Mechanisms of Development 124 (2007) 532–542
www.elsevier.com/locate/modo
Cis-regulation of the amphioxus engrailed gene: Insights into
evolution of a muscle-specific enhancer
Laura Beaster-Jones
a
a,1
, Michael Schubert b, Linda Z. Holland
a,*
Marine Biology Research Division, Scripps Institution of Oceanography, University of California, San Diego, La Jolla, CA 92093-0202, USA
b
Laboratoire de Biologie Moléculaire de la Cellule, Ecole Normale Supérieure de Lyon, Lyon, France
Received 19 December 2006; received in revised form 4 June 2007; accepted 5 June 2007
Available online 12 June 2007
Abstract
To gain insights into the relation between evolution of cis-regulatory DNA and evolution of gene function, we identified tissue-specific
enhancers of the engrailed gene of the basal chordate amphioxus (Branchiostoma floridae) and compared their ability to direct expression
in both amphioxus and its nearest chordate relative, the tunicate Ciona intestinalis. In amphioxus embryos, the native engrailed gene is
expressed in three domains – the eight most anterior somites, a few cells in the central nervous system (CNS) and a few ectodermal cells.
In contrast, in C. intestinalis, in which muscle development is highly divergent, engrailed expression is limited to the CNS. To characterize
the tissue-specific enhancers of amphioxus engrailed, we first showed that 7.8 kb of upstream DNA of amphioxus engrailed directs
expression to all three domains in amphioxus that express the native gene. We then identified the amphioxus engrailed muscle-specific
enhancer as the 1.2 kb region of upstream DNA with the highest sequence identity to the mouse en-2 jaw muscle enhancer. This amphioxus enhancer directed expression to both the somites in amphioxus and to the larval muscles in C. intestinalis. These results show that
even though expression of the native engrailed has apparently been lost in developing C. intestinalis muscles, they express the transcription factors necessary to activate transcription from the amphioxus engrailed enhancer, suggesting that gene networks may not be completely disrupted if an individual component is lost.
Ó 2007 Elsevier Ireland Ltd. All rights reserved.
Keywords: Enhancer; Cis-regulation; Branchiostoma; Muscle evolution; Conserved non-coding DNA; Ciona; Lancelet; Amphioxus; Engrailed
1. Introduction
The ancestral role of the homeobox gene engrailed in
bilaterian development has long been debated. Because
engrailed is expressed in developing neurons in both protostomes and deuterostomes, (Patel et al., 1989) proposed
that the original function was in neuronal specification.
However, the expression of engrailed genes in mesoderm,
non-neural ectoderm and endoderm as well has led to
continuing discussions concerning whether particular
expression domains represent inheritance from a common
ancestor or co-option for new functions (Holland et al.,
*
Corresponding author. Tel.: +1 858 534 5607; fax: +1 858 534 7313.
E-mail address: [email protected] (L.Z. Holland).
1
Present address: Department of Biology, Augustana College, Sioux
Falls, SD 57197, USA.
1997; Iwaki and Lengyel, 2002; Prud’homme et al., 2003;
Mooi et al., 2005). In deuterostomes, engrailed genes are
chiefly expressed in subsets of neurons in the CNS (Fjose
et al., 1992) and in mesodermal derivatives such as the
walls of the developing coeloms in starfish (Byrne et al.,
2005), the anterior muscular somites of amphioxus (Holland et al., 1997), and the developing jaw muscles and paraxial muscles of vertebrates (Hatta et al., 1990; Ekker et al.,
1992).
The engrailed protein has been highly conserved
throughout evolution. For example, Drosophila engrailed
can rescue brain and skeletal defects in mouse en-1
mutants, although it cannot rescue the limb defects (Hanks
et al., 1998). Therefore, the evolutionary changes in
engrailed function are most likely due to changes in cis-regulation, as has occurred in a number of genes during the
speciation of Drosophila and the radiation of echinoderms
0925-4773/$ - see front matter Ó 2007 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.mod.2007.06.002
L. Beaster-Jones et al. / Mechanisms of Development 124 (2007) 532–542
(Gibert and Simpson, 2003; Hinman et al., 2003; Wittkopp
et al., 2004). Regulation of engrailed genes has been studied
in both D. melanogaster and the mouse. For each organism, alignments of upstream and/or intronic DNA between
closely related species identified highly conserved regulatory sequences, most of which direct tissue-specific expression (Kassis et al., 1989; Kassis, 1990; Logan et al., 1992;
Americo et al., 2002). For example, comparisons of mouse
en-2 with human and chicken engrailed sequences identified
a conserved region 6 kb upstream of the ATG start codon
containing two separate enhancers, a 1.5 kb fragment
directing expression to the midbrain/hindbrain boundary
and a 1.0 kb region directing expression to mandibular
myoblasts (Logan et al., 1993). Further dissection of the
muscle enhancer revealed a 356 bp fragment that is necessary but not sufficient for directing en-2 expression to the
jaw muscle precursors (Degenhardt et al., 2002).
