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First published online 6 June 2007
doi: 10.1242/dev.001065
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Review |
National Research Centre `Frontiers in Genetics', Department of Zoology and Animal Biology, University of Geneva, Sciences III, Switzerland. School of Life Sciences, Ecole Polytechnique Fédérale, Lausanne, Switzerland.
e-mails: Denis.Duboule{at}zoo.unige.ch; Denis.Duboule{at}epfl.ch
SUMMARY
Although all bilaterian animals have a related set of Hox genes, the genomic organization of this gene complement comes in different flavors. In some unrelated species, Hox genes are clustered; in others, they are not. This indicates that the bilaterian ancestor had a clustered Hox gene family and that, subsequently, this genomic organization was either maintained or lost. Remarkably, the tightest organization is found in vertebrates, raising the embarrassingly finalistic possibility that vertebrates have maintained best this ancestral configuration. Alternatively, could they have co-evolved with an increased `organization' of the Hox clusters, possibly linked to their genomic amplification, which would be at odds with our current perception of evolutionary mechanisms? When discussing the why's and how's of Hox gene clustering, we need to account for three points: the mechanisms of cluster evolution; the underlying biological constraints; and the developmental modes of the animals under consideration. By integrating these parameters, general conclusions emerge that can help solve the aforementioned dilemma.
"See my son, here time becomes space"Gurnemanz, in Parsifal (R. Wagner)
Introduction
The discovery and study of Hox gene clusters have been central to the
development of many conceptual tools, now widely applied, regarding the
structure, function and regulation of animal genomes. For example, the concept
that various animals not only share their genes, but also complex genetic
systems and that these systems are used at different times and places within
the same organism [see references in Kirschner and Gerhart
(Kirschner and Gerhart,
2006
)]. On an even more basic level, the relatively recent
evolution of vertebrate genomes, as well as important revisions of animal
phylogenies, have also largely relied upon the composition of these gene
clusters (de Rosa et al.,
1999). The heuristic value of this genetic system is in itself a
remarkable and fascinating topic, which lies outside the scope of this review.
However, new paradigms are often associated with undesirable side effects, and
the discovery that mice and flies have evolutionary and functionally related
Hox `clusters' (Duboule and Dollé,
1989; Graham et al.,
1989) is no exception to this rule. It was indeed quickly assumed
that all other animal species would contain a cluster of Hox genes, and many
subsequent publications reported the existence, in some species, of a Hox gene
`cluster', when in fact only isolated Hox genes (or fragments thereof) had
been obtained (e.g. Duboule,
1994a).
This misleading perception of the prevalence of the Hox cluster has been
reinforced by the common, erroneous graphical representation of these loci, in
particular in reviews and textbooks, whenever inter-species comparisons are
shown (e.g. de Rosa et al.,
1999; Lemons and McGinnis,
2006). The reductionism of the classical scheme, inherited from
the first alignments between the mouse and Drosophila genes, usually
conveys four wrong messages. (1) The horizontal alignment of individual genes,
according to which paralogous group they belong to, suggests that they are
structurally linked, when this is not always the case. (2) The representation
of genes as small boxes suggests that Hox genes are identical to each other,
which is rarely the case. (3) The absence of scale suggests that Hox loci from
various species are of the same genomic size, which is rarely so. (4) The
absence of any other information regarding the DNA content (e.g. the presence
or absence of repeats) suggests that intergenic sequences are not
important.
An example, albeit not the most striking, of this biased perception is given in Fig. 1, which shows a comparison between the Drosophila, the amphioxus and one of the four murine Hox clusters, using two different levels of resolution. A schematic alignment is shown (Fig. 1A) as it usually appears in the literature, with colors to illustrate paralogous groups and with Hox genes represented by boxes. The same comparison is also shown at the correct scale (Fig. 1B), which clearly illustrates the discrepancy between the traditional representation and the physical reality. Although this issue might appear somewhat anecdotal, it is of key importance whenever the functional genomic evolution of the Hox cluster(s) is considered.
In this review, I discuss why this lack of precision has contributed to the failure to appreciate a crucial problem associated with our understanding of the evolution of vertebrate Hox clusters: that some extant Hox clusters are probably `better organized' than their ancestral forms. I propose a potential solution to this problem, which relies upon the counter-intuitive view that genome duplications, in some cases, might increase regulatory constraints, thus leading to the consolidation of genetic loci, rather than to their relaxation.
Collinearity: myth or reality?
Ever since the first alignment between vertebrate Hox and
Drosophila homeotic (HOM) clusters was proposed (see
Akam, 1989), confusion has
surrounded the nature of these clusters. As discussed above, this confusion
largely stems from the simplified graphical representation of the Hox clusters
in these two species, which has subsequently been used to extract conclusions
concerning the evolution of this gene family. The fact that the two
Drosophila gene clusters [the Antennapedia (ANT-C) and Bithorax
(BX-C) complementation groups] were artificially juxtaposed to properly align
with their vertebrate counterparts, contributed to the perception that a
single Hox gene cluster exists in insects. This was somehow then formalized
when this collection of HOM genes, distributed at two different loci, became
known as a `HOM complex' (Akam,
1989). Furthermore, not only do Drosophila have two
separate HOM clusters, but they are different from one another: whereas ANT-C
is rather disorganized, with `foreign' (non-HOM) genes interspersed amongst
the HOM genes and with homeotic genes found in both transcriptional
orientations, BX-C is somewhat better organized, resembling to some degree
vertebrate Hox clusters (Duboule,
1992). Why do such details matter?
