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Developmental Biology 223, 399 – 410 (2000)
doi:10.1006/dbio.2000.9756, available online at on
Pax1/Pax9-Related Genes in an Agnathan
Vertebrate, Lampetra japonica: Expression Pattern
of LjPax9 Implies Sequential Evolutionary Events
toward the Gnathostome Body Plan
Michio Ogasawara,* Yasuyo Shigetani,† Shigeki Hirano,‡ Nori Satoh,*
and Shigeru Kuratani† 1
*Department of Zoology, Graduate School of Science, Kyoto University, Oiwake-cho,
Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan; †Department of Biology, Faculty of Science,
Okayama University, 3-1-1 Tsushimanaka, Okayama 700-8530, Japan; and ‡Department of
Anatomy, Niigata University School of Medicine, 1 Asahimachi, Niigata 951-8510, Japan
Among the transcription factor gene families, Pax genes play important and unique roles in morphological patterning of
animal body plans. Of these, Group I Pax genes (Pax1 and Pax9) are expressed in the endodermal pharyngeal pouches in
many groups of deuterostomes, and vertebrates seem to have acquired more extensive expression domains in embryos. To
understand the evolution of Pax1/Pax9-related genes in basal groups of vertebrates, their cognates were isolated from the
Japanese marine lamprey, Lampetra japonica. RT-PCR of larval lamprey cDNA yielded two different fragments containing
vertebrate Pax1- and Pax9-like paired domains. The Pax9 orthologue was isolated and named LjPax9. Whole-mount in situ
hybridization revealed that this gene was expressed in endodermal pharyngeal pouches, mesenchyme of the velum (the oral
pumping apparatus) and the hyoid arch, and the nasohypophysial plate, but not in the somitic mesoderm of the lamprey
embryo. These expression patterns could be regarded as a link between the basal chordates and the gnathostomes and are
consistent with the phylogenetic position of the lamprey. Especially, the appearance of neural crest seemed to be the basis
of velar expression. Homology of the velum and the jaw is also discussed based on the LjPax9 expression in the first
pharyngeal pouch and in the velar mesenchyme. We conclude that Pax9 genes have sequentially expanded into new
expression domains through evolution as more complicated body plans emerged. © 2000 Academic Press
Key Words: Pax genes; lamprey; embryo; pharynx; endoderm; evolution.
The genes of the Pax family encode transcription factors
containing a paired domain and are involved in various
aspects of animal morphogenesis (reviewed by Goulding,
1992; Gruss and Walther, 1992; Strachan and Read, 1994;
St-Onge et al., 1995; Dahl et al., 1997). Nine members of
this gene family are recognized in mammals, and these can
be further classified into four subgroups (Groups I to IV)
The first (M.O.) and second (Y.S.) authors contributed equally to
this study.
To whom correspondence should be addressed at the Department of Biology, Faculty of Science, Okayama University, 3-1-1
Tsushimanaka, Okayama 700-8530, Japan. Fax: ⫹81-86-251-7876.
E-mail: [email protected]
0012-1606/00 $35.00
Copyright © 2000 by Academic Press
All rights of reproduction in any form reserved.
based on structural features and amino acid sequences. This
suggests hierarchical duplications in the evolutionary history of this family, providing an ideal topic for studies of
evolutionary developmental biology.
In vertebrates, Pax genes occupy a central position in
developmental cascades to pattern the body plan. For example, Pax3, Pax7 (Group III), and Pax6 (Group IV) genes
are involved in regional specification of the neural tube
(Walther and Gruss, 1991; Jostes et al., 1991; Goulding et
al., 1993; Conway et al., 1997; Ericson et al., 1997; reviewed
by Chalepakis et al., 1993), Pax2, -5, and -8 (Group II) genes
are expressed in the mid– hindbrain boundary as isthmic
organizer components in anteroposterior patterning of the
neuraxis (Asano and Gruss, 1992), and many of the Pax
family members also function in establishment of a number
Ogasawara et al.
of specific cell lineages and in organogenesis (Koseki et al.,
1993; Barnes et al., 1996; Wallin et al., 1994).