To gain insight into how cis-regulation of engrailed has
been modified as the domains of engrailed expression have
evolved, we focused on the invertebrate chordates amphioxus (Branchiostoma floridae) and a tunicate (Ciona intestinalis). Although initial phylogenetic analyses placed
tunicates basally in the chordates with amphioxus as the
sister group of vertebrates, more recent analyses with large
gene sets reverse these positions, placing tunicates as the
sister group of vertebrates with amphioxus as the basal
chordate (Blair and Hedges, 2005; Philippe et al., 2005;
Bourlat et al., 2006; Delsuc et al., 2006). Both amphioxus
and the tunicate C. intestinalis have a single engrailed gene.
In amphioxus, this gene is expressed in two groups of a few
neurons each in the CNS, in a weak stripe in the ectoderm
and in each of the anterior-most eight somites, which
derive from the dorsolateral walls of the archenteron (Holland et al., 1997). However, engrailed is not expressed in
either the posterior somites that pinch off from the tail
bud or in the notochord, which is a modified muscle as
shown by electron microscopy (Flood et al., 1969; Flood,
1970) and expression of muscle-specific genes (Suzuki and
Satoh, 2000; Urano et al., 2003). In C. intestinalis, engrailed
is only expressed in two groups of neurons in the larval
CNS. There is no expression in the notochord, which is
non-muscular, in the larval tail musculature, which develops chiefly from the myoplasm that is set aside shortly after
fertilization, or in precursors of the adult musculature,
which derives from the trunk mesenchyme arising from
the A7.6-, B8.5- and B7.7-cells (Imai et al., 2002; Tokuoka
et al., 2005).
Most vertebrates have two engrailed genes, although
there have been independent duplications in teleosts. Like
amphioxus engrailed, vertebrate en-1 and en-2 are
expressed in the CNS and in mesodermal derivatives,
including the muscle pioneer cells in the zebrafish somites,
while en-2 is also expressed in precursors of certain jaw
muscles (Davidson et al., 1988; Hatta et al., 1990; Ekker
et al., 1992). In the lamprey, en-2 is expressed in the velar
muscles, which are derived from the mandibular coelom
(Holland et al., 1993). It has been suggested that engrailed
533
expression in the gnathostome jaw muscles may represent
the common evolutionary heritage of the jaw and velar
muscles from the engrailed-expressing anterior somites of
an amphioxus-like ancestor (Holland et al., 1993). Taken
together, the expression of engrailed in coelomic derivatives
in both echinoderms and amphioxus and in the jaw and
velar muscles of vertebrates suggests that the lack of mesodermal expression of engrailed in ascidians may represent a
loss of this domain in connection with the loss of coeloms
in the larval head.
In the present work, we characterized the cis-regulation
of the amphioxus engrailed gene and showed that the
1.2 kb of upstream sequence with the highest identity to
the mouse en-2 jaw muscle enhancer directs engrailed
expression to the amphioxus somites. To gain insight into
the evolution of engrailed regulation in basal chordates,
we expressed amphioxus engrailed reporter constructs in
the tunicate C. intestinalis. Although expression of C. intestinalis engrailed is limited to the CNS, the amphioxus
engrailed muscle enhancer directs expression in C. intestinalis to both the tail muscle and to the precursors of the
adult musculature. These results demonstrate that even
though the C. intestinalis engrailed gene may have lost a
muscle-specific enhancer, C. intestinalis muscles contain
the transcription factors necessary to direct expression
from the amphioxus muscle-specific enhancer.
2. Results
2.1. Identification of a muscle-specific enhancer in amphioxus
engrailed
When injected into amphioxus embryos, a reporter construct of amphioxus engrailed (AmphiEn) that included the
0.7 kb AmphiEn minimal promoter plus another 7.8 kb of
upstream DNA, directed expression to all the domains
where the endogenous gene is expressed – the CNS, ectoderm and somites (Fig. 1A; compare Fig. 2A–D with
Fig. 2E–H). As is typical with transient transgenics, expression was mosaic (Yuh and Davidson, 1996; Koster and
Fraser, 2001; Romano and Wray, 2003). Since expression
of this construct was relatively weak, we sub-cloned the distal 7.8 kb upstream of the relatively strong FoxD minimal
promoter (Yu et al., 2004) (Fig. 1A). Expression of this
construct in the somites and ectoderm was identical to that
of the 8.5 kb construct with the AmphiEn minimal promoter, except that the level of expression was higher
(Fig. 2I–L), and was significantly greater than in controls
injected with the construct containing the FoxD minimal
promoter alone (Table 1). A few embryos expressed the
7.8 kb construct in the CNS; although the number was
not significant, the presence of a CNS enhancer is not ruled
out since the native engrailed gene is expressed in very few
cells in the CNS.
