Division of Biology, 139-74, California Institute of Technology, Pasadena, CA 91125, USA
* Author for correspondence (e-mail: mbronner{at}caltech.edu)
Accepted 30 July 2002
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SUMMARY |
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Key words: AP-2, Amphioxus, Lamprey, Neural crest, Migration
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INTRODUCTION |
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In the most basal vertebrate studied, lamprey, an essentially modern neural
crest generates almost all of the derivatives seen in gnathostomes
(Langille and Hall, 1988). By
contrast, the most vertebrate-like invertebrates, the cephalochordates, appear
to lack even a rudimentary neural crest. Furthermore, the fossil record offers
no obvious intermediate forms that display features suggestive of a primitive
neural crest.
Within the gnathostomes, the molecular mechanisms that underlie neural
crest induction are largely conserved (for a review, see
LaBonne and Bronner-Fraser,
1999). Input from BMP, Wnt and FGF signaling pathways activate a
complement of transcription factors at the neural plate border, including
Snail, Twist, Zic, Id, AP-2, FoxD3, Distal-less, Msx and Pax genes. A subset
of these factors has been shown to cross- and autoregulate, such that a rough
outline of their regulatory relationships is emerging
(Sasai et al., 2001
).
Amphioxus and lamprey are useful organisms for investigating neural crest
evolution as they both diverged near the time neural crest first appeared.
Amphioxus, a cephalochordate, separated from the vertebrate lineage before the
origin of neural crest and is thought to approximate the ancestral
pre-vertebrate chordate. Expression studies in amphioxus reveal that some of
the genetic machinery needed to create neural crest cells (including BMP-4,
Snail, Pax-3, Wnt7B, Distalless and Msx) was in place before bona fide neural
crest cells appeared (reviewed by Holland
and Holland, 2001). Lamprey diverged from other vertebrates
relatively soon after the neural crest arose and is thought to display
primitive features lost or masked in gnathostomes.
In this study, we focus on the regulatory evolution of the transcriptional
activator AP-2 as a starting point for dissecting the molecular history of
neural crest cells. AP-2 is a robust neural crest marker shown to be essential
for cranial neural crest development in vertebrates. The vertebrate AP-2
family consists of four genes (AP-2, ß,
and
) that
have dynamic and largely overlapping patterns of expression during
embryogenesis (reviewed by
Hilger-Eversheim et al., 2000
)
[for description of AP-2 delta see Zhao et al.
(Zhao et al., 2001
)]. At
gastrula stages, AP-2 transcripts are initially observed in non-neural
ectoderm. As neurulation proceeds, AP-2 expression is extinguished in
non-neural ectoderm and upregulated in the neural folds, marking neural crest
cells before, during and after their migration. AP-2
is functionally
important for neural crest cells, as null mice almost completely lack cranial
neural crest derivatives (Schorle et al.,
1996
; Zhang et al.,
1996
). In addition, AP-2 is necessary for expression of HoxA2 in
the neural crest, indicating an indirect role for AP-2 genes in neural crest
patterning (Maconochie et al.,
1999
).
We describe the isolation of amphioxus and lamprey AP-2 homologs and compare their expression patterns with that of AP-2 in the gnathostome axolotl. Using this broad comparative base, we span two important evolutionary transitions: the divergence of vertebrates from invertebrates and the divergence of jawed vertebrates from agnathans. Across each transition, we observe differences in the deployment of AP-2 genes that are suggestive of key genetic and developmental changes during early vertebrate evolution. Taken together, our observations suggest a crucial role for AP-2 during neural crest evolution.
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MATERIALS AND METHODS |
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AP-2 gene isolation
Amphioxus and lamprey embryonic cDNA libraries were the generous gifts of
Jim Langeland. 500 and 200 bp fragments of amphioxus and lamprey AP-2 genes,
respectively, were amplified directly from diluted lambda phage libraries by
degenerate PCR with the following primers: for amphioxus, 5' primer
GTRTTCTGYKCAGKYCCYGGICG and 3' primer GWKATVAGGKWGAAGTGSGTCA; for
lamprey, 5' primer CCVCCIGARTGCCTSAAYGC and 3' primer
GAAGTCICGVGCSARRTG. Amplified fragments were used to screen the libraries at
high stringency (final wash 0.2xSSC) to isolate full-length clones.
