Developmental Biology 226, 18 –33 (2000)
doi:10.1006/dbio.2000.9810, available online at http://www.idealibrary.com on
Evolutionary Conservation of the Presumptive
Neural Plate Markers AmphiSox1/2/3 and
AmphiNeurogenin in the Invertebrate
Chordate Amphioxus
Linda Z. Holland,* M. Schubert,* ,1 N. D. Holland,* and T. Neuman†
*Marine Biology Research Division, Scripps Institution of Oceanography, University of
California at San Diego, La Jolla, California 92093-0202; and †Department of Surgery,
Burns and Allen Research Institute, Cedars–Sinai Medical Center, University of
California at Los Angeles School of Medicine, Los Angeles, California 90048
Amphioxus, as the closest living invertebrate relative of the vertebrates, can give insights into the evolutionary origin of the
vertebrate body plan. Therefore, to investigate the evolution of genetic mechanisms for establishing and patterning the
neuroectoderm, we cloned and determined the embryonic expression of two amphioxus transcription factors, AmphiSox1/
2/3 and AmphiNeurogenin. These genes are the earliest known markers for presumptive neuroectoderm in amphioxus. By
the early neurula stage, AmphiNeurogenin expression becomes restricted to two bilateral columns of segmentally arranged
neural plate cells, which probably include precursors of motor neurons. This is the earliest indication of segmentation in
the amphioxus nerve cord. Later, expression extends to dorsal cells in the nerve cord, which may include precursors of
sensory neurons. By the midneurula, AmphiSox1/2/3 expression becomes limited to the dorsal part of the forming neural
tube. These patterns resemble those of their vertebrate and Drosophila homologs. Taken together with the evolutionarily
conserved expression of the dorsoventral patterning genes, BMP2/4 and chordin, in nonneural and neural ectoderm,
respectively, of chordates and Drosophila, our results are consistent with the evolution of the chordate dorsal nerve cord
and the insect ventral nerve cord from a longitudinal nerve cord in a common bilaterian ancestor. However, AmphiSox1/2/3
differs from its vertebrate homologs in not being expressed outside the CNS, suggesting that additional roles for this gene
have evolved in connection with gene duplication in the vertebrate lineage. In contrast, expression in the midgut of
AmphiNeurogenin together with the gene encoding the insulin-like peptide suggests that amphioxus may have homologs
of vertebrate pancreatic islet cells, which express neurogenin3. In addition, AmphiNeurogenin, like its vertebrate and
Drosophila homologs, is expressed in apparent precursors of epidermal chemosensory and possibly mechanosensory cells,
suggesting a common origin for protostome and deuterostome epidermal sensory cells in the ancestral bilaterian.
© 2000 Academic Press
Key Words: body plan evolution; neural patterning; pancreas; olfactory placodes; motor neurons; Sox; neurogenin; tap;
dichaete; embryogenesis; neural plate.
INTRODUCTION
Comparisons of developmental gene expression patterns
between vertebrates and their closest living invertebrate
relative, amphioxus, have suggested that the genetic
mechanism for distinguishing neuro- and nonneuroectoderm in chordates evolved before the amphioxus/vertebrate
split (Holland and Holland, 1999). In the vertebrate gastrula,
secreted proteins from the organizer (e.g., follistatin, nog1
18
Contributed the work on AmphiSox1/2/3.
gin, and chordin in Xenopus), bind to and inhibit the TGF-␤
family members BMP2 and BMP4 to ensure a neural fate in
the dorsal ectoderm (Sasai and De Robertis, 1997; Weinstein and Hemmati-Brivanlou, 1997). In amphioxus, as in
vertebrates, BMP2/4 is expressed in the nonneural ectoderm of the gastrula, becoming down-regulated in the
neural plate (Panopoulou et al., 1998). In Drosophila as
well, the expression and function of decapentaplegic and
short gastrulation are similar to those of their respective
vertebrate homologs (BMP2/4 and chordin), suggesting ancient roles for these genes in dorsoventral patterning of
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19
Amphioxus Presumptive Neural Plate Markers
bilaterian organisms (Holley et al., 1995; Marqués et al.,
1997). In contrast, the early molecular events downstream
of chordin/sog that establish neural fates in the neuroectoderm are less well understood (Streit and Stern, 1999).
In Xenopus, chordin, follistatin, and noggin secreted by
the organizer appear to act together with other factors in the
induction and maintenance of several early neuroectodermal markers, including Sox2 and 3 (Mizuseki et al., 1998;
Streit et al., 1998). Expression of these markers together
with that of genes such as FGF leads to the initiation of
nerve cell differentiation. Genes in the Sox1/2/3 group are
Sry-related HMG box transcription factors most closely
related to Drosophila Dichaete (⫽fish-hook, ⫽Sox70D)
(Collignon et al., 1996; Nambu and Nambu, 1996; Russell
et al., 1996; Vriz et al., 1996; Penzel et al., 1997; Rex et al.,
1997; Sakai et al., 1997; Y. Ma et al., 1998). Vertebrate Sox2
is first expressed throughout the presumptive neuroectoderm of the very late blastula, turning on after chordin and
before the proneural gene neurogenin-1 (Xngnr-1), a homolog of Drosophila tap (biparous) (Bush et al., 1996;
Gautier et al., 1997; Rex et al., 1997; Mizuseki et al., 1998;
Wood and Episkopou, 1999). neurogenins encode basic
helix-loop-helix (bHLH) transcription factors that bind to
E-box sequences of target genes (Gradwohl et al., 1996).
neurogenin-1 is first expressed in the vertebrate gastrula in
broad domains of presumptive neuroectoderm that give rise
to both neurons and nonneuronal cells (Ma et al., 1996,
1997; Blader, 1997; Korzh et al., 1998; Eisen, 1999). In
Drosophila, the Sox1/2/3 homolog Dichaete and the neurogenin homolog tap (biparous) are also initially expressed
quite broadly in the neuroectoderm (Bush et al., 1996;
Nambu and Nambu, 1996; Russell et al., 1996). The relation between these genes and short gastrulation (sog) has
not been tested, although in sog mutants, expression of
several neuroectodermal markers is nearly normal (Jaźwińska et al., 1999).
To date, no markers of presumptive neuroectoderm have
been cloned from amphioxus, and it is not clear to what
extent gene networks acting downstream of chordin in the
neuroectoderm are conserved across major taxonomic
groups. To address this question, we have cloned and
determined the embryonic expression of amphioxus homologs of Sox1/2/3 and neurogenin. Amphioxus is
vertebrate-like, but genomically and structurally simpler.
Homologs of most vertebrate gene families are probably
represented in the amphioxus genome, but amphioxus has
not undergone the genome duplications that occurred early
in vertebrate evolution (Holland et al., 1994). Structurally,
amphioxus and vertebrates share features such as a dorsal
hollow nerve cord, notochord, muscular somites, and pharyngeal gill slits. However, amphioxus lacks paired appendages, paired eyes, auditory organs, and a skeleton. Microanatomical and genetic studies have suggested that the
amphioxus nerve cord contains regions comparable to the
vertebrate diencephalon and hindbrain and possibly the
anteriormost portion of the midbrain, although an isthmo-
cerebrellar region appears to be lacking (reviewed in Holland and Holland, 1999).
Amphioxus embryology differs in some respects from
that of vertebrates. The blastula has but a single layer of
cells, gastrulation consists of a flattening and invagination
of one side of the blastula, and there is little involution of
cells around the lips of the blastopore (Zhang et al., 1997).
At the end of gastrulation as the neural plate forms, the
amphioxus embryo becomes more vertebrate-like. However, neural folds do not form; instead, the nonneural
ectoderm detaches from the edges of the open neural plate,
moves laterally over the neural plate, and fuses in the dorsal
midline. Only then do the lateral edges of the neural plate
curl up dorsally into the neural tube (Hirakow and Kajita,
1994; N. D. Holland et al., 1996). Nevertheless, despite
these differences, our results show that AmphiSox1/2/3 and
AmphiNeurogenin, like their vertebrate homologs, are
broadly expressed very early in presumptive neuroectoderm. Furthermore, expression of AmphiNeurogenin, like
that of vertebrate neurogenin-1, persists in a segmental
subset of neural plate cells in the presumptive hindbrain,
which probably includes presumptive motor neurons. Expression of Sox1/2/3 and neurogenin homologs in the developing CNS also appears to be somewhat conserved
between amphioxus and Drosophila. These results suggest
evolutionary conservation of genetic mechanisms for establishing the neural plate, for maintenance of neural fate, and
for neural induction in amphioxus and the vertebrates and,
to some extent, in Drosophila as well.
MATERIALS AND METHODS
Amphioxus Material
Adults of the Florida amphioxus (Branchiostoma floridae) were
collected from Old Tampa Bay, Florida. Genomic DNA was extracted in guanidinium isothiocyanate and purified as previously
described (L. Z. Holland et al., 1996). Ripe adults were electrically
stimulated to spawn in the laboratory, and embryos and larvae
were raised at 23°C (Holland and Holland, 1993).
Cloning
For amphioxus Sox genes, 250,000 clones of a cDNA library in
Lambda ZAP II (Stratagene, Inc., La Jolla, CA) made from 5- to 18-h
embryos of B. floridae were screened under moderate stringency
(hybridization in 7.25⫻ SSC, 0.24% SDS, 12.2⫻ Denhardt’s at
60°C; washes in 1⫻ SSC, 0.1% SDS at 60°C) with mixed probes for
two amphioxus Wnt genes—a 354-bp fragment of Wnt6 (Wnt-B;
Holland et al., 1994) and a 405-bp piece of Wnt 7A, which was
obtained in screening a gridded cDNA library from 26-h embryos in
pSport1 (Gibco BRL, Inc., Rockville, MD) under moderately low
stringency with mixed probes for the amphioxus Wnt6 fragment
plus a 360-bp piece of amphioxus Wnt4 (Wnt-A; Holland et al.,
1994). Two very weakly positive clones obtained corresponded to
full-length cDNAs of the same Sox gene.
