Department of Molecular Biology, Massachusetts General Hospital and Department of Genetics, Harvard Medical School, Wellman 8, Boston, MA 02114, USA
* Author for correspondence (e-mail: ruvkun{at}molbio.mgh.harvard.edu)
Accepted 17 July 2003
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SUMMARY |
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Key words: Evolution, Enhancer, C. elegans, C. briggsae, D. melanogaster, Co-evolution
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Introduction |
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To test the extent of conservation of cis-regulatory elements from
distantly related organisms we generated transgenic C. elegans
expressing the green fluorescent protein (GFP) under the control of
tissue-specific enhancers from D. melanogaster. The nematode and
arthropod lineages separated very early in animal evolution, prior to the
`Cambrian explosion' around 530 million years ago
(Morris, 2000). Expression
patterns of enhancer elements have been described in both species;
furthermore, in the worm, expression pattern resolution is possible at the
single-cell level. If functional conservation of enhancers is as prevalent as
suggested by the many transcription factor genes that are functionally
conserved across species, we would anticipate detecting expression of GFP
driven by a variety of Drosophila regulatory elements in homologous
cell types in the worm.
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Materials and methods |
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Worm strains, injections and microscopy
Fusion genes were injected according to standard protocols
(Mello et al., 1991) into
either Bristol N2 or pha-1 (e2123) animals.
Enhancer-containing fusion genes were always injected at 50 ng/µl; whenever
pha-1 (e2123) worms were used, these were co-injected with a
pha-1 rescuing construct (Granato
et al., 1994
) at 2 ng/µl. Injection methods used for C.
briggsae (AF16) were identical to those used for C. elegans N2.
Multiple independent lines were examined for consistency of expression
patterns. We established that fusion genes injected into pha-1
(e2123) and N2 animals produce identical expression patterns. We
noticed that animals from a number of transgenic lines displayed diffuse
expression in the gut, primarily in the most anterior and posterior
compartments, the PVT neuron, which has a projection as described by Aurelio
et al. (Aurelio et al., 2002
),
not White et al. (White et al.,
1986
), and in several muscle cells of the pharynx
(Fig. 1E). We observed this
pattern of nonspecific expression for a number of fusion genes, including
pPD95.75 and pPD122.53 vectors alone, in both the N2 and pha-1
(e2123) genetic backgrounds. We therefore consider it to represent
the `background' pattern associated with the GFP transgene expression in the
worm likely caused by the promiscuous transcriptional control in these tissues
and/or a cryptic enhancer element within vector DNA. Occasionally this
`background' expression was also observed in vulva muscles and three rectal
epithelial cells. All animals were initially evaluated under a dissecting
microscope, and later examined in detail on a compound Zeiss Axioplan
microscope; images were captured with the Open Lab software package and
processed with Adobe Photoshop.
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Results |
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The structure of Cha gene, including its enhancers and its close
linkage with the acetylcholine transporter (unc-17), is highly
conserved between worms, Drosophila and mammals, suggesting
conservation of regulatory mechanisms
(Rand and Nonet, 1997). Yet,
faint expression of Drosophila Cha::GFP was only detected in several
glial cells of labial neurons, several pharyngeal muscle cells and in the
hypoderm (Fig. 1B), not in
cholinergic neurons, whereas the same 3.3 kb fragment located immediately
upstream of Drosophila Cha gene was shown to drive expression of
lacZ in a subset of cholinergic neurons in the fly brain
(Kitamoto et al., 1992
).
Extensive analysis of transcriptional regulation of Drosophila ddc
gene revealed that the cis-regulatory sequences required for endogenous gene
expression in the nervous system are located within the 2.6 kb immediately
upstream of the translation initiation site
(Johnson et al., 1989). A
Drosophila ddc::GFP construct containing this fragment was relatively
strongly expressed in most pharyngeal muscle cells, a single amphid neuron, a
single head interneuron (likely RICL or RIAL) and in PVT, but not in
catecholaminergic neurons. Finally, Drosophila FMRF::GFP construct
contained a 3.6 kb fragment upstream of the translation initiation site of
Drosophila FMRFamide gene which was previously shown to be expressed
in nearly all FMRFergic neurons in the fly
(Benveniste and Taghert, 1999
).
