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First published online 2 October 2008
doi: 10.1242/dev.025817
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1 Center for Advanced Research in Environmental Genomics, Department of Biology,
University of Ottawa, Ottawa, Ontario K1N 6N5, Canada.
2 Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa,
Ontario K1H 8M5, Canada.
3 Centre de recherche en cancérologie de l'Université Laval,
Centre Hospitalier Universitaire de Québec, L'Hôtel-Dieu de
Québec, Québec G1R 2J6, Canada.
* Author for correspondence (e-mail: mekker{at}uottawa.ca)
Accepted 5 September 2008
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: CNS development, Gene duplication, Mouse embryogenesis, Transcriptional regulation, Zebrafish embryogenesis, cis-acting regulatory elements
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). Duplicated genes can be lost by accumulating deleterious
mutations (nonfunctionalization), or they may acquire a new adaptive function
(neofunctionalization) or divide an ancestral function (subfunctionalization).
The duplication-degeneration-complementation (DDC) model predicts that
subfunctionalization is the most common mechanism for the preservation of
duplicated genes (Force et al.,
1999). According to the DDC model, functional modules of
duplicated genes are likely to undergo complementary degeneration. The
division of a single functional unit into separate subunits makes the complete
loss of the original function through nucleotide substitutions, deletions or
other types of DNA alterations, an unlikely event. Moreover, the
subfunctionalized duplicates are under relaxed evolutionary constraints
compared with the ancestral gene. Therefore, subfunctionalization of the
duplicates increases an organism's chances of survival and, in rare cases, may
be a transitional state leading to neofunctionalization
(Lynch and Force, 2000).
The Hox gene family underwent significant expansion during evolution and is
ideally suited to address the question of how genes acquire new functions. Hox
genes are involved in the control of regional identity of the embryonic axes
of metazoans. They are organized in clusters with the most anteriorly
expressed genes located at the 3' end of the cluster and the posteriorly
expressed genes at the 5' end. All vertebrates contain several Hox
clusters that are thought to have resulted from the sequential duplications of
a proHox cluster early in metazoan evolution. Thus, mammals have four Hox gene
clusters (HoxA, HoxB, HoxC and HoxD), whereas zebrafish
possess seven (hoxaa, hoxab, hoxba, hoxbb, hoxca, hoxcb and
hoxd). The additional clusters in zebrafish are likely to have
resulted from a duplication event that happened after the divergence of the
fish and tetrapod lineages, somewhere between 300 and 450 million years ago
(Amores et al., 1998;
Taylor et al., 2001). The
presence of additional Hox clusters is common, but not ubiquitous, in
teleosts, including those species with a small genome such as Takifugu
rubripes and Spheroides nephelus
(Amores et al., 2004;
Chiu et al., 2004). Comparison
of the structure of the teleost Hox clusters with those of mammals reveals the
loss of individual genes within some of the duplicated clusters. For instance,
the genome of the pufferfish, Takifugu rubripes, contains two
hoxb3 genes, hoxb3a and hoxb3b, whereas only
hoxb3a is found in the zebrafish genome, suggesting the loss of one
of the hoxb3 duplicates following the divergence of zebrafish and
Takifugu lineages (Amores et al.,
2004).
There are also examples in which both paralogous genes were retained after
the duplication event, as is the case for the hoxb5 genes in both
zebrafish and Takifugu (Amores et
al., 1998; Amores et al.,
2004). Bruce and colleagues
(Bruce et al., 2001) determined
that two zebrafish hoxb5 genes, hoxb5a and hoxb5b,
are expressed in overlapping yet distinct domains in the notochord, neural
tube and somites. Thus, their combined expression domains are strikingly
similar to that of the single Hoxb5 gene in the mouse. Furthermore,
the same study reported the biochemical equivalence of the Hoxb5a and Hoxb5b
proteins (Bruce et al., 2001).
Combined, these results suggest that the hoxb5a and hoxb5b
genes underwent subfunctionalization through loss of cis-acting regulatory
elements. To determine whether the subfunctionalization of Hoxb5
genes is reflected in changes in non-coding regions, we have compared the
Hoxb5 loci of human, mouse, zebrafish and Takifugu. This
allowed us to identify blocks of highly conserved non-coding elements (CNEs).
We compared the regulatory activity of the CNEs in transgenic assays in which
they were tested collectively and individually. This led to the finding that
one CNE specific to the hoxb5a locus, named J3, accounts for the
differences in expression between the paralogs, but that interactions between
J3 and other CNEs are essential to achieve correct expression. This has
important implications for the experimental approaches chosen to determine the
patterns of regulatory evolution of duplicated genes.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Multiple sequence alignments were computed with MultiPipMaker
(http://bio.cse.psu.edu/pipmaker)
or with the Pretty Results program of the GCG Wisconsin package. The boundary
of each CNE was determined as the longest region of high sequence similarity
between mammalian and teleost sequences. Phylogenetic and molecular
evolutionary analyses were conducted with the Kimura two-parameter model using
the neighbor-joining method and bootstraping
(Kumar et al., 2001). Searches
for potential transcription factor binding sites in the CNEs were performed
with Patch_Search
(http://www.gene-regulation.com/pub/programs.html).