To isolate a 174-bp fragment of amphioxus neurogenin, PCR was
used with degenerate primers corresponding to the bHLH region of
mammalian helix-loop-helix transcription factors and genomic DNA
of B. floridae. The forward primer corresponded to the amino acid
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20
Holland et al.
sequence 5⬘-ANA(D,N)RERR(N,T)R, nucleotide sequence 5⬘GCIAAC(T)G(A)C(A)IC(A)GIGAA(G)C(A)G(A,C)IC(A)G-3⬘, and the
reverse primer to amino acid sequence NYIWALA(T,S)E, nucleotide
sequence 5⬘-TCIC(G)C(T,A)IAG(A)IGCCCAT(G,A)ATG(A)TAG(A)TT-3⬘ (I is inosine). PCR conditions were denaturation—93°C, 1 min;
annealing— 45°C, 3 min; synthesis—72°C, 2 min. PCR products were
cloned, and one, corresponding to neurogenin, was used to screen a
cDNA library of 2- to 4-day larvae in Lambda ZAP II (Stratagene Inc.)
under conditions of medium stringency (hybridization in 2⫻ SSC at
42°C).
Southern Blot Analysis
Sixteen 10-␮g 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) according to L. Z. Holland et al. (1997). The Southern blot was
hybridized at low stringency (55°C) with a 447-bp EcoO109I/EcoRV
fragment of the AmphiSox1/2/3 cDNA located in the coding region
164 bp 3⬘ of the HMG box. Washes were 2⫻ 20 min at 50°C in 2⫻
SSC, 0.1% SDS. These conditions are comparable to those previously used for demonstrating the presence of two muscle actin
genes in amphioxus (Kusakabe et al., 1997). After being probed, the
blot was stripped in boiling 0.5% SDS and reprobed under identical
conditions with a 347-bp SmaI/SphI fragment of AmphiNeurogenin, located 5⬘ of the bHLH domain, overlapping the 5⬘ end of the
HMG domain by 40 bp.
Electrophoretic Mobility Shift Assay (EMSA)
For expression of AmphiNeurogenin, a full-length cDNA was
cloned into the eukaryotic expression vector pcDNA3 (Invitrogen,
Inc., Carlsbad, CA). Amphioxus neurogenin protein was synthesized
in vitro using the TNT Quick Coupled Transcription/Translation
System (Promega, Inc., Madison, WI). The resulting translation product was analyzed by SDS–polyacrylamide gel electrophoresis. EMSA
were carried out as described by Chiaramello et al. (1995a). Briefly, 3
␮l of the translation product was incubated for 15 min at room
temperature in binding buffer (10 mM Tris–HCl, pH 7.5, 50 mM KCl,
0.1 mM EDTA, 1 mM dithiothreitol, 1 mM MgCl2, 5% glycerol) with
100 ng of poly(dI– dC) (Roche Molecular Biochemicals, Indianapolis,
IN) and 40 fmol of a 32P-labeled double-stranded oligonucleotide probe
corresponding to the E-box sequence of the rat p75 gene promoter.
Complementary single-stranded probes [5⬘-GCATTGCCTTCACCCAGCTGCTCCCGCCCGC (E-box sequence is underlined)] were
synthesized, allowed to anneal, and labeled at the 3⬘ recessed ends
with [␣- 32P]dCTP and the large fragment of DNA polymerase I
(Klenow). Unlabeled competitor DNA was prepared by annealing
complementary oligonucleotides. After incubation, the binding reaction was subjected to electrophoresis on a 5% native polyacrylamide
gel. After electrophoresis the gel was dried and exposed to X-ray film.
Cell Culture, Transfection, CAT Assay, and in Situ
Hybridization
Transcriptional activity of AmphiNeurogenin was measured in
an in vitro cell culture system. The rat p75 promoter CAT
construct (1.4 CAT) (Metsis et al., 1992) and GAP-43 promoter
CAT construct (GXC; Chiaramello et al., 1996) were used as
reporter plasmids. Mouse neuroblastoma Neuro2A cells (ATCC)
were grown in Dulbecco’s modified Eagle’s medium containing
10% fetal bovine serum (Gibco). Cells in 35-mm diameter dishes at
a density of 5 ⫻ 10 4 cells per dish were transfected with 5 ␮g of total
plasmid DNA, via the calcium phosphate precipitation method.
The medium was changed to normal growth medium 16 h after
transfection. Cells were harvested 48 h later, and CAT assays were
performed as previously described (Chiaramello et al., 1995b).
Quantification of acetylation ratios was obtained by PhosphorImager (Molecular Dynamics, Sunnyvale, CA) analysis. To normalize
transfection efficiencies, cells were cotransfected with 0.5 ␮g of the
plasmid pRcRSVlacZ. All reported CAT activities were normalized
to total protein and lacZ activity. Values represent the means of at
least three independent transfections.
Embryos and larvae were fixed and processed for whole-mount in
situ hybridization by the method of L. Z. Holland et al. (1996).
Antisense riboprobes were synthesized from full-length cDNA
clones. Hybridized embryos and larvae were photographed as
whole mounts, counterstained with Ponceau S, embedded in plastic, and sectioned at 3 ␮m.
Phylogenetic Analysis and Amino Acid Alignments
Phylogenetic trees were constructed heuristically with 100 random stepwise additions (PAUP 3.1.1). Outgroups were mouse SRY
for Sox proteins and MATH-1 for neurogenins. Bootstrap values
were calculated in 1000 cycles with 10 random stepwise additions
per cycle. For the Sox tree, 13 proteins were used. Because of the
large number of amino acid identities in the HMG box among
different Sox proteins, it was necessary to base the tree on the
HMG box (80 characters) plus conserved regions C-terminal of the
HMG box (45 characters). Only one most parsimonious tree, which
had a length of 561, was retained. The tree of neurogenins is based
only on the 67 amino acids of the bHLH domain. The analysis
yielded a single tree with a length of 121. All sequences used and
their GenBank accession numbers are listed in the figure legends.
Amino acid alignments were performed with ClustalW (Baylor
College of Medicine) and manually adjusted.
RESULTS
Characterization of AmphiSox1/2/3
There are seven groups (A–G) in the vertebrate Sox gene
family. Screening a cDNA with mixed probes for two Wnt
genes (405 and 354 bp long) yielded two identical clones
coding for an amphioxus Sox B group gene. Fortuitously, the
nucleotide sequence between bases 156 and 590 of this Sox
gene is 56% identical to the sequence of heterologous
probes used for screening (data not shown). Both Sox clones
are 1878 bp long, including an open reading frame of 735 bp
coding for a protein of 245 amino acids. The aminoterminal half of the protein includes an HMG box of 79
amino acids characteristic of Sox proteins. The HMG box is
a DNA-binding domain, which resembles that in highmobility group nonhistone chromosomal proteins (Wegner,
1999). As Fig. 1 shows, the amphioxus Sox protein shares 7
of the 14 amino-terminal amino acids that are conserved in
the vertebrate Sox B group proteins Sox1, Sox2, and Sox3. In
addition, the HMG box shares 75% identities with chick
Sox1, 2, and 3 proteins. Several domains C-terminal to the
HMG domain, including the 12-amino-acid group B homology domain adjacent to the HMG box, are also conserved
between the amphioxus Sox protein and the vertebrate
homologs. However, the C-terminal 54 amino acids, rich in
Copyright © 2000 by Academic Press. All rights of reproduction in any form reserved.
21
Amphioxus Presumptive Neural Plate Markers
FIG. 1. Amino acid alignment of Sox1/2/3 proteins. Double underlining indicates the HMG domain. Single underlining indicates regions used
in addition to the HMG domain for constructing the phylogenetic tree in Fig. 3. Identities are indicated by dark shading, conserved substitutions
by light shading. Accession numbers are as in Fig. 3 except for chickSox1 (AB01802), chickSox2 (U12532), and chickSox3 (U12467).
proline (15.5%), serine (15.5%), and threonine (11%), suggestive of a transactivation domain, are not conserved
between the amphioxus Sox B protein and the homologs of
other species. Furthermore, this region is over 40 amino
acids shorter in our amphioxus Sox protein than the corresponding region in other species (Fig. 1).
Southern blot analysis demonstrates a single major band
with some enzymes, e.g., BglI, EcoRI, NcoI, XhoI (Fig. 2).
However, one or more fainter bands are present in all lanes
with major bands less than 10 kb. Some of these bands may
be due to a restriction site in an intron or to polymorphism,
which is very high in amphioxus. Since AmphiSox1/2/3 has
many identities with vertebrate Sox1/2/3 genes over the
region of the probe (Fig. 1), we would expect the additional
bands to be quite intense if there were another AmphiSox1/
2/3 homolog. It is most likely, therefore, that the fainter
bands are due to the recognition of a more distantly related
Sox B class gene, e.g., homologs of sea urchin SoxB2 (Fig. 3)
or chick and human Sox14 and Sox21 (Malas et al., 1999)
or to unrelated genes like the Wnts; the AmphiSox1/2/3
cDNAs were obtained by screening with Wnt4/6
probes, and sequence alignment of AmphiWnt4 and the
AmphiSox1/2/3 probe used for the Southern blot demonstrates 48% nucleotide identity.
Phylogenetic analysis of the Sox B clade (Fig. 3) shows
that the amphioxus Sox B protein is located at the base of
the vertebrate Sox1, Sox2, and Sox3 proteins. We, therefore,
FIG. 2. Genomic Southern blot analysis of pooled amphioxus
DNA. Numbers at top indicate digestion in 1, BamHI; 2, BglI; 3,
BstEII; 4, EcoO109I; 5, EcoRI; 6, BstXI; 7, HindIII; 8, KpnI; 9, PstI;
10, NotI; 11, NcoI; 12, PvuI; 13, SalI; 14, StuI; 15, XbaI; 16, XhoI.
