1723
Development 124, 1723-1732 (1997)
Printed in Great Britain © The Company of Biologists Limited 1997
DEV1168
Sequence and embryonic expression of the amphioxus engrailed gene
(AmphiEn): the metameric pattern of transcription resembles that of its
segment-polarity homolog in Drosophila
Linda Z. Holland1,*, Mamata Kene2, Nic A. Williams3 and Nicholas D. Holland1
1Marine Biology Research Division, Scripps Institution of Oceanography, La Jolla, CA 92093-0202, USA
2Department of Biology, University of Southern California, Los Angeles, CA 90089-0371, USA
3Department of Animal and Microbial Sciences, University of Reading, Whiteknights, Reading RG6 2AJ,
UK
*Author for correspondence (e-mail: [email protected])
SUMMARY
Vertebrate segmentation has been proposed as an evolutionary inheritance either from some metameric protostome or from a more closely related deuterostome. To
address this question, we studied the developmental
expression of AmphiEn, the engrailed gene of amphioxus,
the closest living invertebrate relative of the vertebrates. In
neurula embryos of amphioxus, AmphiEn is expressed
along the anteroposterior axis as metameric stripes, each
located in the posterior part of a nascent or newly formed
segment. This pattern resembles the expression stripes of
the segment-polarity gene engrailed, which has a key role
in establishing and maintaining the metameres in embryos
of Drosophila and other metameric protostomes. Later,
amphioxus embryos express AmphiEn in non-metameric
patterns — transiently in the embryonic ectoderm and
dorsal nerve cord. Nerve cord expression occurs in a few
cells approximately midway along the rostrocaudal axis
and also in a conspicuous group of anterior cells in the
cerebral vesicle at a level previously identified as corresponding to the vertebrate diencephalon. Compared to vertebrate engrailed expression at the midbrain/hindbrain
boundary, AmphiEn expression in the cerebral vesicle is
relatively late. Thus, it is uncertain whether the cerebral
vesicle expression marks the rostral end of the amphioxus
hindbrain; if it does, then amphioxus may have little or no
homolog of the vertebrate midbrain. The segmental
expression of AmphiEn in forming somites suggests that the
functions of engrailed homologs in establishing and maintaining a metameric body plan may have arisen only once
during animal evolution. If so, the protostomes and
deuterostomes probably shared a common segmented
ancestor.
INTRODUCTION
metameres, are developmental compartments. However,
arguments for such a common segmented ancestor appeared
undermined by the balance of the molecular genetic evidence.
For example, Hox gene homologs are expressed along the rostrocaudal axis of non-metameric animals like Caenorhabditis
(Dickinson, 1995). Moreover, insect metamerism is established
via pair-rule and segment-polarity genes, whereas vertebrate
homologs of such genes are expressed in metameres only after
these structures have become well defined morphologically
(Patel, 1994; De Robertis and Sasai, 1996). Even so, the
question of a common segmented ancestor for vertebrates and
insects has not been laid to rest. Müller et al. (1996a) reported
that her-1, a zebrafish homolog of hairy, a Drosophila pair-rule
gene, is expressed in a pair-rule pattern in posterior mesoderm
preceding any morphological differentiation of somites. This
intriguing result led Kimmel (1996) to suggest anew that insects
and vertebrates shared a common, metameric ancestor. Should
this be so, the question is: why are most vertebrate homologs
of Drosophila pair-rule and segment-polarity genes apparently
not involved in establishing vertebrate segmentation?
The evolutionary origin of rostrocaudal segmentation in vertebrates is a central question in schemes deriving vertebrates
from invertebrates. Some authors (e.g. Leydig, 1864; Dohrn,
1875; Patten, 1912) speculated that vertebrate segmentation is
inherited from some protostome, often an arthropod or annelid.
In contrast, others (e.g. Bateson, 1886; Garstang, 1928;
Jefferies, 1986) argued that vertebrate segmentation originated
in some deuterostome group, a scheme that connotes independent origins for vertebrate and protostome segmentation.
Advances in molecular biology have strongly impacted ideas
about the origin of the vertebrates in general and of vertebrate
metamery in particular (Gee, 1996). The evolution of insects
and vertebrates from a common segmented ancestor was
proposed following landmark discoveries in developmental
genetics: first by McGinnis et al. (1984) after the discovery of
conserved homeoboxes in Drosophila homeotic genes and vertebrate Hox genes, and again by Lawrence (1990) after the
demonstration that vertebrate rhombomeres, like insect
Key words: amphioxus, engrailed, segmentation, metamery,
AmphiEn, Drosophila
1724 L. Z. Holland and others
To address this question, we are studying amphioxus
(phylum Chordata; subphylum Cephalochordata), widely
believed to be the closest living invertebrate relative of the vertebrates (Wada and Satoh, 1994) and a useful stand-in for the
proximate invertebrate ancestor of the vertebrates. The
genomic organization of amphioxus is vertebrate-like, but
simpler since amphioxus lacks the extensive gene duplications
correlated with the marked increase in vertebrate structural
complexity (Garcia-Fernàndez and Holland, 1994; Holland et
al., 1994b). In addition, because the overall body plan of
amphioxus is also vertebrate-like, the expression domains of
developmental genes can help indicate homologies between
body parts (Holland, 1996). For example, expression domains
of amphioxus homologs of Pax-1, Brachyury, Distal-less, Otx,
Hox-1, and Hox-3 support homologies of amphioxus structures
with vertebrate gill slits, notochord, forebrain and hindbrain
(Holland et al., 1992; Holland et al., 1994a; N. D. Holland et
al., 1995; P. W. H. Holland et al., 1995; Holland and Holland,
1996; N. D. Holland et al., 1996; Williams and Holland, 1996).
The present paper concerns the engrailed gene of
amphioxus. Homologs of engrailed are widespread in animals
generally, constitute a distinctive class of homeobox genes
encoding DNA-binding transcription factors and are expressed
at several developmental stages in diverse tissues in all three
germ layers. engrailed genes have been most studied in their
earliest expression domains — the midbrain/hindbrain junction
of vertebrates (Ekker et al., 1992; Danielian and McMahon,
1996) and the forming segments of insects (Kornberg and
Tabata, 1993) — as well as in one later expression domain, the
forming wing of insects (Hidalgo, 1996). Biochemical and
genetic studies of engrailed have concerned upstream
promoters of the gene (Logan et al., 1993; Song et al., 1996)
or modulators and downstream targets of the gene product
(Mann, 1994; Serrano et al., 1995; Peltenburg and Murre,
1996), which is typically a repressor of activated transcription
(Jaynes and O’Farrell, 1991; Han and Manley, 1993).
