BIO181, Fall 1995 DROSOPHILA MUTATIONS AND THE HOMEOBOX Vanderbilt, Matthew C

DROSOPHILA MUTATIONS AND THE HOMEOBOX

by
Matthew C. Vanderbilt

Introduction

S
tarting as a fertilized egg with a homogeneous appearance, an embryo made of skin,

muscles, nerves, and other tissues gradually arises through the division of cells (De

Robertis, et al, 1990). This process by which the one aboriginal cell carves out the

brilliant and varied futures for its progeny has been an enigma since biology became a science

two-hundred years ago. Long before most cells in the emerging body begin to specialize, a plan

that designates major regions of the body—the head, the trunk, the tail, and so on—is already

established (De Robertis, et al, 1990). This plan helps seemingly identical combinations of

tissues arrange themselves into distinctly different anatomical structures, such as arms and legs.

Recently, embryologists have made great progress in uncovering the mechanisms that control

this once mysterious process thanks to three major scientific breakthroughs. The first of these

was the discovery in 1953 of the structure of DNA by James Watson and Francis Crick. That led

to quick progress in understanding how genes might direct the growth and fate of cells in a

developing embryo (Caldwell, 1992). The second breakthrough has come in the past decade,

where the powerful techniques of molecular biology have made it possible to isolate and

characterize individual genes that mediate some of the developmental decisions involved in

establishing the embryonic body plan. The third came when, in 1983, biologists working with

Drosophila found a bit of DNA they called a homeobox—named such because it first surfaced as

a leitmotif in homeotic genes, which were known to specify body-part formation. The

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homeobox was identified as a common region in several genes that control the division of the

embryo of Drosophila into segments and help to determine the segments’ fate (which segment

becomes a leg as opposed to becoming a wing, for example). Since that discovery, many

additional homeobox-containing genes have been found in Drosophila, and similar DNA

segments have been implicated in the embryonic development of worms, frogs, and mice

(Benditt, 1989). In all these cases, genes containing the homeobox seem to serve as “master”

genetic elements, interacting with a cascade of other genes to send a cell down a specific

pathway of development (Benditt, 1989). By 1990, it was clear that the key to embryonic body-

plan development was the family of homeobox genes, that subdivide the early embryo into fields

of cells with the potential to become specific tissues and organs. Thus, the homeobox was found

to be part of a basic genetic switch, turning on (whenever it is appropriate) other genes in the

cascade of events by which initially featureless cells are trained for the thousands of different

roles they will eventually assume.

When the homeobox genes were first identified in the mid-1980s, the discovery was hailed as

a major achievement in developmental biology. Since their discovery, homeoboxes have

attracted a great deal of attention, to the point of engendering what some researchers have called

“homeo-madness” (Marx, 1988). A vast amount of research has shown that these genes are

extraordinary in many respects. First, they encode a sequence of sixty amino acids (the

homeobox) that has been evolutionarily conserved in many organisms. The gene sequences are

so widely distributed, that they occur in species ranging from sea urchins to mammals—

including mice and men. (Proof of how closely related genes line up vertically in mice and

human homeobox gene clusters is shown in Figure 1.) The hypothesis is that the mammalian

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homeobox-containing genes, like their Drosophila counterparts, are developmental control genes

(Marx, 1988).

Another extraordinary aspect of the homeobox genes is that they have turned out to be key

regulators of embryonic development. And now, as if those roles were not important enough,

work in several labs is suggesting that homeobox genes may have contributed to evolution as

well. Many developmental biologists believe that homeobox genes may even have been

responsible for helping generate the enormous diversity in organisms alive today.

