Introduction

Developmental biology is entering into a synergism with evolutionary biology. This synergism has been termed ìevo-devoî biology. Developmental biology contributes to evolutionary understanding because evolutionary changes in morphology result from evolutionary changes in development. Thus, a full understanding of evolution requires knowledge of these developmental processes (Futuyma 1998). To answer certain evolutionary questions, therefore, research must dive deep into the development of organismsí different parts, and genes that control body plan development. The expression of different genes in different cells causes much of development (Futuyma 1998). Differences in gene expression result in differences in proteins and enzymes which control cellular functions or make up cell parts. Effector genes cause the differential expression of proteins and are controlled by regulatory genes. One important type of regulatory gene is the homeotic gene. Homeotic genes control the development of an organismís body parts. The largest volume of work on homeotic genes comes from Drosophila, yet researchers have been able to extrapolate that data to perform meaningful studies on human genes.

Some studies show that homeotic genes control wound healing in human fetuses and that the manipulation of these genes may lead to advances in scarless healing. Could humans be able to regenerate tissue like a newt does? Amazingly, we already do, except such regeneration only occurs during early fetal development. What implications might such advancement have in an evolutionary context? How and to what extent to homeotic genes control evolutionary change? There are opposing answers to this question, but before describing them, it is necessary to discuss in more detail what homeotic genes are.

Homeotic Genes: Definition and Background

Homeotic genes control the transcription of numerous other genes, including the effector genes that build the features typical of a particular body structure. Homeotic genes are not required to form a specific part of the pattern, but rather they assign an identity to these regions once they are established. At a critical stage in early development, different combinations of homeotic genes are transcribed at different positions along the anterior-posterior axis of an organismís body. The homeotic genes that control this type of patterning are called Hox genes. Hox genes have been found in every animal phyla examined. These genes do not encode for a structure to form, but they provide positional information: they evoke or repress activity in other genes according to the position of the cell in the developing body (Futuyma 1998).

Homeotic genes were discovered by Edward Lewis in the late 1970s through the observance of mutations in Drosophila melanogaster. Lewis found that homeotic gene mutations change the location of certain structures that are normally found on one part of the body to a location somewhere else on the body. Lewis observed that a mutation in a single gene called Antennapedia causes a structurally proper leg to grow where antenna normally grow (See Fig 1). The Antennapedia gene has two transcription products, one of them initiating within an intron of the other (Frischer et al 1986). One of the products is necessary for dorsal thorax development, while the other is required for leg development and for embryonic viability (Bermingham et al 1990).

Another example of a homeotic gene mutation discovered by Lewis is the Ultrabiothorax (Ubx) gene. In the wild-type Drosophila, the wings are found on the second segment of the thorax, and the third segment contains the halteres. In the Ubx mutant, the third thoracic segment becomes another second thoracic segment, and thus another set of wings are present instead of halteres (Futuyma 1998).

Initially, Lewis thought that there was one gene for each segment, but molecular analysis indicates that there are three regions that encode homeotic genes in Drosophila. Those regions are called Abdominal-A (abdA), Abdominal-B (AbdB), and the aforementioned Ubx. The genes are large and have very complex regulatory regions. Most of Lewis' "genes" are actually cis-acting regulatory elements that control the pattern of expression of the three protein coding regions in each segment. The regulatory regions are defined by a large number of mutations affecting the expression of one or more of the genes in different regions of the body. For example: abx, bx, bxd, and pbx are 4 classes of recessive mutations that all act on the Ubx transcription unit. A mutation in bx causes a transformation of the anterior part of the haltere to anterior part of the wing. Regulatory elements that affect the expression of these genes are spaced along the DNA in the same order as the segments of the abdomen that they affect (Browder et al 1991).

It is important to explain how homeotic gene research in Drosophila gave way to research and conclusions about homeotic genes in humans. Several of the genes involved in human diseases were identified originally in Drosophila because of the wealth of genetic data and the formidable array of genetic techniques developed for use in this organism. Most of the genetic and molecular mechanisms operating to direct development are shared by all animals from insects to more complex animals such as ourselves, so studies in Drosophila have helped us to understand how all animals develop.