Phagemids were excised and inserts sequenced completely from both ends. Low
stringency screens of the amphioxus cDNA library and an arrayed amphioxus
genomic library were performed as described for Southern blot analysis.
Phylogenetic analysis
The conceptual protein products of the amphioxus and lamprey AP-2
transcripts were aligned with vertebrate and Drosophila AP-2 protein
sequences. Axolotl AP-2 was not used for analysis as only a partial sequence
is available. A phylogenetic tree was created within the ClustalX program
(Thompson et al., 1997) using
the neighbor-joining method of Saitou and Nei
(Saitou and Nei, 1987
).
Bootstrap values were determined by 1000 resamplings of alignment data.
GenBank Accession Numbers for the aligned sequences are: mouse AP-2
,
NP035677, mouse AP-2ß, Q61313, mouse AP-2
, Q61312, mouse
AP-2
, AAL16940, chicken AP-2
, AAB65081, chicken AP-2ß,
AAC26111, human AP-2
, NP003211, human AP-2ß, NP003212, human
AP-2
, XP009543, Xenopus AP-2
, S34449,
Drosophila AP-2, CAA07279.
Hox2 in silico cis regulatory analysis
Genomic sequences surrounding the transcriptional start of
Drosophila proboscipedia and AmphiHox2 were scanned for consensus
AP-2 binding sites using the MatInspector v2.2 program. Core and matrix
similarities were set at the default values of 0.75 and 0.85, respectively.
Accession Numbers are NG000110 for proboscipedia and AB050888 and AB050887 for
AmphiHox2 genomic sequences.
In situ hybridization
In situ hybridization on amphioxus embryos was performed as described by
Holland (Holland, 1996a) with the omission of deacetylation and RNAse
treatments. In addition, post-hybridization washes were in PBS-Tween 0.1%,
rather than SSC, and the blocking solution was 2 mg/ml BSA/2% sheep serum in
PBS-Tween 0.1%. Riboprobes against the DNA binding/dimerization domain and
full-length cDNA yielded identical staining patterns.
In situ hybridization on axolotl and lamprey embryos were as described by
Henrique et al. (Henrique et al.,
1995) with the addition of an extra 12 hour wash in MAB-Tween.
Tween-20 concentrations for PBS and MAB solutions were increased to 0.2%.
Proteinase K treatments were also adjusted to 50 µg/ml for 15 minutes for
lamprey embryos and 10 µg/ml for 4 minutes for axolotl embryos.
Hybridization was at 65°C. For lamprey, the riboprobe was generated
against a 500 bp region of the DNA binding/dimerization domain. The axolotl
AP-2 riboprobe was prepared as previously described
(Epperlein et al., 2000
).
Southern blot analysis
Genomic DNA from five adult amphioxus was purified and digested with four
restriction enzymes (ApaI, ClaI, EcoRV and
HindIII). Genomic DNA from a single adult lamprey was isolated and
digested with six restriction enzymes (ApaI, EcoRI,
HindIII, NcoI, PstI and StuI). Digests
were electrophoresed on 0.7% agarose gels and blotted onto GeneScreen Plus
filters (NEN Life Science Products). Homologous 200 bp probes were designed to
intra-exonic regions of the DNA binding and dimerization domains of the
amphioxus and lamprey AP-2 genes. Intron-exon boundaries were deduced from
human AP-2 genomic sequences (Bauer
et al., 1994
) and partial sequencing of amphioxus AP-2 cosmids.
Southern blots were hybridized in 6xSSC/5% SDS/100 µg/ml sheared
herring sperm DNA/5xDenhardt's solution at 60°C to
32P-labeled probes. Washes were in 2xSSC, 0.5% SDS at
55°C.
Plastic sectioning
Embryos were dehydrated in ethanol and embedded in Epon-Araldite. After
polymerization for 72 hours at 60°C, the embryos were sectioned to 10-15
µm using a glass knife, coverslipped in Gelmount and photographed.
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RESULTS |
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Southern blot analysis
Low-stringency Southern blot analysis was used to estimate the number of
AP-2 genes in the amphioxus and lamprey genomes. In both cases, probes were
created that recognized part of the highly conserved DNA-binding domain, but
were likely to be intra-exonic based upon the genomic structure of human and
amphioxus AP-2 genes.
