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The Foundations of Developmental Biology
From Sperm and Egg to Embryo
Genetic Regulation of Development
Organizing the Multicellular Embryo
Generating Cell Diversity
Dynamic Development at a Glance
The Developmental Biology Journal Club
Developmental Biology Tutorial
Establishment of Spatial Patterns of Gene Expression
During Early Vertebrate Development: Hox Genes
by Dr. Derrick Rancourt
Department of Medical Biochemistry
University of Calgary
We shall now continue from the discussion of homeotic genes in Drosophila and
discuss parallels that have been observed in higher eukaryotes, namely vertebrates. Much
of the material that I shall be discussing is new and not in the text because this
particular field has advanced considerably in the past five or so years.
Take Home Lesson:
The homeotic (Hox) complex governs both the overt and non-overt
segmentation that occurs in vertebrates.
Key Features of this lecture are :
1) The vertebrate homeotic complex comprises four distinct Hox
gene clusters (Hox A, B, C, D) that are organized into thirteen homology (or paralogue)
2) The chromosomal organization of the genes in each Hox cluster reflects its
anterior-posterior expression in the body plan (spatial colinearity). Unlike in Drosophila,
vertebrate Hox genes are also temporally colinear and are expressed in an anterior to
posterior direction. In general, members of the same paralogue group are expressed at the
same time and have the same anterior boundary of expression.
3) Homeotic genes are expressed within segmented and unsegmented structures within the
body plan. Hox gene expression in some unsegmented structures arise from segmented
precursors. The specification of segmented structures may be due to a specific combination
of homeotic genes (or Hox Code).
4) Modern genetic methods such as targeted mutagenesis in mouse have begun to reveal the
function of homeotic genes in vertebrates. In general, homeotic phenotypes observed in
specific hox gene mutations are restricted to the anterior boundary of expression.
5) The genetic redundancy of the vertebrate homeotic complex may have enabled the rapid
evolution of vertebrates.
Evolution of the Homeotic Complex
Homeosis was first defined by Bateson (1894) to describe natural
variants of a species where certain parts of the body plan displayed morphological
features typical of other regions. This idea was reiterated by the work of Lewis, who
found several homeotic mutants affecting segment identity in Drosophila.
After the cloning of the Drosophila homeotic complex in Drosophila, these
genes were found to be transcription factors and coined to be homeotic selector
genes, meaning that the fate of a given segment was selected by the expression of specific
homeotic genes. Of course, it was immediately important to see if mouse and other
vertebrates had a similar set of genes - especially because vertebrates only appear to be
partially segmented animals.
Once this was confirmed, a mad race began in the mid eighties to clone all of the homeotic
genes, which only became complete about two years ago. Now, in the mouse and human (and
presumably other vertebrates including chicken and Xenopus), there are thirty nine
genes that are organized on four different Hox clusters (A, B, C, D) found at four
distinct chromosomal loci. Based on homologies, between themselves and their Drosophila
counterparts, these genes have been organized into thirteen homology groups called paralogues.
There are several interesting points that can be made simply by comparing the Drosophila
and vertebrate complexes. First, paralogue groups 9-13 all appear to derived from Abd-A
through some gene duplication event, while similarly paralogue groups 2 and 3 are derived
from proboscapedia. Unlike in Drosophila, where the Antennapedia and
Bithorax complexes were separated in evolution, each vertebrate homeotic complex is
intact. However, within each cluster, there are genes missing, which is thought to be an
important feature in our evolution. (We shall return to this point.) In Amphioxus,
a near vertebrate ancestor, there is only one complex that resembles a complete Hox A
cluster. That means, for instance, that hoxa-8 is present this animal and that it has
since disappeared. Similarly, that ancestral homeotic cluster first quadruplicated itself
four times, after which individual genes were lost in evolution. As we shall discuss
later, there is considerable genetic redundancy within the homeotic gene complex, and this
redundancy made it relatively easy to lose specific homeotic genes in evolution without
drastically affecting the body plan.