Constructs
Reporter constructs encompassing large regions of the zebrafish
hoxb5a and hoxb5b loci were generated by homologous
recombination in bacteria (Copeland et al.,
2001). Recombination was carried out in Escherichia coli
DY380 and EL350 strains provided by Dr Neal Copeland (US National Cancer
Institute, Frederick, MD).
PACs containing the hoxb5a and hoxb5b zebrafish loci were
obtained from RZPD (plate positions 254O17 and 227H9, respectively). For
hoxb5a, a PAC fragment spanning from 1353 bp upstream of the
hoxb5a initiation codon to 1316 bp downstream of the hoxb4a
termination codon (total size 18,495 bp), was first inserted into a plasmid
vector using homology arms of 
200 bp. Similarly, we created a plasmid
containing a 12,169 bp fragment from the hoxb5b locus that spans from
1154 bp upstream of the hoxb5b initiation codon to 9763 bp downstream
of the hoxb5b termination codon. We inserted the lacZ and
EGFP reporter genes, encoding β-galactosidase and enhanced green
fluorescent proteins, respectively, 33 bp downstream of either the
hoxb5a or the hoxb5b initiation codon, in frame, to generate
the hoxb5alacZ, hoxb5aEGFP, hoxb5blacZ and hoxb5bEGFP
constructs (Fig. 1A,C).
Constructs containing lacZ and EGFP were used for the
production of transgenic mice and zebrafish, respectively.
Additional reporter constructs derived from hoxb5alacZ and
hoxb5aEGFP, from which the J3 enhancer sequence was deleted, were
produced through an additional round of homologous recombination. The homology
arms in the targeting cassette corresponded to sequences located directly
upstream and downstream of J3. The deletion constructs are referred to as
hoxb5a
J3lacZ and
hoxb5aJ3EGFP (Fig.
1B).
Finally, the J3 sequence from hoxb5a was introduced into the
hoxb5blacZ and hoxb5bEGFP plasmids to generate
hoxb5binsJ3lacZ and hoxb5binsJ3EGFP. The
J3 sequence was inserted at a position that would correspond to that of J3 if
this enhancer existed in the hoxb5b locus
(Fig. 1D). Although the
hoxb5b locus appears to have diminished in size after duplication,
elements existing in both zebrafish loci (J1, J2 and mir10a) are
present in the same order and at the same relative positions. By
extrapolation, if present, J3 would be located 3.1 kb downstream of the
hoxb5b termination codon. To excise the neo gene adjacent to
the introduced J3 element, EL350 cells hosting
hoxb5binsJ3lacZ or hoxb5binsJ3EGFP were
grown in arabinose-containing medium to induce recombination between loxP
sites flanking the neo gene.
To produce transgene constructs containing individual CNEs, these were
PCR-amplified and inserted downstream of the reporter gene in a way that
mimics their position and orientation in their original genomic context
(Fig. 1E-G). The p1229
and βpEGFP vectors, which contain a human β-globin minimal
promoter coupled to either lacZ or EGFP, respectively, were
used for the production of transgenic mice and zebrafish
(Cormack et al., 1996;
Yee and Rigby, 1993;
Zerucha et al., 2000).
Production and genotyping of transgenic animals
All experiments were performed according to the guidelines of the Canadian
Council on Animal Care and were approved by the institutional animal care
committees. Transgene preparation and microinjection into fertilized eggs were
according to standard procedures (Hogan, 1986). Genotyping of animals and
analysis of lacZ expression were as previously described
(Fraidenraich et al., 1998;
Larochelle et al., 1999).
Production of transgenic zebrafish and monitoring of EGFP expression were as
described (Amsterdam et al.,
1995). EGFP reporter constructs were microinjected at 100
ng/µl into more than 100 zebrafish embryos and those showing at least two
EGFP-expressing cells were referred to as primary transgenic
embryos.
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) or on paraffin sections
(Jaffe et al., 1990).
Quantitative RT-PCR
One hundred transient-transgenic zebrafish embryos were generated for each
of the constructs and were randomly divided in three pools of 33 embryos.
Total RNA was extracted from each pool using the RNAeasy Kit (Qiagen) and cDNA
synthesis was carried out using oligo(dT) primers. Relative amounts of
EGFP, hoxb5a and hoxb5b transcripts were estimated by PCR
amplification of 150 bp regions of each gene using the QuantiTect SYBR
Green PCR Kit (Qiagen) with standard curves inferred from five different
concentrations of a hoxb5aEGFP cDNA standard. Relative EGFP
transcript numbers were quantified for each group and normalized to endogenous
hoxb5a expression.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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120 to 260 bp. J1 and J2
sequences are both present in all the Hoxb5 loci we examined. In
mouse and human, the J1 and J2 sequences are 1.7 kb and 5.5 kb downstream of
the Hoxb5 stop codon, respectively
(Fig. 2). The sequence identity varied from 56 to 99% for the J1 element, depending on the species compared (see Table S1 in the supplementary material). Identity for J2 sequences varied between 46 and 90% in pairwise comparisons. The J2 sequences from Takifugu were more divergent and not initially detected by the Pipmaker algorithm (Fig. 2 and see Table S1 in the supplementary material). The third conserved sequence, J3, was found further downstream of the Hoxb5 genes of mammals and teleosts, but was absent from the teleost hoxb5b loci (Fig. 2). This element showed identity that varied from 47 to 91% in pairwise comparisons.