We detected consistent expression of this fusion gene in most muscle cells in
the pharynx, a single head interneuron (RMDDL, RMDL, RMF, or RMH) and three to
five neurons in the ventral cord (DA or VA). Again, none of these detected
expression patterns could be considered homologous to the endogenous patterns
in the fly.
In C. elegans, a gene unc-119 is expressed throughout the
nervous system (Maduro and Pilgrim,
1995). Its ortholog in Drosophila is also expressed in
essentially all neurons and is functionally conserved
(Maduro et al., 2000
). We
therefore tested the expression pattern in C. elegans of a GFP fusion
gene containing 2.5 kb upstream of the Drosophila gene, which,
although not previously tested in flies, was expected to contain at least some
of the regulatory elements. As shown in
Fig. 1C, strong expression can
be seen in up to 10 neurons in the head, four to six in the tail, and several
in the body (including HSN and SDQs). Although not all neurons expressed GFP,
and additional expression was seen in glial cells, the pharynx and the vulva,
it may be significant that the nervous system was the predominant site of
expression. Our studies of the 5' regulatory region of the C.
elegans unc-119 suggest that the pan-neural expression pattern of this
gene is assembled in a `piecemeal' fashion, probably mediated by the action of
independent cis-regulatory elements (I.R. and G.R., unpublished). It is
plausible that the Drosophila unc-119 gene is similarly regulated and
that some of its enhancer elements are recognized by the same transcription
factors that regulate expression of C. elegans unc-119 gene.
Encouraged by this observation, we tested upstream sequences (2.2 and 2.4 kb)
of Drosophila orthologs of two additional C. elegans
pan-neural genes, Drosophila ric-19 and sng-1
(synaptogyrin), which are expected to be expressed throughout the nervous
system (Pilon et al., 2000
;
Zhao and Nonet, 2001
). The
former was faintly expressed in the pharynx, whereas the latter was restricted
to four to six glial cells in the head; neither therefore was expressed
pan-neurally. Thus, unc-119 is unique in the conservation of its
neuronal regulation.
Although C. elegans displays light sensing behaviors
(Burr, 1985), it lacks
morphologically defined eyes. Because a key transcription factor in the eye
specification program is highly conserved among all animals - eyeless
in Drosophila, Pax6 in vertebrates and vab-3 in C.
elegans (Carroll et al.,
2001
; Davidson,
2001
) - we also tested enhancers of Drosophila
eye-specific genes in C. elegans. In addition, an evolutionary
connection has been proposed to exist between thermosensory neurons of
nematodes and photoreceptors of other animals
(Satterlee et al., 2001
;
Svendsen and McGhee, 1995
). We
generated fusion genes containing enhancer elements of eyeless (0.5
kb) and eyes absent (0.3 kb) genes; both of these sequences were
previously shown to direct reporter gene expression in eye primordia in
Drosophila (Zimmerman et al.,
2000
; Hauck et al.,
1999
). Worms carrying Drosophila ey::GFP transgene showed
strong and consistent expression in a pair of labial neurons in the head, IL1D
(L, R), and the PVT neuron (Fig.
1D), cells that probably do not express vab-3 (A.
Chisholm, personal communication), although it is tantalizing that IL1D (L, R)
are a pair of anterior sensory neurons. Drosophila eya::GFP-carrying
worms showed no expression in any head neurons
(Fig. 1E). Therefore, two
enhancers that are expressed in the same cells in the fly are not co-expressed
in the worm.