Hox Gene Expression and Colinearity
Whereas the colinearity that occurs between the homeotic complex
and anterior-posterior expression was first recognized by Lewis in his genetic
experiments, it was formally demonstrated in mouse using in situ hybridization.
Here, members of the hoxB cluster have different anterior boundaries
of expression in the CNS and prevertebra, which reflected their relative chromosomal
organization. In 1989, hox genes were given names according to the order in which they
were discovered. In 1997, we know that the anteriorly expressed genes reside at the 3' end
of the complex and that unlike in Drosophila (whose homeotic genes are all
expressed at the same time), the mouse homeotic complex is also temporally colinear,
meaning that as the body plan develops in an anterior-posterior direction during
gastulation, there is a sequential expression of the homeotic complex. We also know that
the paralogue groups are expressed with roughly the same anterior boundaries in segmented
tissues and that the next more posterior group is expressed one or two segments more
(See Fig. 14.39, Browder et al., 1991; Gilbert, 1997,
Fig. 16.3; Kalthoff, 1996, Fig. 22.6; Wolpert et al., 1998, Fig. 4.8)
There are some interesting differences in the regulation between vertebrate and Drosophila
- First, each vertebrate hox cluster is the size of the Ubx
gene in Drosophila. This observation has been used to argue that the regulation of
the Drosophila complex is more complex. Indeed, whereas we have observed shared
regulatory elements in the vertebrate genes, in Drosophila each homeotic gene
appears to function more or less independantly.
- Second unlike in Drosophila, there are no equivalent pair
rule and segment polarity genes setting up segmentation for the homeotic genes. While many
homologues of these genes exist, such as even skipped and paired, these homologues are
often expressed after the homeotic genes are expressed. Thus, it is still a mystery how
vertebrate hox genes are turned on in embryogenesis.
In vertebrates, the main evidence of segmentation is in the
vertebral column. The head, the fore- and hindlimbs are later adaptations onto the early
vertebrate skeleton. The earliest expression studies focused on the expression of hox
genes in the prevertebra of mid-gestation embryos. In these experiments, it was realized
that specific paralogue groups had anterior boundaries within prevertebra and that each
vertebra had its own hox address or code. Whereas the anterior boundaries are well
defined, the posterior boundaries of hox genes are not; suffice it to say that many more
homeotic genes are expressed within posterior segments than in anterior segments.
Hox Gene Expression and Embryogenesis
Recall that during neurulation, the vertebral precursors, somites,
condense out of axial medoderm as the neural folds emerge from Hensen's node. Whereas the
somites are the earliest and most overt evidence of the segmentation at this time, there
is a theory that the nervous system is segmented as well. As the neural folds are emerging
and before we begin to see somites, electron microscopists have observed transient
stuctures within the neural folds called somitomeres. Although these structures will
disappear at somitogenesis, it is believed that the basic neural tube is segmented,
although there is little overt evidence to demonstrate this. However, the existence of
somitomeres helps to explain the boundaries of hox gene expression within the midgestation
However, if we look at the CNS earlier in embryogenesis, shortly after neurulation, and
during somitogenesis, segmentation is obvious in the rhombencephalon, which is the CNS
precursor of the hindbrain. In this structure and at this time there is a segmental
periodicity to the expression of the most anteriorly expressed genes. So, each rhombomere
has its own hox code as well (i.e., except R1 and R2, which do not express hox genes.
(Browder et al., 1991, Fig. 14.40; Gilbert, 1997, Fig.
16.4; Kalthoff, 1996, Figs. 22.7-22.8; Wolpert et al., 1998, Figs. 4.23-4.24)
Hox gene are not expressed anterior to the hindbrain. The forebrain and midbrain are
more advanced structures in vertebrate and although there is evidence that these
structures are also segmented, the homeotic gene family had nothing to do with the
elaboration of these structures. An important feature of the rhombencephalon is that
cranial neural crest cells delaminate from this structure as the neural tube is closing.