To help identify the muscle-specific enhancer, we used
ClustalW to align the 8.5 kb of AmphiEn upstream of the
ATG start codon with the 1.0 kb mouse en-2 jaw muscle
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L. Beaster-Jones et al. / Mechanisms of Development 124 (2007) 532–542
Fig. 1. Schematic diagrams of engrailed reporter constructs expressed in
(A) amphioxus and (B) Ciona intestinalis. Thin lines indicate amphioxus
engrailed regulatory sequence, bold lines specify the AmphiFoxD or
C. intestinalis forkhead (Cifkh) minimal promoter sequence. Boxes
designate the lacZ reporter gene in vector, p72-1.27.
enhancer (Logan et al., 1993) (Supplemental Fig. S1).
Although the mouse en-2 jaw muscle enhancer is located
about 6 kb upstream of the ATG start codon, the region
of AmphiEn that aligns with it is from 1.4 to 2.6 kb.
The same region of AmphiEn aligned when an additional
12 kb of upstream AmphiEn sequence was included in the
alignment (data not shown). Although the percentage of
identity of the aligned amphioxus and mouse sequences is
less than 50%, they contain similar constellations of potential transcription factor binding sites, albeit in somewhat
different positions (Supplemental Fig. S1). Given the low
level of identity of the aligned regions, it is not surprising
that such programs as LAGAN and Family Relations
did not reveal any highly conserved regions between the
10–20 kb of upstream DNA of AmphiEn and mouse en-2
(data not shown). Such low identity would be expected
since the corresponding regions of mouse and human en2 are only 53% identical and have the conserved potential
transcription factor binding sites in different locations
(Degenhardt et al., 2002). The conserved sites between
the mouse en-2 jaw muscle enhancer and the corresponding
regions of human and amphioxus engrailed include those
for myogenic factors (MEF2, myogenin, MyoD) and
NFAT, which have been shown to cooperate in mediation
of transcriptional activation in slow muscle fibers (Chin
et al., 1998), as well as a b-motif (CTAATTA), which contains the core homeodomain binding motif, and is required
for directing expression of Otx to the mandibular mesenchyme of the mouse (Kimura et al., 1997). The density of
the potential binding sites for myogenic factors in the
1.2 kb region of AmphiEn is about twice as high as elsewhere in the 7.8 kb region. Moreover, no other copies of
the b-motif are present in the 20 kb of AmphiEn upstream
to the adjacent gene, in the intron or downstream of the
coding region to 6 kb. The 1.2 kb region of AmphiEn also
has three potential high affinity binding sites for Pax3
(twice the density as elsewhere in the upstream DNA),
compared to one in the mouse en-2 jaw muscle enhancer.
Pax3/7 is expressed before AmphiEn in the developing
amphioxus somites (Holland et al., 1999); however, it does
not appear to be involved in myogenesis in the mouse head
(Horst et al., 2006).
To determine if this 1.2 kb region of amphioxus
engrailed directs expression to muscle as suggested by the
conservation of potential transcription factor binding sites
– in particular the b-motif – with the mouse en-2 jaw muscle enhancer, we made three additional reporter constructs:
one corresponding to the 1.2 kb region that aligned with
the mouse en-2 jaw muscle enhancer, a 2.0 kb construct
that spanned the 1.2 kb region and one including only
the central 0.4 kb of the 1.2 kb region, which corresponded
to the 0.4 kb of the mouse en-2 jaw muscle enhancer that
was found to be essential, but not sufficient, for jaw muscle
expression (Degenhardt et al., 2002) (Fig. 1A). When
injected into amphioxus eggs, both the 2.0 and 1.2 kb
AmphiEn constructs directed significant expression to the
somites (Fig. 2M–Q) (Table 1). Expression in the ectoderm
and CNS was not significantly different from the controls
(Table 1). These results together with the results from
injecting the 7.8 kb construct suggest that the region
between 1.4 and 2.6 kb (the 1.2 kb region) is a muscle-specific enhancer and that an ectoderm enhancer is
located upstream of the 2.0 kb region between 2.7 and
8.5 kb. The percentage expressing the 2.0 kb construct
in the somites is significantly higher than for the 1.2 kb
construct, and approximately the same as for the 7.8 kb
construct (Table 1). These results suggest that there are
enhancer(s) that potentiate expression in the somites
between 2.6 and 8.5 kb as well as between 1.4 and
2.6 kb. The 0.4 kb construct did not express in any tissue
at levels significantly higher than that of the vector alone
(Table 1). Thus, we conclude that this region is insufficient
to direct muscle-specific expression in amphioxus.