 

Homeotic Genes and Evolutionary Change

Homeotic genes are ubiquitously expressed throughout all animal phyla. There are three main changes that help explain evolutionary change due to homeotic genes. Changes in the number of homeotic genes over time contributed to morphological evolution of species. Drosophila has 8 homeotic genes controlling all body segments. The vertebrate amphioxus has 10 homeotic genes, while mice and humans both have 38 (but only 13 paralogous clusters which are similar to the 10 genes in amphioxus ñ in mice and humans the clusters have been quadrupled) (See Figs. 2 and 3) (Futuyma 1998). Increasing the number of homeotic genes may have contributed to the greater differentiation of body parts and more complex body plans of vertebrates. Other factors that probably led to the morphological evolution include changes in spatial expression and changes in gene interactions.

Changes in spatial expression of homeotic genes are caused by either changes in genes that regulate the homeotic genes, or changes in the homeotic genesí response to those regulators (Futuyma 1998). Changes in gene interactions occur when homeotic gene expression stays the same, but different genes are regulated by their products. For example, in Drosophila the normal function of the Ubx gene is required to develop halteres on the third thoracic segment, while in butterflies, the Ubx gene controls the development of the second set of wings in the third thoracic segment. Thus, the difference between the two is due to differing responses by downstream genes, not because of differences in the Ubx gene. Another example of changes in gene interactions is illustrated by the following three mutations: the Eyeless mutation in Drosophila, the Small eye mutation in mice, and the Aniridia mutation in humans. The protein products between the vertebrate and fly genes are 94 percent identical. Moreover, Georg Halderís lab, (1995) was able to express anatomically normal (yet nonfunctional) eyes on Drosophila in various locations, such as legs and wings. They concluded that the normal Eyeless gene that encodes for normal eyes must be like a switch that turns on several genes that cause eye production. The normal mouse (normal Small eye) gene was transferred into Drosophila and the function was retained. The gene has evolved very little in sequence and function in millions of years (Futuyma 1998). Other studies support the idea that mammals and insects share control genes such as the Six3 gene (Oliver et al 1995).

Homeotic Genes in Human Fetal Development

Through the above overview of the past and more recent work on homeotic genes, it becomes clear that homeotic genes are essential to the development of an organismís body. While much of the knowledge of homeotic genes comes from studies on non-human organisms, there have been advancements in understanding the implications of specific homeotic genesí roles in human development. More specifically, Krumlauf (1994) showed that homeotic genes play an integral role in the embryonic development of vertebrates. Furthermore, homeotic genes are expressed strongly in an organ- and stage-specific pattern in human embryos, which suggests their involvement in the control of early fetal development of organs and body parts (Mavilio et al 1986). Specifically, the development of the skin is controlled by homeotic genes.

Homeotic Genes in Skin Development

The morphogenesis of the skin is controlled by a coordination of several genes. This is to be expected because of the complexity of the organ. The expression of a number of homeobox genes in both fetal and adult skin suggests that these genes play an important role in skin development. ìAs skin homeobox gene expression may be associated with the constant differentiation necessary to maintain the epidermis, it may therefore play an important role in the regeneration of the dermis/epidermis following cutaneous injury, where the coordinated expression of a number of genes is required to achieve successful regeneration of the skinî (White et al. 2003). Additional studies confirm the roles of homeotic genes in skin development. Komuves et al (2002) showed that the protein products of the homeotic gene HOXB4 were uniformly detected in all regions of fetal and adult skin. It was found that HOXB4 proteins were present in both nuclear and cytosolic domains, in both adult and fetal cells located in all areas of the body, including scalp, back, and arm.

While these skin studies focused mainly on the role of homeotic genes controlling skin cells in relation to skin diseases and cancers, a logical connection can be made between the knowledge of homeotic genes in fetal development and skin development. Combining the information gained from these studies yields ideas and questions about fetal wound healing.