Blot probed under low stringency with a 447-bp EcoO109I, EcoRV
fragment of AmphiSox1/2/3 located in the coding region just 3⬘ of
the HMG box. Molecular size standards in kilobases at left.
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22
Holland et al.
FIG. 3. Phylogenetic tree of Sox1/2/3 proteins. Drosophila Dichaete is used as an outgroup. A single most parsimonious tree was obtained.
Accession numbers are Drosophila Dichaete (X96419), sea urchin SoxB1 (AF157389), mouse Sox1 (X94126), mouse Sox2 (X94127), mouse
Sox3 (X94125), mouse SRY (AF070933), mouse Sox15 (AB014474), mouse Sox4 (S37303), mouse Sox17 (Q61473), mouse Sox10 (Q048880),
and AmphiSox1/2/3 (AF271787).
term this amphioxus gene AmphiSox1/2/3. This tree suggests that the Sox1, Sox2, and Sox3 genes arose from gene
duplication in the vertebrate lineage. The analysis further
indicates that sea urchin SoxB1 is more closely related to
AmphiSox1/2/3 than is sea urchin SoxB2 and suggests that
these sea urchin genes arose from an independent gene
duplication within the echinoderm lineage.
Embryonic Expression of AmphiSox1/2/3
AmphiSox1/2/3 is a highly specific marker for presumptive neuroectoderm. Its expression is first detectable by in
situ hybridization in the early gastrula (cap-shaped stage) in
the dorsal epiblast, including the presumptive neuroectoderm (Figs. 4A and 4B). Expression remains uniformly
strong in the presumptive neuroectoderm throughout gastrulation (Figs. 4C– 4E). Whether the expression domain
includes any cells in adjacent nonneural ectoderm at these
early stages is not clear because of the lack of anatomical
markers. However, at the onset of neurulation when the
neural plate flattens and becomes distinct from nonneural
ectoderm (Figs. 4F and 4G), it is evident that expression is
limited to neural ectoderm and is excluded from nonneural
ectoderm. As the edges of the neural plate curl dorsally,
expression initially remains panneural (Figs. 4H and 4I).
Later, before the edges of the neural plate have fused
dorsally, expression becomes down-regulated along the
midline and in the anterior part of the CNS (Figs. 4J– 4L). At
the midneurula stage (Fig. 4M), expression is limited to the
extreme posterior-dorsal portion of CNS and then ceases
entirely. However, in much later larvae at the one- and
two-gill-slit stages (2–5 days), prolonged staining revealed
extremely weak expression in a few cells scattered in the
CNS rostral to the pigment spot at the level of somite 5
(data not shown). Expression was not detected in tissues
other than neuroectoderm.
Characterization of AmphiNeurogenin
Of 52 clones obtained by amplification of genomic DNA
of B. floridae with degenerate primers to the bHLH region
and sequenced, 14 corresponded to a 174-bp fragment of a
single amphioxus neurogenin gene. No other clones corresponding to neurogenins were obtained. cDNA library
screening with the neurogenin fragment yielded four amphioxus neurogenin clones. The longest was 1240 bp, including the entire coding region but lacking the C-terminal
portion of the 3⬘ UTR. The gene codes for a protein of 255
aa, including a bHLH region of 60 aa. C-terminal of the
bHLH domain there are several proline, serine, and glycine
residues conserved among the amphioxus, vertebrate, and
Drosophila homologs (Fig. 5).
Southern blot analysis with a probe 5⬘ of the bHLH region
reveals a single major band with most enzymes (Fig. 6). Two
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23
Amphioxus Presumptive Neural Plate Markers
FIG. 4. AmphiSox1/2/3 expression during development. For side and dorsal views of whole mounts, anterior is to the left. (A) Animal pole
view of an early gastrula with the blastopore opening away from viewer; expression is in the epiblast at the presumptive dorsal side of the
embryo. (B) Cross section of very early gastrula with blastopore opening toward the right (approximate posterior end of embryo). Expression
is in the dorsal epiblast (arrow). (C) Whole mount of midgastrula in dorsal view; expression is in the dorsal epiblast (presumptive
neuroectoderm) including the dorsal lip of the blastopore, opening toward the right. (D) Cross section of midgastrula in side view.
Expression is in the presumptive neuroectoderm. (E) Side view of late gastrula/early neurula with expression throughout early neural plate.
(F) Same gastrula as in E in dorsal view showing expression in early neural plate. (G) Cross section at the level of the arrowhead in F showing
expression throughout early neural plate. (H) Early neurula with expression limited to neural plate. (I) Cross section through level of
arrowhead in H showing expression in the neural plate but not in the dorsal epidermis (arrows) overgrowing the neural plate. (J) Side view
of an early to midneurula with expression in neural plate. (K) Dorsal view of neurula in J. Expression is becoming restricted to the edges
of the neural plate. (L) Optical cross section though level of arrowhead in K. Expression is down-regulated in the midline of the neural plate
(floor plate). (M) Side view of midneurula. Expression is down-regulated except in the most posterior region of the neural tube (arrow). Scale
for whole mounts, 50 ␮m, and for cross sections (counterstained pink), 25 ␮m.
bands of equal intensity obtained with some enzymes, e.g.,
SalI and StuI, are probably due to polymorphism or to the
presence of introns. Thus, the amphioxus neurogenin gene
we cloned probably has no close relatives, a conclusion
supported by the results from PCR amplification of
genomic DNA with degenerate primers. Phylogenetic analysis shows that this gene is located at the base of the
vertebrate neurogenins (Fig. 7). We, therefore, term this
amphioxus gene AmphiNeurogenin. The tree indicates that
the vertebrate neurogenins arose from gene duplication in
the vertebrate lineage. In addition, Drosophila tap groups
with the neurogenins and, thus, NeuroD and Neurogenin
evidently separated before the deuterostome/protostome
split.
To examine the ability of AmphiNeurogenin to activate
transcription from two promoters of neuronal genes that
contain E-box sequences, rat p75 and GAP-43, transient
CAT assays were performed with mouse neuroblastoma
Neuro2A cells. Cotransfection of an AmphiNeurogenin
expression vector with a reporter plasmid resulted in significant increases in CAT activity in transfected cells (Fig.
8A), indicating that AmphiNeurogenin can act as a tran-
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24
Holland et al.
FIG. 5. Amino acid alignment of neurogenin proteins. Single underlining indicates the bHLH, used for constructing the phylogenetic tree
in Fig. 7. Identities are indicated by dark shading, conserved substitutions by light shading. Accession numbers as in Fig. 7.
scriptional activator in mammalian cells. To determine if
AmphiNeurogenin can bind to E-box sequences and activate transcription we performed EMSA and transient CAT
assays. Binding of AmphiNeurogenin to an oligonucleotide
carrying the p75 gene promoter E-box (CAGCTG) produced
a shifted AmphiNeurogenin–DNA complex (Fig. 8B), suggesting that AmphiNeurogenin functions as a DNA binding
protein.
FIG. 6. The same Southern blot as in Fig. 2, stripped and reprobed
with a 347-bp SmaI, SphI fragment of AmphiNeurogenin located in
the coding region just 5⬘ of the bHLH region. Restriction enzymes
numbered at top as in Fig. 2. Molecular size standards in kilobases
at left.
Embryonic Expression of AmphiNeurogenin
Expression of AmphiNeurogenin is first detectable in the
very early gastrula about the stage at which AmphiSox1/2/3
is first expressed (Figs. 9A and 9B). Expression is restricted
to the presumptive neural plate in a domain that largely
overlaps that of AmphiSox1/2/3; however, AmphiNeurogenin, unlike AmphiSox1/2/3, is not expressed in the dorsal
lip of the blastopore (Fig. 9B). As gastrulation proceeds,
expression remains strong in the presumptive neural plate
(Figs. 9C–9F). In the early neurula, while AmphiSox1/2/3 is
still uniformly expressed in the neural plate, AmphiNeurogenin becomes down-regulated in the future floor plate and
expression begins to break up into transverse stripes (Figs.
9G–9I). In addition, expression becomes down-regulated in
the anteriormost portion of the neural plate. By the midneurula stage, expression in the CNS is in dorsolateral rows
of transverse stripes except in the cerebral vesicle and in the
posteriormost portion of the nerve cord (Figs. 9J–9L). There
is no expression in the floor plate. A dorsal view (Fig. 9K)
shows that cells strongly expressing AmphiNeurogenin are
in approximate register with somite boundaries; however,
there are two areas of strong expression on each side at the
level of somite 2. In the late neurula (Fig. 9M), these stripes
of expression persist in dorsolateral cells of the nerve cord
(Figs. 9N and 9O); in addition, expression has become
detectable in the ventral endoderm cells of the midgut (Figs.
9M, arrowhead, and 9N). In embryos with an incipient
mouth opening, expression in the nerve cord has been
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25
Amphioxus Presumptive Neural Plate Markers
FIG. 7. Phylogenetic tree of neurogenin and neuroD proteins. The distantly related bHLH protein MATH1 (mouse atonal-related protein
1) is used as an outgroup. GenBank accession numbers are MATH-1 (D43694), mouse NeuroD-1 (D83507), mouse NeuroD-2 (U58471),
zebrafish NeuroD-1a (AF115772), mouse neurogenin-1 (U76207), mouse neurogenin-2 (U76207), mouse neurogenin-3 (U76208), zebrafish
Neurogenin-1 (AF036149), AmphiNeurogenin (AF271788), Drosophila Tap (O16867).
largely down-regulated anteriorly and now occurs mostly
posterior to the primary pigment spot (Fig. 9P, arrow).