L. Beaster-Jones et al. / Mechanisms of Development 124 (2007) 532–542
535
Fig. 2. Endogenous amphioxus engrailed expression visualized by in situ hybridization (A–D) and expression of amphioxus engrailed reporter constructs
as visualized by b-galactosidase activity with X-Gal (E–Q). For whole mounts (A–F, I–K, N–Q) anterior is left; scale = 50 lm. For cross-sections (G, H;
L, M) scale = 25 lm. For schematic diagrams of constructs, red = amphioxus upstream engrailed sequence. Green = FoxD minimal promoter.
Black = lacZ coding region. (A) Dorsal view of a 9-h neurula showing expression in posterior halves of the first three forming somites. (B) Dorsal view of a
12-h neurula with expression in the four anterior somites. (C) Dorsal view of a 14-h neurula showing expression in the eight developing somites. (D) Side
view of a 28-h embryo with expression in anterior part of the nerve cord (cerebral vesicle) (arrow) and a few cells in the hindbrain (double arrow). (E and
F) Side views of mid-neurula embryos expressing the 8.5 kb construct in the somites and nerve cord. (G) Cross-section through level g in E showing
expression in the myotomal compartment of the somite. (H) Cross-section through level h in (F) showing expression in the nerve cord. (I and J) Side views
of 22-h neurulae expressing the 7.8 kb construct in the ectoderm, somites and nerve cord. Ectopic gut expression also observed in (I). (K) Dorsal view of
20-h embryo expressing the 7.8 kb construct in the somites. (L) Cross-section through level l in (J) showing expression in the ectoderm. (M) Cross-section
of the 22-h embryo in (P) through level m showing somitic expression of the 1.2 kb construct. (N) Dorsal view of 15-h neurula expressing the 2.0 kb
construct in somites. (O) Side view of 15-h neurula expressing the 2.0 kb construct in somites. (P) Dorsal view of 22-h neurula expressing the 1.2 kb
construct in somites. (Q) Side view of a 24-h embryo expressing the 1.2 kb construct in somites.
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L. Beaster-Jones et al. / Mechanisms of Development 124 (2007) 532–542
Table 1
Expression of engrailed reporter constructs analyzed in amphioxus
Reporter construct
Minimal promoter
7.8 kb (0.7 to 8.5 kb)
2.0 kb (0.7 to 2.7 kb)
1.2 kb (1.4 to 2.6 kb)
0.4 kb (1.8 to 2.2 kb)
Somite
Ectoderm
Nerve cord
Total injected No.
No.
Per cent
No.
Per cent
No.
Per cent
2
67
43
22
2
0.9
15.5
12.3
4.0
1.5
3
31
9
6
0
1.3
7.2
2.6
1.1
0.0
0
4
0
0
0
0.0
0.9
0.0
0.0
0.0
224
433
351
555
130
Percentages in bold are significantly different from those of the vector plus minimal promoter alone. For the 1.2 kb construct P 6 0.00275 for expression in
ectoderm. For all other significant numbers P 6 0.001; v2, not applicable to zero values.
2.2. Amphioxus engrailed regulatory DNA directs expression
in C. intestinalis embryos to tissues that do not express the
native C. intestinalis engrailed gene
To investigate the evolution of engrailed regulation, we
also determined the expression of amphioxus engrailed
reporter constructs in C. intestinalis. To optimize expression, rather than using the amphioxus FoxD minimal promoter, we used the basal promoter from the C. intestinalis
forkhead gene, which is commonly used for transient transgenics in C. intestinalis (Christiaen et al., 2005; Harafuji
et al., 2002) (Fig. 1B). Although C. intestinalis engrailed
is only expressed in the CNS (Fig. 3A and B) (Imai et al.,
2002), none of the AmphiEn constructs directed expression
there. Instead, all constructs directed expression to tissues
that do not normally express engrailed. All constructs
directed expression to the C. intestinalis ectoderm at levels
significantly higher than that of the control (Table 2,
Fig. 3C–M). However, ectodermal expression of constructs
lacking the region between 2.7 and 8.5 kb was significantly lower than that of the 7.8 kb construct, suggesting
that as for amphioxus, ectodermal expression is driven
chiefly by sequences between 2.7 and 8.5 kb and/or that
the 0.4 kb region may contain a suppressor of ectodermal
expression (Table 2). Surprisingly, the 7.8 kb construct also
directed expression to the notochord even though the comparable construct does not direct expression to the amphioxus notochord (Fig. 3D and L; Table 2). Such
notochordal expression may be an artifact created by the
minimal promoter, since the original amphioxus engrailed
8.5 kb construct with the amphioxus engrailed minimal
promoter did not direct expression to the notochord in
C. intestinalis, although it did express in ectoderm and
muscles (data not shown). Similar artifactual expression
of reporter constructs in the ascidian notochord has also
been seen with a different minimal promoter in cross-species experiments between two ascidians (Oda-Ishii et al.,
2005).