These specialized neuroectodermal cells are a more recent adaptation of jawed vertebrates
and will give rise to, or contribute to, the formation of the jaw, the cranial nerves and
soft tissues of the neck and throat, including the thyroid and thymus. Anterior hox genes
that are expressed in rhombencephalon are also expressed in their derivative neural crest
cells. These neural crest cells will migrate to the branchial arches and give rise to
these specialized structures. Each branchial arch therefore has its own hox code, and - as
we shall see - mutations within the anterior genes can affect the emergence of these
Whereas hox gene expression has been closely studied in cranial neural crest, little is
known about the role of hox genes in trunk neural crest. Although various hox gene have
been found to be expressed in soft tissues such as the lung, gut, etc., little is known
about whether this expression is the result of segmentation (i.e., being derived from
trunk neural crest migration) or whether this expression is de novo.
The Hox Code
The hox code model predicts that combinations of hox genes
specify the development of the anterior-posterior axis. Evidence for the code comes from
three lines of experimentation:
- Comparative Anatomy indicates that the appearance of
specific vertebrae such as the cervico-thoracic junction is correlated with the expression
of specific hox genes. HoxC6 expression is invariantly in the cervicothoracic prevertebra
in mouse, chicken, goose, Xenopus and zebrafish. Interestingly, the length of the
neck is different in each species. Similarly, the positioning of the lumbral-sacral
junction may be a function of members of the hox10 paralogous family.
- Retinoic acid induced homeosis during mouse embryogenesis.
Retinoic acid, a developmental steroid and teratogenenic agent, has been found to alter
the axial expression of homeotic genes when administered during somitogenesis. Each hox
cluster has cis elements that respond to retinoic acid and thus normal
developmental regulation is perturbed. The anterior expression boundaries of several
anterior hox genes were shifted posteriorly, resulting in the anteriorization of
vertebrae. Similarly, posterior gene expression was shifted anteriorly, resulting in a
posteriorization of fate. Similar results were observed in cranial neural crest
derivatives of the hindbrain: Expression boundaries shifted anteriorly, and R2 and R3
resembled the characteristics of R4 and R5.
(See Gilbert, 1997, Fig. 16.5; Kalthoff, 1966, Fig. 22.18; )
- Transgenic induced homeosis in mouse. Gain of
function mutations, in which a posterior gene is expressed more anteriorly, resulted in a
posteriorization of anterior vertebrae. This obsevation led to the suggestion of posterior
prevalence, meaning that when moved to an anterior location, a posterior gene had
priority over its anterior collegues. Loss of function mutations, in which a gene is
removed by targeted mutagenesis, resulted in an anteriorization of vertebrae. In this
situation, the hox code of the mutant prevertebra or rhombomere would now partially
resemble its near-anterior neighbour. Following the posterior prevalence model, the
respecified vertebra no longer expresses the posterior hox gene, which made this segment
different than its anterior neighbour.
Targeted Mutagenesis in Mice
More recently, other types of manipulations have been possible.
Reporter genes such as lacZ can be integrated into a locus so that it is under the control
of native elements (see Targeted Modifications in Mice). Gene swaps can be performed so that when applied to the homeotic
complex, a posterior gene can be made more anterior and visa versa. Very subtle mutations
can be made such as point mutations. In this case, the neo cassette is placed within an
intron so that expression of the gene is not perturbed. We can also make small and large
deletions within the complex to destroy regulatory elements. Although people have been
able to delete an entire hox complex in ES cells, they have thus far been unable to
generate these mice.
I was a postdoctoral fellow in the laboratory where gene targeting was developed. Part of
our interest was to apply this new technology to understanding the homeotic complex. There
were approximately 30 genes known in the complex at the time, and we undertook the
insurmountable task of knocking out each and every gene in the complex. By the time I
left, this work was about three quarters complete. I want to highlight a few interesting
experiments that shed light on the complexity of this multigene family.