Probing of genomic DNA from a single adult amphioxus revealed two strongly hybridizing fragments when digested with seven out of eight enzymes (data not shown). This raised the possibility that there was more than one AP-2 family member in the amphioxus genome. To test this, we re-probed the cDNA library at low stringency and detected no additional AP-2 cDNAs. Low stringency screening of an arrayed amphioxus genomic library also yielded no new AP-2 gene sequences. We then investigated whether the multiple fragments were due to polymorphism at the AP-2 locus. Genomic DNA from five individual adult amphioxus were digested with four enzymes. All five adults had different restriction fragment length profiles (Fig. 2A). For each enzyme, two to four different fragments were observed in total, with each animal possessing only one or two fragment types per enzyme. Collectively, the results are consistent with various homo- and heterozygotic combinations of several restriction fragment length alleles at a single highly polymorphic locus. Based upon this, and the fact that low-stringency screens of cDNA and genomic libraries consistently yielded a single gene, we conclude that there is a single AP-2 gene in the amphioxus genome.
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Low-stringency Southern blot of genomic DNA from an individual adult lamprey showed a single band in four out of five digests. Probing of lamprey genomic DNA with an amphioxus AP-2 probe yielded no discernable signal above background (data not shown).
AP-2 gene phylogeny
Amphioxus, lamprey, mouse, chicken, frog, human and Drosophila
AP-2 sequences were aligned, and a phylogenetic tree was generated using the
neighbor joining method (Fig.
1B). Axolotl AP-2 was not used for analysis as only a partial
sequence is available. The deduced phylogeny shows amphioxus AP-2 falling
outside of the vertebrate AP-2 clade, which includes lamprey AP-2 and
gnathostome AP-2, ß and
. Within the vertebrate clade,
lamprey AP-2 fails to group with any one gnathostome AP-2 isoform. This
general topology is maintained when the DNA binding/dimerization domain alone
is used for alignment.
Unexpectedly, the recently described mouse AP-2 fails to group with
vertebrate AP-2 proteins when full-length sequences are aligned. When only the
conserved DNA binding/dimerization domains are used for alignment, AP-2
also falls outside of the amphioxus/vertebrate clade (data not shown). Both
phylogenetic positions are poorly supported by low bootstrap values and may
reflect rapid evolution of AP-2
in gnathostomes or early divergence of
AP-2
in the vertebrate lineage.
Pattern of AP-2 expression in amphioxus
Amphioxus development proceeds in a simplified vertebrate-like manner, with
the neural plate forming from dorsal ectoderm at 8-9 hours post-fertilization.
In 9 hour neurulae, AP-2 transcripts are detected throughout the non-neural
ectoderm (Fig. 3A,B). No
expression is seen in the open neural plate or mesendoderm. After the onset of
somitogenesis at 9.5-10 hours, non-neural ectoderm begins closing over the
invaginating neural plate. In 11.5 hour neurulae, AP-2-expressing ectoderm
cells appear to be migrating over the closing neural plate
(Fig. 3C,D,G). Upon hatching at
12 hours, the neurula is covered in ciliated AP-2-positive epidermis.
Neurulation is completed under the epidermis by hour 18. During this period,
AP-2 ectodermal expression begins to recede from the anterior- and
posterior-most ends of the larva (Fig.
3E). At 20 hours, a small spot of staining appears in the anterior
gut, probably presaging formation of the left gut diverticulum. At 24 hours,
this expression sharpens, marking the endodermal portion of the developing
preoral pit (Fig. 3F,H).
Simultaneously, strong staining appears in the ventrolateral walls of the
cerebral vesicle and expression in the epidermis fades
(Fig. 3H,I,L). At 36 hours, the
embryo has elongated to roughly twice its 18-hour length, and the mouth and
first gill slit begin to form (Fig.
3K). Both the cerebral vesicle and pre-oral pit staining become
markedly reduced after this time (Fig.
3J), but persist weakly until 4 days.
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Pattern of AP-2 expression in lamprey
At 4 days, the neural plate of the lamprey embryo is a flattened area of
dorsal ectoderm. At this stage, AP-2 staining is observed in non-neural
ectoderm (Fig. 4A,F). As the
neural plate condenses towards the dorsal midline around day 5, AP-2
transcripts are detected at the edges of the neural plate and broadly in the
adjacent ectoderm (data not shown). AP-2 is downregulated in the non-neural
ectoderm at ventral and lateral levels.