Next, we tested fusion genes containing putative enhancers of
nompC (Walker et al.,
2000) and nompA (Chung
et al., 2001
), which in Drosophila are expressed by
ciliated sensory neurons and their glial support cells, respectively. As the
endogenous enhancer of Drosophila nompC was not characterized in
detail, our construct included a 1.7 kb fragment immediately upstream of the
translation initiation site, covering the interval between nompC and
the upstream gene. This fusion gene was not expressed above the background
level in C. elegans. The putative enhancer (2 kb) included in the
Drosophila nompA fusion gene also extended between the site of
translation initiation and the upstream gene and covered the sequence capable
of directing GFP expression in the endogenous pattern
(Chung et al., 2001
), although
coding sequences and downstream introns were absent from our construct. We
detected strong GFP expression in six to eight amphid neurons and several
cells of anterior hypoderm (Fig.
1F). Neither pattern could be considered homologous to the
endogenous expression domain in the fly, nor, in the case of nompC
construct, to the pattern of the putative worm ortholog
(Walker et al., 2000
).
Finally, we tested enhancers of four olfactory/gustatory receptors
expressed in sensory neurons in Drosophila
(Scott et al., 2001;
Gao et al., 2000
;
Vosshall et al., 2000
). These
constructs, or23a (2.6 kb), or46a (2.0 kb), or47a
(4 kb) and gr32d (3.8 kb), contained sequences immediately upstream
of receptor genes and were previously demonstrated to be sufficient to drive
reporter gene expression in the endogenous pattern. We therefore expected them
to be expressed in ciliated sensory neurons, where worm olfactory receptors
are expressed. However, we observed no expression of these fusion genes in
C. elegans.
Expression of Drosophila heart-specific enhancers is not
confined to C. elegans pharynx
To test whether enhancers of genes expressed in tissues other than neurons
are conserved between worms and flies, we examined expression patterns of
fusion genes containing Drosophila heart-specific enhancers. Although
nematodes do not have a heart, there are some functional similarities between
the nematode pharynx and the vertebrate and the insect heart
(Okkema et al., 1997). Aspects
of heart patterning are highly conserved in evolution
(Fishman and Olson, 1997
),
including members of the tinman family of transcription factors,
which are functionally interchangeable between worms and vertebrates
(Haun et al., 1998
).
We generated fusion genes containing entire heart-specific enhancers of
Drosophila tinman (0.9 kb) (Yin
et al., 1997), even-skipped (1 kb)
(Halfon et al., 2000
),
teashirt (1.2 kb) (McCormick et
al., 1995
) and Mef2 (5.5 kb)
(Cripps et al., 1999
) genes.
All four of these enhancers have been previously demonstrated to direct
expression of reporter genes in the Drosophila heart. Because all
four genes are involved in a conserved pathway of cardiomyocyte
differentiation, we expected that the constructs would be expressed in the
pharynx. Drosophila Mef2::GFP was strongly expressed throughout the
pharynx and in a single interneuron - AVG
(Fig. 1G). Two other fusion
genes, Drosophila tin::GFP and Drosophila tsh::GFP, were
expressed in the `background' pattern and in seam cells, whereas
Drosophila eve::GFP was consistently and strongly expressed in up to
six glial cells of labial neurons (Fig.
1H). Therefore, one of four heart-specific enhancers,
Drosophila Mef2, displayed an expression pattern consistent with the
conservation of transcriptional control between insects and nematodes. It is
possible that this enhancer element is functionally conserved between worms
and flies, although it is also possible that this instance represents a
convergently acquired similarity of expression patterns.
Orthologous enhancers from C. briggsae and C.
elegans produce similar, yet distinct, expression patterns
Because many Drosophila enhancers showed little or no conservation
of tissue-specific expression in C. elegans, we assessed the
functional conservation of enhancer elements between more closely related
species. We compared expression patterns driven by orthologous enhancers from
C. elegans and C. briggsae, two nematode species that retain
nearly identical morphology (Fitch and
Thomas, 1997), but are estimated to have diverged about 50-120
million years ago (Coghlan and Wolfe,
2002
), or about 10 times more recently than arthropods and
nematodes. We chose enhancers of two genes, unc-25 and
unc-47, because they are relatively short and well characterized. As
shown in Fig. 3A, in C.