We identified two additional conserved non-coding sequences in the
Hoxb5 loci. The first, located between J2 and J3, is found in all the
mammalian and teleost species examined. It is found in hoxb5a, but
not in hoxb5b loci (Fig.
2). This conserved sequence is smaller than the others (<50
bp). Another conserved region is located downstream of J3
(Fig. 2) and represents a
previously identified microRNA gene (mir10a) that codes for a 22 nt
non-coding RNA molecule that is suggested to be involved in the regulation of
Hox gene expression (Mansfield et al.,
2004).
Finally, the immediate 5' flanking region of Hoxb5 genes was well conserved in all species examined (69 to 94%, Fig. 2; see Table S1 in the supplementary material).
The conserved non-coding elements found in the 3' flanking region of
Hoxb5 fall within three large fragments (3-4.5 kb) of the mouse
Hoxb5/Hoxb4 intergenic region previously shown to have regulatory
activity that recapitulates aspects of endogenous Hoxb5 and
Hoxb4 expression (Sharpe et al.,
1998). The zebrafish J1, J2 and J3 CNEs also correspond to
sequences reported by Hadrys and colleagues in their comparative analysis of
the Hoxb clusters in mammals and teleosts
(Hadrys et al., 2004).
Transgenic analysis of cis-regulatory sequences found near the zebrafish hoxb5 loci
We analyzed the activity of non-coding elements found near the zebrafish
hoxb5 loci by inserting the lacZ or EGFP reporter
genes in frame with the first exon of either hoxb5a or
hoxb5b (see Materials and methods). The constructs encompassed 18.5
kb and 12.2 kb of the respective loci (Fig.
1A,C) and included 1.2-1.3 kb of 5' flanking sequences.
In the 3' direction, the hoxb5a construct spanned 12 kb
and encompassed the J1-J3 conserved non-coding sequences. It also contained
the hoxb4a exons and intron, as well as 1.3 kb of hoxb4a
3' flanking sequence. Similarly, the hoxb5b construct extended
over a distance of 8 kb downstream of the last exon and encompassed the
J1 and J2 conserved non-coding sequences.
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). At
E10.5, hoxb5alacZ embryos exhibited β-galactosidase activity
along the embryonic neural tube, with the anterior border of reporter gene
expression reaching the caudal hindbrain
(Fig. 4A,B, arrows). By E12.5,
the anterior boundary of lacZ expression shifted rostrally, extending
to the caudal limit of the otic vesicle
(Fig. 4I,J, arrows). This
rostral extension of the expression domain might be mediated by a retinoic
acid response element (RARE), previously characterized by Sharpe and
colleagues (Sharpe et al.,
1998), that resides within the J2 CNE. Furthermore, the
hoxb5alacZ construct consistently targeted reporter gene expression
to the paraxial mesoderm/anterior somites at all developmental stages examined
(Fig. 4A,B,I,J, asterisks).
Analysis of the hoxb5alacZ expression patterns on sagittal sections
of E13.5 embryos revealed β-galactosidase activity in the cartilage
primordia of embryonic ribs (Fig. 5A,B,
cp). At E10.5, the anterior boundary of hoxb5blacZ reporter expression in the central nervous system (CNS) was located at the caudal limit of the embryonic forelimbs, that is, more posteriorly than the CNS expression boundary observed for hoxb5alacZ (Fig. 4A-D, arrows). By E12.5, the rostral boundary of hoxb5blacZ reporter gene expression in the CNS extended up to the caudal limit of the otic vesicle, similar to that detected in the hoxb5alacZ transgenic embryos (Fig. 4J,L, arrows). In addition to the CNS, hoxb5blacZ was expressed in neural crest derivatives such as cranial and dorsal root ganglia and associated nerve fibers. At E10.5 hoxb5blacZ expression was detected in the nodose, tenth cranial nerve, facio-acoustic and dorsal root ganglia (Fig. 4C, ng, fg). Expression persisted in these domains until at least E13.5 (Fig. 5D,E).
Dissection of internal organs from hoxb5alacZ mouse embryos at
E13.5 revealed β-galactosidase activity in the midgut in a punctuate
distribution (Fig. 5C,I). This
might reflect a dot-like pattern of Hoxb5 expression in the enteric
nervous system similar to that reported for the Hoxa5 gene
(Larochelle et al., 1999). In
addition, hoxb5alacZ was expressed in the stomach, meta- and
mesonephros and adrenal gland (Fig. 5I, st,
k, ag). A β-galactosidase signal of lower intensity was
detected in the stomach, midgut and adrenal gland in hoxb5blacZ
embryos (Fig. 5F,J).
Expression of hoxb5alacZ was stable and consistent between embryos from established lines at all embryonic stages examined (Fig. 4 and data not shown). By contrast, embryos obtained from hoxb5blacZ males showed patterns of transgene expression that differed between embryos collected from the same mating (see Fig. S2 in the supplementary material). Only embryos with the strongest X-Gal staining are shown in Figs 4, 5. This phenomenon was observed until at least the F2 generation obtained from an outcross of transgenic F1 males to wild-type females.