Fetal Wound Healing

            Studies by White et al (2003) and Stelnicki et al (1998) explain the possible implications of knowing more about fetal wound healing and the homeotic genes that regulate tissue regeneration in human fetuses. Fetal wound healing is different from adult wound healing because the former is a regenerative process without scarring, yet the basis of the difference is poorly studied (White et al 2003). There are, however, a few studies that have shown fibroblasts to be integral to scarless fetal wound healing (Lorenz et al 1995). The other factors that may have a role in scarless fetal wound healing are certain unique characteristics of inflammatory cells, extra-cellular matrix, cytokine profile, and developmental gene regulation. (Dang et al 2003). The key in this list may be developmental gene regulation, according to White et al (2003). In their study, the homeotic gene Prx-2 significantly decreased important fetal fibroblast responses implicated in fetal wound healing. It is important to note, however, that the deletion of Prx-2 stimulated the extra-cellular matrix reorganization. Still, this study provides further evidence for the importance of homeotic genes in the regulation of fetal wound healing responses.

            Another interesting study by Stelnicki et al (1998) notes that homeotic gene expression increases when amphibian limb regeneration takes place, suggesting that they play a major role in this process. Because homeotic genes are so conserved (as noted earlier in this paper), it seems plausible that genes of similar type could be functioning in fetal wound healing. Stelnicki et al (1998) found that Prx-2 and HOXB13 exhibit expression patterns consistent with a role in fetal wound healing. Thus, both genes were expressed in proliferating fibroblasts and showed differential expression patterns in scarless fetal skin wounds compared with adult scarring wounds.

            What is the significance? Researchers hope that their work can be used to develop methods to reduce scarring in healing, and in the distant future to somehow allow for the regeneration of mammalian tissues. This means that changes in homeotic genes will cause differences in humansí body plans, like it did in that of Drosophila experiments. Obviously, homeotic genes can be involved in evolutionary change, but how and to what extent? There is some controversy surrounding this topic.

Controversy

According to the University of Utah Genetic Science Learning Center, organisms that have homeotic gene mutations and consequently different phenotypes can still potentially reproduce if the mutation is not lethal. This means that homeotic mutations can be an effective means for evolutionary change. The Center provides an example in which a mammal with a single homeotic mutation has, say, an arm that is broader than the wild type organism. If the broader arm still works and looks like an arm, then there is a chance that that organism might have a selective advantage of the wild type organisms. This might happen if the organism can then swim faster because of the broader arm. If this occurred, then the mutant organism would have greater fitness, and its genes would be preferentially passed along to the next generation, thus influencing the course of evolution.

It seems that this suggestion for evolution through homeotic gene mutations is flawed for the following reasons. First, neutral theory holds that although a small minority of mutations in DNA or protein sequences are advantageous and fixed by natural selection, and although some are disadvantageous and are eliminated by ìpurifyingî natural selection, the majority of mutations that are fixed are effectively neutral with respect to fitness and are fixed by genetic drift. The theory states that most genetic variation at the molecular level (like mutation) is selectively neutral and lacks adaptive significance. It is important to note that this theory acknowledges that many mutations are deleterious and are eliminated by natural selection so that they contribute little to the variation we observe (Futuyma 1998). Thus, if a mutation in a homeotic gene did not cause an organismís death, it seems unlikely that it would be an ìeffective means for evolutionary change.î Second, there seems to be too much diversity in body plans over the past 550 million years for the differences to have arisen by this method. It would require that chance mutations create nonlethal differences with some functionality that persist over time. How might this occur if homeotic genes are regulatory genes that control entire body segments? It seems that if a homeotic gene that codes for heads or legs or arms gets ìturned off,î that the resultant mutant would not be selected for by natural selection. How do we account for the variation in body plans among organisms?

Michael Akam (1998) offers a different view on evolutionary changes due to homeotic genes. He suggests that we abandon the view that homeotic genes are ìbinary switches,î but rather that mutations in homeotic genes cause more subtle changes. Homeotic genes (such as Ubx and Abdominal-B) ìshow dosage effects, suggesting that quantitative variation in the levels of Hox gene products can affect segment morphology in subtle ways. Recent studies suggest that these effects are not just artifacts of laboratory mutations, but rather that they parallel the complex role that regulation of the Hox genes actually plays in the control of morphogenesis. Allelic variation at Hox loci does exist in natural populations though the morphological consequences of this variation await studyî (Akam 1998). Akam (1998) also suggests that models for homeotic gene function move beyond a rigid hierarchical view of gene action in development, towards a model that recognizes the complexity of regulatory information that can be integrated by single promoters. The organization of such promoters will allow for a more suitable explanation of evolution through homeotic genes.