Expression in the midgut endoderm has expanded (Fig. 9P,
arrowhead), and new domains have appeared in a few dorsal
cells of the cerebral vesicle (Fig. 9Q, arrowhead) and in a few
epidermal cells that may be precursors of the ciliary tuft
cells, which differentiate just ventral to the mouth (Fig. 9Q,
arrow). At 2 days of development, expression continues in
the cerebral vesicle, in the neural tube posterior to the
primary pigment spot, and in the midgut endoderm, but is
down-regulated in the presumed ciliary tuft precursors.
However, expression is up-regulated in the rostral ectoderm
(Figs. 9R and 9S, tandem arrowheads). At 6 days of age, the
larvae have elongated considerably, and expression continues in the cerebral vesicle, in the nerve cord (in a few widely
spaced cells anteriorly and more closely spaced cells posteriorly), and in the midgut endoderm (Fig. 9T). In larvae more
than a week old, expression persists only in the endoderm
of the midgut, where it is both dorsal and ventral (Fig. 9T).
Expression in a subset of midgut cells continues at least
until metamorphosis (data not shown).
DISCUSSION
Structure, Function, and Evolution of Sox1/2/3 and
Neurogenin Genes
Vertebrate Sox1/2/3 proteins are transcriptional activators. The transactivation domain is serine, threonine, and
FIG. 8. (A) Transactivation potential of AmphiNeurogenin. AmpiNeurogenin stimulates activity of rat p75 and GAP-43 promoters in
Neuro2A cell line. Plasmids p75 CAT and GAP43 CAT were
transfected together with pRcCMV (control, C) or pRcCMVAmphiNeurogenin (A) into neuroblastoma Neuro2A cells. CAT
activities are corrected for transfection efficiency and expressed
relative to the value obtained by transfection of p75-CAT and
GAP-43-CAT promoter reporter plasmids and expression plasmid
pRcCMV. CAT activities are presented as means ⫾ standard
deviations and represent results of at least three independent
experiments. (B) Electrophoretic mobility shift assay showing binding of AmphiNeurogenin to the E-box sequence. 1, control; 2,
AmphiNeurogenin.
Copyright © 2000 by Academic Press. All rights of reproduction in any form reserved.
26
Holland et al.
Copyright © 2000 by Academic Press. All rights of reproduction in any form reserved.
27
Amphioxus Presumptive Neural Plate Markers
proline rich and comprises the C-terminal 90 amino acids
(Kamachi et al., 1998; Uchikawa et al., 1999). The corresponding domain of AmphiSox1/2/3 is also serine, proline,
and threonine rich (44%), including several regions with a
high percentage of identity to vertebrate Sox1, Sox2, and
Sox 3 sequences. Thus, although AmphiSox1/2/3 lacks 40
amino acids at its C-terminus compared to its vertebrate
and sea urchin homologs, its C-terminal region is also
probably a transactivation domain. Since this region in
vertebrate Sox1, but not Sox2, Sox3, or AmphiSox1/2/3,
also includes several stretches of alanine repeats, these
repeats probably evolved only in vertebrate Sox1.
Vertebrate Sox1/2/3 proteins typically bind to target
genes, such as Fgf-4, in concert with POU domain proteins
like Oct (Ambrosetti et al., 1997; Fraidenraich et al., 1998;
reviewed in Bianchi and Beltrame, 1998; Uchikawa et al.,
1999) and initiate pathways leading to expression of neurogenins (Mizuseki et al., 1998), which in turn results in
activation of a cascade of downstream bHLH factors, including NeuroD, leading to neuronal differentiation (Q. Ma
et al., 1998). DNA-binding studies have shown that mouse
neurogenin-2 (MATH4A) can bind as a heterodimer with
the ubiquitous bHLH protein E12 to the insulin E-box
sequence (Gradwohl et al., 1996). Activation studies of
neurogenins have not been done; however, the related
NeuroD genes are transcriptional activators (McCormick et
al., 1996). Similarly, our results show that AmphiNeurogenin can bind to E boxes and act as a transcriptional activator
in a mammalian cell culture system. Given the sequence
conservation between AmphiNeurogenin and vertebrate
neurogenins, it is likely that the binding and activation
characteristics of vertebrate neurogenins are like those of
AmphiNeurogenin.
Southern blot and phylogenetic analyses suggest that
AmphiSox1/2/3 and AmphiNeurogenin are the amphioxus
homologs, respectively, of three vertebrate Sox1/2/3 genes
and three vertebrate neurogenin genes. Although the bootstrap values for the neurogenin tree are low due to the high
percentage of identities in the bHLH region, the presence of
a single amphioxus neurogenin gene is additionally supported by the failure of PCR with degenerate primers to
amplify more than one neurogenin gene. Thus, the three
vertebrate homologs of each gene probably evolved by
duplication of a single gene in the common ancestor of
amphioxus and the vertebrates. This presumably reflects
the two rounds of genome duplication that occurred early in
vertebrate evolution (Holland et al., 1994). Unexpectedly,
however, the analyses show that Drosophila tap (biparous)
is more closely related to amphioxus and vertebrate neurogenins than it is to NeuroD. Until the discovery of tap
(Gautier et al., 1997), it was thought that Drosophila had no
close relative of neurogenin and NeuroD, the nearest relative being atonal, most closely related to vertebrate
MATH-1 (Akazawa et al., 1995; reviewed in Lee, 1997).
However, our tree shows that Drosophila tap groups with
neurogenins, not with NeuroD, suggesting that neurogenin
and NeuroD arose by gene duplication before the
deuterostome–protostome split. Thus, both amphioxus and
Drosophila may have homologs of NeuroD which have yet
to be discovered.
FIG. 9. AmphiNeurogenin expression during development. For side and dorsal views of whole mounts, anterior is to the left. (A) Animal
pole view of a very early gastrula with blastopore opening away from viewer; expression in the dorsal epiblast (presumptive neuroectoderm).
(B) Cross section of very early gastrula as in A with blastopore opening toward right (approximate posterior end of embryo). Expression is
in the presumptive neuroectoderm (arrow) except just anterior to the blastopore. (C) Midgastrula in dorsal view with expression in the
dorsal epiblast. Blastopore opens to the right. (D) Midgastrula in side view. Expression is in the epiblast except just anterior to the blastopore
(arrowhead). (E) Dorsal view of midgastrula with expression in dorsal epiblast (presumptive neuroectoderm) except just anterior to the
blastopore. (F) Gastrula in E viewed from the blastopore (approximate posterior side). Expression is in dorsal epiblast. (G) Side view of very
early neurula with nearly closed blastopore (arrow). Expression is in the anterior 7/8 of the neural plate. (H) Dorsal view of neurula in G.
Expression is restricted to two rows of cells with a segmental arrangement on either side of the midline of the neural plate. (I) Cross section
through x in H showing the dorsal nonneural ectoderm (arrowheads) overgrowing the neural plate; cells in the midline of the neural plate
(presumptive floor plate) do not express the gene. (J) Side view of an early/midneurula showing segmental expression in two rows of cells
on either side of the floor plate. (K) Dorsal view of embryo in J. Segmental expression in cells (probable motor neuron precursors) on either
side of the midline in approximate register with the somites (numbered). (L) Cross section through x in K. Expression is at either side of
the neural plate, which has been overgrown by the epidermis dorsally. The notochord (n) is dorsal to the gut (g) and flanked by somites. (M)
Side view of late neurula with expression in the neural tube and in the midgut endoderm. (N) Cross section through x in M. Expression is
in the nerve cord, but not the floor plate and in ventral endoderm cells of the gut. (O) Cross section through y in M. Expression is in
dorsolateral cells of the neural tube. (P) Side view of embryo with incipient mouth opening. Expression is in scattered groups of cells in the
neural tube and in ventral endodermal cells of the midgut (arrowhead). The arrow indicates the primary pigment spot in the nerve cord. (Q)
Enlargement of the anterior end of an embryo with incipient mouth opening; expression is in a few cells of the cerebral vesicle (single
arrowhead) and in likely precursor cells of the ciliary tuft (arrow). (R) Side view of 2-day larva with primary pigment spot (arrow). Expression
is in the neural tube, including the cerebral vesicle (single arrowhead), in midgut cells, and in the anterior epidermis (tandem arrowhead).
(S) Enlargement of the anterior end of a 2-day larva with expression in the anterior epidermis (tandem arrowhead) and in a few cells of the
cerebral vesicle (single arrowhead). (T) Side view of a 6-day larva with primary pigment spot (arrow). Expression is in endoderm cells of the
midgut (inset) and in cells of the neural tube. Scale for whole mounts, 50 ␮m, and for cross sections (counterstained pink), 25 ␮m.
Copyright © 2000 by Academic Press. All rights of reproduction in any form reserved.
28
Holland et al.
Conservation of Early Expression of Sox1/2/3
Genes and neurogenins in Amphioxus and
Vertebrates
Conservation of Expression of AmphiSox1/2/3 and
AmphiNeurogenin during Neurulation in
Amphioxus and Vertebrates
AmphiSox1/2/3 and AmphiNeurogenin are the earliest
known markers for the presumptive neuroectoderm in
amphioxus, being up-regulated in the very early gastrula
as soon as the mesendoderm has invaginated. The
panneural expression of AmphiSox1/2/3 resembles that
of vertebrate Sox2. In vertebrates, initial expression of
Sox2 may be more extensive than presumptive neuroectoderm (Rex et al., 1997) and may be so in amphioxus.
The panneural expression of Sox1/2/3 genes was at first
thought to confer neural competence on ectoderm (Streit
et al., 1997). However, more recent evidence suggests
that it may modulate the responsiveness to additional
signals in cells which have gained neural competence but
are not yet committed to a neural fate (Mizuseki et al.,
1998; Pevny et al., 1998).