Suggested functions for engrailed include roles in neurogenesis, axon targeting, establishment/maintenance of compartment
boundaries, cell cycle control and cytodifferentiation (Logan et
al., 1993; Condron et al., 1994; Friedman and O’Leary, 1996;
Hidalgo, 1996; Loomis et al., 1996; Rétaux et al., 1996).
During Drosophila development, the earliest expression of
engrailed is in a banded pattern in the ectoderm (and more transiently in the early mesoderm), where it acts as a segmentpolarity gene establishing and maintaining the metameres; later
expression is in a subset of neural cells and in a few other
tissues (Lawrence, 1992). In contrast, during vertebrate development, the earliest expression of engrailed homologs is not
banded, but occurs in a single broad stripe in the neuroepithelium at the midbrain/hindbrain boundary (Patel et al., 1989b;
Davis et al., 1991). Later in development, vertebrate engrailed
genes are transcribed in widely distributed neural cells as well
as in several other tissues. Some of these later expression
domains of vertebrate engrailed may appear in an iterated
pattern (e.g. the muscle pioneer cells), but only in structures
that have already become morphologically segmented (Hatta
et al., 1991; Ekker et al., 1992). Thus, comparisons of
engrailed expression between vertebrates and other animals
have been consistent with an independent origin of segmentation in protostomes and deuterostomes. In contrast to the vertebrate pattern, our present results show that amphioxus
engrailed has an early developmental expression in the forming
segments and a later expression in the nervous system and
ectoderm. The early transcription is in reiterated stripes along
the rostrocaudal axis; each stripe is in the posterior part of a
nascent or newly formed segment in a pattern resembling the
expression domains of engrailed homologs of metameric protostomes (e.g. annelids and arthropods). This pattern is consistent with suggestions that protostomes and deuterostomes
evolved from a common segmented ancestor.
MATERIALS AND METHODS
Obtaining amphioxus and purification of DNA and RNA
Adults of the Florida amphioxus (Branchiostoma floridae) were
collected in Tampa Bay, Florida. Ripe males and females were
spawned and the embryos were cultured at 23°C as previously
described (Holland and Holland, 1993). Genomic DNA for library
construction and Southern blotting was extracted from 25 adults in
GuSCN and purified by ultracentrifugation (L. Z. Holland et al.,
1996). For cDNA library construction, RNA was extracted from
embryos by the method of Chomczynski and Sacchi (1987).
DNA probes; construction and screening of libraries
The polymerase chain reaction (PCR) was used to amplify a 233 base
pair (bp) fragment (including the homeobox) from pooled genomic
DNA of Branchiostoma floridae. The forward primer B and reverse
primer D were, respectively: GA(A/C)AAGCGGCCGCGCAC(A/G)GCCTTC and TGGTTGTCAG(A/C/G/T)CCCTG(A/C/G/T)GCCATGAG (Holland and Williams, 1990). The PCR product was cloned
into pUC18 and subcloned into pT7T3 (Pharmacia, Piscataway, NJ,
USA). Eight clones were sequenced.
For genomic library construction, DNA digested with Sau3A was
cloned into the XhoI site of Lambda Fix II (Stratagene, La Jolla, CA,
USA). About 250,000 clones of the once-amplified library were
screened with the insert of one PCR clone labeled with 32P by random
priming. Hybridization was at 65°C in 6× SSC, 10× Denhardt’s, 0.1%
SDS, 100 µg/ml tRNA, 1 mM EDTA with 106 cts/minute/ml of probe
(specific activity of 108 cts/minute/µg). After hybridization, plaquelifts were washed 3× 20 minutes in 1× SSC, 0.1% SDS at 60°C.
A cDNA library was constructed in the pSPORT vector (GIBCOBRL, Gaithersburg, MD, USA) from RNA pooled from 26-hour
embryos. Approximately 60,000 unamplified clones were screened
with a SalI-Eco0109 fragment (862 bp) of a genomic clone; the
fragment, comprising the coding region 3′ of the intron (including the
homeobox) and the most 5′ two-thirds of the 3′ untranslated region
(UTR), was labeled with 32P by random priming. Colony lifts were
hybridized in 6.95% SDS, 1 mM EDTA, 0.1 mg/ml tRNA, 0.5 M
sodium phosphate buffer (pH 7.2) at 65°C with 106 cts/minute/ml of
32P-labeled probe and washed as already described for the genomic
screening.
Southern blot analysis
Fifteen 10 µg aliquots of genomic DNA were each digested with a
different restriction enzyme, subjected to electrophoresis and transferred to Hybond N+ (Amersham Life Sciences, Cleveland, OH, USA)
according to L. Z. Holland et al. (1995). The Southern blot was
hybridized at low stringency (50°C) with the same probe used for
screening the CDNA library and in the same hybridization solution
for genomic library screening. Washes were 2× 20 minutes at 55°C
in 2× SSC, 0.1% SDS (L. Z. Holland et al., 1995).
In situ hybridization and histology
Expression of amphioxus engrailed was determined by in situ hybridizations on embryos of Branchiostoma floridae fixed at intervals
Amphioxus engrailed gene 1725
during the first 2 days of development (L. Z. Holland et al., 1996).
Fertilization envelopes were removed from prehatching stages to
facilitate penetration of reagents. Two antisense riboprobes were
combined to maximize the signal. A 3′ probe was synthesized with a
SalI genomic subclone as a template and a 5′ probe was synthesized
from a partial cDNA clone (see results). After being photographed as
whole mounts, labeled embryos were prepared as histological
sections. In addition, some embryos were hybridized with riboprobes
for both engrailed and for the amphioxus Distal-less homolog (N. D.
Holland et al., 1996).
RESULTS
Sequence analysis
Amplification by PCR of the engrailed homeobox of Branchiostoma floridae yielded two types of clones with identical
deduced amino acid sequences, but somewhat different base
sequences. Screening a B. floridae genomic library with one of
the PCR clones yielded 12 strongly hybridizing clones.
Although no two clones gave exactly the same restriction
pattern when mapped with 6 different restriction enzymes,
sequencing with a primer to the homeobox revealed that all
were engrailed. More extensive sequencing of three clones
showed that 3′ of the intron splice site, all had the same
deduced amino acid sequence, although the base sequences
differed somewhat (approximately one out of 100 positions in
the coding region and one out of 50 in the 3′ UTR and intron).