Although the engrailed gene of C. intestinalis has apparently lost muscle-specific expression, all of the constructs
directed expression to the tail musculature at levels significantly greater than controls (Fig. 3C–M; Table 2). However, only the 0.4, 1.2 and 2.0 kb constructs directed
expression to the trunk mesenchyme that gives rise to the
adult musculature at levels significantly higher than in the
controls (Fig. 3E–J, K, M). In fact, expression of the
7.8 kb construct in the mesenchyme was less than that of
the controls, and its expression in the tail muscle, though
greater than that of the controls was significantly lower
than that of the other constructs (Fig. 3C, D, and L).
Moreover, expression of the 2.0 kb construct in both the
tail muscle and mesenchyme was also less than that of
the 1.2 and 0.4 kb constructs. Taken together, these results
suggest that, in contrast with the situation in amphioxus, a
suppressor of expression in both types of C. intestinalis
muscle precursors lies between 2.7 and 8.5 kb and that
the 0.4 kb construct contains an enhancer that directs
expression to both the adult muscle precursors and tail
muscle in C. intestinalis.
3. Discussion
3.1. Sequence alignments over wide phylogenetic distances
can reveal functional enhancers
The upstream amphioxus engrailed sequence with the
best match to the mouse en-2 jaw muscle enhancer (Logan
et al., 1993; Degenhardt et al., 2002) directs expression to
c
Fig. 3. Endogenous Ciona intestinalis engrailed expression visualized by in situ hybridization (A and B) and expression of amphioxus engrailed reporter
constructs in Ciona embryos as visualized by b-galactosidase activity with X-Gal (C–M). In whole mounts (A–J), anterior is at left; scale = 50 lm. Crosssections (K–M) scale = 25 lm. For schematic diagrams of constructs, red = amphioxus upstream engrailed sequence. Blue = C. intestinalis fkhd minimal
promoter. Black = lacZ coding region. (A) Side view of early tailbud C. intestinalis embryo with engrailed expression in the central nervous system. The
native gene is not expressed at older stages. (B) Enlarged dorsal view of embryo in (A). (Images of C. intestinalis embryos kindly provided by K. Imai.) (C
and D) C. intestinalis larvae expressing the 7.8 kb construct in the ectoderm, head mesenchyme, tail muscle and notochord. l–l 0 shows level of section
through notochord and tail muscle in (L). (E and F) Early and late tailbud stages with the 2.0 kb construct expressed in the head mesenchyme (arrows) and
tail muscle. (G and H) The 1.2 kb construct is expressed in the head mesenchyme and tail muscle. k–k 0 indicates level of section through head mesenchyme
in (K). (I) Expression of 0.4 kb construct in the head mesenchyme. (J) Expression of the 0.4 kb construct in tail muscle. (K) Cross-section through level
k–k 0 in (H) showing expression in the head mesenchyme. (L) Cross-section through level l–l 0 in (D) showing expression in notochord and tail muscle. (M)
Cross-section through level m–m 0 in (J) showing expression in tail muscle.
L. Beaster-Jones et al. / Mechanisms of Development 124 (2007) 532–542
amphioxus muscles, suggesting a degree of evolutionary
conservation despite less than 50% sequence identity
between these sequences. Most notably, the amphioxus
and mouse muscle enhancers both have a b-motif and
potential binding sites for MyoD, MEF2 and NFAT as
537
well as several transcription factors including TCF/LEF,
which mediates Wnt/b-catenin signaling (Supplemental
Fig S1). In vertebrates, proteins in the NFAT and MEF2
families have been shown to mediate transcriptional activation of genes involved in the development of slow skeletal
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Table 2
Summary of engrailed reporter constructs analyzed in ascidians
Reporter construct
Minimal promoter
7.8 kb (0.7 to 8.5 kb)
2.0 kb (0.7 to 2.7 kb)
1.2 kb (1.4 to 2.6 kb)
0.4 kb (1.8 to 2.2 kb)
Tail muscle
Mesenchyme
Ectoderm
Notochord
No.
Per cent
No.
Per cent
No.
Per cent
No.
Per cent
23
32
128
95
152
2.3
9.8
15.3
28.0
24.8
52
7
139
187
223
5.2
2.1
16.2
55.2
36.7
59
186
68
32
53
5.9
56.9
8.0
9.4
8.7
1
114
0
0
0
0.0
34.9
0.0
0.0
0.0
Total electroporated
997
327
851
339
608
Numbers in bold are significantly different by v2 test from those of the vector plus minimal promoter alone. For all significant values, P 6 0.001 except for
ectodermal expression of the 2.0, 1.2 and 0.4 kb constructs for which P 6 0.005; and for mesenchymal expression of the 7.8 kb construct for which
P 6 0.025.
muscle fibers (Chin et al., 1998), while the b-motif is
required for directing expression of Otx2 to premandibular
and mandibular mesenchyme (Kimura et al., 1997).