Although early expression of amphioxus and vertebrate
Sox1/2/3 genes are similar, initial expression of amphioxus and vertebrate neurogenins differ. The vertebrate
genes are never expressed throughout the entire anterior
7/8 of the dorsal epiblast. This difference, however, may
simply be quantitative, not qualitative. Although neurogenins were initially thought to be strictly neuronal
determination genes, acting downstream of Sox1/2/3
(Sommer et al., 1996; Fode et al., 1998; Q. Ma et al., 1998,
1999), at least in vertebrates, neurogenin-expressing cells
can be mitotic and biased toward, but not irreversibly
committed to, a neural fate (Ma et al., 1996; Blader et al.,
1997; Kim et al., 1997; Korzh et al., 1998). Similarly, in
amphioxus, since nonneuronal cells as well as neurons
differentiate anteriorly in the nerve cord (Lacalli et al.,
1994), the neuroectodermal cells initially expressing AmphiNeurogenin presumably differentiate into both cell
types.
In vertebrates, the number of cells expressing neurogenin is regulated by lateral inhibition mediated by Notch/
Delta signaling. Consequently, overexpression of vertebrate neurogenin outside the neural plate, where Notch/
Delta signaling does not operate, induces ectopic
neurons, while overexpression within the neural plate
can reduce the number of neurons (Ma et al., 1996; Blader
et al., 1997; Takke et al., 1999) evidently due to activation of Delta/Notch signaling (Takke et al., 1999). Thus,
the differences between amphioxus and vertebrates in the
percentage of cells initially expressing neurogenins in the
presumptive neuroectoderm may be due to differences in
levels of Delta/Notch signaling. Delta has not been
cloned from amphioxus, but the expression of AmphiNotch in presumptive neuroectoderm is complementary
to that of AmphiNeurogenin (our unpublished data),
suggesting that Notch/Delta signaling might be involved
in modulating the expression of AmphiNeurogenin in the
neural plate.
As neurulation begins, AmphiSox1/2/3 and AmphiNeurogenin are both down-regulated in specific areas of the
neural plate. AmphiSox1/2/3 turns off in the midline,
becoming restricted to the edges of the neural plate, while
AmphiNeurogenin expression becomes restricted to cells in
two rows on either side of the midline. Within each row,
the pattern of expression is striped, indicating a segmental
organization in the very early neural plate. The striped
pattern extends posteriorly after the nerve cord has formed.
In later larvae, cells in the cerebral vesicle and several
domains outside the CNS also express the gene. In contrast,
expression of AmphiSox1/2/3 largely ceases at the four- to
five-somite stage, well before the neural tube closes dorsally, and it is not expressed in any other tissues during
development.
These expression patterns are similar to those of all three
of their respective vertebrate homologs considered together.
Vertebrate Sox1, Sox2, and Sox3 typically have overlapping
patterns of expression in the early vertebrate CNS and may
have considerable functional redundancy (Collignon et al.,
1996; Vriz et al., 1996; Rex et al., 1997; Sakai et al., 1997;
Streit et al., 1997; Mizuseki et al., 1998; Pevny et al., 1998;
Zygar et al., 1998; Uchikawa et al., 1999; Wood and
Episkopou, 1999). Like AmphiSox1/2/3, the vertebrate
genes turn off ventrally in the floor plate (Penzel et al.,
1997; Pevny et al., 1998; Zygar et al., 1998; Uchikawa et al.,
1999; Wood and Episkopou, 1999). However, expression of
AmphiSox1/2/3 in the dorsal part of the CNS ceases early
compared to that of its vertebrate counterparts. The difference may be due to early differentiation of some neuronal
cell types in amphioxus. Amphioxus embryos display a
positive phototropism as soon as they hatch at 10 h, and
nerve cell bodies of the peripheral nervous system in
amphioxus have already begun to differentiate by the 8- to
10-somite stage (15–17 h), as shown by labeling with
anti-acetylated ␣-tubulin (Yasui et al., 1998). In addition,
the embryos begin muscular movements by 18 –20 h (10 –12
somites) (Stokes, 1997), indicating that motor neurons
differentiate relatively early.
While vertebrate Sox1/2/3 genes apparently become
down-regulated as cells cease dividing and embark on
differentiation pathways, neurogenins apparently turn off
in cells that have lost the competence to become neurons
and adopted nonneuronal fates. Although the function of
AmphiNeurogenin in vitro has not been determined, its in
vivo behavior and expression in the neural plate are similar
to those of its vertebrate counterparts. Thus, by comparison, the two rows of cells with a segmental organization
expressing AmphiNeurogenin in the neural plate are probably neuronal precursors. The pattern in 18-h embryos
strongly suggests that the most intensely expressing cells
are the dorsal compartment (DC) somatic motor neurons,
which have been mapped in late larvae by Lacalli and Kelly
(1999) and which express the motor neuron marker Islet
Copyright © 2000 by Academic Press. All rights of reproduction in any form reserved.
29
Amphioxus Presumptive Neural Plate Markers
(Jackman et al., 2000). Like the most intensely labeled cells
in Fig. 5K, the DC cells are located in pairs that are
staggered left to right on either side of the floor plate. There
are two pairs in somite 2 and one pair per somite in 3, 4, and
5. The DC cells innervate the superficial muscle cells,
which are responsible for slow movement. The ventral
compartment somatic motor neurons are less segmentally
arranged and extend farther forward than the DC cells.
These may be the more weakly AmphiNeurogeninexpressing cells visible in Fig. 5K.
In vertebrates, neurogenin is also one of the first transcription factors expressed in prospective motor neurons
(Eisen, 1998, 1999). In addition, other genes, like islet, have
conserved expression in presumptive motor neurons in
amphioxus, vertebrates, and also Drosophila (Thor and
Thomas, 1997; reviewed in Eisen, 1998, 1999; Jackman et
al., 2000). Moreover, Sonic hedgehog (shh), which can
induce neurogenin expression and is important for motor
neuron formation (Blader et al., 1997; reviewed in Eisen,
1998), is expressed in the floor plate and underlying notochord in both amphioxus (Shimeld, 1999) and vertebrates.
Nevertheless, the expression of some downstream genes
differs between amphioxus and vertebrates, suggesting that
downstream genetic programs operating in motor neurons
may differ between amphioxus and vertebrates. For example, Nkx2.2 and Pax6 are expressed in subsets of motor
neurons in vertebrates (Eisen, 1998), but not in amphioxus
(Glardon et al., 1998; Holland et al., 1998).
Vertebrate neurogenins are also expressed in precursors of
sensory neurons and interneurons (Ma et al., 1996). Similarly, AmphiNeurogenin is expressed in many dorsal cells
which are unlikely to be motor neurons (Figs. 5M–5O and
5R). Dorsal cells in the cerebral vesicle develop into the
lamellar body, a likely homolog of the pineal eye in vertebrates, and neurons just posterior to it receive input from
the frontal photoreceptor (Lacalli, 1996). More posteriorly
are the photoreceptive Joseph cells (Welsch, 1968); Rhode
cells, apparently involved in swimming; and several types
of apparent sensory neurons, some similar to Rohon–Beard
cells in lamprey larvae (Bone, 1960). Since only the cerebral
vesicle and the motor neurons just posterior to it have been
mapped in detail (Lacalli et al., 1994; Lacalli, 1996; Lacalli
and Kelly, 1999), many of the AmphiNeurogenin-expressing
cells cannot be correlated with particular cell types.
Expression of Sox1/2/3 and neurogenins
outside the CNS
Unlike vertebrate Sox1/2/3 genes, AmphiSox1/2/3 is not
expressed outside the amphioxus CNS during embryogenesis. Vertebrate Sox1/2/3 genes are expressed in some
tissues with likely amphioxus counterparts, including the
endoderm of the gut, branchial arches, nasal epithelium,
and gonad (Collignon et al., 1996; Uwanogo et al., 1995;
Ishii et al., 1998; Uchikawa et al., 1999; Wood et al., 1999).
We did not determine AmphiSox1/2/3 expression in amphioxus gonads since they do not develop until after metamorphosis. In addition, vertebrate Sox1/2/3 genes are ex-
pressed in tissues for which amphioxus apparently lacks a
counterpart, such as the lens and retina, the otic placode,
and several neural crest derivatives (vertebrate Sox1/2/3
genes are not expressed in migrating neural crest) (Vriz et
al., 1996; Zygar et al., 1998; Uchikawa et al., 1999; Wood
and Episkopou, 1999).
The Drosophila Sox1/2/3 homolog Dichaete is also expressed outside the CNS, where it is involved in segmentation, regulating pair-rule genes (Nambu and Nambu, 1996;
Russell et al., 1996; Y. Ma et al., 1998). In contrast, neither
amphioxus nor vertebrate Sox1/2/3 genes are segmentally
expressed in any tissue. Thus, the extraneural expression
domains of Dichaete and vertebrate Sox1/2/3 genes do not
appear comparable. Sea urchins also express SoxB1 in the
larval ectoderm (Kenny et al., 1999); however, this expression seems more comparable to expression of amphioxus
and vertebrate homologs in the dorsal presumptive neuroectoderm than to expression outside the CNS (see below).
Thus, expression of vertebrate Sox1/2/3 homologs outside
the CNS may signify new functions for these genes.
Unlike Sox1/2/3 genes, vertebrate neurogenins are expressed in migrating neural crest cells, as well as in epibranchial placodes and cranial sensory ganglia (Sommer et
al., 1996; Anderson et al., 1997; Q. Ma et al., 1998, 1999;
Perez et al., 1999). In contrast, AmphiNeurogenin is not
expressed outside the CNS in a pattern suggestive of migrating neural crest or neural crest derivatives. Although
amphioxus appears to have an evolutionary precursor of
neural crest in the cells at the edges of the neural plate and
adjacent nonneural ectoderm (reviewed in Baker and
Bronner-Fraser, 1997), cells in that region do not appear to
migrate as individuals (N. D. Holland et al., 1996; reviewed
in Holland and Holland, 1999). In contrast, neurogenins are
expressed in epidermal chemosensory tissues not only in
vertebrates but also in amphioxus and Drosophila. For
example, both the timing and the expression of AmphiNeurogenin and AmphiPax6 (but not, as noted above,
AmphiSox1/2/3) in the anterior epidermis, which may be
chemosensory (Baatrup, 1981; Lacalli et al., 1999a), are
similar to those of their vertebrate homologs in the olfactory epithelium (Cau et al., 1997; Glardon et al., 1998).