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At six days, AP-2 is expressed solely in the dorsal neural rod (Fig. 4C,G), forming a stripe that is disrupted anteriorly by a gap in expression near the protruding head (Fig. 4B). Although expression in non-neural ectoderm is extinguished in 6-day-old embryos, a new phase of epidermal expression begins at 7 days in the head (Fig. 4D). Scattered AP-2-positive cells appear throughout the head ectoderm, but are conspicuously absent from the otic placode. Also at 7 days, the anterior gap in neural rod expression sharpens, and sections reveal staining in surrounding head mesenchyme highly reminiscent of early migrating neural crest in other vertebrates (Fig. 4H).
At 7.5 days, separations in the head staining become discernable, suggestive of neural crest-free spaces between AP-2 positive streams (Fig. 4E). Horizontal sections reveal the initial outpocketing of first arch endoderm at this time with AP-2 transcripts in the mesenchyme and dorsal neural tube (Fig. 4P). Sections at the level of the otic vesicle show accumulation of AP-2 signal in the space dorsal to the vesicle as well as in the mesenchyme below it, but never medial to the otic vesicle. (Fig. 4I)
At 8 days, divisions in the head staining become more obvious, and three broad areas of AP-2 expression can be distinguished an anterior band and two more caudal swathes straddling the putative otic placode (Fig. 4K). Ventrally, in the region of the nascent pharyngeal arches, the two posterior streams fuse into one continuous mass, while the anterior stream splits into three smaller streams. The rostral-most stream sits just anterior to the area of the optic cup and probably represents the ophthalmic neural crest stream (Fig. 6B). Around the mouth, the rest of the anterior stream forks, marking cells in the mandibular arch and maxillary (anterior lip) region. Horizontal sections at 8.5 days show formation of the first three pharyngeal arches with AP-2 transcripts detected in the ectoderm, superficial to the ectoderm, and adjacent to pharyngeal endoderm (Fig. 4Q). From 8.5-9.0 days, staining in the area of the pharyngeal arches accumulates (Fig. 4L,M). In the trunk, staining in the dorsal neural tube and dorsal fin, and weak staining between the somites are apparent (data not shown).
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At 10-11 days, a new phase of AP-2 neural expression begins in a subset of cells in the anterior neural tube (Fig. 4O). From days 11 to 12, gaps appear in the AP-2-positive arch mesenchyme where the pharyngeal endoderm and ectoderm meet to create the pharyngeal slits (Fig. 4N). Horizontal sections at 12 days show the formed arches, with AP-2 signal present medial and lateral to the pharyngeal mesoderm (Fig. 4R).
Pattern of AP-2 expression in axolotl
To facilitate comparison of lamprey and amphioxus AP-2 gene usage with that
of gnathostomes, a developmental series of axolotl embryos was probed for AP-2
transcripts. AP-2 expression in the axolotl has been described for stages just
preceding and following neural crest migration and was found to mirror that of
mouse, chicken and frog (Epperlein et al.,
2000). Staining patterns in early neurulae, however, have not been
previously described. At open neural plate stages, AP-2 transcripts are
detected in the non-neural ectoderm, and are strongly expressed at the neural
plate border (Fig. 5A, far
right panel). As neurulation proceeds, AP-2 is further upregulated in the
protruding neural folds and downregulated in the non-neural ectoderm. Upon
neural tube closure, AP-2 staining in the dorsal aspect of the neural tube is
maximal, while non-neural ectoderm has only a residual AP-2-positive signal
(data not shown). Little or no staining is apparent in non-neural ectoderm at
later stages.
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DISCUSSION |
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Chordate AP-2 genes
Single representatives of the AP-2 gene family were isolated from amphioxus
and lamprey embryonic cDNA libraries. The presence of a single AP-2 gene in
each genome was suggested by low-stringency genomic Southern blotting and
phylogenetic analyses. This follows with gene numbers in amphioxus where a 1:3
or 1:4 correspondence of amphioxus to gnathostome gene homologs is usually
observed, and supports the proposed scheme of two whole or partial genome
duplications in the vertebrate lineage (for a review, see
Holland, 1999). Limited data
from lamprey indicate a homolog ratio closer to 1:2 when taking into account
lamprey-specific gene duplication events
(Sharman and Holland, 1998
;
Ueki et al., 1998
;
Ogasawara et al., 2000
;
Myojin et al., 2001
;
Neidert et al., 2001
;
Force et al., 2002
). Thus,
there is a chance that another lamprey AP-2 gene exists that was not detected.