Expression of the relevant transcription factors (MyoD
family members as well as TCF/LEF, Wnt8 and Pax3) in
developing amphioxus muscles before engrailed turns on
(Holland et al., 1999; Lin et al., 2006; Schubert et al.,
2000, 2003) suggests that these sites may indeed be
functional.
This conservation of binding sites in a background of
rather poorly conserved sequence is similar to that found,
for example, in the Otx genes of two distantly related
ascidian tunicates (Oda-Ishii et al., 2005). Within distantly
related vertebrates, close sequence conservation of noncoding elements is relatively common (Woolfe et al.,
2005; Venkatesh et al., 2006). However, between fairly distantly related organisms like amphioxus and tunicates or
amphioxus and vertebrates, functionally conserved enhancers with quite different sequences but conserved transcription factor binding sites may be more the rule than the
exception. Moreover, even when the enhancer sequences
are conserved, the enhancers themselves may occupy
different positions in relation to the gene that is regulated
and, therefore, may not be detected in alignments of large
DNA segments. For example, comparisons of the housefly
and fruitfly, showed that even though the domain of tll
expression is conserved, the enhancer sequences that direct
expression cannot be aligned, and neither the sequence of
the enhancer nor the sequence, number, spacing and position of the binding sites that direct expression are conserved (Wratten et al., 2006). Therefore, it is perhaps not
surprising that there are no alignable regions of upstream
engrailed DNA between amphioxus and C. intestinalis even
though both genes are expressed in the CNS. As the differences in Otx regulation noted above between two species of
ascidian tunicates exemplify, tunicate genomes are evolving
rapidly. Thus even though tunicates are now placed as the
sister group to vertebrates, the amphioxus genome, which
has neither undergone extensive gene duplications as in
vertebrates nor the gene loss characteristic of tunicates, is
probably the best genome for comparison with vertebrate
genomes to address such questions as the evolution of
new regulatory elements subsequent to gene duplication
and the conservation/non-conservation of gene regulation
as genes are co-opted for new functions.
3.2. Expression of the amphioxus muscle-specific enhancer in
C. intestinalis suggests a conserved gene network in muscle
Although engrailed is not part of the muscle-specific
gene network in C. intestinalis, expression of the amphioxus 1.2 kb engrailed muscle enhancer in C. intestinalis
muscles suggests evolutionary conservation of the musclespecific transcription factors directing engrailed expression
in amphioxus and tunicate muscle. The lack of native
engrailed expression in C. intestinalis muscle suggests that
the C. intestinalis engrailed gene may have lost the muscle-specific enhancer that is conserved in amphioxus and
the mouse. Such a loss is not too surprising, since tunicates
have compact genomes and have discarded many genes
during evolution (Holland and Gibson-Brown, 2003). In
C. intestinalis, the tail muscles develop largely from cytoplasm that is set aside shortly after fertilization and partitioned into muscle precursors at each cleavage. In
contrast, in amphioxus, the anterior somites pinch off from
the dorsolateral walls of the archenteron, and engrailed is
expressed in the posterior portion of each one, suggesting
a function in somite segmentation (Holland et al., 1997).
In light of such differences in the mode of muscle formation, it is perhaps surprising that the 1.2 kb amphioxus
engrailed enhancer directs expression to C. intestinalis
muscle as well as to amphioxus muscle. However, recent
large-scale bioinformatic analyses have suggested that
developmental gene regulatory networks are very well
conserved (Iwama and Gojobori, 2004; Kimura-Yoshida
et al., 2004; von Bubnoff et al., 2005; Woolfe et al.,
2005). When evolutionary changes occur in these networks,
they are often limited to specific cis-regulatory elements
(Gibert and Simpson, 2003; Hinman et al., 2003), indicating that there are constraints on gene networks directing
expression to specific tissues that allow them to lose or gain
some components but remain largely intact. Therefore,
although C. intestinalis engrailed has evidently lost this
muscle-specific enhancer, components of the gene network
in C. intestinalis muscle are still able to direct expression of
an exogenous muscle-specific enhancer.