AmphiNeurogenin is also probably expressed in precursor
cells of the ciliary tuft, near the mouth, for which a sensory
function has been proposed (Stokes and Holland, 1995;
Lacalli et al., 1999b), while tap, the Drosophila neurogenin
homolog, is expressed in chemosensory organs (Gautier et
al., 1997; Ledent et al., 1998). These comparisons suggest an
inheritance of an ancient and conserved program for differentiation of chemosensory cells in chordates and arthropods.
In addition, expression of AmphiNeurogenin and the
amphioxus insulin-like peptide (P. W. H. Holland et al.,
1997; L. Z. Holland and S. Chan, unpublished) suggests that
cells in the middle region of the simple tubular gut of
amphioxus may be homologous to the pancreatic islet cells
of vertebrates, which express insulin and neurogenin3
(Sommer et al., 1996; Apelqvist, 1999). However, the Dro-
Copyright © 2000 by Academic Press. All rights of reproduction in any form reserved.
30
Holland et al.
sophila neurogenin homolog, tap, is apparently not expressed in the gut and its derivatives (Gautier et al., 1997;
Sommer et al., 1996), suggesting that new roles for neurogenins in gut development may have evolved within the
chordates.
Sox1/2/3 and neurogenin Genes: Insights into
Evolution of Bilaterian Nervous Systems
It is now generally accepted that the genetic mechanism
involving antagonism of BMP2/4/dpp by chordin/sog for
distinguishing neuroectoderm and nonneural ectoderm is
evolutionarily conserved in Drosophila and vertebrates.
The present study shows that expression of two genes
(Sox1/2/3 and neurogenin) with very early roles in the
presumptive neuroectoderm is conserved between amphioxus and vertebrates and to some extent between amphioxus and Drosophila as well (Bush et al., 1996; Nambu
and Nambu, 1996; Russell et al., 1996; Q. Ma et al., 1998).
Factors controlling expression of Sox1/2/3 homologs in
amphioxus and Drosophila have not been determined.
However, in vertebrates, Sox2 transcription is up-regulated
by chordin and suppressed by BMP4 (Sasai et al., 1995;
Mizuseki et al., 1998; reviewed in Chitnis, 1999). Even in
sea urchins, which are nonchordate deuterostomes without
a neural plate, Sox1/2/3 and BMP2/4 homologs appear to
play roles in axial patterning (Angerer and Angerer, 1999,
2000; Kenny et al., 1999).
In vertebrates, neurogenin appears to be not far downstream of Sox1/2/3. In amphioxus the two genes are coexpressed in most cells of the presumptive neuroectoderm. In
vertebrates, Sox1/2/3 homologs cooperate with partners
like Oct3 in activating genes such as FGF which in turn can
induce expression of neurogenin-1 and nerve cell differentiation (Vriz et al., 1996; Ambrosetti et al., 1997; Mizuseki
et al., 1998; Streit et al., 1998). Correspondingly, the Drosophila Sox1/2/3 homolog Dichaete also appears to function in nerve cell induction (Nambu and Nambu, 1996).
Thus, it seems likely that in the presumptive neuroectoderm, genetic pathways leading from chordin to Sox1/2/3
and thence to neurogenin predate the deuterostome–
protostome split. In contrast, expression of some genes in
presumptive neuroectoderm is not conserved between Drosophila and vertebrates. For example, neural expression
patterns of Snail and Distalless are similar in vertebrates
and amphioxus (N. D. Holland et al., 1996; Langeland et al.,
1998), but not in Drosophila (Mann, 1994). Moreover, while
ZicR-1 is induced by chordin and coexpressed with Sox2 in
the vertebrate neuroectoderm, its Drosophila homolog,
odd-paired, is a segmentation gene, which influences expression of genes such as en and wg involved in specifying
neuroblast identity in each segment (Benedyk et al., 1994;
Natkata et al., 1997; Bhat, 1999). Zic/odd-paired homologs
have not been cloned from amphioxus, but neither Wnt nor
engrailed genes are segmentally expressed in the nerve cord
(L. Z. Holland et al., 1997; Schubert et al., 2000). Thus,
while the present research helps to address the question
raised by Bier (1997) as to what degree conserved versus
organism-specific pathways are involved in the first steps of
neurogenesis, a more thorough comparison of the expression and function of genes in presumptive neuroectoderm
in amphioxus and other nonmodel systems is in order.
In summary, expression of Sox1/2/3 and neurogenin in
the CNS appears to be highly conserved in evolution.
Together with the evolutionary conservation of genetic
pathways involving BMP2/4 and chordin for distinguishing
neural from nonneural ectoderm, these results lend additional support to the idea that the dorsal nerve cord of
chordates and the ventral nerve cord of insects are homologous structures that evolved from the nerve cord of an
ancestral bilaterian (Holley et al., 1995; Schmidt et al.,
1995; De Robertis and Sasai, 1996; Arendt and Nübler-Jung,
1997).
ACKNOWLEDGMENTS
We thank J. M. Lawrence of the University of South Florida,
Tampa, for providing facilities for summer field research on amphioxus. This research was supported by NSF Grants IBN 96309938 and INT 97-07861 to N.D.H. and L.Z.H and NASA–Ames
Grant NAG 2-376 to L.Z.H.
REFERENCES
Akazawa, C., Ishibashi, M., Shimizu, C., Nakanishi, S., and
Kageyama, R. (1995). A mammalian helix-loop-helix factor structurally related to the product of Drosophila proneural gene
atonal is a positive transcriptional regulator expressed in the
developing nervous system. J. Biol. Chem. 270, 8730 – 8738.
Ambrosetti, D.-C., Basilico, C., and Dailey, L. (1997). Synergistic
activation of the fibroblast growth factor 4 enhancer by Sox2 and
Oct-2 depends on protein–protein interactions facilitated by a
specific spatial arrangement of factor binding sites. Mol. Cell.
Biol. 17, 6321– 6329.
Anderson, D. J., Groves, A., Lo, L., Ma, Q., Rao., M., Shah, N. M.,
and Sommer, L. (1997). Cell lineage determination and the
control of neuronal identity in the neural crest. Cold Spring
Harbor Symp. Quant. Biol. 62, 493–504.
Angerer, L. M., and Angerer, R. C. (1999). Regulative development
of the sea urchin embryo: Signalling cascades and morphogen
gradients. Semin. Cell Dev. Biol. 10, 327–334.
Angerer, L. M., and Angerer, R. C. (2000). Animal–vegetal axis
patterning mechanisms in the early sea urchin embryo. Dev.
Biol. 218, 1–12,
Apelqvist, Å., Li, H., Sommer, L., Beatus, P., Anderson, D. J., Honjo,
T., Hrabe de Angelis, M., Lendahl, U, and Edland, H. (1999).
Notch signalling controls pancreatic cell differentiation. Nature
400, 877– 881.
Arendt, D., and Nübler-Jung, K. (1997). Dorsal or ventral: Similarities in fate maps and gastrulation patterns in annelids, arthropods and chordates. Mech. Dev. 61, 7–21.
Baatrup, E. (1981). Primary sensory cells in the skin of amphioxus
Branchiostoma lanceolatum (P). Acta Zool. 62, 147–157.
Baker, C. V. H., and Bronner-Fraser, M. (1997). The origins of the
neural crest. Part II. An evolutionary perspective. Mech. Dev. 69,
13–29.
Benedyk, M. J., Mullen, J. R., and DiNardo, S. (1994). Odd-paired:
A zinc finger pair-rule protein required for the timely activation
Copyright © 2000 by Academic Press. All rights of reproduction in any form reserved.
31
Amphioxus Presumptive Neural Plate Markers
of engrailed and wingless in Drosophila embryos. Genes Dev. 8,
105–107.
Bhat, K. M. (1999). Segment polarity genes in neuroblast formation
and identity specification during Drosophila neurogenesis.
Trends Genet. 21, 472– 485.
Bianchi, M. E., and Beltrame, M. (1998). Protein complexes ‘98.
Flexing DNA: HMG-box proteins and their partners. Am. J.
Hum. Genet. 63, 1573–1577.
Bier, E. (1997). Anti-neural-inhibition: A conserved mechanism for
neural induction. Cell 89, 681– 684.
Blader, P., Fischer, N., Gradwohl, Guillemot, F., and Strähle, U.
(1997). The activity of neurogenin1 is controlled by local cues in
the zebrafish embryo. Development 124, 4557– 4569.
Bone, Q. (1960). The central nervous system in amphioxus.
J. Comp. Neurol. 115, 27–51.
Bush, A., Hiromi, Y., and Cole, M. (1996). Biparous: A novel bHLH
gene expressed in neuronal and glial precursors in Drosophila.
Dev. Biol. 180, 759 –772.
Cau, E., Gradwohl, G., Fode, C., and Guillemot, F. (1997). Mash1
activates a cascade of bHLH regulators in olfactory neuron
progenitors. Development 124, 1611–1621.
Chiaramello, A., Neuman, K., Palm, K., Metsis, M., and Neuman,
T. (1995a). Helix-loop-helix transcription factors mediate activation and repression of the p75LNGFR gene. Mol. Cell. Biol. 15,
6036 – 6045.
Chiaramello, A., Soosaar, A., Neuman, T., and Zuber, M. X.