In addition, there were short (up to 3 bp) deletions and insertions in the 3′ UTRs and longer (up to 20 bp) insertions and
deletions in the intron. The base sequences of the two most
similar genomic clones corresponded to the two types of clones
obtained by PCR. Sequencing of one genomic clone revealed
that the intron exceeded 4,000 bp. Therefore, to obtain the
coding region 5′ of the intron plus the 5′ UTR, we screened a
cDNA library with part of one genomic clone. One cDNA
clone was obtained — a partial clone including the entire
cDNA 5′ of the intron splice site plus 72 bp 3′ of the splice
site. Since the base sequence of the cDNA in the overlap zone
1
62
143
AACCTAGTTTCCTACAATCACTGTCAGGGAGAGGGGAAACACTGAGCTAAGTGACTCAAGC
GCTCCGACCCCAGGGCAGTTGTTTATAGCCGGCGAACTCTGAGCACCGGGAGATTATAAGTAGGAGTAATAGTGACTGACA
TTTTGGATTCGCGTGCGTCTTCGGTCCTTTCAATATAGGGTCGAAGTTCTTGGAGACAGAAGCGAGTAGGAGGACCGTAAG
61
142
223
224
1
ATGGCGAACAGTAGCGCGGATGAGAACGGGAACCCACCGGCGAGCCCGCCGCCTCCTCACAGCCCGGTCCAACCGGAGGAC
M A N S S A D E N G N P P A S P P P P H S P V Q P E D
304
27
305
28
AGTCCGAGCCCTGACGGCCCGCGCACCACCAACTTTTCCATCGCGAACATCCTCCGGCCGGAGTTCGGCGCTCGCAAGAGA
S P S P D G P R T T N F S I A N I L R P E F G A R K R
385
54
386
55
GACAGTAAAAGTTGCGACTCCTCTCCCGTGTCCAGTCCGGGGAAACCGGCACCGGGAGACCTCTCCCCCTCCCTGCCGGCC
D S K S C D S S P V S S P G K P A P G D L S P S L P A
466
81
467
82
AGCCCGGGCGGAGTCCCGCAGGAGAACACGACGACAGCCGATGGACTCCGCCTCGAGGATGACCCCGCCAAGCCCGTTACG
S P G G V P Q E N T T T A D G L R L E D D P A K P V T
547
108
548
109
EH2
GCAGACGATAAGGACGGTAAACCGGTGCCCCAGCCTATGATCTGGCCAGCGTGGGTCTACTGTACCCGCTACTCGGACCGC
A D D K D G K P V P Q P M I W P A W V Y C T R Y S D R
628
135
629
136
EH3
CCTTCTTCTGTTCGCACCGCAGGACCCCGAACGCGGAAAGCTCGGCCCAAAGATCCGAAGAAAGCCGAGGAGAAGCGGCCG
P S S V R T A G P R T R K A R P K D P K K A E E K R P
709
162
710
163
EH4 = homeobox
AGAACGGCGTTCACTTCCGAACAGCTCCAGCGCCTGAAAAAGGAGTTCCAAGAGAACCGTTACCTCACAGAGCAACGGCGG
R T A F T S E Q L Q R L K K E F Q E N R Y L T E Q R R
790
189
791
190
EH4 = homeobox
CAGGATTTGGCAAGAGAGCTGAAGCTAAACGAGTCACAGATCAAGATTTGGTTTCAGAATAAGCGTGCGAAAATCAAGAAG
Q D L A R E L K L N E S Q I K I W F Q N K R A K I K K
871
216
872
217
EH5
GCGGCTGGAGTACGGAACGGACTAGCACTCCACCTCATGGCGCAGGGGCTCTACAACCATTCCACCATGCCGACAATGGGA
A A G V R N G L A L H L M A Q G L Y N H S T M P T M G
952
243
953
244
GACGAGCACGGGCTCGATATGCATGACTAGTGCGGCAAACAAACCTCGGCGGCGAACCGGCCCGTGAAATTGTGGACAGCC 1033
D E H G L D M H D
252
1034
GGACATCAGAAACATCTTACGGACAAGCATTGTCTGGACATATCGTCTAACTGACCAACTGAATCTCAGAGCTATGTTCCT 1114
1115
1196
1277
1358
1439
1520
TGTTTCAATACAAAAAAGCATCAAGTAAGCTTAATGGTTTAGAAGGTAAGGTCTCAAAATTTTTGCTAGTGTCTTAATTTA
TTGTGACGATATTTTTGGCAACAGAAAAGGGTAGATTCTTTTGCGGACGAAAAAAGAATAATGAATGGACAGTACAAAAGA
AGGAATCGTTTGATCAGTCTACCTTGATATATTTACATATCCATAGCGGACAGAAGAGAGATATTATTTTGTCAGTCAGTC
TCTGAATTCTGTGATCTTTTGACAAGTGAAATTTGTGTTTTTCTGTTCGTGTGTCTATAGAATGAAATAGTTGTAAATACC
CGTACAATTGCACGTCGTTGTCACCGTGCGAGCAAGTTAGTCAATATCTCTTTTTTCTCTTCTTTATCTCTTTTTTTGTTG
TAGTCGACGGATCGATAAGCTTATATCTAAAGGGAGTCGACTCGATCCTCTATCTGTTCGAATTCCTGCAGCCCGGGGGAT
1601
1682
CCACTGAGTTCTTCTAGAGCGGCCGCCACCGGTGAGCTCAATTAACCCTCATGTATGGGGGCTCAAAAGTGACAATGGAAG 1681
1760
TCAATTCTATCATAACGCTGTTATCTGTTACTGACCGTCCAGATTTAATAAACCAAACCGTAATATATTCAATAAAAAA
EH1
Fig. 1. Base and deduced amino
acid sequences of amphioxus
AmphiEn (GenBank accession
number U82487). Composite base
sequence from genomic (3′ of the
intron) and cDNA clones (5′ of
intron). Location of poly(A) tail is
assumed to coincide with
consensus (t)5 21 bp downstream
of the polyadenylation signal
(underlined) in genomic clone. Inframe stop codons upstream from
translational start site also
underlined. Boxed regions (EH1,
EH2, EH3, EH4 = homeobox, and
EH5) are highly conserved among
most metazoan engrailed
homologs. Open arrowhead, most
5′ possible site for intron; closed
arrowhead, nearest consensus
sequence for 3′ intron splice site.