Similar constructs containing EGFP as the reporter were tested in primary transgenic zebrafish embryos. The hoxb5aEGFP and hoxb5bEGFP constructs recapitulated aspects of the endogenous hoxb5a and hoxb5b expression. In most cases, the anterior border of reporter gene expression corresponded to the posterior hindbrain, reflecting the rostral restriction of endogenous hoxb5 gene expression detected by in situ hybridization (Fig. 3E-J, Fig. 6A-I).
At 24-36 hours post-fertilization (hpf), hoxb5aEGFP expression was observed in cells of the developing CNS (Fig. 6A, arrows) and somites (Fig. 6A, arrowheads), mirroring hoxb5a expression (Fig. 3 and data not shown). At later stages, strong reporter expression persisted in the developing CNS and was also observed in muscle cells along the anterior-posterior axis (Fig. 6B,C). At 48 hpf, 10% of primary hoxb5aEGFP embryos expressed the transgene in the heart and the cardial veins (data not shown).
In zebrafish, the hoxb5b sequences directed reporter gene expression preferentially to cells of neuronal origin at all developmental stages examined (Fig. 6D,E), similar to what was found in transgenic mouse embryos. This contrasted with the mesodermal activity recorded for the paralogous hoxb5a locus (Fig. 6A-C, arrowheads). Occasionally, hoxb5bEGFP expression was observed in cells of hematopoietic origin (data not shown).
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Combined, the results obtained from the mouse and zebrafish transgenesis
experiments demonstrate that the DNA sequences included in the constructs are
able to recapitulate most of the hoxb5a and hoxb5b
expression domains except for the notochord and branchial arches. Furthermore,
the differences in expression of the hoxb5a and hoxb5b
constructs appear to be associated with those aspects of expression that
differ between paralogs. For example, one of the most striking differences
observed between hoxb5a and hoxb5b expression is the
exclusive expression of hoxb5b in structures of the peripheral
nervous system, such as the nerve ganglia, and this was mimicked by
hoxb5blacZ but not by hoxb5alacZ
(Fig. 4C,
Fig. 5D,E). Similarly, the high
expression levels of hoxb5a in somites and their derivatives
(Bruce et al., 2001) were
recapitulated by hoxb5aEGFP (Fig.
4B,J, asterisks; Fig. 5A,B,
cp; Fig. 6A,C,
arrowheads).
As the J3 element is only present in hoxb5a, and presumably
degenerated in hoxb5b, we hypothesized that it might be involved in
directing unique aspects of hoxb5a expression. To test this, we
deleted J3 from the hoxb5alacZ and hoxb5aEGFP reporter
constructs, yielding hoxb5aJ3lacZ and
hoxb5aJ3EGFP. Conversely, we also inserted J3 into
the hoxb5blacZ and hoxb5bEGFP constructs to examine whether
this would render hoxb5b expression more similar to that of
hoxb5a; the resulting constructs were named
hoxb5binsJ3lacZ and hoxb5binsJ3EGFP.
In mouse, the hoxb5aJ3lacZ construct targeted
lacZ expression to several domains of Hoxb5 expression where
its counterpart, hoxb5alacZ, was also expressed. However, expression
levels were markedly diminished. Thus, at E10.5,
hoxb5aJ3lacZ was expressed in the spinal cord, with
anterior limits of expression reaching the base of the hindbrain
(Fig. 4G,H, arrows), but the
expression was reduced compared with that observed in hoxb5alacZ
embryos (Fig. 4A,B; data not
shown). Similar to hoxb5alacZ, the
hoxb5aJ3lacZ construct was expressed in somites and
their derivatives at E10.5 and E12.5 (Fig.
4G,H,O,P, asterisks).
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J3EGFP construct
(Fig. 6F,G) exhibited both
neuronal and mesodermal expression similar to that recorded for its intact
counterpart, hoxb5aEGFP (Fig.
6B; data not shown). However, considerably fewer
EGFP-expressing cells were observed. Furthermore, transgene
expression appeared significantly diminished in cells of mesodermal origin as
compared with that observed in hoxb5aEGFP zebrafish embryos (data not
shown). When quantified by RT-PCR, transcript levels for
hoxb5aJ3EGFP resembled those seen in
hoxb5bEGFP transgenic embryos, rather than those characteristic of
hoxb5aEGFP embryos (Fig.
6J). The rostral boundary of hoxb5binsJ3lacZ expression in the E10.5 mouse neural tube was shifted anteriorly compared with that of hoxb5blacZ (Fig. 4E,F). Furthermore, high levels of hoxb5binsJ3lacZ expression were detected in the gut and stomach (Fig. 5K, st, g), similar to hoxb5alacZ. Finally, the hoxb5binsJ3lacZ construct drove reporter gene expression in somites and their derivatives (Fig. 4E,F,M,N, asterisks), whereas the hoxb5blacZ did not (Fig. 4C,D,K,L).
Levels of the hoxb5binsJ3lacZ reporter were apparently higher than those driven by the hoxb5blacZ construct (Fig. 4K-N). Moreover, the hoxb5binsJ3lacZ construct drove more consistent patterns of reporter gene expression than its parental counterpart, and the variegated patterns of expression seen with hoxb5blacZ were only observed in 10% of hoxb5binsJ3lacZ embryos (data not shown). When injected into zebrafish embryos, the hoxb5binsJ3EGFP construct drove reporter expression in both neural and mesodermal cells (Fig. 6I, arrows and arrowhead), whereas hoxb5bEGFP only exhibited neural expression (Fig. 6D,E, arrows). The hoxb5binsJ3EGFP construct also produced greater numbers of EGFP-positive embryos (Fig. 6H,I) and levels of EGFP expression were increased 2.4-fold compared with hoxb5bEGFP (Fig. 6K).