Furthermore, phylogenetic analysis leaves open the possibility that lamprey
has an AP-2
, as lamprey AP-2 groups with gnathostome
, ß
and
, but not AP-2
. Whether this is due to rapid evolution of
AP-2
in mammals or early divergence of AP-2
in vertebrates is
unclear. Alternately, a second lamprey AP-2 may have been lost during
evolution or double duplication of an ancestral AP-2 gene occurred after the
divergence of agnathans. Outside of phylum chordata, it is likely that having
one AP-2 gene is the primitive condition for bilateria, as only a single AP-2
gene is found in Drosophila (Bauer
et al., 1998
; Monge and
Mitchell, 1998
). Overall, our data are consistent with the
vertebrate genome double-duplication hypothesis, but are inconclusive as to
the timing of these duplications relative to gnathostome origins.
Early non-neural ectoderm AP-2 expression is ancestral
A striking feature of AP-2 expression common to amphioxus, lamprey and
axolotl, is robust expression in non-neural ectoderm at open neural plate
stages (Fig. 5A). Similar early
ectodermal expression has been reported for chick and mouse
(Shen et al., 1997;
Mitchell et al., 1991
). These
data suggest an ancient role for AP-2 in the chordate non-neural ectoderm, and
strong conservation of an early ectodermal regulatory element in the AP-2
promoter. Interestingly, Dlx and BMP-4 are also co-expressed in the non-neural
ectoderm of gnathostomes and amphioxus
(Panopoulou et al., 1998
;
Holland et al., 1996b
), and a
regulatory relationship between the two has been proposed in frog
(Feledy et al., 1999
). It is
possible that all three genes interact in an evolutionarily ancient pathway
for specification of non-neural ectoderm in chordates.
Neural tube expression differs between amphioxus and vertebrates
Before neural tube formation in vertebrates, AP-2 expression at the
neural/non-neural interface increases
(Mitchell et al., 1991;
Chazaud et al., 1996
;
Moser et al., 1997
;
Shen et al., 1997
).
Simultaneously, expression in the remaining non-neural ectoderm begins to
fade. By the completion of neurulation, AP-2-positive cells have become
incorporated into the dorsal neural tube and epidermal staining is reduced.
AP-2 expression in the neural folds and neural tube at these stages mirrors
that of neural crest markers Snail, Slug and Id-2, and overlaps with the
cranial neural crest markers Dlx-2, Msx-1 and Msx-2
(Robinson and Mahon, 1994
;
Martinsen and Bronner-Fraser,
1998
; Sefton et al.,
1998
; Bendall and Abate-Shen,
2000
).
During parallel stages in amphioxus, the non-neural ectoderm has closed
over the forming neural tube and AP-2 transcripts are detected strongly
throughout the epidermis. After neurulation, AP-2 is downregulated in the
epidermis, but no AP-2-positive cells become incorporated into the dorsal
neural tube. Subsequent AP-2 expression includes only a few cells in the
ventrolateral cerebral vesicle and preoral pit. During these stages, the
amphioxus homologs of Snail and Msx are expressed at the edges of the neural
plate and then expand throughout the neural tube
(Langeland et al., 1997;
Sharman et al., 1999
). Unlike
vertebrates, neither AmphiSnail or AmphiMsx gene expression overlaps with AP-2
in the dorsal neural tube.
Differences in amphioxus and vertebrate AP-2 neural expression imply
divergent modes of AP-2 regulation in the two subphyla. A simplistic
explanation is the presence of a neural crest enhancer in vertebrate AP-2 gene
promoters that is absent in the homologous amphioxus promoter. Candidate
regulators for a putative novel enhancer would include genes such as Snail and
Msx, which are co-expressed with AP-2 in vertebrate neural crest, but not in
the amphioxus neural tube. Provocatively, the murine Msx-1 promoter contains a
consensus AP-2-binding site (Kuzuoka et
al., 1994). Thus, it is possible that new regulatory relationships
between these genes resulted in novel deployment of AP-2 to neural crest early
in vertebrate evolution. An alternative explanation is novel deployment of the
upstream regulators of AP-2 in vertebrates. Furthermore, secondary loss of a
neural enhancer, or differential deployment of trans-acting regulators may
have resulted in a loss of AP-2 expression in the amphioxus dorsal neural
tube. Expression data from the third chordate subphylum, Urochordata, may
clarify the direction of this evolutionary change.