1195
1276
1357
1438
1519
1600
1726 L. Z. Holland and others
Fig. 2. Amino acid sequences
of amphioxus AmphiEn and
engrailed proteins of some
other animals. Alignment with
Clustal V; amino acid identities
indicated by colons; other
notations as in Fig. 1. Zebrafish
(zf) sequences (Ekker et
al.,1992); mouse (mo)
sequences (Logan et al., 1992);
Drosophila sequence (Poole et
al., 1985); brine shrimp
(Artemia) sequence
(Manzanares et al., 1993);
flatworm (Schistosoma)
sequence (Webster and
Mansour, 1992).
AmphiEn
zfEn-1
zfEn-2
zfEn-3
mo En-1
mo En-2
Drosoph
Artemia
fluke
MANSSADENG----------------------------------------------------------------NPPASPPPPH
MEDQRRGQGEEEDDSGS-------------------------------------------------------LPS::LLPA--MDENEQSARDVEQRGASDESNSAIRPL--------------------------------------------------------MEENDHSNRDVERQDSGDESNRAILPL--------------------------------------------------------MEEQQPEPKSQRDSGLGAVAAAAPSGLSLSLSPGASGSSGSDGDSVPVSPQPAPPSPPAAPCLPPLAHHPHLPPH::PP::::P
MEEKDSKP--------SETAAEAQRQ------PEPSSGGGSGGGSSPSDSDTGRRRALMLP----------------------M:LEDRCSPQSAPSPITLQMQHLHHQQQQQQQQQQQMQHLHQLQQ...79 aa...DMSFHNQTHTTNEEEEAEEDDDIDVDVD
MGSAIFEPGPLSLLNLACSNLTERYDGPSPLSASTPGPSPDRPGS...40 aa...TGFPTLAAIQSGHLAFRQLV:TL:FNTMFKLLDNF:EKSKSIMIEQMKQQYCLLQYDKE...78 aa...TTTTTSTTTTTSGIIPLSSTIIPSTIISPVKTSLSSLSMMT
EH1
AmphiEn
zfEn-1
zfEn-2
zfEn-3
mo En-1
mo En-2
Drosoph
Artemia
fluke
SPVQPEDSPSPDG------PRTTNFSIANILRPEFGARK--------RDSKSC---------------DSSPVSSPGKPAPGDL
-------------------H:N:D:F:D:::::D::CKRERE--KVT:::GVRPTALPDSRSDGVSSSA::T::::VSSR--------LQA:GNLQLP----H:I:::F:D:::::D::RK:EANIT:YED--------------------------------NHGA
-----LQA:GNV-LP----H:I:::Y:D:::::D::R::EGS-R:DEI--------------------------------NIVE
P:P:HLAA:AHQPQPAAQLH:::::F:D:::::D::CK:EQPLPQLLVASAAAGGGAAAGGGSRVERDRGQTGAGRDPVHSLGT
---EVLQA:GNHQHP----H:I:::F:D::::::::R::DAG-----TCCAGAGGARGGEGGAGTTEGGGGGAGGAEQL--LGA
DTSAGGRLPPPAHQQQSTAKPSLA:::S:::SDR::DVQKPGKSIENQASIFRPF...87 aa...KSALGSLCKAVSQIGQPA
VKSSEGQVKEVVSTQ--SQKKPLA:::DS::::D::K-------------------------ETNEVKRRHASPHREEPKKK-IDDLQNDISKINYEEDFFSKLLKCTL:R:ENSYSNEIEQLSLSSSSSSS:SSSSSSSSSSCSTNSSSSVYMSEKKANGFFVKDI
AmphiEn
zfEn-1
zfEn-2
zfEn-3
mo En-1
mo En-2
Drosoph
Artemia
fluke
-----------------------SPSLPASPGGVPQENTTTADGLRLEDDPAKPVTADDKDG-----------KP--------QSNKVEQGSSKSSSPS-------------------------------------------------------------------RENHNPT---------GPSTG-----QVGSTVPAEEAS::HTSSGGK:AEIES----------------EEPL::RGENVDQ-RENRCPS---------APGSG-----QV:P-VSGEGTSSPRAVNASKKT:IST----------------DESL:SRAETGDQ-RASGAASLLCAPDANCGPPDGSQPATAVGAGASKAGNPAAAAAAAAAAAAA:VAAA:AAASKPSDGGGSGGNAGSPGAQGAKFP
RESR-------PNPACAPSAG-------GTLSAAAGDPAVDGE:GSKTLSLH----GGAKKPGDPGGSLDGVL:ARGLGGGDLS
APTMTQPPLSSSASSLASPPPASNASTISSTSSVATSSSSSSSGCSSAASSLNSSPSSRLGASGSGVNASSPQPQPIPPPSAVS
VQYIEQMKKKEEIKEEARTESRL:SSSKD:VPDNDKI:P--------------------------------------------LSFDKHKVIRRQKTDDSFEKEVLVKGRDEEKK...52 aa...VGSEEEEDNDDIN:AAKNNNTNYLRKTVSDDGMKLKSNRNH
Amphien
zfEn-1
zfEn-2
zfEn-3
Mo En-1
Mo En-2
Drosoph
Artemia
fluke
---------------------VPQPMIWPAWVYCTRYSDRPSSVRTAG--PRTRKARPKDPKKA-EEKRPRTAFTSEQLQRLKK
-------------------KDSQKQIL::::::::::::::::----:--:::::LKK:NNNTESDD::::::::A:::::::A
-----CLGSESDSSQSNSNGQTG:G:L::::::::::::::::----:--::S::PKK:A--ASK:D::::::::A:::::::A
-----CLSSDSDCSQRC-AAQAK:::L::::::::::::::::----:--::S::PKK:T--PTK:D::::::::A:::::::N
EHNPAILLMGSANGGPVVKTDSQ::LV::::::::::::::::----:--:::::LKK:K--NEK:D::::::::A:::::::A
-------VSSDSDSSQASATLGA:::L::::::::::::::::----:--::S::PKK:N--PNK:D::::::::A:::::::A
RDSGMESSDDTRSETGSTTTEGGKNEM::::::::::::::::----:--::Y:RPK-QPKDKTND::::::::S::::A:::R
-------------------PLP:EASK:::::F::::::::::----:RS::C:RMKK:KAITP-D:::::::::A:::S:::H
NKKLGSRGRIHEITSSKLNTND:SRLHL::::F::::::::::----:--::I::P:MNRSNDELNL:::::S::VP::K::SQ
AmphiEn
zfEn-1
zfEn-2
zfEn-3
mo En-1
mo En-2
Drosoph
Artemia
fluke
EFQENRYLTEQRRQDLARELKLNESQIKIWFQNKRAKIKKAAGVRNGLALHLMAQGLYNHSTMPTMGDEHG--------LDMHD
:::TS::I::::::A:::::G:::::::::::::::::::SS:FK:A::MQ:::::::::::TTIQEE:D-------------N
:::T::::::::::S::Q::G::::::::::::::::::::S::K::::I::::::::::::TSKEDKSDS------------H
:::N::::::::::A::Q::G::::::::::::::::::::T:NK:T::V::::::::::A:VTKDDKSDS------------:
:::A:::I::::::T::Q::S::::::::::::::::::::T:IK:::::::::::::::::TTVQDKDES------------E
:::T::::::::::S::Q::S::::::::::::::::::::T:NK:T::V::::::::::::TAKE:KSDS------------E
::N:::::::R:::Q:SS::G:::A:::::::::::::::ST:SK:P:::Q:::::::::T:V:LTKE:EE-LEMRMNGQ--IP
::N:::::::R:::::::::G:H:N::::::::N:::L::SS:QK:P:::Q:::::::::::I::ED:EDD-EISSTSLQARIE
::EK::::D:L::KK::T::D:R:::V::::::::::T:::S:AQ:C:::::::E::::::VRVRSDI:EDEEDSDDMNTSEKE
EH2
EH4 = homeodomain
matched most closely the base sequence of the genomic clone
used to screen the cDNA library, we chose the latter clone to
make a composite cDNA sequence of the amphioxus engrailed
gene, which we named AmphiEn.