(1995b). Differential expression and distinct DNA-binding specificity of ME1a and ME2 suggest a unique role during differentiation and neuronal plasticity. Mol. Brain Res. 29, 107–118.
Chiaramello, A., Neuman, T., Peavy, D. R., and Zuber, M. X.
(1996). The GAP-43 gene is a direct downstream target of the
basic helix-loop-helix transcription factors. J. Biol. Chem. 271,
22035–22043.
Chitnis, A. B. (1999). Control of neurogenesis—Lessons from frogs,
fish and flies. Curr. Opin. Neurobiol. 9, 18 –25.
Collignon, J., Sockanathan, S., Hacker, A., Cohen-Tannoudji, M.,
Norris, D., Rastan, S., Stevanovic, M., Goodfellow, P. N., and
Lovell-Badge, R. (1996). A comparison of the properties of Sox-3
with Sry and two related genes, Sox-1 and Sox-2. Development
122, 509 –520.
De Robertis, E. M., and Sasai, Y. (1996). A common plan for
dorsoventral patterning in Bilateria. Nature 380, 37– 40.
Eisen, J. S. (1998). Genetic and molecular analyses of motoneuron
development. Curr. Opin. Neurobiol. 8, 697–704.
Eisen, J. S. (1999). Patterning motor neurons in the vertebrate
nervous system. Trends Neurosci. 22, 321–326.
Fraidenraich, D., Lang, R., and Basilico, C. (1998). Distinct regulatory elements govern Fgf4 gene expression in the mouse blastocyst, myotomes, and developing limb. Dev. Biol. 204, 197–209.
Fode, C., Gradwohl, G., Morin, X., Dierich, A., Lemeur, M.,
Goridis, C., and Guillemot, F. (1998). The bHLH protein neurogenin 2 is a determination factor for epibranchial placode-derived
sensory neurons. Neuron 20, 483– 494.
Gautier, P., Ledent, V., Massaer, M., Dambley-Chaudière, and
Ghysen, A. (1997). Tap, a Drosophila bHLH gene expressed in
chemosensory organs. Gene 191, 15–21.
Glardon, S., Holland, L. Z., Gehring, W. J., and Holland, N. D.
(1998). Isolation and developmental expression of the amphioxus
Pax-6 gene AmphiPax-6: Insights into eye and photoreceptor
evolution. Development 125, 2701–2710.
Gradwohl, G., Fode, C., and Guillemot, F. (1996). Restricted
expression of a novel murine atonal-related bHLH protein in
undifferentiated neural precursors. Dev. Biol. 180, 227–241.
Hirakow, R., and Kajita, N. (1994). Electron microscopic study of
the development of amphioxus Branchiostoma belcheri tsingtauensei: The neurula and larva. Acta Anat. Nippon 69, 1–13.
Holland, L. Z., Holland, P. W. H., and Holland, N. D. (1996).
Revealing homologies between body parts of distantly related
animals by in situ hybridization to developmental genes: Amphioxus versus vertebrates. In “Molecular Zoology: Advances,
Strategies, and Protocols” (J. D. Ferraris and S. R. Palumbi, Eds.),
pp. 267–282. Wiley–Liss, New York.
Holland, L. Z., Kene, M., Williams, N. A., and Holland, N. D.
(1997). Sequence and embryonic expression of the amphioxus
engrailed gene (AmphiEn): The metameric pattern of transcription resembles that of its segment-polarity homolog in Drosophila. Development 124, 1723–1732.
Holland, L. Z., Venkatesh, T. V., Gorlin, A., Bodmer, R., and
Holland, N. D. (1998). Characterization and developmental expression of AmphiNk2-2, an NK2 class homeobox gene from
amphioxus (Phylum Chordata; Subphylum Cephalochordata).
Dev. Genes Evol. 208, 100 –105.
Holland, L. Z., and Holland, N. D. (1999). Chordate origins of the
vertebrate central nervous system. Curr. Opin. Neurobiol. 9,
596 – 602.
Holland, N. D., and Holland, L. Z. (1993). Embryos and larvae of
invertebrate deuterostomes. In “Essential Developmental Biology: A Practical Approach” (C. Stern and P. W. H. Holland, Eds.),
pp. 21–32. IRL Press, Oxford.
Holland, N. D., Panganiban, G., Henyey, E. L., and Holland, L. Z.
(1996). Sequence and developmental expression of AmphiDll, an
amphioxus Distal-less gene transcribed in the ectoderm, epidermis and nervous system: Insights into evolution of craniate
forebrain and neural crest. Development 122, 2911–2920.
Holland, P. W. H., Garcia-Fernàndez, J., Williams, N. A., and
Sidow, A. (1994). Gene duplications and the origins of vertebrate
development. Development Suppl. 120, 125–133.
Holland, P. W. H., Patton, S. J., Brooke, N. M., and GarciaFernàndez, J. (1997). Genetic patterning of the ectoderm and
endoderm in amphioxus: From homeobox genes to hormones. In
“Advances in Comparative Endocrinology: Proceedings of the
13th International Congress on Comparative Endocrinology” (S.
Kawashima and S. Kikuyama, Eds.), pp. 247–252. Bologna.
Holley, S. A., Jackson, P. D., Sasai, Y., Lu, B., De Robertis, E. M.,
Hoffmann, F. M., and Ferguson, E. L. (1995). A conserved system
for dorsal–ventral patterning in insects and vertebrates involving
sog and Chordin. Nature 376, 249 –377.
Ishii, Y., Rex, M., Scotting, P. J., and Yasugi, S. (1998). Regionspecific expression of chicken Sox2 in the developing gut and
lung epithelium: Regulation by epithelial–mesenchymal interactions. Dev. Dyn. 213, 464 – 475.
Jackman, W. R., Langeland, J. A., and Kimmel, C. B. (2000). islet
reveals segmentation in the amphioxus hindbrain homolog. Dev.
Biol. 220,16 –26.
Jaźwińska, A., Rushlow, C., and Roth, S. (1999). The role of brinker
in mediating the graded response to Dpp in early Drosophila
embryos. Development 126, 3323–3334.
Kamachi, Y., Uchikawa, M., Collignon, J., Lovell-Badge, R., and
Kondoh, H. (1998). Involvement of Sox1, 2 and 3 in the early and
subsequent molecular events of lens induction. Development
125, 2521–2532.
Kenny, A. P., Kozlowski, D., Oleksy, D. W., Angerer, L. M., and
Angerer, R. C. (1999). SpSoxB1, a maternally encoded transcription factor asymmetrically distributed among early sea urchin
blastomeres. Development 126, 5473–5483.
Copyright © 2000 by Academic Press. All rights of reproduction in any form reserved.
32
Holland et al.
Kim, C.-H., Bae, Y.-K., Yamanaka, Y., Yamashita, S., Shimizu, T.,
Fujii, R., Park, H.-C., Yeo, S.-Y., Huh, T.-L., Hibi, M., and Hirano,
R. (1997). Overexpression of neurogenin induces ectopic expression of HuC in zebrafish. Neurosci. Lett. 239, 113–116.
Korzh, V., Sleptsova, I., Liao, J., He, J., and Gong, Z. (1998).
Expression of zebrafish bHLH genes ngn1 and nrd defines distinct stages of neural differentiation. Dev. Dyn. 213, 92–104.
Kusakabe, R., Kusakabe, T., Satoh, N., Holland, N. D., and Holland,
L. Z. (1997). Differential gene expression and intracellular
mRNA localization of amphioxus actin isoforms throughout
development: Implications for conserved mechanisms of chordate development. Dev. Genes Evol. 207, 203–215.
Lacalli, T. C. (1996). Frontal eye circuitry, rostral sensory pathways
and brain organization in amphioxus larvae: Evidence from 3D
reconstructions. Philos. Trans. R. Soc. London B 351, 243–263.
Lacalli, T. C., Holland, N. D., and West, J. E. (1994). Landmarks in
the anterior central nervous system of amphioxus larvae. Philos.
Trans. R. Soc. London B 344, 165–185.
Lacalli, T. C., Gilmour, T. H. J., and Hou, S. (1999a). A reexamination of the epithelial sensory cells of amphioxus (Branchiostoma). Acta Zool. 80, 125–134.
Lacalli, T. C., and Kelly, S. J. (1999). Somatic motoneurons in
amphioxus larvae: Cell types, cell position and innervation
patterns. Acta Zool. 80, 113–124.
Lacalli, T. C., Gilmour, T. H. J., and Kelly, S. J. (1999b).The oral
nerve plexus in amphioxus larvae: Function, cell types and
phylogenetic significance. Proc. R. Soc. Biol. Sci. Ser. B 266,
1461–1470.
Langeland, J. A., Tomsa, J. M., Jackman, W. R., and Kimmel, C. B.
(1998). An amphioxus snail gene: Expression in paraxial mesoderm and neural plate suggests a conserved role in patterning the
chordate embryo. Dev. Genes Evol. 208, 569 –577.
Ledent, V., Gaillard, F., Gautier, P., Ghysen, A., and DamblyChaudiere, C. (1998). Expression and function of tap in the
gustatory and olfactory organs of Drosophila. Int. J. Dev. Biol. 42,
163–170.
Lee, J. E. (1997). Basic helix-loop-helix genes in neural development. Curr. Opin. Neurobiol. 7, 13–20.
Ma, Q., Kintner, C., and Anderson, D. J. (1996). Identification of
neurogenin, a vertebrate neuronal determination gene. Cell 87,
43–52.
Ma, Q., Sommer, L., Cserjesi, P., and Anderson, D. J. (1997). Mash1
and neurogenin1 expression patterns define complementary domains of neuroepithelium in the developing CNS and are correlated with regions expressing notch ligands. J. Neurosci. 17,
3644 –3652.