Hox gene clustering is neither a topographic oddity, nor the mere trace of
how this gene family originated through local gene duplications. In several
cases, it reflects a more profound level of functional organization, which was
originally described by Ed Lewis in genetic terms
(Lewis, 1978). The collinear
correspondence between gene order and the body levels where these genes are
expressed during development has, for many years, provided a convenient
explanation as to why Hox genes had remained `clustered'. Recently, however,
this explanation has been challenged in several studies, coinciding with the
detailed description of additional animal model systems, such as urochordates
(e.g. Seo et al., 2004), where
at least some level of coordination in Hox gene expression is observed despite
the absence of gene clustering (see
Galliot, 2005;
Monteiro and Ferrier, 2006).
To make sense of these apparently paradoxical datasets (see
Lemons and McGinnis, 2006), we
need to integrate several parameters, such as the kind of cluster under
consideration for a given animal, the precise definition(s) of
collinearity(ies), and the relationship between the developmental strategies
of different animals and the type of cluster used.
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In addressing the first issue, a tentative definition of what is meant by
`clustering' is required, as the use of `clustered' versus `non-clustered' Hox
genes has become arguably limiting. Without abusing an exhaustive number of
qualifications [nicely clustered, clustered but separated, clustered in pairs,
tightly or loosely clustered (see Payre
and Ternell, 1994)], I propose to define a minimal number of
structural organizations to help us to think about the problem. For clarity's
sake, let us consider only four possibilities as a first-line classification
of Hox `clusters' (Fig. 2): (1)
organized clusters (or `type O clusters', as in the prototypic vertebrate);
(2) disorganized clusters (`type D', e.g. the sea urchin cluster); (3) split
clusters (`type S', e.g. the Drosophila HOM `cluster'); and
(4) atomized clusters (`type A', e.g. the urochordate Oikopleura
`cluster'), where genes are mostly scattered throughout the genome. Multiple
combinations of types can, of course, be found, particularly in `type S'
animals, which may have, for example, both type O and type D sub-clusters.
Other combinations will undoubtedly be reported as additional genomes are
sequenced. Moreover, only a few genomes have been analyzed to the extent that
a firm assignment can be given to a cluster type. Also, a bias might exist in
the selection of protostome model systems as most of these animals have rather
small genomes and develop rapidly. Yet, the final picture might not differ
drastically from what we can now contemplate. With this simple analytical tool
at hand, we can reconsider the animal phylogeny and superimpose the
appropriate types of clusters to hypothesize about their structural evolution
(Fig. 3).
Cnidarians have Hox genes (e.g. Gauchat
et al., 2000) that are organized in `type A clusters'. Although
some Hox genes are still found in pairs, the general organization of the few
Hox genes is atomized (Chourrout et al.,
2006; Kamm et al.,
2006). This situation is similar to that found in the flatworm
Schistosoma mansoni (Pierce et
al., 2005) and in nematodes, and can be regarded as type A,
although some genes remain in pairs (i.e. they maintain some degree of genomic
organization) (Aboobaker and Blaxter,
2003). The prototype of the type S (split) cluster is found in
Drosophila, where a chromosomal breakpoint can be either between
Antp and Ubx, or between Ubx and abdominal
A (abd-A) (Von Allmen et
al., 1996; Negre and Ruis,
2007). Other non-dipteran insects show a type S cluster, as in the
moth Bombix mori, although a breakpoint lies closer to the `anterior
extremity' of the gene series (Yasukochi
et al., 2004). By contrast, some insect species have the full
complement of Hox genes at a single locus. In these cases, clusters can
nevertheless be classified as type D, mostly on account of their large size
and apparent high level of `disorganization'. For example, in the mosquito
Anopheles gambiae, a very large cluster that exceeds 700 kb is found
(Holt et al., 2002), which
contains many interspersed repeats that are mostly absent from type O
clusters. Similarly, a large but unique cluster appears to exist in
Tribolium castaneum (Brown et al.,
2002).
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) but
also in nemerteans (Kmita-Cunisse et al.,
1998) or brachiopods (de Rosa
et al., 1999). Yet, so far, the genomic organization of only a
flatworm (see above) and the annelid polychaete Platynereis is known,
and these probably contain a rather intact cluster (D. Ferrier, personal
communication). The situation in protostomes is equally heterogeneous. The sea
urchin Hox cluster is large and contains genes in opposite transcriptional
orientations and at unexpected positions with respect to their paralogous
groups, revealing the occurrence of important rearrangements
(Cameron et al., 2006) that are
typical of type D clusters. By contrast, analyses of Ciona
intestinalis and Oikopleura dioica indicate that urochordates
have atomized `clusters' of Hox genes
(Ikuta et al., 2004;
Seo et al., 2004). In marked
contrast, the cephalochordate amphioxus has a well-defined cluster, although
some aspects of it make the distinction between type D and type O difficult -
for example, its rather large size (at least 450 kb) as compared with
vertebrates (Garcia-Fernandez and Holland,
1994) [see references in Minguillon et al.