Fig. 1 shows the complete cDNA and deduced amino acid
sequences of AmphiEn. The cDNA is 1,760 bp long with a
polyadenylation signal 21 bp upstream from the poly(A) tail.
There are several in-frame stop codons upstream of the
indicated translational start site. The open triangle in Fig. 1
shows where the base sequences of the cDNA (CCCTTCT↓TCTGTTC) and genomic DNA clones (CCGATGC↓TCTGTTC) diverge. This position is, therefore, the most 5′ possible
for the intron. However, the base sequence at this location does
not correspond to the consensus sequence for the 3′ intron
splice site (TXCAG↓), the nearest CAG being 14 bp downstream at a site corresponding to an intron in engrailed genes
in other species (Figs 1, 2, closed arrowhead). If the 3′ intron
splice site in AmphiEn is at this consensus sequence, then an
unusually large number of bases (16) must be repeated at the
3′ end of the preceding exon. Although the available data do
not rule out the presence of an additional, more 5′ intron, such
a second intron is unlikely, because more 5′ introns are lacking
in engrailed genes of other animals.
The longest open reading frame codes for a protein of 252
amino acids (Fig. 1) that includes a homeodomain (EH4) of
EH3
EH5
the engrailed class as well as four additional conserved regions,
EH1, EH2, EH3 and EH5 (Logan et al., 1992) (Fig. 2). In
Drosophila, EH1 and EH5 are required for full repression
activity, and EH2-EH3 influence targeting of engrailed (Peltenberg and Murre, 1996; Smith and Jaynes, 1996). AmphiEn
contains an insert of four amino acids in EH2, VRTA (Fig. 2),
which, being located in EH2, might influence targeting of the
protein. AmphiEn, like mouse En-1, includes a proline-rich
region upstream from EH1 that has been suggested as a
potential activation domain (Logan et al., 1992).
Southern blot analysis
Fig. 3 shows a Southern blot hybridized at low stringency with
an 862 bp stretch of AmphiEn comprising the coding region 3′
of the intron (including the homeobox and regions EH3 and
EH5) plus the most 5′ two-thirds of the 3′ untranslated region.
Digestion by six out of 12 restriction enzymes yielded a single
major hybridization band. This result, together with the results
from sequencing, suggests that AmphiEn is the sole engrailed
gene in Branchiostoma floridae. The two bands from digestion
by KpnI and SpeI, as well as the faint band at 6 kb from SmaI
are presumably due to polymorphism.
Developmental expression of AmphiEn
No AmphiEn transcripts are detectable in the blastula or gastrula
Amphioxus engrailed gene 1727
Fig. 3. Genomic Southern blot analysis of pooled amphioxus DNA.
Numbers at top indicate digestion in: 1, BglII; 2, BstEII; 3, BstXI; 4,
Eco0109I; 5, EcoRI; 6, HindIII; 7, KpnI; 8, NcoI; 9, PstI; 10, SalI;
11, SmaI; 12, SpeI. Blot probed at low stringency with 862 bp
fragment of AmphiEn including homeobox. Size markers in
kilobases at left.
only in the dorsal nervous system. Expression continues in the
two small groups of neural tube cells (Fig. 4J,M, arrowheads)
midway along the rostrocaudal axis just caudal of the first
pigment spot. Within the anterior end of the neural tube, the
medullary canal has dilated slightly, thus defining a region
called the cerebral vesicle. There is a conspicuous zone of
AmphiEn expression in the wall of the cerebral vesicle about
50 µm caudal of the neuropore (Fig. 4J,K). Histological cross
sections show that the expressing cells are concentrated ventrolaterally on either side of the cerebral vesicle (Fig. 4L). In
24-hour embryos hybridized simultaneously with riboprobes
recognizing AmphiEn and amphioxus Distal-less (N. D.
Holland et al., 1996), the zone of AmphiEn expression is offset
slightly rostrally from the strong zone of dorsal expression of
the Distal-less gene (Fig. 4N). By 30 hours of development,
AmphiEn transcripts are no longer detectable anywhere in the
embryo by whole-mount in situ hybridization.
DISCUSSION
stages. Expression is first seen in early neurulae at 9.5 hours
(Fig. 4A), as paraxial mesoderm evaginates from the archenteron to form presomitic grooves on either side of the midline.
Anteriorly, the first myogenic somite (numbering system of
Conklin, 1932) has been formed by a constriction of the wall
of the groove, and the second somite has partly formed.
AmphiEn is expressed in three stripes — the most anterior in
cells constituting the posterior third of the first somite, the next
in posterior cells of the second somite as it is forming and the
most posterior in the wall of the presomitic groove at a rostrocaudal level where the posterior part of the third somite will be
located after constricting off from the groove. Significantly, at
this last site, AmphiEn expression precedes any morphological
sign of a posterior boundary for the third somite.
