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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 that 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 began with a geneticist 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. (Gap genes)
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 repress 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 protein 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 |