The first gene that we disrupted was hoxA3,and the phenotype that was observed was soft
tissue defects in the neck and upper chest. For example, the glottis and epiglottis were
aberrant, the thymus and thyroid were reduced and there were significant alterations of
the aortic arteries. Because all of these structures were derived from the neural crest,
which invaded the fourth branchial arch, these results were the first to demonstrate that
hox gene expression was important for specifying the fate of neural crest cells.
This result has been confirmed with mutations in other anterior genes such as hoxB2 ,which
is expressed up to the R1 boundary. Interestingly in this mutation, bones of the jaw,
which arise from neural crest in the second branchial arch, are duplicated posteriorly as
a result of an anteriorizing homeotic mutation. This is "heady" stuff here, but
the interesting part of this mutation, is that the jaw structure of this mutant resembles
that of reptiles and thus this hoxB2 mutation is considered to be atavistic -
meaning that it is found in ancestral animals.
An interesting observation that was made following the A3 mutation was that mutations in
the paralogue D3 did not result in a soft tissue defect but resulted in a skeletal defect,
suggesting that - whereas paralogues appeared to have similar expression boundaries -
their roles were distinct in different tissues. In this case, the first cervical vertebra
was fused to the occipital bone, which was considered to be an anteriorizing homeotic
At this time, of course, we wondered what the A3, D3 double mutant might look at. What was
emerging in the gene targeting field was the idea of genetic redundancy; i.e., that
phenotypes in mice could be made less severe because they were masked by the function of
related genes. Genetic redundancy was confirmed when A3, D3 double mutants were generated.
Interestingly, one or two mutant copies of the D3 gene were found to exacerbate the A3
soft tissue phenotype, whereas one or two mutant copies of the A3 were found to exacerbate
the D3 skeletal phenotype. Thus, it appears that - whereas both hox genes have a primary
responsibility in soft tissue or skeletal development - they appear to be able to
contribute independently to each function.
Genetic redundancy was not restricted to the paralogous member, however, because in
experiments that I performed, we observed similar genetic interactions between
neighbouring hox genes. Now we know that a complex web of interactions govern the homeotic
complex and that neighbouring paralogues and even next to neighbouring paralogues can
augment each others' phenotypes just like in the case of A3, D3.
As a result, a considerable amount of genetics will still be required to understand the
complexity of the homeotic complex.
Genetic Redundancy of the Homeotic Complex Permits Evolution of
the Body Plan
With the exception of some of the more anterior genes in the
complex, mutations in many hox genes resulted in only minor phenotypes that did not affect
morphology greatly. Often, in mutations affecting vertebral structure, only one side of
the animal was affected, suggesting that there is some plasticity in the response hox
code. Hox genes have long thought to play an important role in limb development. However,
when limb genes such as A11 or D11 were made, the phenotypes were hardly detectable. On
the other hand, once two mutations were put together, the effects were dramatic. For
example, the absence of both A11 and D11 results in a severe and life threatening
reduction in the radius and ulna. What this result tells us is that the redundancy of the
Hox complex permits some flexibility in the response to mutational change.
Redundancy permits a rich potential to allow for evolutionary alterations of the body
plan, through the following mutational changes:
- variations in the number of homeotic genes, by deletion and or
- increases in the number of hox complexes through whole complex
- mutations affecting the timing, position or level of homeotic gene
activation, which may be most relevant to generate small adptive changes;
- alterations in the regulatory interactions between Hox proteins
and their targets, through muations of the coding sequenc of Hox genes.
Recently, heterochronic mutations have been made within the hox
gene complex by changing the position of a given gene within the complex. When hoxD11 is
targeted into D13, a significant limb phenotype results - not from the inactivation of
hoxD13 itself but from the inappropriate activation of D11 at the wrong time.
Mann, R.S. 1997. Why are Hox genes clustered? BioEssays 19: 661-664.
Morrison, A., Ariza-McNaughton, L., Gould, A., Featherstone, M. and Krumlauf, R. 1997. HOXD4
and regulation of the group 4 paralog genes. Development 124: 3135-3146.
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