(Minguillon et al., 2005)] and
the presence of internal repeats within two intergenic regions (C. Amemiya,
personal communication). By and large, vertebrates display the most tightly
organized Hox gene clusters, with all genes in the same transcriptional
orientation, spanning 
100 kb. These clusters are very rich in conserved
non-coding DNA sequences, they are mostly devoid of any repetitive sequences
(e.g. Lander et al., 2001) and
the genes typically have very short introns (not visible at the scale used in
Fig. 1), thus reinforcing their
`compacted' nature, as if only minimal sequence requirements have been
conserved along with a fully optimized genetic structure. Construction versus destruction
Despite the many animal groups for which genomic data are not available and for which the status of their Hox `clusters' remains unknown, when considering the distribution of cluster types shown in Fig. 3, a surprising conclusion is reached and an embarrassing question raised. Firstly, it becomes clear that most bilateral animals will have, at best, a largely disorganized cluster, most probably a split cluster. Furthermore, a complete fragmentation of the `cluster' is seen in very different groups of animals, and might thus be expected for many species; the textbook Hox cluster might thus be the exception and not the rule. Secondly, whereas various groups display a single Hox gene `cluster' (types O/D), those that can be classified as `organized' are exclusively found in chordates, or even within vertebrates, if one considers the amphioxus cluster to resemble type D, for reasons mentioned above. With this in mind, we can now reconsider the question of the ancestral bilaterian Hox cluster, as well as the potential sequence of structural modifications leading to the situation shown in Fig. 3.
The fact that our bilaterian ancestor had at least one set of clustered Hox
genes can be inferred, given that the vertebrate cluster and the
Drosophila counterpart correspond to each other. Although the exact
composition of such an ancestral cluster, in terms of how many genes and which
paralogous groups are represented, is still open to debate (see
de Rosa et al., 1999;
Ryan et al., 2007;
Garcia-Fernandez, 2005),
Drosophila Hox genes, clearly orthologous to vertebrate counterparts,
were found in both sub-clusters of the fly, indicating that the cluster had
been split at some point in the evolution of Diptera
(Duboule and Dollé,
1989; Graham et al.,
1989). This explanation is indeed more parsimonious than the
opposing scenario, wherein two original clusters would have repeatedly and
independently merged into a unique and comparably contiguous series. In this
context, and provided the associated constraints (as discussed below) were
released, one can imagine how a cluster can progressively become disorganized,
or even split into two pieces or more, through recurrent events leading to the
atomized situation.
Yet if we assume that a unidirectional logic prevailed in this process, i.e. from an organized state towards a less organized state, we must naturally conclude that the `best organized' cluster is the closest relative in terms of general structure to the ancestral bilaterian cluster, while all others suffered an evolutionary erosion. Interestingly, whichever criteria are applied to define the `best organized Hox cluster' (see, for example, Fig. 1), vertebrates always score highest, indicating that a direct relationship exists, at least at the level of the structural organization of Hox genes, between the ancestral bilateria and ourselves, from which all other animals are derived. In other words, vertebrates, amazingly, would be the only animals in which the original genomic structure of this crucial gene family has persisted throughout evolution (Fig. 4).
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) are generally used to qualify the evolution of
Hox gene clusters, rather than `organization' or `consolidation'. Under this
proposed scenario, the ancestral cluster would be type D, and would evolve
into a type O along the vertebrate lineage (Figs
4,
5). What kind of mechanism(s)
could have underpinned such counter-intuitive processes, assuming that the
necessary selective potential existed? Global is beautiful
It is difficult to imagine why or how a large gene cluster (say 500 kb)
that contains about ten genes in various orientations, together with some
repeats, would be progressively transformed into a 100 kb cluster, with the
same ten genes encoded now by the same DNA strand and without any repeats.
This process of `consolidation' must have represented an `added value', in
evolutionary terms, that was selectively favored over either a stabilization
or a `simplification' of the cluster (Fig.
5). The various solutions that might account for such
consolidation all rely upon the integration of the functions of single genes
into a more global mode of operation. Such a communal ability to fulfil a
functional task that cannot be fulfilled by any of the genes in isolation was
called `meta-genic', and, accordingly, the Hox clusters should be regarded as
meta-genes (Duboule, 1994b).
For example, a source of novel protein products could result from splicing
patterns that become more complex, once neighboring genes are encoded by the
same DNA strand. Also, the emergence of global enhancer sequences, located
outside the cluster itself, might favor increased gene proximity to facilitate
and secure a coordinated transcriptional response
(Spitz et al., 2001;
Spitz et al., 2003).
Optimized, coordinated responses to global regulations might themselves be the
favored functional approach of this gene family, in which gene dosage effects
and compensatory mechanisms are often observed amongst neighboring genes,
helped by largely redundant protein functions (e.g.
Zakany et al., 1997;
Wellik and Capecchi,
2003).
In this context, the acquisition of global, cluster-wide regulations might
have triggered a progressively increasing level of structural organization
between neighboring genes, to allow them to respond at the transcriptional
level in a more coordinated way (Fig.
6). For example, the reduction of intergenic distances and the
elimination of `foreign' sequences (non-Hox transcription units or repeats)
can be understood where a group of contiguous Hox genes is recruited to
achieve a novel meta-genic function; for example, the recruitment of several
genes of the mammalian Hoxd cluster by a digit-specific global
regulation (Kmita et al.,
2002; Spitz et al.,
2003). In turn, this process of consolidation is intrinsically
directional, as it paves the way for other regulatory co-options to occur, for
at least two reasons: first, the functional potential of a coordinated series
of regulators is largely greater than that of a single transcription unit, as
it may provide more integrated possibilities, including dosage effects and
redundancy; second, it is conceivable that strong and remote global enhancer
regions might foster the emergence of novel regulatory controls at the same
site, owing to the presence of various specific or general transcription
factors, an increased accessibility or a particular chromosomal architecture,
a process referred to as `regulatory priming'
(Gonzalez et al., 2007)
(Fig. 6). In other words,
cluster consolidation may merely illustrate the evolution of a meta-gene
structure and its associated meta-cis regulations
(Duboule, 1994b), in a way
that is similar to our current views of how a single transcription unit might
have appeared and be further stabilized, for example by `consolidating' exonic
sequences or recruiting various regulations.