Taken together, the above results suggest that the J3 element is necessary
to maintain higher levels of hoxb5 gene expression and is at least
partially responsible for the exclusive expression of hoxb5a in cells
of mesodermal origin (Bruce et al.,
2001). Deleting the J3 sequence from the hoxb5a locus
reduced overall reporter transgene expression, whereas introduction of J3 into
the hoxb5b locus resulted in higher expression levels and induced
reporter expression in cells uniquely associated with hoxb5a
expression.
Transgenic analysis of individual non-coding elements
To assess the individual contributions of the J1, J2 and J3 CNEs to overall
Hoxb5 expression, we generated reporter constructs carrying
individual CNEs directing expression of lacZ or EGFP from a
β-globin minimal promoter (Fig.
1E-G). Primary transgenic mouse embryos were examined at
E12.5-14.5 and primary transgenic zebrafish embryos were examined at 24-96
hpf.
When tested in transgenic mouse embryos, each of the conserved sequences
from the mouse Hoxb5 locus or from the zebrafish hoxb5a and
hoxb5b loci drove reporter transgene expression in domains
representative of endogenous Hoxb5 expression
(Oosterveen et al., 2003;
Sakach and Safaei, 1996). A
transgene containing the mouse J1 element (MmJ1) directed lacZ
expression in structures of the paraxial mesoderm
(Fig. 7A) and neural tube
(Table 1, data not shown). The
mouse J2 element, MmJ2, induced reporter gene expression in the neural tube
and in prevertebrae along the entire anterior-posterior axis
(Fig. 7D,
Table 1). Finally, the
construct containing MmJ3 drove lacZ expression in the neural tube,
developing vertebrae and posterior somites
(Fig. 7G,
Table 1). Individual zebrafish
elements from either hoxb5a or from hoxb5b induced transgene
expression in prevertebrae, in the neural tube and, occasionally, in the
dorsal root ganglia and/or associated nerves
(Fig. 7,
Table 1). No major differences
were observed between the activity of the zebrafish CNEs and their mouse
counterparts, except for an inability of zebrafish J3 (DrJ3) to target
expression to the somites/prevertebrae
(Table 1).
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The above comparative analysis revealed that regulatory activity conferred by the cognate CNEs from duplicated zebrafish loci extensively overlapped and, in general, corresponded to expression patterns common to both hoxb5 orthologs.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). The
model makes the following predictions with regards to molecular mechanisms of
subfunctionalization. (1) Subfunctionalization happens by means of changes in
the non-coding regions of duplicated genes. (2) Resolution of functional
redundancy occurs through complementary degeneration of individual regulatory
elements. This requires the regulation of an ancestral gene to be modular in
nature; the gene subfunctions have to be independent from each other and
executed by discrete regulatory elements. (3) The process of
subfunctionalization should be completed within 12.5 million years after a
duplication event.
This model has received a lot of attention in the field of evolutionary
developmental biology. The fates of several gene duplicates were investigated
in an attempt to bridge the mathematical predictions of the DDC model with
experimental observations. Studies of the paralogous Nudt10 and
Nudt11 genes in mammals, the sox9a and sox9b and
the mitf-m and mitf-b genes in teleost
(Altschmied et al., 2002;
Hua et al., 2003;
Kluver et al., 2005) confirmed
that complementary loss of independent subfunctions (subfunctionalization) may
indeed constitute a common mechanism of resolution of functional redundancy
between duplicated genes. However, these studies did not address the molecular
changes that occurred at the level of the regulatory elements that instigated
subfunctionalization.
To clarify how the dynamics of cis-regulatory sequence evolution support
the DDC model, Santini et al. (Santini et
al., 2003) and Wolfe and Elgar (Wolfe and Elgar, 2007), performed
comparative sequence analyses of multiple gene loci that are duplicated in
teleosts but are present at single copy in mammals. They found that paralogous
genes in teleost often retain differential subsets of putative regulatory
elements, consistent with the notion of regulatory subfunctionalization
(Santini et al., 2003;
Woolfe and Elgar, 2007).
We characterized the structure and function of regulatory elements from the
subfunctionalized zebrafish hoxb5a and hoxb5b genes to
determine whether the evolution of these paralogs occurred in accordance with
the predictions of the DDC model. We identified conserved non-coding elements
and tested their activity collectively in a context that is close to their
natural environment. Changes occurring in CNEs from duplicated genes were
previously shown to be associated with the differential domains of expression
of co-paralogs (Kleinjan et al.,
2008; Tumpel et al.,
2006). In these two studies, CNEs were tested individually in
reporter constructs that were used to produce transgenic animals in the
endogenous species or in more-amenable model species. Although this approach
can reveal changes in cis-regulatory elements that might account for some
aspects of the differential expression patterns of the co-paralogs, it does
have distinct limitations, such as a failure to reveal regulatory interactions
as demonstrated by the results of the present study.