Figure 1: How They Stack Up (Marx, 1992)

Adapted From M.T. Murtha, T.F. Leckman, and F.H. Ruddle, Proc. Natl. Acad. Sci., 88, 10711 (1991)
Rpt. in Marx, Jean. “Homeobox Genes Go Evolutionary.” Science. 255:24 (Jan. 1992)

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Discussion

O nce the basic coordinates of the embryo are established, normal development depends

on the actions of segmentation genes that control the development of the proper number

of segments in an organism (Dorit, et al, 1991). 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, but even in vertebrates, the muscular regions and the backbone are

segmented (Singer). Occasionally, a segment takes on the identity of another segment of the

organism. This is called homeosis and leads to homeotic mutations. Homeotic mutations are

most common in the highly segmented organisms, such as annelids, and the greatest amount of

documentation on this phenomenon has been done with Drosophila, one of the greatest of

laboratory specimens. The reason there is so much documentation on Drosophila is that they are

prolific breeders, producing hundreds of offspring in a single mating, and the new generations

can be bred every two weeks (Campbell 1993). Another advantage of the fruit fly is that it has

only four pairs of chromosomes, which are easily distinguishable under the light microscope

(Campbell 1993).

Mutations in ‘homeotic’ genes of Drosophila cause the development of the ‘wrong’

segmental appendages (Smith 1975). A number of these genes have now been isolated, and

their nucleotide sequences and times of action determined (Smith 1975). All of these homeobox

genes contains one-hundred eighty nucleotides coding for sixty amino acids (Benditt 1989). As

stated above, the role of homeosis in elucidating genetic control of overall body plans is best

illustrated in the development of Drosophila Melangaster; the common fruit fly (Singer).

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Although they have been found to cause many mutations, homeotic selector genes are not Dr.

Frankenstein’s helpers. The homeotic selector genes, and homeoboxes, are the genes that

specify segment identity. Homeotic genes, also called segment identity genes, control the

eventual fate and determine the types of structures that will develop from each segment (Dorit, et

al, 1991). As was implied in the introduction, all of the embryonic cells have the exact same

DNA sequences; they’re (basically) all exactly alike. What is it that controls which of all those

cells are going to become nerve cells, or part of the epithelial tissue, or even which cells will

make up the head and which cells will make the foot? This is where the homeobox genes have

their jobs cut out for them. The homeotic genes “tell” specific cells what part of the developing

body to go to, and what they will do there. Therefore, even though there are many mutations

caused by these genes, as will be seen later, the amino acid sequence of the homeobox, or

homeodomain, is extraordinarily well conserved across vast evolutionary distances: the

homeodomain in a given Drosophila homeotic gene is virtually identical (fifty-nine of sixty

amino acids) with the homeodomain in the equivalent frog homeotic gene, even though six-

hundred million years have elapsed since those two lineages last shared a common ancestor

(Dorit, et al, 1991). Thus, it can be easily concluded that the homeodomain is performing a

crucial developmental function that depends critically on its amino acid sequence (Dorit, et al,

1991).

Homeotic selector gene mutations result not in missing body parts, as with segmentation gene

mutations, but in a normal number of segments, but segments with abnormal identities (Singer).

Based on mutant phenotypes, homeotic selector genes are found in two specific Drosophila gene

clusters, the Antennapedia complex and the bithorax complex. Genes associated with the

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Antennapedia complex appear to determine the fate of segments associated with the anterior

body segments, whereas the bithorax complex appears to be responsible for the more posterior

segments, such as the abdominal segments.

The Antennapedia and bithorax gene complexes have been identified because of the

mutations that have been produced by them. Interestingly, the Antennapedia and bithorax

complexes appear to be lined up in the same order that 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

(Singer). These interactions between the homeotic selector genes and other genes may, in part,

be controlled by the homeodomain protein coded for by the “homeobox” associated with many

of these genes (Singer).