The pinching-off of definitive somites from the presomitic
groove progresses in a caudal direction. Just before constriction produces a new somite, expression of AmphiEn begins in
the future posterior cells of the somite. By hatching (11 hours),
there are three or four definitive somites on either side (Fig.
1B,C). The posterior region of each somite expresses AmphiEn
with an intensity progressively decreasing from rostral to
caudal. By 12 hours, AmphiEn is expressed in the posterior
portion of each of the first six somites on either side of the
midline (Fig. 4D,E) and, by 13.5 hours, AmphiEn is expressed
posteriorly in the first eight somites on either side (Fig. 4F).
At later developmental times, the subsequently forming
somites never express AmphiEn. Further, in the late (15-hour)
neurula, as the ninth somite begins to form, expression in the
first eight somites diminishes rapidly, although it lingers
longest in the posterior part of the first somite on the right side
(Fig. 4G,H, arrow). At 15 hours, expression of AmphiEn begins
in two small groups of cells (one to three cells each) located
in the ventrolateral wall of the neural tube about midway along
the rostrocaudal axis (Fig. 4G,H, arrowhead). By 17 hours,
AmphiEn expression is no longer detectable in the right first
somite; however, transcripts are still found in a few neural tube
cells (out of the plane of focus in Fig. 4I) midway along the
rostrocaudal axis, and weak, transient expression appears in a
band of epidermal cells approximately in register with the
second somite (Fig. 4I, arrow).
In the 24-hour embryo, AmphiEn expression is detectable
Sequence comparisons between AmphiEn and
engrailed of other animals
In the genome of Branchiostoma floridae, AmphiEn is
evidently the only member of the engrailed family. A single
engrailed gene appears to be a plesiomorphic feature of
animals generally (reviewed by Wray et al., 1995); the known
exceptions are higher insects (two engrailed genes), a cephalopod mollusc (two engrailed genes) and most vertebrates (two
or sometimes three engrailed genes). The gene duplications
almost certainly arose independently in the insects, molluscs
and vertebrates, and the presence of a single engrailed gene in
amphioxus, the most likely sister group of the vertebrates, is
consistent with this conclusion.
A comparison of the amino acid sequences of engrailed
proteins among the animals in Fig. 2 shows that AmphiEn
shares the most identities with vertebrate engrailed proteins,
somewhat fewer with arthropod engrailed and considerably
fewer with flatworm engrailed. AmphiEn is probably a fair representative of the single engrailed protein present in the earliest
vertebrates before gene duplications took place. However,
AmphiEn is not obviously related more closely to any one vertebrate homolog (En-1, En-2 or En-3) than to any other.
In spite of structural similarities among engrailed proteins
of different animals (Fig. 2), AmphiEn-containing cells in
amphioxus were not labeled by antibodies against engrailed
proteins of foreign species. The monoclonal antibody against
Drosophila engrailed (4D9) evidently fails to recognize
AmphiEn, because recognition requires a glycine at homeodomain position 40, where AmphiEn has a lysine. Polyclonal
antibodies raised against mouse (αEnhb-1) and leech (αht-en)
engrailed proteins did recognize proteins in some neural tube
cells, but in a pattern unrelated to cells transcribing AmphiEn.
Moreover, each polyclonal recognized a distinctly different
subpopulation of cells.
Neural expression compared between AmphiEn and
engrailed homologs of other animals
The most straightforward comparison of expression of
engrailed homologs between amphioxus and vertebrates is the
relatively late transcription observed in scattered cells in the
central nervous system. In amphioxus, transcripts occur in rel-
1728 L. Z. Holland and others
atively few cells in a region that Holland et al. (1992) proposed
as equivalent to part of the vertebrate hindbrain. In vertebrates,
the later neural expression is in cells scattered within the
hindbrain and spinal cord (Davis et al., 1991; Hatta et al., 1991;
Gardner and Barald, 1992). Davis et al. (1991) proposed that
engrailed expression in such cells may be involved in the
differentiation of a set of spatially defined neurons.
Neural expression of engrailed homologs is also observed
as a conspicuous stripe in the cerebral vesicle of amphioxus
(present results) and at the midbrain/hindbrain boundary of
vertebrates (Patel et al., 1989b; Davis et al., 1991; Hatta et al.,
1991; Ekker et al., 1992; Gardner and Barald, 1992). However,
this major neural expression domain of AmphiEn differs in
three ways from that of vertebrate engrailed: AmphiEn (1) is
transcribed relatively late in development, (2) is not transcribed
dorsally in the neural tube and (3) is expressed only 50-100
µm from the rostral end of the nerve cord — within a region
thought (on the basis of neuroanatomy and expression of
amphioxus homologs of the vertebrate forebrain markers Otx
and Dll) to be equivalent to diencephalon (Lacalli, 1996; N. D.
Holland et al., 1996; Williams and Holland, 1996). Thus, if the
conspicuous AmphiEn expression in the cerebral vesicle of
amphioxus truly marks a homolog of the vertebrate
midbrain/hindbrain junction, one would be forced to conclude
Amphioxus engrailed gene 1729
Fig. 4. AmphiEn expression in amphioxus embryos; all except L are
whole-mount in situs; anterior toward left; unless otherwise noted, all
scale lines are 50 µm (A) Frontal view of 9.5 hour neurula; on either
side of midline, first somite has formed from rostral end of
presomitic groove; second somite is pinching off; arrows indicate
AmphiEn expression in first two somites and presumptive third
somite. (B) Side view of 11 hour neurula; single arrows indicate
AmphiEn expression in first four somites; tandem arrow indicates
neuropore. (C) Frontal view of 11 hour neurula; single arrows
indicate AmphiEn expression in first four somites. (D) Frontal view
of 12 hour neurula expressing AmphiEn in first six somites.
(E) Enlargement of D showing AmphiEn expression in posterior
region of each somite; scale line 25 µm. (F) Frontal view of 13.5
hour neurula expressing AmphiEn in first eight somites. (G) Partial
frontal view and (H) side view of 15 hour neurula; AmphiEn
expression persists only in first somite (arrow) on right side and is
also detectable in a few neural tube cells (arrowhead) midway along
rostrocaudal axis. (I) Side view of 17 hour neurula; arrow indicates
epidermal cells weakly expressing AmphiEn. (J) Side view of 24
hour embryo; AmphiEn expressed inconspicuously in a few neural
tube cells (arrowheads) just posterior to first pigment spot (p);
conspicuous expression near rostral end of cerebral vesicle (top left).
