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Control of Segmental Identity in Drosophila: Homeotic Genes

by Dr. William Brook
Department of Medical Biochemistry
University of Calgary

Insects are composed of a series of separate segments. Some of the evolutionary ancestors of insects are composed of essentially indistinguishable segments (think centipede!!). Insect segments are more advanced and have become specialized for specific functions. For example, the thoracic segments are often specialized for locomotion and develop wings and legs. This topic will focus on how segments acquire unique characteristics.

The organization of the body plan along the AP axis begins with the establishment of the non-periodic pattern of gap gene expression where domains have discrete identities. The gap domains are multiple segments in width. The gap gene expression is converted to a periodic through pair-rule and segment polarity genes expression. Unlike the pattern of gap gene expression, the stripes of pair-rule and segment polarity gene expression do not provide any information about position along the AP axis. These genes merely subdivide the embryo into parasegments and organize short-range pattern within a parasegment (PS).

(For diagrams of the relationship between segments and parasegments, see Browder et al., 1991,Figures 14.13 and 14.14; Gilbert, 1997, Fig. 14.17; Kalthoff, 1996, Fig. 21.2; Wolpert et al., 1998, Fig. 5.20)

Homeotic genes

Another set of genes is required to assign distinct identities to different parasegments. These are called the homeotic genes. The homeotics differ from the other classes of genes we have considered, in that they are not required to form a specific part of the pattern but rather they assign an identity to these regions once established. Mutations in homeotic genes do not eliminate elements of the pattern but rather cause these elements to develop with inappropriate identities.

Homeotic genes were first identified as dominant mutations that changed the identity of structures. One of the best studied examples of these is the gene Antennapedia(Antp), which as the name suggests causes the transformation of antennal structures to the corresponding (homologous) leg structures.

(See Browder et al., 1991, Figure 14.11; Gilbert, 1997, Fig. 14.28; Kalthoff, 1996, Color Plate 5; Wolpert, 1998, Fig. 10.34)

The dominant phenotype of Antp mutants is a result of inappropriate expression of the normal Antp gene product. Antp is normally required in PS4 which corresponds to the posterior part of the first thoracic segment (T1p) and the anterior part of the second thoracic segment (T2a). If ANTP is expressed in the antenna (far anterior to PS4) transformation of antennal structures to leg structures occurs. This can result from a mutation that brings normal coding sequences under inappropriate control or by regulated mis-expression of a cDNA encoding the protein under heat shock control.

(See Figure 14.27b, Browder et al., 1991)

The homeotic genes can be thought of as genetic switches turn different programs of cellular differentiation on or off. The activities of a number of homeotic genes are required to establish the identity of parasegments in the trunk region (posterior head, thorax, and abdomen) of the embryo. These genes are clustered in two major groups called the Antennapedia complex (ANT-C) and the bithorax complex (BX-C).

The bithorax complex

Studies of homeotics really begin with a genticist named Ed Lewis. During a period spanning several decades, he worked out the genetics of the bithorax complex. Lewis made the interesting observation that within the BX-C, the order of the genes on the chromosome was the same as the order of segments that they affect along the embryonic axis. The same turned out to be largely true for the ANT-C and as you will hear later in other species as well (this is sometimes referred to as the "colinearity principle".

Initially, Lewis thought that 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). The genes are large (they span approx. 300 kb) 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 of the BX-C 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. abx and bx are required for normal development of PS5. bxd and pbx are required for the correct development of PS6. As an example in bx mutations there is a transformation of the anterior part of the haltere (a balance organ on the third thoracic segment that is evolutionarily related to the wing) such that it develops as the anterior part of the wing. There is also a transformation of the anterior part of the third leg such that it develops as the anterior part of the second leg (T3a develops as T2a). pbx mutations for example affect another set of structures and result in the transformation of the posterior part of the haltere into the posterior part of the wing and the posterior part of the third leg into the posterior part of the second leg (T3p -> T2p). The same situation exists for AbdA and AbdB. 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. These elements are called infra-abdominal 2 although iab-2 controls AbdA in Abdominal segment 2, iab-3 controls expression in abdominal 3 etc. These regulatory elements behave as enhancers.

(See Figure 14.28, Browder et al., 1991; Gilbert, 1997, Fig. 14.34; Wolpert et al., Fig. 5.37)

Regulation of homeotic gene expression

Regulation by segmentation genes: The control elements described above are likely to be complex integrators of a variety of information from the gap, pair-rule, and segment polarity genes. The domains of expression of the homeotic genes are parasegmental. Therefore the limits of expression must be controlled at least partially by the pair-rule and segment polarity genes. Mutations in pair rule genes do affect homeotic gene expression. However, since various pair-rule stripes are not molecularly distinguishable from one another, additional information is required to determine where along the AP axis particular homeotic genes are active. This type of information already exists at the time the homeotic genes are activated in the spatially ordered domains of gap gene expression. As an example, hunchback seems to set the anterior limits of Ubx expression. In hunchback mutant embryos, Ubx expression expands anteriorly. hunchback protein can bind to cis-acting control regions of the Ubx gene and mutations of hunchback binding sites in the enhancer region obviates hunchback mediated repression.

Cross-regulation between homeotic genes: homeotic genes also regulate each other. Cross-regulatory interactions are important in defining the domains of expression. Genetic analysis shows that more posterior acting genes function as negative regulators of their more anterior neighbors. AbdB represses AbdA, both represses Ubx, all three repress Antp. The hierarchy can be deduced from the phenotypes of mutant embryos as follows:

  • in AbdB mutant embryos: PS 13 and PS 14 resemble PS7-12 (the AbdA domain)
  • in AbdA mutant embryos: PS 7-12 resemble PS 6 (the UBX domain)
  • in Ubx mutant embryos PS 5 and 6 resemble PS 4 (the Antp domain)
  • in Antp mutant embryos PS 3 and 4 resemble PS 2 (the Scr domain; n.b., Scr is another member of ANT-C)

Figure. 1. Expression and lethal embryonic phenotypes of homeotic genes. (Adapted from Akam, 1987.)

