 2001. <http://www.nobel.se/medicine/laureates/1995/illpres/more-anaridia.html>

Brook, William. Control of Segmental Identity in Drosophila: Homeotic Genes. University of Calgary. 22 Mar. 2003 <http://www.ucalgary.ca/UofC/eduweb/virtualembryo/homeotics.html>.

Chan S.K., and R.S. Mann. ≥A structural model for a homeotic protein-extradenticle-DNA complex accounts for the choice of HOX protein in the heterodimer.≤ Proceedings of the National Academy of Sciences 93.11 (1996): 5223-5228.

≥Consequences of the Discoveries.≤ Nobel e-Museum. 2002. Nobel Foundation. 10 May 2001. <www.nobel.se/medicine/laureates/1995/illpres/consequences.html>.

Hallick, Richard B. Homeotic genes. University of Arizona. 22 Mar. 2003 <http://www.blc.arizona.edu/courses/181gh/rick/development1/homeotic. html>.

≥Homeotic Genes Determine Specialization of Segments.≤ Nobel e-Museum. 2002. Nobel Foundation. 10 May 2001. <www.nobel.se/medicine/laureates/1995/illpres/more-l-segmspec.html>.

≥Homeotic Mutations Could be Involved in Evolutionary Change.≤ Genetic Science Learning Center. 2003. University of Utah. 23 Mar. 2003. <gslc.genetics.utah.edu/units/basics/bodypatterns/evolutionary.cfm>.

≥Hunting for the Genes Behind Pattern Formation in Drosophila's Early Embryonic Development.≤ Nobel e-Museum. 2002. Nobel Foundation. 10 May 2001. <www.nobel.se/medicine/laureates/1995/illpres/more-nw-disc.html>.

≥Lewisπ Discoveries.≤ Nobel e-Museum. 2002. Nobel Foundation. 10 May 2001. <www.nobel.se/medicine/laureates/1995/illpres/lewis.html>.

Li, X., and W. McGinnis. ≥Activity regulation of Hox proteins, a mechanism for altering functional specificity in development and evolution.≤ Proceedings of the National Academy of Sciences 96 (1999): 6802-6807.

Lohmann, I., and W. McGinnis. ≥Hox Genes: It's All a Matter of Context.≤ Current Biology 12 (2002):R514-R516.

McGinnis, W. ≥A Century of Homeosis, A Decade of Homeoboxes.≤ Genetics 137 (1994): 607-611.

McGinnis, W., and R. Krumlauf. ≥Homeobox genes and axial patterning.≤ Cell 68 (1992): 283-302.

≥Mutations in Mammalian Homeotic Genes.≤ Genetic Science Learning Center. 2003. University of Utah. 23 Mar. 2003. <gslc,genetics.utah.edu/units/basics/bodypatterns/mutation.cfm>.

≥N¸sslein-Volhard's and Wieschaus' Discoveries.≤ Nobel e-Museum. 2002. Nobel Foundation. 10 May 2001. <www.nobel.se/medicine/laureates/1995/illpres/nuss-weisch.html>.

Pederson, J.A., J.W. LaFollette, C. Gross, A. Veraksa, W. McGinnis, and J.W. Mahaffey. ≥Regulation by Homeoproteins: A Comparison of Deformed-Responsive Elements.≤ Genetics 156 (2000): 677-686.

Reuter R., G.E. Panganiban, F.M. Hoffmann and M.P. Scott. ≥Homeotic genes regulate the spatial expression of putative growth factors in the visceral mesoderm of Drosophila embryos.≤ Development 110.4 (1990): 1031-1040.