(K) Anterior of 24 hour embryo in side view showing AmphiEn
expression in cerebral vesicle a short distance caudal to neuropore
(tandem arrow). (L) 24 hour embryo cross-sectioned in plane x-x′ in
K; AmphiEn expressed ventrolaterally within wall of cerebral vesicle,
which encloses neural canal (arrow) and is dorsal to notochord (n);
dark granules in epidermal cells are pigment, not positive reaction for
AmphiEn transcripts. (M) Enlarged side view of 24 hour embryo
showing AmphiEn-expressing neural tube cells (arrowheads) just
posterior to first pigment spot (p). (N) Side view of anterior end of 24
hour embryo double-labelled for both AmphiEn and amphioxus
Distal-less; within cerebral vesicle, the latter (d) is strongly
expressed dorsal and slightly caudal to the former (e); tandem arrow,
neuropore.
that amphioxus has little or no homolog of the vertebrate
midbrain. Such a conclusion would conflict with neuroanatomical data suggesting that amphioxus has a midbrain
as well as a diencephalon (Lacalli, 1996). This discrepancy
emphasizes the need for further mapping of the amphioxus
brain with genetic markers specific for vertebrate midbrain,
such as amphioxus homologs of Pax-7 (Jostes et al., 1991) or
her-5 (Müller et al., 1996b). Alternatively, the expression
domain of AmphiEn in the amphioxus cerebral vesicle might
be comparable to one of the three bands of engrailed
expression that appear in arthropod brains relatively late in
development (Hirth et al., 1995; Scholtz, 1995).
When engrailed genes are compared across the animal
kingdom, their most widespread function appears to be in neurogenesis. This commonality has led many to assume that,
during animal evolution, engrailed originally functioned in
neurogenesis and was only later co-opted for other functions,
such as establishing a segmented body plan (Patel et al., 1989b;
Joyner and Hanks, 1991; Holland, 1992; Webster and Mansour,
1992; Lans et al., 1993; Condron et al., 1994; Gee, 1996). The
validity of this evolutionary scenario still requires testing by
studies of the structure and function of engrailed homologs in
a wide spectrum of lower invertebrate phyla.
Early segmental expression compared between
AmphiEn and engrailed homologs of other animals
The genetic basis of segmentation is best understood for
Drosophila, an advanced insect (Lawrence, 1992): sequential
expression of maternal, gap, pair-rule and segment-polarity
genes subdivides the anteroposterior axis into successively
smaller regions, culminating in a virtually simultaneous
appearance of a series of parasegments. In each nascent
parasegment, the anteriormost cells begin to express the
segment-polarity gene, engrailed. The morphologically
obvious segments appearing later in development are out of
register with the parasegments such that the engrailed-expressing cells are posteriorly located in each segment. In studies of
other protostomes, it is usually presumed that parasegments are
the initial lineage units of metameres, although this is rarely
established rigorously and the metameres actually described
are usually segments. The general rule, to which AmphiEn
conforms, is that engrailed homologs playing a role in establishing segments are expressed posteriorly in each metamere.
From one group of metameric protostomes to the next,
different germ layers express engrailed homologs during
segment formation. In centipedes (Whitington et al., 1991) and
annelids (Wedeen and Weisblat, 1991; Lans et al., 1993),
expression is both mesodermal and ectodermal. In molluscs
(Jacobs and De Salle, 1994; Jacobs, personal communication),
crustaceans (Patel et al., 1989a; Scholtz et al., 1994; Manzanares et al., 1993, 1996) and insects expression is ectodermal — if one disregards transient transcription of engrailed
homologs in the mesoderm at the onset of insect segmentation
(Baylies et al., 1995). In comparing these germ layer differences in engrailed expression during segment formation, Lans
et al. (1993) concluded that the mesodermal site of action was
ancestral and an ectodermal site was derived. This implies a
transfer of the site of action from one germ layer to another,
which is an instance of ‘homeogenetic induction’ as defined by
De Robertis et al. (1989). In this process, cells transcribing
genes specifying position somehow induce neighboring cells
in a different germ layer to express the same genes — in this
instance, mesoderm cells homeogenetically induce adjacent
ectoderm cells. Presumably, in some animals (e.g. arthropods),
once the genetic fiducial marks for segmentation were activated
in the ectoderm, those in the mesoderm became inactive.
The virtually simultaneous formation of all the segments at
once, as in Drosophila, is atypical of animals in general. Most
metameric protostomes, including lower insects, add many or
all segments one at a time in an orderly rostrocaudal sequence
from a growth zone near the posterior end of the body (Brown
et al., 1994; Klingler, 1994; Nagy, 1994; Patel, 1994). In vertebrates, early somites usually take shape sequentially within
the paraxial segmental plates (Tam and Trainor, 1994); later
somites may be generated sequentially from a posterior growth
zone associated with the tail bud (De Robertis et al., 1994). In
amphioxus, the formation of the first eight somites on either
side of the midline appears comparable to the segmental plate
stage of vertebrate somite formation, and the later somites arise
sequentially from a posterior growth zone. Perhaps it is significant that AmphiEn is expressed only in the genesis of the
first eight somites, which are formed by enterocoely, whereas
the more posterior somites are formed from solid blocks of
mesoderm that later hollow out by schizocoely (Jefferies,
1986).
During vertebrate development, as already mentioned,
engrailed homologs can be expressed in metameric structures,
but only after such structures have already become morpho-
1730 L. Z. Holland and others
logically segmented (Hatta et al., 1991; Ekker et al., 1992);
thus vertebrate engrailed homologs are not considered
candidate genes for controlling segmentation. Unlike vertebrates, many metameric protostomes begin to express
engrailed homologs in presumptive segmental regions that
only later become morphologically distinguishable as
segments. In this respect, amphioxus resembles not vertebrates,
but metameric protostomes, because AmphiEn expression
commences in the nascent somites before they become morphologically distinct. The possibility that AmphiEn has homologous functions with the Drosophila segment-polarity gene
engrailed should be addressed by studies of the relations of
amphioxus AmphiEn to its upstream promoters and downstream targets.
Evolutionary questions raised by a common
segmented ancestor for metameric protostomes and
deuterostomes
Substantive questions arise from the proposal that metameric
protostomes and deuterostomes evolved from a common
ancestor in which mesodermal expression of engrailed played
a role in segment development. For example: what became of
the ancestral segmentation in some lower deuterostomes, and
why doesn’t engrailed play a role in the establishment of vertebrate segmentation?