Ma, Q., Chen, Z., Del Barco Barrantes, I., De La Pompa, J.-L., and
Anderson, D. J. (1998). neurogenin1 is essential for the determination of neuronal precursors for proximal cranial sensory ganglia. Neuron 20, 469 – 482.
Ma, Q., Fode, C., Guillemot, F., and Anderson, D. J. (1999).
Neurogenin1 and Neurogenin2 control two distinct waves of
neurogenesis in developing dorsal root ganglia. Genes Dev. 13,
1717–1728.
Ma, Y., Niemitz, E. L., Nambu, P. A., Shan, X., Sackerson, C.,
Fujioka, M., Goto, T., and Nambu, J. R. (1998). Gene regulatory
functions of Drosophila Fish-hook, a high mobility group domain
Sox protein. Mech. Dev. 73, 169 –182.
Malas, S., Duthie, S., Deloukas, P., and Episkopou, V. (1999). The
isolation and high-resolution chromosomal mapping of human
SOX14 and SOX21, two members of the SOX gene family related
to SOX1, SOX2, and SOX3. Mamm. Genome 10, 934 –937.
Mann, R. S. (1994). Engrailed-mediated repression of Ultrabithorax
is necessary for the parasegment 6 identity in Drosophila.
Development 120, 3205–3212.
Marqués, G., Musacchio, M., Shimell, M. J., WünnenbergStapleton, K., Cho, K. W. Y., and O’Connor, M. B. (1997).
Production of a DPP activity gradient in the early Drosophila
embryo through the opposing actions of the SOG and TLD
proteins. Cell 91, 417– 426.
McCormick, M. B., Tamimi, R. M., Snider, L., Asakura, A.,
Bergstrom, D., and Tapscott, S. J. (1996). NeuroD2 and neuroD3:
Distinct expression patterns and transcriptional activation potentials within the neuroD gene family. Mol. Cell. Biol. 16,
5792–5800.
Metsis, M., Timmusk, T., Allikmets, R., Saarma, M., and Persson,
H. (1992). Regulatory elements and transcriptional regulation by
testosterone and retinoic acid of the rat nerve growth factor
receptor promoter. Gene 121, 247–254.
Mizuseki, K., Kishi, M., Matsui, M., Nakanishi, S., and Sasai, Y.
(1998). Xenopus Zic-related-1 and Sox-2, two factors induced by
chordin, have distinct activities in the initiation of neural
induction. Development 125, 579 –587.
Nakata, K., Nagai, T., Aruga, J., and Mikoshiba, D. (1997). Xenopus
Zic3, a primary regulator both in neural and neural crest development. Proc. Natl. Acad. Sci. USA 94, 11980 –11985.
Nambu, P. A., and Nambu, J. R. (1996). The Drosophila fish-hook
gene encodes a HMG domain protein essential for segmentation
and CNS development. Development 122, 3467–3475.
Panopoulou, G. D., Clark, M. D., Holland, L. Z., Lehrach, H., and
Holland, N. D. (1998). AmphiMBP2/4, an amphioxus bone morphogenetic protein closely related to Drosophila decapentaplegic
and vertebrate BMP2 and BMP4: Insights into evolution of
dorsoventral axis specification. Dev. Dyn. 213, 130 –139.
Penzel, R., Oschwald, R., Chen, Y., Tacke, L., and Grunz, H. (1997).
Characterization and early embryonic expression of a neural
specific transcription factor xSox3 in Xenopus laevis. Int. J. Dev.
Biol. 41, 667– 677.
Perez, S. E., Rebelo, S., and Anderson, D. J. (1999). Early specification of sensory neuron fate revealed by expression and function
of neurogenins in the chick embryo. Development 126, 1715–
1728.
Pevny, L. A., Sockanathan, S., Placzek, M., and Lovell-Badge, R.
(1998). A role for SOX1 in neural determination. Development
125, 1967–1978.
Rex, M., Orme, A., Uwanogho, D., Tointon, K., Wigmore, P. M.,
Sharpe, P. T., and Scotting, P. J. (1997). Dynamic expression of
chicken Sox2 and Sox3 genes in ectoderm induced to form neural
tissue. Dev. Dyn. 209, 323–333.
Russell, S. R. H., Sanchez-Soriano, N., Wright, C. R., and Ashburner, M. (1996). The Dichaete gene of Drosophila melanogaster encodes a SOX-domain protein required for embryonic
segmentation. Development 122, 3669 –3676.
Sakai, Y., Hiraoka, Y., Konishi, M., Ogawa, M., and Aiso, S. (1997).
Isolation and characterization of Xenopus laevis xSox-B1 cDNA.
Arch. Biochem. Biophys. 346, 1– 6.
Sasai, Y., Lu, B., Steinbeisser, H., and De Robertis, E. M. (1995).
Regulation of neural induction by the Chd and Bmp-4 antagonistic patterning signals in Xenopus. Nature 376, 333–336.
Sasai, Y., and De Robertis, E. M. (1997). Ectodermal patterning in
vertebrate embryos. Dev. Biol. 182, 5–20.
Schmidt, J., Francois, V., Bier, E., and Kimelman, D. (1995).
Drosophila short gastrulation induces an ectopic axis in Xenopus: Evidence for conserved mechanisms of dorsal–ventral patterning. Development 121, 4319 – 4328.
Copyright © 2000 by Academic Press. All rights of reproduction in any form reserved.
33
Amphioxus Presumptive Neural Plate Markers
Schubert, M., Holland, L. Z., and Holland, N. D. (2000). Characterization of two amphioxus Wnt genes AmphiWnt4 and
AmphiWnt7b with early expression in the developing central
nervous system. Dev. Dyn. 217, 205–215.
Shimeld, S. M. (1999). The evolution of the hedgehog gene family in
chordates: Insights from amphioxus hedgehog. Dev. Genes Evol.
209, 40 – 47.
Sommer, L., Ma, Q., and Anderson, D. J. (1996). Neurogenins, a
novel family of atonal-related bHLH transcription factors, are
putative mammalian neuronal determination genes that reveal
progenitor cell heterogeneity in the developing CNS and PNS.
Mol. Cell. Neurosci. 8, 221–241.
Stokes, M. D. (1997). Larval locomotion of the lancelet Branchiostoma floridae. J. Exp. Biol. 200, 1661–1680.
Stokes, M. D., and Holland, N. D. (1995). Embryos and larvae of a
lancelet, Branchiostoma floridae, from hatching through metamorphosis: Growth in the laboratory and external morphology.
Acta Zool. 76, 105–120.
Streit, A., Sockanathan, S., Pérez, L., Rex, M., Scotting, P. J., Sharpe,
P. T., Lovell-Badge, R., and Stern, C. D. (1997). Preventing the
loss of competence for neural induction: HGF/SF, L5 and Sox2.
Development 124, 1191–1202.
Streit, A., Lee, K. J., Woo, I., Roberts, C., Jessell, T. M., and Stern,
C. D. (1998). Chordin regulates primitive streak development
and the stability of induced neural cells, but is not sufficient for
neural induction in the chick embryo. Development 125, 507–
519.
Streit, A., and Stern, C. D. (1999). Neural induction. A bird’s eye
view. Trends Genet. 15, 20 –24.
Takke, C., Dornseifer, P., Weizäcker, V., and Campos-Ortega, J. A.
(1999). Her4, a zebrafish homologue of the Drosophila neurogenic gene Espl, is a target of Notch signalling. Development 126,
1811–1821.
Thor, S., and Thomas, J. A. B. (1997). The Drosophila islet gene
governs axon pathfinding and neurotransmitter identity. Neuron
18, 397– 409.
Uchikawa, M., Kamachi, Y., and Kondoh, H. (1999). Two distinct
subgroups of Group B Sox genes for transcriptional activators and
repressors: Their expression during embryonic organogenesis of
the chicken. Mech. Dev. 84, 103–120.
Uwanogho, D., Rex, M., Cartwright, E. J., Pearl, G., Healy, C.,
Scotting, P. J., and Sharpe, P. T. (1995). Embryonic expression of
the chicken Sox2, Sox3 and Sox11 genes suggests an interactive
role in neuronal development. Mech. Dev. 49, 23–36.
Vriz, S., Joly, C., Boulekbache, H., and Condamine, H. (1996).
Zygotic expression of the zebrafish Sox-19, an HMG boxcontaining gene, suggests an involvement in central nervous
system development. Mol. Brain Res. 40, 221–228.
Weinstein, D. C., and Hemmeti-Brivanlou, H. (1997). Neural induction in Xenopus laevis: Evidence for the default model. Curr.
Opin. Neurobiol. 7, 7–12.
Wegner, M. (1999). From head to toes: The multiple facets of Sox
proteins. Nucleic Acids Res. 27, 1409 –1420.
Welsch, U. (1968). Die Feinstruktur der Josephschen Zellen im
Gehirn von Amphioxus. Z. Zellforsch. 86, 252–261.
Wood, H. B., and Episkopou, V. (1999). Comparative expression of
the mouse Sox1, Sox2 and Sox3 genes from pre-gastrulation to
early somite stages. Mech. Dev. 86, 197–201.
Yasui, K., Tabata, S., Ueki, T., Uemura, M., and Zhang, S.-C. (1998).
Early development of the peripheral nervous system in a lancelet
species. J. Comp. Neurol. 393, 415– 425.
Zhang, S.-C., Holland, N. D., and Holland, L. Z. (1997).Topographic
changes in nascent and early mesoderm in amphioxus embryo
studies by Dil labeling and by in situ hybridization for a
Brachyury gene. Dev. Genes Evol. 206, 532–535.
Zygar, C. A., Cook, T. L., and Grainger, R. M. (1998). Gene
activation during early stages of lens induction in Xenopus.
Development 125, 3509 –3515.
Received for publication November 30, 1999
Revised May 3, 2000
Accepted May 23, 2000
Copyright © 2000 by Academic Press. All rights of reproduction in any form reserved.