This phenotypic analysis has been confirmed by the expression patterns of the various genes in various mutant backgrounds. The anterior limits of the domain of expression of a particular homeotic gene are presumably set by a collaboration between gap genes and pair-rule genes (see above). Removal of an anteriorly-acting homeotic has no effect on expression or phenotype in the domain of a more posteriorly acting homeotic gene.

  • Antp expands posteriorly from PS4 to PS6 in a Ubx mutant
  • Antp expands posteriorly from PS4 to PS12 in a Ubx AbdA mutant
  • Antp expands posteriorly from PS4 to PS14 in a Ubx AbdA AbdB mutant
  • Ubx expands posteriorly from PS6 to PS12 in a AbdA mutant
  • Ubx expands posteriorly from PS6 to PS14 in a AbdA AbdB mutant
  • etc.

Figure 2. Homeotic gene expression in homeotic mutant embryos. (Adapted from Akam, 1987.)

Interactions between homeotic genes

The function of a given homeotic gene is epistatic to its anterior neighbors (a similar phenomenon in vertebrates is called "posterior prevalence"). Under normal circumstances, the dominance of a posterior gene in repressing its anterior is obvious in molecular terms as the more posterior genes will repress the transcription of the more anterior genes. However, it is possible to force the expression of homeotic genes outside their normal domain of expression either using transgenes or mutations. The results from these experiments are quite surprising.

Using a heat shock construct (the heat shock promoter driving a Ubx cDNA) it is possible to drive high levels of Ubx protein throughout the embryo. In the AbdA and AbdB domains there is no phenotypic consequence of Ubx overexpression. In more anterior regions, i.e the Antp domain, hs-Ubx has the expected consequence of transforming segments to PS6 identity. Similarly, hs-Antp has no effect in the more posterior domains but can transform more anterior domains. The epistasis of Ubx over Antp can be strikingly demonstrated by experiments where both are heat-shocked together. Each alone transforms the anterior segments to its own character. When co-expressed, Ubx dominates and all structures anterior to the Ubx domain are transformed to PS6, despite the presence of high levels of Antp protein(Gonzalez-Reyes et al., 1990; Mann and Hogness, 1990).

Figure 4

In other words, more posteriorly-acting genes are able to block not only the transcription of more anteriorly-acting genes but can also block their activity if both are present.

How do the homeotics specify segmental identity?

In short we really don't know.

In general, we think of the homeotics as genetic switches that control the choice between different developmental pathways but it is not well understood in any specific detail how the homeotics work. All the members of the BX-C and ANT-C encode homeodomain proteins and several have been proven to act as transcription factors in vitro. It is generally assumed that the homeotic genes act by turning on or off the expression of downstream target genes. The data from the co-expression of Ubx and Antp transgenes suggests that posterior acting genes can block the ability of anterior acting genes to regulate downstream target genes.

There have been very few examples target genes identified. One example is the gene Distal-less, which is repressed in the abdominal segments by the BX-C (Vachon et al., 1992). One of the most obvious differences between thoracic and abdominal segments is an absence of legs. Distal-less is a homeodomain expressed in the leg primordia in the thoracic segments. and its expression is required for limb development. Both Ubx and AbdA have been shown to bind to the Distal-less enhancer and can act as repressors of Distal-less expression. In BX-C mutant embryos, Distal-less is expressed in all abdominal segments. This demonstrates that Distal-less is one of the genes regulated by the BX-C genes in order to promote abdominal development.

Figure 4. (Adapted from Vachon et al., 1992)

Learning Objectives

What are homeotic genes?

Describe the molecular organization of the bithorax complex and how it relates to the function and regulation of the genes Ubx, AbdA and AbdB.

How do the different classes of segmentation genes control the spatial patterns of homeotic gene expression?

A given homeotic gene is epistatic or dominant over its anterior neighbors. Explain how the homeotic mutant phenotypes, the expression patterns of the homeotic genes in homeotic mutants, and the results of ectopic homeotic gene expression on transgenes support that statement.

How are the homeotic genes thought to control segmental identity?

Digging Deeper:

Links to Related Material

Multiple regulatory modes for homeodomain proteins (in Zygote)

References to Related Material

See Peifer et al. (1987) for a review of the bithorax complex.

See McGinnis and Krumlauf (1992) for a review of homeotic gene organization, regulation and function.

Reviews* and References

*Akam, M. 1987. The molecular basis for metameric pattern in the Drosophila embryo. Development 101, 1-22.

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

Gonzalez-Reyes, A., Urquia, N., Gehring, W. J., Struhl, G., and Morata, G. 1990. Are cross-regulatory interactions between homoeotic genes functionally significant? Nature 344, 78-80.

Mann, R. S., and Hogness, D. S. 1990. Functional dissection of Ultrabithorax proteins in D. melanogaster. Cell 60, 597-610.

*McGinnis, W., and Krumlauf, R. 1992. Homeobox genes and axial patterning. Cell 68, 283-302.

*Peifer, M., F., K., and Bender, W. 1987. The bithorax complex: control of segmental identity. Genes Dev. 1, 891-898.

Vachon, G., Cohen, B., Pfeifle, C., McGuffin, M. E., Botas, J., and Cohen, S. M. 1992. Homeotic genes of the Bithorax complex repress limb development in the abdomen of the Drosophila embryo through the target gene Distal-less. Cell 71, 437-450

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Last revised Monday, July 20, 1998