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Why us?
The vertebrate lineage is the only one in which multiple complete Hox
clusters have been described, i.e. containing both anterior and posterior
types of Hox genes. All vertebrates thus contain a minimum of four Hox
clusters, with additional copies present in fish
(Hurley et al., 2005) (see
below). The mechanisms that operated at the genesis of this cluster
amplification are still a matter of debate; in particular, whether or not
these four copies were produced by full-genome duplications or by
more-restricted DNA segmental amplification. It is likely that the first
possibility will be validated, once more genomes are sequenced, in particular
those of animals that might provide a link between early chordates and
gnathostomes (vertebrates with jaws) such as agnathans species (vertebrates
without jaws). Although this mechanistic question is not necessarily relevant
to the problem of Hox cluster functional evolution, it raises another paradox,
which, once clarified, might help us to understand the emergence of
consolidation: how can one explain that highly consolidated Hox clusters are
found in those species that evolved several copies of them? One obvious
possibility is that a highly organized (i.e. already fully consolidated)
cluster had evolved before the first genome duplication event. If correct,
this cluster, under `consolidation constraints', would no longer be
represented in those early chordates such as urochordates or cephalochordates,
for which genomic sequence is available. Alternatively, a semi-consolidated
cluster could have been duplicated, with some further consolidation occurring
thereafter, independently on both duplicated copies.
It is difficult to imagine how convergent cluster organizations may have
followed large-scale genome duplication, to reach such a level of similarity
amongst, for example, the four human Hox clusters. However, the existence of
an already fully consolidated cluster before genome amplification does not
make the explanation easier. Our current views regarding the selective
advantages of a genome (a gene; a meta-gene) being duplicated arguably predict
that the constraints that maintain genes in close proximity would be relaxed
in one or other of the two duplicated copies
(Ohno, 1970;
Holland, 1999). This should
favor fragmentation and disorganization, rather than maintenance or further
consolidation. Yet, if we consider the emergence of global regulation (and the
associated regulatory hubs) as major factors of consolidation, as discussed
above, we might be able to explain why cluster duplication did not lead to
fragmentation but, instead, to further internal organization (see Figs
5,
6).
In vertebrates, Hox gene functions are essential for the development of
various morphological features that are generally considered to be late
evolutionary novelties, associated either with the emergence of this lineage
or with important steps of its own evolution, such as the development of
skeletal appendages, external genital organs, metanephric kidneys and the
branchial apparatus. Much in the same way that duplicated genes can acquire
some evolutionary flexibility and be recruited for divergent functions, Hox
meta-genes, following their duplication, might have offered novel
possibilities for regulations to be co-opted, thus triggering the emergence of
these various vertebrate features [see Wagner et al.
(Wagner et al., 2003) and
references therein].
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)
(Fig. 6), together with
more-local, gene-specific controls, as in the case of hindbrain patterning
(Trainor and Krumlauf, 2001).
It is indeed conceivable that a single cluster rapidly experienced serious
constraints in its capacity to recruit novel global regulations, without
interfering with pre-existing ones. In this view, cluster duplication would
favor consolidation, not only by widening the range of regulatory
possibilities, but also by actively triggering potential regulatory
innovations owing to the presence of an already `competent' locus
architecture. The apparent co-evolution of important regulatory controls for
both limbs and genitals in tetrapods may illustrate this effect
(Kondo et al., 1997). This
increase in the general organization of the clusters, as triggered by the
maintenance or re-enforcement of a consolidation process, might have required
some internal rearrangements, and there are many examples of paralogous genes
being lost in a single, or in several, clusters. This phenomenon is usually
explained by the existence of redundancy, occurring once clusters have been
duplicated. Yet the possibility that some Hox genes were lost by positive
selection owing to their incompatibility with a newly recruited global
regulatory control should not be overlooked.
Although this scenario might account for some convergence in the
consolidation of Hox gene clusters after duplication, it is unlikely that all
four clusters independently evolved from an ancestral type D to type O
organization. Consequently, this process must have started to occur early on,
during the early chordate-to-vertebrate transition. In this respect, the
actual structure of the cephalochordate amphioxus cluster, although testifying
to the existence of a single entire cluster in an early chordate ancestor, is
in fact of limited significance, as it cannot be considered to be a direct
ancestral form of the vertebrate clusters. First, the phylogenetic position of
cephalochordates as the closest relatives of vertebrates has recently been
challenged (Delsuc et al.,
2006). Second, this cluster itself might have succumbed to either
some disorganization or consolidation after cephalochordates separated from
the vertebrate (or the vertebrate-urochordate) lineage. Functional analyses
will hopefully reveal whether or not global Hox regulation is at work in these
animals.
Most importantly, the functional characterization of Hox clusters in
agnathans should indicate whether consolidation does indeed go hand-in-hand
with duplication, i.e. whether or not the consolidation process in ancestral
vertebrate clusters started before the full complement was reached, as would
be expected from the above discussions. A detailed structure-function analysis
of the vertebrate Hox clusters may also help in this endeavor, as it might
reveal the remnants of pre-duplication global regulatory mechanisms, as
exemplified by a remnant of the mouse Hoxd global control region
(GCR), which is located at a similar relative position upstream of the
Hoxa cluster (Lehoczky et al.,
2004). The GCR, located upstream of the Hoxd cluster
(Spitz et al., 2003), contains
several globally acting enhancers
(Gonzalez et al., 2007), in
contrast to its reduced and simplified counterpart on the Hoxa
cluster (Lehoczky et al.,
2004). Analysis of this element(s) in an ancestral cluster, prior
to duplication, might indicate whether GCR-associated regulations were lost in
the Hoxa cluster after duplication, or acquired in the Hoxd
cluster.