L. Beaster-Jones et al. / Mechanisms of Development 124 (2007) 532–542
3.3. Analyzing cis-regulatory DNA over large taxonomic
distances
Expression of conserved non-coding DNA between closely related organisms (e.g. within different vertebrates or
different species of sea urchins or Drosophila) can identify
functional regulatory regions (Yuh and Davidson, 1996;
Woolfe et al., 2005; Prud’homme et al., 2006). However,
our results show that results from cross-species experiments, especially when phylogenetic distances are large,
should be interpreted cautiously. Previous studies of the
expression of reporter constructs of amphioxus genes in
other species have not included expression in amphioxus
as well, because techniques for microinjection were only
recently developed and the breeding season is limited
(Manzanares et al., 2000; Holland and Yu, 2004; Wada
et al., 2005, 2006). For example, Wada et al. (2005) argued
that since an amphioxus Hox2 reporter construct with Ets
binding sites directs expression to the pharynx in C. intestinalis and since amphioxus Ets1/2 and Hox2 are transiently co-expressed in the amphioxus preoral pit, a
pharyngeal structure, Ets1/2 probably activates Hox2
there. However, expression of the 7.8 kb amphioxus
engrailed construct in the C. intestinalis notochord, but
not the amphioxus notochord shows that constellations
of binding sites that can function as an enhancer in another
species may not be functional in the native species. Even
when there is indirect evidence that an enhancer is probably functional in the native species, expression of the
enhancer in the native species can strengthen the conclusions. For example, since retinoic acid regulates Hox
expression in amphioxus (Schubert et al., 2004, 2005,
2006) it is likely that the retinoic acid response elements
in the amphioxus Hox1 and Hox3 genes act as enhancers
in amphioxus. These enhancers were shown to direct
expression to both the neural tube and neural crest in the
chick and mouse (Manzanares et al., 2000), but not to precisely the same domains that express the corresponding
vertebrate Hox genes. Therefore, although the results suggest evolutionary conservation of Hox regulation by retinoic acid in amphioxus and vertebrates, the argument
would have been stronger had it been shown that the constructs also direct expression to the amphioxus neural tube.
In sum, although expression of amphioxus reporter constructs in other chordates can give insights into the evolution of gene regulation, expression in amphioxus as well
can strengthen the conclusions.
4. Conclusions
As genes evolve, their functions are probably altered
chiefly by changes in the control regions, although changes
in the coding regions may also be important. It has been
noted that for inferring homologies, overly similar features
are not particularly interesting, and very divergent features
are uninterpretable, whereas moderately divergent ones can
reveal interesting changes over evolutionary time in homol-
539
ogous structures, whether these are molecular or anatomical features (Wagner, 1989; Holland and Holland, 1999).
Although the rate of evolutionary change in the control
regions of genes is high, we have found that regions of regulatory DNA are sufficiently conserved between the
engrailed genes of amphioxus and vertebrates, even though
these lineages split about half a billion years ago, to permit
useful comparisons. Because an outstandingly important
evolutionary event – namely the origin of the vertebrates
from the invertebrates – has taken place within the Phylum
Chordata, it is encouraging that useful comparisons of
gene regulatory regions are possible across the chordate
subphyla.
5. Materials and methods
5.1. Collection of animals
Ripe adult amphioxus (Branchiostoma floridae) were collected in
Tampa Bay, Florida, and induced to spawn by electrical stimulation. Eggs
and sperm were obtained as described in Yu et al. (2004). Embryos were
cultured at 22.5 °C as previously described (Holland and Holland, 1993;
Holland and Yu, 2004).
Adult Ciona intestinalis were collected from San Diego Bay and Mission Bay in San Diego, California and maintained in a natural seawater
aquarium at 18–20 °C under constant light to prevent the spawning.
Sperm and eggs were dissected from adults within 10 days of animal
collection.
5.2. Identification of conserved non-coding regions and preparation
of reporter constructs
The cosmid library MPMGc117, available from the (RZPD) at http://
www.rzpd.de, was screened with a partial engrailed (AmphiEn) clone that
included the 5 0 coding sequence (Holland et al., 1997). One positive clone,
MPMGc117L1019, was digested with restriction enzymes and subjected to
Southern blotting with the same clone used for library screening. A 9.0 kb
fragment including the engrailed ATG start codon and upstream regulatory region was cloned into pBluescript and sequenced. The initial reporter
construct was made by cloning the 8.5 kb upstream of the ATG start
codon into the p72-1.72 plasmid (Corbo et al., 1997). For all other reporter constructs, fragments of amphioxus engrailed regulatory sequence were
amplified by PCR with the Expand Long Template PCR System (Roche
Applied Science, Indianapolis, IN) and cloned into the p72-1.72 plasmid
with either amphioxus-specific or C. intestinalis-specific minimal promoters from the FoxD (Yu et al., 2004) and fkh genes, respectively (Di Gregorio et al., 2001; Harafuji et al., 2002). To aid in identifying a likely muscle
enhancer, ClustalW (blosum scoring matrix; opening and end gap penalties of 10; extending and separation gap penalties of 0.05) was used to
align this 8.5 kb sequence of AmphiEn with the 1.0 kb jaw muscle enhancer
of mouse en-2. The 1.2 kb construct included the amphioxus sequence that
aligned with the mouse sequence. The 2.0 kb construct overlapped this
region, and the 0.4 kb construct included the central portion corresponding to the 356 bp region of the mouse en-2 jaw muscle enhancer known to
be essential, but insufficient for directing expression (Logan et al., 1993).