Given its essential role in cranial neural crest cell differentiation and
proliferation, it is tempting to speculate that co-option of AP-2 by the
dorsal neural tube was a crucial event in neural crest evolution. Knockout
studies of AP-2 demonstrate the necessity of AP-2 activity in
post-migratory cranial neural crest
(Morriss-Kay, 1996
;
Schorle et al., 1996
;
Zhang et al., 1996
). Mice that
lack AP-2 have relatively normal neural crest migration, but most neural crest
derivatives in the head (including the rostral-most parts of the skull, the
first and second arch cartilages, and cranial sensory ganglia) are missing or
reduced. Thus, AP-2 expression in the dorsal neural tube may have been a
prerequisite for the evolution of neural crest cells
If AP-2 use in the dorsal neural tube is indeed a vertebrate apomorphy, an intriguing issue is whether its new roles in neural crest involved evolution of the AP-2 protein itself, or simply redeployment of a functionally conserved gene. Although all described AP-2 protein sequences are highly conserved in the DNA-binding/dimerization domains, novel motifs in the more divergent transactivation domain may confer additional regulatory properties onto vertebrate AP-2 genes. In vivo and in vitro comparisons of amphioxus and lamprey AP-2 gene function may shed light on the biochemical features important for AP-2 function in the neural crest.
Later AP-2 expression in amphioxus
Shortly after expression has faded from the epidermis in amphioxus, AP-2 is
upregulated in cells of the ventrolateral cerebral vesicle and forming preoral
pit. In axolotl and lamprey, AP-2 has a potentially homologous late phase of
expression in neurons of the anterior neural tube
(Fig. 5B). AP-2 expression in
the developing cerebellum of mouse also has been reported
(Moser et al., 1997).
Furthermore, AP-2 expression in the developing fly brain is a prominent
feature of Drosophila AP-2 expression
(Monge and Mitchell, 1998
),
suggesting an ancient function for AP-2 genes in the anterior nervous system
of bilaterians.
Enrichment in the developing pre-oral pit is harder to relate to any aspect
of vertebrate expression. It may reflect, however, an evolutionarily conserved
regulatory relationship between AP-2 and Hox2 genes. AmphiHox2 expression
temporally and spatially overlaps with AP-2 in the preoral pit
(Wada et al., 1999), and AP-2
genes are essential for HoxA2 expression in cranial neural crest
(Maconochie et al., 1999
).
Amphioxus AP-2 may similarly regulate Hox2 in the preoral pit, as two
consensus AP-2 binding sites are present in the 5' genomic sequence of
AmphiHox2. AP-2 expression in Drosophila also overlaps with
proboscipedia (Hox2) (Monge and Mitchell,
1998
). Three consensus AP-2 binding sites are found clustered
within a 1 kb intronic region shown to direct reporter expression to the
maxillary lobe. Taken together, these observations suggest an ancient role for
AP-2 in Hox class 2 gene regulation.
AP-2 and neural crest migration patterns in lamprey
Our results show that AP-2 expression in lamprey closely resembles AP-2
expression in axolotl, chicken and mouse. Early deployment in ectoderm is
followed by expression in the neural folds and dorsal neural tube. AP-2
transcripts are then seen throughout the head mesenchyme, in a pattern
consistent with expression in early migrating neural crest. Later, AP-2
staining in the head is confined to apparent streams or blocks of cells.
Finally, lamprey AP-2 appears in mesenchyme surrounding pharyngeal arch
mesoderm. The similarity of lamprey AP-2 and gnathostome AP-2 staining,
together with the anatomical context of lamprey AP-2 expression, strongly
suggests that AP-2 marks neural crest cells in lamprey.