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In the above scenario, cluster duplications facilitated the evolution of
global regulations, which in turn accompanied the emergence of crucial
vertebrate features, leading to increased organism complexity. However, this
view seems to be contradicted in the case of teleost fishes, which are of
particular interest in this context. It is well accepted that crown teleostei
experienced an additional round of genome duplication
(Prince et al., 1998;
Amores et al., 1998). As a
consequence, all teleost fish genomes analyzed so far bear seven to eight Hox
gene clusters, depending on the species [e.g. Amores et al.
(Amores et al., 2004) and
references therein]. These clusters are often referred to as having been
`further amplified', much in the same way as ancestral vertebrates had an
`amplified' complement of four clusters derived from two successive genome
duplications. Yet, despite the expected loss of several newly duplicated fish
Hox genes, as occurred during previous rounds of duplications, cluster
consolidation has not been seen. Also, if the passage from one to four
clusters was associated with increased complexity in vertebrates (e.g.
Holland and Garcia-Fernandez,
1996), what could have been the adaptive value, for teleostei, to
have twice as many Hox clusters as us?
Here again, a closer look at the teleost Hox clusters reveals an unexpected
picture, as illustrated by the zebrafish, Fugu and medaka genomes
displaying similar global organizations
(Kurosawa et al., 2006),
although with some differences that are not relevant to this argument. In
contrast to what is commonly believed, a comparison between zebrafish and
murine Hox clusters at the same scale (Fig.
7) reveals that teleostei have not experienced a further
`amplification' of their complement of Hox clusters, but rather an important
reorganization of this gene family, which accompanied (was made possible by)
an additional round of genome duplication. In fact, the overall number of Hox
genes in fishes (around 48) is close to that in mice (39), despite the
existence of seven clusters, not four. This is obviously not due to cluster
splitting, as illustrated by paralogous and syntenic relationships, but
instead to a massive elimination of Hox genes after the additional
duplication. What about consolidation?
Only two of the seven fish clusters (hoxba and hoxca)
resemble their mouse counterparts in terms of both gene number and cluster
size (Fig. 7). The other five
clusters show clear signs of consolidation, as suggested by their compacted
sizes. Whereas together the sizes of the four murine Hox clusters come to
415 kb (not including Hoxb13, see
Fig. 7 legend), the seven fish
clusters together give an overall size of 430 kb. Therefore, a 20% increase in
gene number correlates with only a 3% increase in the overall size of the
clusters. In this case, again, cluster duplication, if anything, leads to an
increased `organization' rather than to cluster atomization.
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100 kb) from the rest
of the cluster, such that its expression is late and restricted posteriorly.
These different, yet functionally convergent strategies suggest that these
compact zebrafish meta-genes have maintained the same functional organization
as that found in other vertebrates.
Even though the regulatory modalities associated with fish Hox clusters
have not yet been studied, and hence we do not know to what extent global
regulations are present, the redistribution of the Hox informational content
into numerous, but small and compact, meta-genes must have given teleostei an
increased genetic modularity, allowing for more flexibility in implementing
large-scale regulations (for example the activation or extinction of a
mini-cluster in one particular structure). The impact of these alternative Hox
genomic configurations should perhaps not be considered in an ontogenic
context - that of organism complexity - but instead in a phylogenetic context,
at the level of the very high number of species found in this animal group
owing to their enormous potential for radiation and rapid evolution.
Therefore, the evolution of the Hox cluster complement in teleostei might have
favored their great evolutionary success, perhaps at the expense of a more
robust, but also more constrained, developmental body plan. In this view and
in agreement with Wagner and colleagues
(Crow et al., 2006), it is not
the number of clusters (i.e. the overall number of Hox genes) that correlates
with higher organism complexity and/or species diversity but, instead, the
functionality and regulatory flexibility of meta-genes.
Trans-collinearity versus cis-collinearity
If global (meta-) gene regulation accounts for the evolution and stability
of type O clusters, what mechanisms maintained type D clusters and prevented
them from splitting and further entering into a phase of fragmentation? Ever
since spatial collinearity (the correspondence between the order of Hox genes
on the chromosome and their domains of expression) was discovered in
vertebrates (Gaunt et al.,
1988), it has been considered to be a major constraint that keeps
these genes together. Such coordinated gene expression is indeed most easily
envisaged as occurring via in-cis mechanisms, such as enhancer sharing
(Sharpe et al., 1998), or via
large-scale gene regulation (Kmita et al.,
2000), rather than via solely transacting processes. This view has
been recently challenged, following several reports that show that genes
belonging to type A clusters (for example, the fully fragmented `cluster' of
the larvacean Oikopleura) are, to some extent, also expressed with a
spatially `collinear' distribution (see
Seo et al., 2004;
Lemons and McGinnis, 2006;
Monteiro and Ferrier, 2006).
This apparent paradox is not a surprise, as a similar problem was encountered
early on, when single Hox genes were isolated from mammalian type O clusters
and studied in transgenic mice in vivo. Such transgenes, when integrated
randomly in the genome, could recapitulate part of their spatial expression
patterns, indicating that cluster organization is dispensable for establishing
some of the expected rostral-to-caudal expression boundaries, at least within
a certain spatial window (Krumlauf,
1994).