Execution of complementary subfunctions of the hoxb5a and hoxb5b genes may rely on interactions between multiple cis-regulatory elements
When tested in transgenic animals, large fragments from the zebrafish
hoxb5a and hoxb5b loci targeted reporter gene expression to
domains of endogenous Hoxb5 expression that were consistent with the
differences detected in the expression patterns of the two paralogs (Figs
3,
4,
5)
(Bruce et al., 2001). For
example, the rostral boundary of hoxb5blacZ expression in the neural
tube was shifted posteriorly at E10.5 as compared with hoxb5alacZ
embryos (Fig. 4). We then
questioned if, consistent with the DDC model, complementary degenerative
changes within individual elements are responsible for changes in expression.
The J3 element is only retained in the hoxb5a locus and is lost from
hoxb5b. Transgenic data obtained in both zebrafish and mice suggest
that the loss of J3 might have contributed to divergence in expression between
the zebrafish hoxb5a and hoxb5b genes (Figs
4,
6).
By contrast, functional tests of individual regulatory elements from zebrafish hoxb5a and hoxb5b did not reveal clear differences in activity (Figs 7, 8). MmJ2, DrJ2a and DrJ2b targeted transgene expression to similar domains in the mouse neural tube with a correct anterior border of expression. In addition, when tested individually, hoxb5a elements occasionally showed regulatory activity associated with domains exclusive to hoxb5b. For example, the DrJ3a and DrJ1a elements occasionally drove lacZ expression in the dorsal root ganglia and associated nerve fibers (Table 1, data not shown), an expression domain that correlates with zebrafish hoxb5b rather than hoxb5a expression.
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The dynamics of change in the cis-acting regulatory elements of hoxb5 duplicates are more complex than predicted by the DDC model
The DDC model predicts that regulatory elements of duplicated genes will
undergo rapid complementary degeneration. Partition of an original CNE
regulatory function, although not suggested by our transgenic analysis, could
also be supported by phylogenetic analysis.
We aligned the J1, J2 and the immediate 5' flanking sequences from all Hoxb5 loci and examined the alignments for the presence of short (6-12 bp) highly conserved (>91%) sequences. Such sequences, which are evolving at an exceptionally slow rate, are likely to be functionally important. Although we identified short DNA sequences that were conserved in cognate elements of mammals and teleosts (Table 2), we were unable to find sequences that were specific for one of the two teleost paralogs (e.g. hoxb5a from zebrafish or Takifugu), but divergent or absent in the other teleost paralogs (e.g. hoxb5b) (data not shown). Thus, the data do not provide evidence for a simple complementary degeneration of regulatory subfunctions within the elements present in both teleost hoxb5 loci.
|
) was found within J2. Another putative RARE was found in one
of the highly conserved regions of J3
(Table 2).
The DDC model also predicts that degeneration of redundant subfunctions
should occur within 4.0-12.5 million years after the duplication event
(Force et al., 1999). Thus,
functional specialization of the hoxb5a and hoxb5b genes in
teleosts would have preceded the divergence of the zebrafish and
Takifugu lineages, and one might expect higher sequence conservation
between orthologous functional modules (i.e. hoxb5a from zebrafish
and hoxb5a from Takifugu) than between paralogous modules
(i.e. zebrafish hoxb5a and hoxb5b)
(Fig. 9). We examined
phylogenies based on the analysis of coding sequences of Hoxb5 genes
from human, mouse, zebrafish and Takifugu
(Fig. 9B), as well as sequences
from the 5' flanking regions of Hoxb5 genes
(Fig. 9C) and of the J1 and J2
elements (Fig. 9D,E). The tree
built for the J1 CNE matched the branching pattern of the hypothetical model
(Fig. 9A,D), whereas phylogeny
for other functional modules did not (Fig.
9A-C,E). We also joined the sequences of the CNEs in a continuous
sequence for each locus. The topology of the resulting tree
(Fig. 9F) was similar to that
calculated for the coding sequences and the 5' flanking regions of
Hoxb5 genes, suggesting that the coding region and most regulatory
regions of Hoxb5 loci are under common selective constraints or are
drifting at the same rate. Overall, the dynamics of the molecular changes
involved in the evolution of hoxb5a and hoxb5b teleost genes
differ from those anticipated for duplicated genes that undergo
subfunctionalization (Force et al.,
1999). Alternatively, the DDC model might be imperfect in its
timing aspects. In fact, the regulatory elements of hoxb5a and
hoxb5b duplicates appear to diverge slower than anticipated.
Deviations from the predicted rate of divergence have also been reported
for the duplicated teleost sox9a and sox9b genes
(Cresko et al., 2003).
Differences in the expression patterns of sox9a and sox9b in
zebrafish and stickleback led to the conclusion that, even though partitioning
of most sox9 subfunctions occurred before the divergence of the
teleost lineages, some gene subfunctions might have assorted differently in
the two teleost species.