elegans both genes are expressed exclusively in the 26 GABAergic neurons
- four RMEs, AVL, RIS, six DDs, 13 VDs and DBA
(McIntire et al., 1993
).
|
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In contrast to the unc-25 enhancer, both ce unc-47::GFP
and cb unc-47::GFP were expressed in all 26 GABAergic neurons of both
C. elegans and C. briggsae. Additionally, cb
unc-47::GFP was strongly expressed in SDQ (L, R) in C. elegans
and weakly in SDQL in C. briggsae
(Fig. 3). The two SDQ neurons
are descendants of the Q (L, R) blast cells and are not GABAergic
(Rand and Nonet, 1997;
McIntire et al., 1993
;
Guastella et al., 1991
). We
sought to identify the cis-element(s) within the cb unc-47 enhancer
responsible for SDQ (L, R) expression. We generated two enhancer fusion genes
- one encompassed the most proximal 250 nucleotides containing several highly
conserved sequences upstream the ATG and the other the remaining 580
nucleotides (Fig. 4). When
these were introduced in C. elegans, the former recapitulated almost
the entire pattern of the original cb unc-47 enhancer, with the
exception of RME (D, V), which either did not express GFP or were very faint;
SDQ (L, R) expression was also conspicuously absent. We tested the distal 580
nucleotide fragment alone, in direct and reverse orientation and as two
tandemly repeated copies in direct orientation. Expression patterns of these
three fusion genes were similar, in two GABAergic neurons: RME (D, V), as well
as in two pairs of amphid neurons, in one pharyngeal neuron and the
`background' pattern (PVT and in the gut). We observed no expression in SDQ
neurons. These results therefore suggest that the novel expression pattern
characteristic of the cb unc-47 enhancer probably resulted from a
synergistic interaction between the elements within the distal and the
proximal enhancer fragments, or less likely by an element at the -250
site.
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Discussion |
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Conservation and divergence between enhancers of C. elegans
and C. briggsae
The results of our tests of functional conservation of unc-25 and
unc-47 enhancers between C. elegans and C. briggsae
suggest that despite divergence of primary sequence and substantial changes in
spacing between conserved blocks of sequence, these two sets of enhancers
largely maintained their function over 50-120 million years separating the two
species. We noticed that in the case of both unc-25 and
unc-47, expression was stronger and more consistent in the
homospecific enhancer/host species combination, similar to what was seen in
other enhancer comparisons (Molin et al.,
2000; Ludwig et al.,
1998
). It is of interest that structurally similar
(Fig. 2), orthologous fragments
of unc-47 enhancer from C. elegans and C. briggsae
are functionally nonequivalent (Fig.
3). Specifically, expression in SDQ (L, R) is a property inherent
to the cb unc-47, but not ce unc-47, enhancer. Our results
further suggest that a synergistic interaction between the distal and the
proximal enhancer fragments, rather than the acquisition of a specific site,
results in the SDQ (L, R) expression pattern.
Recently, Romano and Wray (Romano and
Wray, 2003) examined functional conservation of enhancer elements
between two species of sea urchins which are separated by approximately the
same genetic distance as C. briggsae and C. elegans.
Although overall expression patterns observed in heterologous enhancer/host
tests were similar for these two species, there were also notable differences,
including ectopic expression. Remarkable similarity between these results and
our observations (Fig. 3),
further supports the notion of rapid evolution of transcription factors and
cis-regulatory elements and suggests that examination of orthologous enhancers
from intermediately divergent species will likely shed light on molecular
bases of evolutionary change.
Our functional analysis of cb unc-47 enhancer provides evidence
that expression of this gene is regulated by genetically distinct mechanisms
in RME (D, V) versus RME (L, R) cells, because proximal enhancer was
predominantly expressed in the left/right pair, whereas the distal enhancer
only in the dorsal/ventral pair (Fig.
4). Interestingly, in C. elegans, expression of
lim-6, a gene possibly acting in specification of non-D GABA cells,
is detected in the L/R, not the D/V pair
(Hobert et al., 1999), further
indicating that these pairs are genetically distinct. Similarly, in C.
elegans expression of unc-47 may be regulated by different
mechanisms in RME (L, R) and RME (D, V) cells (Y. Jin, personal
communication).