As in the case of clustering (see above), the paradigmatic value of
collinearity has led many of us to describe this process as occurring in
settings in which it failed to exist. Statements that mention that `collinear
expression was maintained in the absence of clustering' illustrate this
problem (see Monteiro and Ferrier,
2006). This confusion understandably reflects the fact that the
rostral-to-caudal progressive expression of Hox genes in animals carrying a
type A cluster is likely to derive from a genuine collinear mechanism
associated with an ancestral type D cluster. Consequently, talking about
collinearity in animals that do not have a Hox gene cluster might not be
entirely incorrect, when considering this phylogenetic view. To clarify this
issue, I suggest that we refer to these distinct situations as either cis- or
trans-collinearity, despite the intrinsic paradox that the latter
qualification presents. Cis-collinearity defines the correspondence between
the physical order of Hox genes and their domains of expression along the body
axis, and hence applies to the original phenomenon as described by Ed Lewis in
the Bithorax cluster of Drosophila
(Lewis, 1978), whereas
trans-collinearity defines the maintenance of the correct sequence of
expression domains along the axis, with respect to paralogous groups, in the
partial or complete absence of genomic clustering. Cis-collinearity applies to
type O and D clusters and to the internal structure of sub-clusters in animals
with type S clusters, whereas trans-collinearity applies to type A `clusters'
and to the `collinear' correspondence that might exist between sub-clusters in
type S animals (e.g. Drosophila). Accordingly, bilateral animals
could be classified as being `cis-collinear' (types O and D),
`cis/trans-collinear' (type S) or `trans-collinear' (type A; see
Fig. 5). This classification
might be of use when discussing the relationship between Hox gene (non-)
clustering and various developmental modes.
Various, non-mutually exclusive explanations have been proposed to account
for trans-collinearity, i.e. the fact that Hox genes maintain their
rostral-to-caudal coordinated expression in the absence of a bona fide gene
cluster. Clustering might be necessary to refine, coordinate and to stabilize
expression domains that are otherwise dictated by regulatory controls lying in
the vicinity of the genes themselves. Also, the different readouts of Hox
expression that are currently used (e.g. the developing vertebrate spinal cord
or sclerotome) might not exert the strongest constraints on the system. For
example, the early collinear expression of Hox genes in epiblast cells during
gastrulation (Forlani et al.,
2003; Iimura and Pourquie,
2006) might require a strictly clustered organization, whereas
subsequent `collinear' domains of expression, such as in the developing spinal
cord, might not have such a requirement. Understandably, type O/D clustering
might be constrained by a unique site of collinear expression, which would
thus require cis-collinearity, other sites having already evolved more
gene-specific types of regulation. In the absence of the former constraint,
such as in a type A `cluster', fragmentation can thus occur, leading to
trans-collinearity.
To give time to time
Although several examples of trans-collinearity have now been reported,
they all are concerned with spatial rather than temporal expression, and the
importance of time in keeping Hox genes clustered
(Duboule, 1992;
Duboule, 1994b) has not yet
been challenged (see Garcia-Fernandez,
2005; Monteiro and Ferrier,
2006). Temporal collinearity [the correspondence between Hox gene
order and their temporal sequence of activation
(Dollé et al., 1989;
Izpisúa-Belmonte et al.,
1991)] might thus have been a major factor in keeping Hox genes
together, whenever type O/D clusters are considered. Accordingly, animals
carrying type S/A clusters are not expected to implement this regulatory
property. Therefore, temporal collinearity can be both crucial and
dispensable, depending upon the animal species under consideration. The
understanding of these contrasting situations requires consideration of both
the biological relevance of this process and the underlying mechanisms.
It is well accepted that all animals with bilateral symmetry use their Hox
gene complement to organize their rostral-to-caudal polarity, and that this
process relies on similar combinations of Hox gene products. For example, Hox
genes related to the Drosophila gene labial (paralogy group
1; Fig. 1) function in the
patterning of the rostral extremities of bilaterian animals, whereas
Abd-B-related gene(s) (paralogy groups 9 to 13;
Fig. 1) pattern caudal
structures. Genetic analyses in mice and flies suggest that this genetic
circuitry, observed in various structures of the same animal (e.g. the
vertebrate limbs or intestine), is unlikely to be strictly based upon a
protein combinatorial system [the notion of `Hox code', as proposed by Kessel
and Gruss (Kessel and Gruss,
1991), taken in its most orthodox meaning]. Instead, some Hox
proteins have intrinsic properties that enable the more-`posterior' proteins
to counteract, or over-rule, the function of the more-`anterior' ones,
whenever both products co-exist. For example, the recent combined inactivation
of all paralogy group-10 Hox genes in mice generated a strong phenotype, with
lumbar segments bearing ribs (Wellik and
Capecchi, 2003). However, this complete functional ablation of
group-10 function did not elicit any remarkable phenotype in more-caudal
regions, in particular where group-11 Hox genes are expressed along with group
10.
This property, called the `posterior prevalence' rule
(Duboule, 1991;
Duboule and Morata, 1994),
requires `posterior' products to be present only at the developing caudal end
to avoid problems of mis-specification at the anterior end of the developing
embryo via the functional suppression of more-`anterior' functions. Various
regulatory strategies have evolved that prevent the antagonizing effect of
posterior Hox products over anterior functions. For example, in
Drosophila, a complex interplay of cis-acting sequences controls the
expression of the posterior Abd-B gene in the most-posterior
parasegments only (see Maeda and Karch,
2006). Alternatively, in those animals where the elaboration of
more-caudal segments is delayed in time, a mere delay in `posterior' Hox gene
activation might be sufficient to restrict their expression posteriorly, thus
preventing their deleterious effects (the `Hox clock')
(Duboule, 1994b). The
importance of posterior prevalence has been verified in many instances; for
example, in the proper determination of pools of motoneurons
(Tarchini et al., 2005) and in
the early sequential migration of epiblast cells during chick gastrulation
(Iimura and Pourquié,
2006).