Using AP-2 as a marker gives valuable insight into the migration patterns
of neural crest cells in a basal vertebrate. Previous studies have analyzed
lamprey neural crest migration using scanning electron microscopy, molecular
markers for subsets of crest cells (Otx, Dlx)
(Tomsa and Langeland, 1999;
Neidert et al., 2001
) or
limited DiI labeling (Horigome et al.,
1999
). The current study is the first time expression of a
pan-neural crest marker has been analyzed in lamprey. Comparing lamprey and
axolotl AP-2 expression patterns illustrates that lamprey cranial neural crest
migrates in typical vertebrate fashion. Three broad areas of AP-2 expression
can be discerned in the lamprey head, which appear equivalent to the
trigeminal, hyoid and branchial streams in gnathostomes
(Fig. 6B,E). Furthermore, the
hyoid and branchial streams appear to lie on either side of the otic vesicle,
as in gnathostomes. This contradicts previous scanning electron microscopy
analyses suggesting that lamprey hyoid neural crest migrates directly under
the otic vesicle (Horigome et al.,
1999
). In sections through lamprey embryos, no AP-2-positive cells
are observed interior to the otic vesicle
(Fig. 4I).
Interestingly, during the early stages of neural crest migration (6-7 days), a gap in AP-2 expression appears in the neural tube just anterior to the otic placode (Fig. 6A). In gnathostomes, similar gaps correspond to rhombomeres 3 and 5, which are depleted of neural crest (Fig. 6D). We cannot be sure if this gap corresponds to a rhombomere as no molecular or anatomical rhombomeric markers are available for this stage in lamprey. However, the presence of only one gap is suggestive of reduced patterning in the early migrating neural crest of lamprey.
At later stages, the putative trigeminal stream appears to divide into the ophthalmic stream rostrally, and `maxillomandibular' caudally (Fig. 6B). The maxillo-mandibular then splits around the mouth, filling the maxillary (anterior lip) and mandibular regions. This subdivision mimics streaming patterns in gnathostomes as illustrated by AP-2 staining in axolotl (Fig. 6E) and supports homology of lamprey and gnathostome mandibular segments. This finding, along with recent studies of engrailed and Otx expression, lend molecular support to the idea that gnathostome jaws evolved from the pumping organ of an agnathan ancestor, rather than anterior gill arch cartilage.
Although initial subdivision of putative neural crest cells in lamprey closely mimics that of gnathostomes, later ventral migration into the nascent pharyngeal arches is somewhat different. Conspicuously, coherent streaming of lamprey cranial neural crest is not maintained as the cells move ventrally, and the three streams appear to fuse as they fill the pharyngeal region (compare Fig. 6B with 6E). Subsequent partitioning of pharyngeal arch neural crest appears to occur only after migration as the outpocketing endoderm divides both the paraxial mesoderm and overlying neural crest (Fig. 4N). This difference in streaming pattern may reflect a heterochrony in arch formation relative to neural crest migration between lamprey and gnathostomes.
A long-recognized difference between lamprey and gnathostome cranial neural
crest is its final destination in the arches
(Graham, 2001;
Kimmel et al., 2001
). In
gnathostomes, cartilages derived from cranial neural crest lie medial to the
arch mesoderm. In lamprey, this support tissue lies lateral to the arch
mesoderm. This is reflected by AP-2 staining in axolotl, showing neural crest
cells internal to the arch mesoderm (Fig.
6F). We find that in lamprey, AP-2 transcripts are similarly
distributed internal to the pharyngeal arch mesoderm
(Fig. 6C), suggesting medial
movement of pharyngeal arch neural crest does indeed occur in lamprey,
although to a lesser degree than in gnathostomes.
Conclusions
In this study, we have documented differences in AP-2 regulation across the
evolutionary transitions from invertebrate to vertebrate and agnathan to
gnathostome. AP-2 expression in amphioxus and vertebrates implies co-option of
AP-2 by neural crest cells in the vertebrate lineage. This was a potentially
crucial event in vertebrate evolution, as AP-2 has essential roles in cranial
neural crest differentiation and proliferation. AP-2 deployment in the neural
tube may have potentiated neural crest evolution by promoting transcription of
downstream effectors of cranial neural crest differentiation. AP-2 expression
patterns in lamprey and axolotl demonstrate an increase in neural crest
patterning in gnathostomes, and elaboration of neural crest migratory behavior
that may relate to the timing of pharyngeal arch formation. As in situ
hybridization is merely a series of static observations of the use of a single
gene, conclusive proof of these differences await the results of detailed cell
tracking experiments. Taken together, the regulatory history of AP-2 genes in
the chordate lineage suggest molecular and developmental mechanisms for the
evolution of the vertebrate head.
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ACKNOWLEDGMENTS |
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