Co-evolution of enhancers and transcription factors maintains
homologous patterns of gene expression
Sequence comparisons of unc-25 and unc-47 enhancers
between C. elegans and C. briggsae
(Fig. 2) suggest that both the
relative spacing of conserved blocks and the sequences within such blocks,
diverge relatively rapidly. Similar patterns of sequence variation were
previously observed in enhancer comparisons of drosophilids
(Ludwig et al., 1998) and
rhabditid nematodes (Webb et al.,
2002
). Apparently, during the initial stages of species divergence
there is a large degree of functional conservation that persists despite the
accumulation of a considerable number of differences within enhancers. In some
instances, functional equivalence is maintained even between highly divergent
regulatory elements with distinct internal organization
(Takahashi et al., 1999
).
The co-evolution between transcription factors and their binding sites is
the most plausible hypothesis to account for these observations. According to
this model, individual binding sites within enhancer elements arise and vanish
on the time scale of a few million years. If one site disappears while another
arises at a different location within the enhancer, there will be an
appearance of `reshuffling' of binding sites. To counterbalance this constant
change, transcription factors co-evolve with their binding targets
(Shaw et al., 2002). Because
concerted action of a number of proteins is required for transcriptional
initiation (Tjian and Maniatis,
1994
), perhaps the most important aspect of this co-evolution is
not the adjustment of binding affinity to a newly evolved site, but the
changes in protein-protein interactions with other transcription factors whose
binding sites are located nearby. Recent studies revealed that while retaining
their overall functions, transcription factors can evolve novel roles by
acquiring amino acid replacements in their protein-protein interaction domains
(Hsia and McGinnis, 2003
;
Galant and Carroll, 2002
;
Ronshaugen et al., 2002
). It
is known that not only orthologous transcription factors, but even more
distantly related family members, often recognize similar DNA sequences
(Conlon et al., 2001
). It is
also well established that DNA-binding domains of transcription factors evolve
considerably slower than the domains involved in protein-protein interactions
(it is true for transcription factors in this study, see Figs S2 and S3 at
http://dev.biologists.org/supplemental/).
Moreover, changes in DNA-binding specificity would affect multiple target
genes, whereas because of the modular nature of transcription factors,
interactions with one partner may be adjusted without compromising other
functions. Over time, `reshuffling' of individual binding sites gives an
appearance of considerable sequence divergence, yet the complex of
transcription factors that assembles on an enhancer may be largely the same,
resulting in the conservation of gene expression patterns.
The co-evolution model can be used to explain a seemingly paradoxical
observation: individual transcription factors are often functionally conserved
over very large phylogenetic distances
(Grens et al., 1995), whereas
our results suggest that enhancer sequences from an arthropod often are not
properly recognized in a nematode. If we consider two distantly related
species A and B, enhancers of orthologous target genes would have little
detectable sequence similarity due to multiple rounds of `reshuffling', yet
two sets of orthologous transcription factors may regulate their expression,
each optimally co-evolved to recognize its target
(Fig. 5A,B). When placed into
species A, which is mutant for a particular transcription factor, an ortholog
from species B could bind to an appropriate target site
(Fig. 5C). Although this
binding may be weaker than to its native target and its interaction with other
transcription factors assembled on the enhancer may be less specific than to
its native binding partners; in the framework of an experiment it may still
rescue the mutation because some binding and some interaction are retained.
If, however, an enhancer from species A is placed into species B
(Fig. 5D), it is unlikely to be
expressed because none of the interactions between transcription factors
required for transcriptional activation are likely occur. Therefore,
co-evolution of rapidly diverging enhancer elements with their transcription
factors may be one of the molecular mechanisms underlying a commonly observed
phenomenon of `developmental systems drift'
(True and Haag, 2001
), in
which apparently homologous traits in distantly related species are determined
by distinct genetic programs.
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ACKNOWLEDGMENTS |
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Footnotes |
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