Even though the mechanisms that underlie temporal collinearity are not yet
fully understood, the use of the linear structure of the DNA molecule to
support a time device is not difficult to conceptualize (see
Deschamps and Van Nes, 2005).
For instance, processes involving the directional spreading, or removal, of
any kind of molecule or relative distance effects between an enhancer and
neighboring target promoters (e.g.
Tarchini and Duboule, 2006),
could impose a collinear temporal regulation on any series of contiguous
transcription units in a way that would be impossible to achieve if a gene's
genomic neighborhood were to disappear. Distinct mechanisms might also
co-exist in the same animal in different contexts (tissues, cell-types,
structures), because once temporal collinearity constrains genes to stay
together, it paves the way for the recruitment of other collinear regulations.
For example, the evolution of a global enhancer positioned outside a gene
cluster might easily lead to a temporal sequence in the response of target
genes, following a relative distance effect or even a stochastic process
(Kmita and Duboule, 2003).
Consequently, a correlation must exist between the existence of a Hox gene
cluster and the implementation of developmental strategies that determine
rostral-to-caudal identities in a temporal sequence. Clustering need not be
complete (for example, in those cases in which only a restricted caudal part
of the embryo would segment following a temporal progression), but ought to
involve those Hox genes that precisely determine these caudal segments. In the
absence of this temporal parameter, a major constraint is released and the
cluster can freely evolve towards a more disorganized state as described
above. An illustration of this process is provided by Drosophila, in
which both splitting and disorganization has occurred. However, the ANT-C
sub-cluster is clearly more disorganized than is the BX-C cluster, which might
be because the thoracic and abdominal parts were more exposed to this temporal
constraint in the lineage that gave rise to Diptera (i.e. in short-germ
insects). Because in Drosophila this constraint has also been
released, owing to the particular mechanism of abdominal segmentation, it has
been proposed that BX-C was permissive and available for rearrangements
(Duboule, 1992). Since then,
several different breakpoints have been isolated within BX-C, which separate
Ubx from the two abdominal genes (abd-A and Abd-B)
in D. melanogaster (Von Allmen et
al., 1996) and in other species such as Drosophila
virilis (Negre and Ruiz,
2007), that support this view.
License to split? Inverting the constraints
Diptera provide a good example of the transition from a time
sequence-dependent segmentation process (short-germ insects) to a time
sequence-independent process, associated with modification of the Hox cluster
following the release of the temporal constraint. This slow and progressive
disorganization in Drosophila was recently explained by the complete
release of all the constraints that keep these genes together in other animals
(except between the pair of genes abd-A and Abd-B, which are
never found separated). In this view, the existence of Hox clusters in
Drosophila reflects the `phylogenetic inertia' of the system, i.e.
the difficulty of finding an acceptable breakpoint and of maintaining it at
the population level (Negre and Ruiz,
2007). Accordingly, one might consider that the more drastic
rearrangements that lead to trans-collinearity, which are observed in other
groups of bilaterian animals, also followed the emergence of developmental
modes that no longer require a precisely timed sequence of Hox gene
activation.
The relationship between cluster organization, the type of collinearity and
the existence of larval stages is more difficult to generalize. Hox genes are
required for the elaboration of the adult body plan, hence their function
might not be crucial in primary larvae, such as in the dipleurula larvae of
those sea urchin that show an indirect developmental mode, or in the
lophotrochozoans polychaete annelid larvae
(Peterson et al., 2000).
Whereas these annelid larvae seem to implement temporal collinearity in the
late activation of their Hox gene complement, the same is unlikely to be true
for sea urchins. In any case, the fact that, in these species, Hox genes may
be somewhat `set-aside' much in the same way that set-aside cells may
contribute to the definitive body plan, as argued by Davidson and colleagues
(Peterson et al., 2000), does
not help us to predict whether cis- or trans-collinearity contribute, and in
which temporal context. In such indirect developmental modes, the necessity to
activate Hox genes in a temporal sequence, and hence to maintain a gene
cluster, might depend upon the fate of these set-aside cells, i.e. whether the
derived rostral and caudal structures will be generated in a time sequence or
concomitantly.
Understandably, the implementation of a completely determinative
developmental strategy, i.e. developmental modes in which fates are generally
invariable and determined early on, as for example in the ascidian
Oikopleura (Seo et al.,
2004) and in nematodes, must have made temporal collinearity
unnecessary (Duboule, 1992).
In addition, animals that display either a poorly segmented body plan, or a
highly heteromeric series (i.e. `segments' that do not bear any similarity or
mechanistic relationship to each other), might no longer require any local
enhancer sharing between pairs of neighboring Hox genes, allowing for a
complete fragmentation of the cluster. Although these various factors point to
a natural tendency towards disorganization, once constraints have been
released, the alternative possibility, wherein the mere existence of a cluster
itself represents a constraint for the evolving potential of an organism,
should not be ignored.
In this somewhat counter-intuitive view, the effects of gene clustering in coordinating gene regulation in time might be detrimental to the implementation of one particular developmental strategy; for example, in the many animals that do develop their rostral and caudal extremities concomitantly. In such cases, the disorganization of the cluster might not reflect a release of some constraints (e.g. the loss of temporal collinearity), but, instead, might have been a necessary step to escape the obligation of activating `caudal' genes only after `rostral' genes, thus favoring, or accompanying, the shift to another developmental mode. In such a scheme, the cluster becomes a constraint, and cluster disorganization, much like the consolidation observed in vertebrates, is seen as an active process that is under positive selection, rather than being the result of tolerated evolutionary erosion.