As stated before, the most startling insight to emerge from DNA sequence analysis of genes

controlling pattern formation in Drosophila is the existence of a short, one-hundred eighty base

pair sequence called the homeobox, which is found with the three prime exon of many homeotic

and segmentation genes, including the Ubx, abd-A, and Abd-B regions of the bithorax complex

and the Dfd, Scr, ftz, and Antp of the Antennapedia complex (Watson, et al, 1988). Likewise,

the homeobox is a one-hundred eighty nucleotide pair sequence that is included in many of the

homeotic selector genes as well as in some segmentation genes and affect its transcription

(Singer). It is a thought that this may allow the genes to control their phenotypic expressions,

along with other related genes. For example, according to Singer, a pair-rule gene known as

fushi tarazu, which is expressed in a seven-stripe pattern during early embryogenesis (Lavorgna

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1991), 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 off. Thus, the ability of the homeodomain to bind to DNA

provides a way for the hierarchical control of homeotic selector genes by segmentation genes

(Singer). Although it is not known how homeotic genes coordinate the differentiation of

segment genes, the homeotic genes are linked with gene expression that leads to the

development of specific structures associated with different segments.

The best-studied segment of Drosophila are the three that make up the thorax and the eight

that make up the abdomen. This is because two remarkable collections of genes — supergenes

— control their development (Wills 1989). These supergenes are called the bithorax and

Antennapedia complexes (BX-C and ANT-C for short) (Wills 1989). According to Wills, these

complexes have fascinated developmental geneticist for decades, because mutations in these

genetic regions have large effects on the fly. Some of the most striking mutations have the

property of allowing structures normally found in one segment to appear in other segments.

Because of these effects, it can be shown that the major function of the bithorax complex is to

control the development of the hind half of the Drosophila body (Wills 1989). Wills continues,

stating that it can be determined what the bithorax complex does by the simple experiment of

removing it and seeing what happens to the fly in its absence. The Spanish geneticist Ginés

Morata did this, and found that when the bithorax complex had been deleted from the

chromosomes, all the segments posterior to the middle thoracic segment began to develop in the

same way as the middle thoracic segment normally does, causing the fly to have legs and wings

on all those segments when there would normally be none (Wills 1989). Therefore, it was

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concluded that the bithorax complex was responsible for modifying the basic structure of the

middle thoracic segment. This does cause death to the larva, but if it had survived to adulthood,

the Drosophila would have had an enormously extended thorax with twenty wings and twenty-

two legs, and it would have had no abdomen at all (Wills 1989). Thus, the deletion of the

bithorax complex produces a classic homeotic mutation — it removes one level of genetic

control from the development of a series making structures appear in inappropriate places (Wills

1989).

As dramatic as that homeotic mutant may seem, it is not as great a sight as those mutations

that can actually be seen in adult flies. One of these, states Wills, the mutant Antennapedia for

which the ANT-C complex was named, has legs instead of antennae growing out of its head.

Wills goes on to state that a fairly good idea of how this grotesquerie can happen is because of

the overproduction of an Antennapedia complex substance. The critical study for this was

carried out by Richard Garber and his group at the University of Washington in Seattle, who

reasoned that if deletion of ANT-C removes control, then turning-on of ANT-C in parts of the fly

where it is usually not expressed should exert this control in inappropriate places (Wills 1989).

To test their hypothesis, the group examined an Antennapedia mutant. The group knew that the

mutation was the result of a small inversion of a piece of the chromosome that included part of

the ANT-C complex, and they found that the inversion had the effect of joining the

Antennapedia gene onto another gene that was normally expressed in the head of the fly and, as

a result, the gene for making legs was expressed inappropriately in one of the head segments

(Wills 1989).

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Stephan Schneuwly, Walter Gehring, and their collaborators at the University of Basel went a

step further and attached ANT-C to a regulator for a different gene in the fly, a gene that is only

expressed if the temperature is raised briefly during development (Wills 1989). According to

Wills, they found that the flies carrying this genetic construct raised at normal temperatures

developed normally, but when such flies were heat-shocked as developing eggs, many of them

had legs instead of antennae — the ANT-C control was indeed being exerted in the wrong place.