If the ancestral deuterostome had extensive, protostome-like
segmentation, this feature must have been partially or entirely
lost in echinoderms, hemichordates and tunicates. For echinoderms, one can propose that a common segmented ancestor
gave rise to the extinct segmented species (homalozoans) and
also, by the reduction of segmentation, to the living species,
which retain only embryonic trimery. In living echinoderms,
developmental expression of engrailed homologs appears
limited to cells scattered in the radial nerves (Popodi et al.,
1996; Wray, 1996). For hemichordates, one can propose that a
segmented ancestor lost most traces of segmentation (except
for trimery and gill slits) in modern species. To date there have
been no studies of hemichordate engrailed expression. For
tunicates, the segmentation of larval ascidians and appendicularians is inconspicuous, if present at all. If one proposes a
segmented ancestor for tunicates, the vague muscular segmentation would be a vestige of an originally metameric condition.
Recent support for this interpretation comes from Wada et al.
(1996), who found iterated ectodermal and neural expression
of tunicate Pax-3/7 and suggested descent from a more
elaborate segmental plan. Clearly, there is a pressing need for
studies of developmental expression of engrailed and other
segment-polarity genes in tunicates.
Compared to the proposed loss of segmentation in some
deuterostomes, the apparent loss of engrailed participation in
forming the segmented body plan of vertebrates is a much more
complex problem. In vertebrates the relationship between segmentation of the nerve cord and mesoderm is far from clear at
both the morphological and the molecular levels. The picture
is complicated by differences between head and trunk segmentation and between lower (lampreys) and higher vertebrates. Morphologically, somite formation in lower vertebrates resembles that in amphioxus, the anterior somites
(called head cavities) being enterocoelic and the trunk somites
being schizocoelic; these relationships led Gilland and Baker
(1993) to homologize the 8-9 somite embryo of amphioxus
with the head of lower vertebrates. In higher vertebrates, head
cavities are lacking and the head mesoderm is transiently
segmented into somitomeres, condensations of mesodermal
cells visible only with scanning electron microscopy. In the
chick, the first true somite is at the level of rhombomere 7 in
the hindbrain. In spite of differences in the mode of formation,
in amphioxus and in both lower and higher vertebrates, the
anterior and posterior mesodermal segments are considered to
be serial homologs. In addition, the head cavities of lower vertebrates and the somitomeres of higher vertebrates are thought
to be homologous even though they differ both in number and
in the way they are formed. To complicate the situation further,
there is not a 1:1 correspondence between mesodermal
segments and segments of the neuroectoderm. Nevertheless,
there have been several schemes for aligning the segments in
amphioxus and vertebrates. The most recent takes into consideration Hox gene expression in the nerve cord as well as
anatomy (Gilland and Baker, 1993). In this scheme, the first
somite of amphioxus is equated with the mandibular head
cavity (second somite) of the lamprey and shark and to the first
three somitomeres of the chick, the second through the fifth
amphioxus somites are equated with somites 3-5 of the
lamprey and shark and to somitomeres 4-7 of the chick, while
the sixth somite of amphioxus is equated with the sixth somite
in the lamprey and shark and to the first true somite in the
chick.
The genes involved in establishing metamerism in the vertebrate nerve cord and mesoderm are poorly understood: En-1
and Wnt-1 evidently have roles in establishing the
midbrain/hindbrain boundary (McMahon et al., 1992; Wurst et
al., 1994); Krox-20 and kreisler appear to be involved in segmentation of the hindbrain (Lumsden and Krumlauf, 1996;)
and a homolog of the Drosophila pair-rule gene hairy may
function in establishing metamerism of the mesoderm (Müller
et al., 1996a). However, there is no evidence of a role for
engrailed in establishing mesodermal segmentation. If vertebrates did arise from amphioxus-like ancestors in which
mesodermal expression of engrailed played a role in establishing segmentation, then a role for engrailed in establishing
vertebrate segmentation has apparently been lost. It is surprising, if the anterior somites of amphioxus are indeed homologous to the mesodermal head segments of vertebrates, that the
genetic basis of vertebrate segmentation is so different from
that in amphioxus. Baker (1992) has suggested that the primary
metameric plan of vertebrates resided in the mesoderm early
in their phylogeny, but became transferred to the neuroectoderm and our present results are compatible with this idea. In
terms of homeogenetic induction, mesodermal cells using
genes (including engrailed) to help specify segmentation
should have induced expression of the same genes in neighboring neuroectodermal cells.
One possible explanation for the loss of a role for engrailed
in segmentation of the vertebrate mesoderm is that specification of segments may have been strongly influenced by the
epithelium-to-mesenchyme transition that precedes the
formation of most somites (Keynes and Stern, 1988). Perhaps
the additional genetic controls needed for such a transition are
somehow incompatible with the establishment of metamery by
segmentation genes. This idea is consistent with the expression
of lamprey engrailed in the posterior wall of the forming
mandibular head cavity, which gives rise to the muscles of the
Amphioxus engrailed gene 1731
mandibular arch (Holland et al., 1993). This engrailed
expression might be a last vestige of the involvement of this
segment-polarity gene in vertebrate somite formation.
In conclusion, the embryo of amphioxus, which is a
deuterostome, becomes striped with zones of AmphiEn
expression, each located in the posterior part of a nascent or
newly formed segment. This resembles the expression pattern
of engrailed homologs in embryos of metameric protostomes.
Therefore, functions of engrailed in establishing and maintaining a metameric body plan may have arisen only once in
evolution — in a common segmented ancestor of protostomes
and deuterostomes. This insight, along with recent genetic
information suggesting a reversal of the dorsoventral axes
between Drosophila and vertebrates (Jones and Smith, 1995;
De Robertis and Sasai, 1996), favors phylogenetic scenarios
deriving the vertebrates from annelid-like or arthropod-like
body plans.
We thank Ray Wilson for laboratory facilities at University of South
Florida (St Petersburg). Antibodies were generously supplied by Clay
Davis (αEnhb-1), Nipam Patel (4D9) and Cathy Wedeen (αht-en).
Our research was supported by NSF research grant IBN 96-309938
(to N. D. H. and L. Z. H.). M. K. was supported by the NSF Research
Experience for Undergraduates Program.
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(Accepted 25 February 1997)