In the beginning
Recent comparisons between sets of Hox genes of various animals, including
cnidarians, suggest that this original Hox cluster contained a rather
substantial number of genes, belonging to all major classes of Hox genes
represented in extant species (e.g. de
Rosa et al., 1999;
Garcia-Fernandez, 2005;
Ryan et al., 2007). The fact
that some particular groups were amplified subsequently - for example,
Abd-B-related genes in vertebrates - does not change the basic
problem, which is: what mechanisms kept these genes together and which
constraint was applied to this genomic structure, either to prevent
fragmentation or to facilitate consolidation.
Although these considerations concerning the evolution of Hox clusters might indeed contribute to our understanding about mechanisms and the relationships between cluster type and developmental modes, they do not help us to understand which type of cluster and, accordingly, which type of collinearity was present, both at the origin of bilaterian animals and just before their radiation. We and others assumed (as discussed above) that the ancestral bilaterian animal had a type D cluster and that spatial cis-collinearity occurred, as virtually all animal classes analyzed to date display this latter property to some extent, either in cis or in trans. Accordingly, it is fair to speculate that such an animal was segmented, or at least displayed some reiteration of modular structures along its rostral-to-caudal axis. It is nevertheless more problematic to infer, either from available experimental data, or from theoretical considerations, whether or not temporal collinearity was implemented and, correspondingly, whether this ancestral animal produced its caudal segments with a time delay with respect to more-rostral structures.
Even though temporal collinearity is considered as the strongest constraint
to maintain Hox genes in a single cluster
(Duboule, 1992;
Monteiro and Ferrier, 2006),
it is conceivable that it emerged as a consequence of clustering, i.e. was
made possible by the existence of a gene cluster, rather than being the
original force that kept the genes together in an ancestral form. To propose
an inverted sequence of events, as suggested by Ferrier and Holland
(Ferrier and Holland, 2002),
would once again, as for clustering itself (see above), support a
vertebro-centrist view that is unlikely to be correct, as it seems that few
animal species implement a full temporal collinear mechanism, from the most
rostral to the most caudal paralogy group. As discussed previously
(Kmita and Duboule, 2003),
collinear mechanisms (whether spatial or temporal) can come in different
flavors, and hence the quest for a universal collinear process might be
futile. In this view, it is perhaps simpler to consider the existence of a
gene cluster that might have subsequently triggered the emergence of other
collinear strategies (temporal or spatial), and that the various forms of Hox
gene clusters that we contemplate today merely testify to particular histories
of successive recruitment and abandonment of collinear mechanisms.
The scenario proposed above (temporal collinearity appearing after spatial
cis-collinearity) would fit with the intuitive perception that regulatory
mechanisms recruited, during evolution, on the top of pre-existing principles
(in this case owing to the existence of a cluster, hence of cis-collinearity),
should be generally less constrained than the former and thus more prone to
being selected against (Duboule and
Wilkins, 1998). The fact that temporal collinearity is certainly
found in a minority of bilaterians, unlike trans- or cis-spatial collinearity,
suggests that the former postdates the emergence of the latter. If true, the
question as to when exactly temporal collinearity was recruited on top of a
cis-colinear system will have to be answered. This problem will be difficult
to address, even with a comprehensive description of many more genomes of
animal species with well-described developmental modes. This is owing to the
versatility of collinearity; once a cis-collinearity cluster is established
and maintained, it can be used as a matrix where various collinear mechanisms
can evolve, based on processes as different as DNA replication, chromatin
spreading and distance effects, to name but a few. A superficial description
of these novel model systems will lead to a simplified conclusion (it is, or
it is not, collinear), which cannot faithfully reflect the evolutionary
history of the mechanisms involved.
Conclusion
The epistemic value of the Hox gene family has arguably not yet reached its full potential. The emergence of new model systems, as well as the availability of additional sequenced genomes, will help us to understand the functional evolution of this amazing gene family and will undoubtedly provide novel, widely applicable conceptual tools. In this review, I have discussed two rather counter-intuitive proposals to account for the functional and structural evolution of Hox gene clusters. First, genomic topographies (gene clusters in this case) can evolve towards `more-organized states' through a process of consolidation. Second, the consolidation of Hox clusters was stimulated, or at least reinforced, after genome duplications, to accompany the emergence of vertebrates, as gene clusters were more readily able to recruit additional global regulatory controls. In the past, several structural, functional or regulatory features of Hox genes were successfully transposed to other genetic systems. Future work will tell us whether similar hypotheses can be proposed for other evolutionarily conserved gene clusters.
ACKNOWLEDGMENTS
I thank M. Akam, C. Amemiya, A. Amores, D. Ferrier, B. Galliot, J. Garcia-Fernandez, S. Kuratani, F. Spitz, L. Wolpert and J. Woltering for communicating data and for discussions; J. Alfred and C. Garvey for careful editing of the manuscript; and G. Perec and the referees for inspiring suggestions. D.D. is supported by funds from the Canton de Genève, the Louis-Jeantet and Claraz Foundations, the Swiss National Research Fund, the National Research Center (NCCR) `Frontiers in Genetics' and the EU programmes `Cells into Organs' and `Crescendo'.
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, A ZFHoxDb `cluster' is shown, even in the absence of Hox
genes, as this cluster was selectively lost, as indicated by the structure of
the remaining genomic locus (Woltering and
Durston, 2006