 

 

Conclusion

            Ultimately, as evolutionary and developmental biology meshes, a deeper understanding of what role homeotic genes have in evolutionary change will result. Although much of the past work on homeotic genes was performed on Drosophila, much work has been done on mammalian and human homeotic genes. Specifically, the roles of homeotic genes in scarless fetal wound healing have been somewhat elucidated. It is hoped that this knowledge will be used to reduce scarring in adult humans. The evolutionary consequences of such changes in homeotic genes are under debate. While theories currently explain homeotic genes as on/off switches that cause abrupt changes that must survive against the odds, some hold a theory that the changes that result from homeotic mutations are much more subtle. Thus, it seems that more research should be conducted in this area. Perhaps as evolutionary and developmental biology synthesize, the answers will become clearer.

 

 

 

 

 

 

 

 

 

 

Fig. 1 ñ Fruit fly with mutation in the Antennapedia gene

Fruit fly with and without mutation

Fig. 2 - Fruit Fly - Homeotic genes in the fruit fly are broken into two clusters on the same chromosome and separated by a long stretch of DNA (blue line). The five genes shown at left (blue and pink boxes) represent the Antennapedia complex. The three genes shown at right (yellow boxes) are part of the Bithorax complex.

fruit fly homeotic complex

 

Fig. 3 ñ Mouse - The mouse has four homeotic gene clusters. All four are similar, but they are located on different chromosomes.

mouse hox genes

All figures courtesy <http:// gslc.genetics.utah.edu/>

Works Cited

 

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Browder, L.W., Erickson, C.A. and Jeffery, W.R. 1991. Developmental Biology. Third ed. Saunders College Pub. Philadelphia, USA

Dang C, Ting K, Soo C, Longaker MT, Lorenz HP. 2003. Fetal wound healing current perspectives. Clin Plast Surg. 30:13-23. Review.

 

Frischer, L. E., Hagen, F. S. and Garber, R. L. 1986. An inversion that disrupts the Antennapedia gene causes abnormal structure and localization of RNAs. Cell 47: 1017 - 1023.

 

Futuyma, Douglas J. 1998. Evolutionary Biology, Third Ed. Sinauer. Sunderland, Massachusetts, USA. 48-50, 665-69.

 

Halder, G., Callaerts, P. and Gehring, W. J. 1995. Induction of ectopic eyes by targeted expression of the eyeless gene in Drosophila. Science 267: 1788-1792.

 

Komuves LG, Michael E, Arbeit JM, Ma XK, Kwong A, Stelnicki E, Rozenfeld S, Morimune M, Yu QC, Largman C. 2002. HOXB4 homeodomain protein is expressed in developing epidermis and skin disorders and modulates keratinocyte proliferation. Dev Dyn 224:58-68.

 

Krumlauf, R. Hox genes in vertebrate development. 1994. Cell 78: 191-201. Review.

 

Lorenz HP, Lin RY, Longaker MT, Whitby DJ, Adzick NS. 1995. The fetal fibroblast: the effector cell of scarless fetal skin repair. Plast Reconstr Surg. 96:1251-61.

 

Mavilio F, Simeone A, Giampaolo A, Faiella A, Zappavigna V, Acampora D, Poiana G, Russo G, Peschle C, Boncinelli E. 1986. Differential and stage-related expression in embryonic tissues of a new human homoeobox gene. Nature 324: 664-668.

 

Oliver G, Mailhos A, Wehr R, Copeland NG, Jenkins NA, Gruss P. 1995. Six3, a murine homologue of the sine oculis gene, demarcates the most anterior border of the developing neural plate and is expressed during eye development. Development 121: 4045-55

 

ìHomeotic Mutations Could Be Involved in Evolutionary Change.î University of Utah. 11 April 2003. Genetic Science Learning Center. 17 April 2003. <http:// gslc.genetics.utah.edu/units/basics/bodypatterns/evolutionary.cfm>

 

White, Philip, David W Thomas, Steven Fong, Eric Stelnicki, Fritz Meijlink, Corey Largman, and Phil Stephens. 2003. Deletion of the Homeobox Gene PRX-2 Affects Fetal but Not Adult Fibroblast Wound Healing Responses. J Invest Dermatol 120: 135.