In addition to its enhancer activity, the J3 element may be required for proper maintenance of hoxb5 expression
The absence of the J3 regulatory element in one of the two hoxb5
loci is consistent with the DDC model. Sharpe and colleagues have previously
referred to a large fragment of the mouse Hoxb5/Hoxb4 intergenic
region, encompassing J3, as the `mesodermal enhancer region'
(Sharpe et al., 1998). Our
experiments revealed that the J3 element is not only responsible for the
mesodermal Hoxb5 expression but may also be required for maintaining
high levels of Hoxb5 expression. Indeed, reporter constructs
containing J3 (hoxb5a and hoxb5binsJ3) appear to show higher
expression levels than their J3-deleted counterparts (hoxb5b and
hoxb5aJ3). This might also be associated with the intriguing
observation that mouse embryos carrying hoxb5blacZ and
hoxb5aJ3lacZ, the two transgenes lacking J3, showed
variegated patterns of transgene expression among embryos from the same mating
(see Fig. S2 in the supplementary material). Primary transgenic embryos also
showed a similar variability in the transgene expression pattern (data not
shown). As the number of independent integration events and the use of
established lines argue against positional effects or mosaicism, we propose
that one of the functions of the J3 element is to maintain gene expression.
This additional function of J3 might involve interactions with proteins such
as members of the Polycomb and Trithorax groups, which are known to regulate
and maintain the transcriptional status of Hox genes through modification of
chromatin structure.
Conclusions
Our data suggest that the patterns of regulatory evolution of teleost
hoxb5 duplicates involve mechanisms additional to those suggested by
the DDC model (Force et al.,
1999). Although phylogenetic filtering and functional tests of
individual elements from the hoxb5a and hoxb5b loci did not
reveal clear signs of complementation between the regulatory elements retained
in both zebrafish loci, our results highlight the importance of interactions
between CNEs in the execution of complementary subfunctions of duplicated
genes.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/21/3543/DC1
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Altschmied, J., Delfgaauw, J., Wilde, B., Duschl, J., Bouneau,
L., Volff, J. N. and Schartl, M. (2002). Subfunctionalization
of duplicate mitf genes associated with differential degeneration of
alternative exons in fish. Genetics
161,259
-267.
Amores, A., Force, A., Yan, Y. L., Joly, L., Amemiya, C., Fritz,
A., Ho, R. K., Langeland, J., Prince, V., Wang, Y. L. et al.
(1998). Zebrafish hox clusters and vertebrate genome evolution.
Science 282,1711
-1714.
Amores, A., Suzuki, T., Yan, Y. L., Pomeroy, J., Singer, A.,
Amemiya, C. and Postlethwait, J. H. (2004). Developmental
roles of pufferfish Hox clusters and genome evolution in ray-fin fish.
Genome Res. 14,1
-10.
Amsterdam, A., Lin, S. and Hopkins, N. (1995).
The Aequorea victoria green fluorescent protein can be used as a reporter in
live zebrafish embryos. Dev. Biol.
171,123
-129.[CrossRef][Medline]
Bruce, A. E., Oates, A. C., Prince, V. E. and Ho, R. K.
(2001). Additional hox clusters in the zebrafish: divergent
expression patterns belie equivalent activities of duplicate hoxB5 genes.
Evol. Dev. 3,127
-144.[CrossRef][Medline]
Chiu, C. H., Dewar, K., Wagner, G. P., Takahashi, K., Ruddle,
F., Ledje, C., Bartsch, P., Scemama, J. L., Stellwag, E., Fried, C. et al.
(2004). Bichir HoxA cluster sequence reveals surprising trends in
ray-finned fish genomic evolution. Genome Res.
14, 11-17.
Copeland, N. G., Jenkins, N. A. and Court, D. L.
(2001). Recombineering: a powerful new tool for mouse functional
genomics. Nat. Rev. Genet.
2, 769-779.[Medline]
Cormack, B. P., Valdivia, R. H. and Falkow, S.
(1996). FACS-optimized mutants of the green fluorescent protein
(GFP). Gene 173,33
-38.[CrossRef][Medline]
Cresko, W. A., Yan, Y. L., Baltrus, D. A., Amores, A., Singer,
A., Rodriguez-Mari, A. and Postlethwait, J. H. (2003). Genome
duplication, subfunction partitioning, and lineage divergence: Sox9 in
stickleback and zebrafish. Dev. Dyn.
228,480
-489.[CrossRef][Medline]
Force, A., Lynch, M., Pickett, F. B., Amores, A., Yan, Y. L. and
Postlethwait, J. (1999). Preservation of duplicate genes by
complementary, degenerative mutations. Genetics
151,1531
-1545.
Fraidenraich, D., Lang, R. and Basilico, C.
(1998). Distinct regulatory elements govern Fgf4 gene expression
in the mouse blastocyst, myotomes, and developing limb. Dev.
Biol. 204,197
-209.[CrossRef][Medline]
Hadrys, T., Prince, V., Hunter, M., Baker, R. and Rinkwitz,
S. (2004). Comparative genomic analysis of vertebrate Hox3
and Hox4 genes. J. Exp. Zoolog. B Mol. Dev. Evol.
302,147
-164.[Medline]
Hogan, B., Constantini, F. and Lacy, E. (1986).
Manipulating the Mouse Embryo: a Laboratory Manual.
Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
Hogan, B. L., Holland, P. W. and Lumsden, A.