Searches for potential transcription factor binding sites in the mouse en2 jaw muscle enhancer and the AmphiEn muscle-specific enhancer were
done by MatInspector (http://www.genomatix.de/products/MatInspector/), which identifies potential high affinity sites and with Match Search
(http://www.gene-regulation.com/pub/programs.html#match), which also
identifies potential sites with moderate affinity. Primers for amplifying the
7.8 kb region from approximately 8.5 to 0.7 kb upstream of the ATG
start site, were LB01: 5 0 -CGGGATCCTATCCGAGAGGGGATTTCTC-3 0
and EnBamR: 5 0 -CGGGATCCACCACTCTTCCATTGTCCTCCTCG
540
L. Beaster-Jones et al. / Mechanisms of Development 124 (2007) 532–542
AC-3 0 . Primers for amplifying the 2.0 kb region from 2.7 to 0.7 kb
upstream of the ATG start site, were LB03: 5 0 -CGGGATCCT
CTCCAAGTATCTCTTCACTTAC-3 0 and EnBamR. The 1.2 kb region
from 2.6 to 1.4 kb upstream of the ATG start site, was amplified with
LB04: 5 0 -CGCTCGAGTGTTGTTCAACTTGCGGTAGGAC-3 0 and
LB05: 5 0 -CCGGCATGCCCGAAAATTGACTCGTATGTC-3 0 . The
0.4 kb region, from 2.1 to 1.7 kb upstream of the ATG start site,
was amplified with MS01: 5 0 -AAGCATGCCGTTGATAGATTTCCTT
CAG-3 0 and MS02: 5 0 -AAGGATCCACACACGGCCTTACACC-3 0 . To
generate the Del 2.0 kb construct, the region from 8.5 to 2.7 kb
upstream of the ATG start site, was amplified with EnPstF: 5 0 -AACTGC
AGTATCCGAGAGGGGATTTCTCGTTGCT-3 0 and LB39: 5 0 -CGGG
ATCCAACTGTAACATATGGTAACAG-3 0 . To make the Del 1.2 kb
construct, which consists of the 7.8 kb fragment minus a 1.2 kb region
from 2.6 to 1.4 kb, the regulatory regions on either side of the deletion
plus the entire vector were amplified with LB40: 5 0 -CGAGGCCTGGCG
CACATACAGTTGTGTG-3 0 and LB41: 5 0 -CGAGGCCTGAGGGA
GATGCCGCGAATTCC-3 0 from a pBluescript vector containing the
entire 7.8 kb regulatory sequence. The amplified plasmid was then religated creating a Del 1.2 construct with a StuI site in the place of
1.2 kb regulatory sequence. The Del 1.2 fragment was then cloned into
the BamHI site of pCES. The Del 0.4 kb construct was generated by
digesting the 7.8 kb construct with XmaI which cuts the plasmid into three
fragments: 9200, 4500 and 400 bp. The 9200 and 4500 bp fragments were
re-ligated to produce a construct which contains the 7.8 kb sequence
minus the 400 bp from 2.1 to 1.7 kb. The identity and orientation of
inserts were confirmed by sequencing. For electroporation into C. intestinalis, plasmid DNA was prepared using a maxiprep protocol and purified
on a CsCl gradient (Sambrook et al., 1989). For microinjection into
amphioxus, circular plasmid DNA was purified on a Qiagen miniprep column (Qiagen, Valencia, CA).
5.3. Microinjection and electroporation
Amphioxus eggs were microinjected with circular plasmid DNA as
described in Yu et al. (2004) except the injection mixes contained
500 lg/ml DNA. Electroporation of C. intestinalis embryos was performed according to the methods of Zeller (2004). Briefly, 50 lg of circular
DNA was mixed with 0.77 M mannitol to a final 500 ll volume. About
300 ll of fertilized, dechorionated eggs was added to the DNA/mannitol
solution and immediately transferred to a 0.4-cm cuvette and pulsed with
125 VDC/cm for 20 ms with an Electro Cell 600 electroporator (BTX, San
Diego, CA, USA). The electroporated eggs were transferred to gelatincoated dishes with micro-filtered seawater plus antibiotics and incubated
at 16–18 °C until the desired developmental stage.
5.4. Detection of b-galactosidase
Ascidian and amphioxus embryos were both fixed and stained for
b-galactosidase activity according to the methods of Yu et al. (2004).
Whole specimens were mounted in 80% glycerol and photographed. After
whole mount photography, the specimens were counterstained with Ponceau S, embedded in Spurr’s resin, and sectioned at 3–4 lm with glass
knives.
Acknowledgements
We are indebted to John Lawrence for providing laboratory facilities at the University of South Florida. We thank
Brad Davidson for providing the Ciona intestinalis expression vector (pCES), Jr-Kai Yu for microinjection guidance,
and Robert Zeller for advice on electroporation. We thank
Eddie Kisfaludy for assistance with animal collection,
Kaoru Imai for providing images of engrailed in C. intestinal-
is and N.D. Holland for comments on the manuscript. This
research was supported NSF Grant IBN 02-3617 to L.Z.H.
Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.mod.
2007.06.002.
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