Many other homeotic mutant in Drosophila are known, such as in proboscipedia, where legs

grow where mouth parts should be, or in engrailed, which makes the front part of the wing into a

mirror image of the hind part (Wills 1989). A change in the environment during an insect’s

development can often mimic these genetic effects, says Wills. The effect produced by

amputation of the antenna, where, in some cases, regeneration is heteromorphous: in Carausius,

if the antenna is cut through at the level of the flagellum, it regenerates as an antenna; if it is

amputated through one of the two basel segments it commonly regenerates as a leg, complete

with tarsal claws (Wigglesworth), though it occurs in a different insect, is similar to that

produced by Antennapedia (Wills 1989).

Classical genetic approaches have been used with homeotic mutants to map genes to

chromosomes and to identify interactions between genes. For example, it can be determined

whether two genes are on the same chromosome by making a series of specific mating between

flies with mutations in these genes and wild-type flies. If the genes are not on the same

chromosome, offspring with one mutation will not necessarily have the other mutation (Massion

1995). If the mutations are both on the same chromosome, they will be inherited together,

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except in rare situations where there is recombination between chromosomes (Massion 1995).

Geneticists use the frequency of recombination to assess how close together two genes are on a

chromosome (Massion 1995). This approach helped geneticists determine the order of genes

within the Antennapedia and bithorax complexes (Singer). According to Singer, matings

between different mutant Drosophila have also established the hierarchy of genetic control

among egg polarity, segmentation, and homeotic selector genes.

Conclusion

T he simplest interpretation of the available evidence is that a homeotic gene like

Antennapedia affects the initial commitment of all or part of one imaginal disc to become

all or part of a particular structure on the surface of the organism. An alternative, but

somewhat more complicated interpretation, is that the homeotic gene product might govern not

the initial commitment of the disc cells in embryogenesis, but rather the ability of the embryonic

cells to retain that commitment through the three larval instars that precede metamorphosis

(Watson, et al, 1988).

A significant fraction of organism homeobox genes can be linked to some aspect of

morphogenetic patterning, ranging from the most general to very specific. At present, almost

half of the homeobox genes have no known genetic function, although only one, z2, appears to

be dispensable for normal development (Dessain, et al, 1993). Many of the known homeobox

genes are arranged in clusters containing from two to nine members (Dessain, et al, 1993). As a

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general rule, within these clusters, the structures of the respective homeodomains, the expression

patterns, and the genetic functions of the genes tend to be related.

Finally, a surprising number of Drosophila homeobox genes have vertebrate homologs.

Using a greater than eighty-percent or more identity in the homeodomain region as a criterion,

less than twenty-one of fifty-five genes, consisting of prd, cad, eve, en inv, Dll, gsb-d and gsb-p,

lab, pd, Dfd, Scr, Antp, Ubx, Abd-A, Abd-B, msh, NK2, 93Bal, S59/NK1, and 66Dem, can be said

to be much more closely related to a vertebrate homeobox gene than to any other Drosophila

genes, and it seems likely that many more of the Drosophila genes will be found to have

vertebrate homologs (Dessain, et al, 1993). Thus, a very significant percentage of the known

homeobox genes are ancient, having arisen prior to the time, approximately six-hundred to

seven-hundred million years ago, of the vertebrate/arthropod common ancestor.

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Benditt, J.
1989 POU! Goes the homeobox.
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Caldwell, M.
1992. How does a single cell become a
whole body?
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Campbell, Neil A.
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De Robertis, E. M.; G. Oliver and C. E.V.

Wright
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vertebrate body plan.
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Dessain, S. and W. McGinnis

1993 Drosophila homeobox genes.
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Dorit, Robert; W.F. Walker, Jr.; and R.D. Barnes

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Wu
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fushi tarazu. Science, Vol.252.

p.848-851
Massion, Dennis D.
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A. Steitz, and A. M. Weiner
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BasicBooks. n.p.: 227-238

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