(1988). Expression of the homeobox gene, Hox 2.1, during mouse
embryogenesis. Cell Differ. Dev. 25
Suppl, 39-44.[CrossRef][Medline]
Hua, L. V., Hidaka, K., Pesesse, X., Barnes, L. D. and Shears,
S. B. (2003). Paralogous murine Nudt10 and Nudt11 genes have
differential expression patterns but encode identical proteins that are
physiologically competent diphosphoinositol polyphosphate phosphohydrolases.
Biochem. J. 373,81
-89.[CrossRef][Medline]
Jaffe, L., Jeannotte, L., Bikoff, E. K. and Robertson, E. J.
(1990). Analysis of beta 2-microglobulin gene expression in the
developing mouse embryo and placenta. J. Immunol.
145,3474
-3482.[Abstract]
Kleinjan, D. A., Bancewicz, R. M., Gautier, P., Dahm, R.,
Schonthaler, H. B., Damante, G., Seawright, A., Hever, A. M., Yeyati, P. L.,
van Heyningen, V. et al. (2008). Subfunctionalization of
duplicated zebrafish pax6 genes by cis-regulatory divergence. PLoS
Genet. 4,e29
.[CrossRef][Medline]
Kluver, N., Kondo, M., Herpin, A., Mitani, H. and Schartl,
M. (2005). Divergent expression patterns of Sox9 duplicates
in teleosts indicate a lineage specific subfunctionalization. Dev.
Genes Evol. 215,297
-305.[CrossRef][Medline]
Krumlauf, R., Holland, P. W., McVey, J. H. and Hogan, B. L.
(1987). Developmental and spatial patterns of expression of the
mouse homeobox gene, Hox 2.1. Development
99,603
-617.
Kumar, S., Tamura, K., Jakobsen, I. B. and Nei, M.
(2001). MEGA2: molecular evolutionary genetics analysis software.
Bioinformatics 17,1244
-1245.
Larochelle, C., Tremblay, M., Bernier, D., Aubin, J. and
Jeannotte, L. (1999). Multiple cis-acting regulatory regions
are required for restricted spatio-temporal Hoxa5 gene expression.
Dev. Dyn. 214,127
-140.[CrossRef][Medline]
Lynch, M. and Force, A. (2000). The probability
of duplicate gene preservation by subfunctionalization.
Genetics 154,459
-473.
Mansfield, J. H., Harfe, B. D., Nissen, R., Obenauer, J.,
Srineel, J., Chaudhuri, A., Farzan-Kashani, R., Zuker, M., Pasquinelli, A. E.,
Ruvkun, G. et al. (2004). MicroRNA-responsive `sensor'
transgenes uncover Hox-like and other developmentally regulated patterns of
vertebrate microRNA expression. Nat. Genet.
36,1079
-1083.[CrossRef][Medline]
Ohno, S. (1970). Evolution by Gene
Duplication. Heidelberg, Germany: Springer-Verlag.
Oosterveen, T., Niederreither, K., Dolle, P., Chambon, P.,
Meijlink, F. and Deschamps, J. (2003). Retinoids regulate the
anterior expression boundaries of 5' Hoxb genes in posterior hindbrain.
EMBO J. 22,262
-269.[CrossRef][Medline]
Sakach, M. and Safaei, R. (1996). Localization
of the HoxB5 protein in the developing CNS of late gestational mouse embryos.
Int. J. Dev. Neurosci.
14,567
-573.[CrossRef][Medline]
Santini, S., Boore, J. L. and Meyer, A. (2003).
Evolutionary conservation of regulatory elements in vertebrate Hox gene
clusters. Genome Res.
13,1111
-1122.
Sharpe, J., Nonchev, S., Gould, A., Whiting, J. and Krumlauf,
R. (1998). Selectivity, sharing and competitive interactions
in the regulation of Hoxb genes. EMBO J.
17,1788
-1798.[CrossRef][Medline]
Taylor, J. S., Van de Peer, Y., Braasch, I. and Meyer, A.
(2001). Comparative genomics provides evidence for an ancient
genome duplication event in fish. Philos. Trans. R. Soc. Lond. B
Biol. Sci. 356,1661
-1679.
Thisse, C., Thisse, B., Schilling, T. F. and Postlethwait, J.
H. (1993). Structure of the zebrafish snail1 gene and its
expression in wild-type, spadetail and no tail mutant embryos.
Development 119,1203
-1215.[Abstract]
Tumpel, S., Cambronero, F., Wiedemann, L. M. and Krumlauf,
R. (2006). Evolution of cis elements in the differential
expression of two Hoxa2 coparalogous genes in pufferfish (Takifugu rubripes).
Proc. Natl. Acad. Sci. USA
103,5419
-5424.
Woolfe, A. and Elgar, G. (2007). Comparative
genomics using Fugu reveals insights into regulatory subfunctionalization.
Genome Biol. 8,R53
.[CrossRef][Medline]
Yee, S. P. and Rigby, P. W. (1993). The
regulation of myogenin gene expression during the embryonic development of the
mouse. Genes Dev. 7,1277
-1289.
Zerucha, T., Stuhmer, T., Hatch, G., Park, B. K., Long, Q., Yu,
G., Gambarotta, A., Schultz, J. R., Rubenstein, J. L. and Ekker, M.
(2000). A highly conserved enhancer in the Dlx5/Dlx6 intergenic
region is the site of cross-regulatory interactions between Dlx genes in the
embryonic forebrain. J. Neurosci.
20,709
-721.
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