Comparative analyses of Pax gene family expression
patterns have suggested that the developmental functions
of each orthologue are rather well conserved within vertebrates (Krauss et al., 1991; Püschel et al., 1992; Glardon et
al., 1997, 1998). In larger scale evolution including changes
in the body plan, however, regulatory genes usually have
acquired additional expression domains in crown groups
that exhibit more complex body architecture (Wada et al.,
1998). Of the Pax genes, Pax1 and -9 (Group I) are expressed
in pharyngeal slits of vertebrate embryos (Wallin et al.,
1996; Müller et al., 1996), a pattern that appears to be
conserved in distantly related animal embryos belonging to
the deuterostomes (Holland and Holland, 1995; Ogasawara
et al., 1999). Similar expression patterns are retained in the
urochordates (Ciona intestinalis; Ogasawara et al., 1999)
and cephalochordates (Holland and Holland, 1995, 1996), in
which branchial arches show diverse patterns of development. A Pax1/Pax9 orthologue was also found in a hemichordate, Ptychodera flava, and its expression in pharyngeal slits supports the homology of gill slits of this animal
with those in chordates (see Ogasawara et al., 1999). In
vertebrates, however, the gene expression domains include
more structures than in other animal groups, such as the
somite-derived skeletogenic mesoderm, limb bud, mandibular arch, and pharyngeal pouch and its derivatives, for
which these genes are involved in organogenesis (Hofmann
et al., 1998; Peters et al., 1998; Henderson et al., 1999). In
this line of evolutionary studies, the expression of the
Pax1/Pax9 group in the embryonic lamprey is now a missing link. Of particular interest is the number of genes or
history of duplication of the genes, since only gnathostomes
are known to possess two members in Group I (Dahl et al.,
Agnathans are regarded as a basal group of the vertebrate
lineage and show a typical bilateral pattern of pharyngeal
pouch development specific to vertebrates. In the present
study, we isolated members of the Pax1/Pax9 gene family
in a Japanese marine lamprey, Lampetra japonica, and
examined expression patterns of the putative orthologue of
the Pax9 gene in a series of developing embryos.
Embryos. Adult male and female lampreys (L. japonica) were
collected in a tributary of the Miomote River, Niigata, Japan,
during the breeding season (late May through June). The eggs were
artificially fertilized and kept in fresh water at 18°C or in 10%
Steinberg’s solution (Steinberg, 1957) below 23°C. Embryos were
fixed with 4% paraformaldehyde in 0.1 M phosphate-buffered
saline (PFA/PBS). Since embryological development is easily affected by temperature, they were staged morphologically according
to the table of Tahara (1988) for L. reissneri, a brook species of L.
Construction of cDNA libraries and isolation of cDNA clones
for lamprey Pax1/Pax9-related gene. Total RNA was extracted
from the head region of larval L. japonica by the AGPC method
(Chomczynski and Sacchi, 1987). Poly(A) ⫹ RNA was purified using
Oligotex dT30 beads (Roche, Tokyo, Japan). Complementary DNA
was synthesized and constructed as described previously (Ogasawara et al., 1996).
A L. japonica larval head cDNA library was constructed using
the Uni-ZAP II vector (Stratagene). The sense-strand oligonucleotide
5⬘-GTNAA(CT)CA(AG)(CT)TNGGNGGNGTNTT-3⬘, which corresponds to the amino acid sequence VNQLGGVF, and the antisense oligonucleotide primer
Pax-R3, 5⬘-A(AG)I(CT)(GT)(AG)TCIC(GT)IAT(CT)TCCCAI(CG)I(AG)AA-3⬘, which corresponds to the amino acid sequence FAWEIRDKL, were synthesized based on conserved paired domains
from Caenorhabditis elegans, Drosophila, tunicates, amphioxus,
and vertebrates. Using the poly(A) ⫹ RNA extracted from the head
region of larval lamprey, target fragments were amplified by
RT-PCR and two types of PCR products were obtained. These PCR
fragments were randomly labeled with [ 32P]dCTP (Amersham,
Buckingham, UK), and 5.5 ⫻ 10 5 phages of a cDNA library were
screened by hybridization in 6⫻ SSPE, 0.1% SDS, 1⫻ Denhardt’s
solution, 50% formamide at 42°C for 16 h and washing in 2⫻
SSC– 0.1% SDS at 50°C for 30 min, 1⫻ SSC– 0.1% SDS at 50°C for
30 min, 0.1⫻ SSC– 0.1% SDS at 50°C for 30 min. Isolated clones
were sequenced using an ABI Prism 377 DNA Sequencer (Perkin–
Elmer, Roche, NJ).
Sequence comparisons and molecular phylogenetic analyses.
Sequences were aligned using the SeqQpp 1.9 manual aligner for
Macintosh (Gilbert, 1993). Phylogenetic analyses were performed
on the amino acid sequences of the paired domain. Estimation of
molecular phylogeny was carried out by the neighbor-joining
method (Saitou and Nei, 1987) using the Clustal V (Higgins et al.,
1992) program. Confidence in the phylogeny was assessed by
bootstrap resampling of the data (⫻100) (Felsenstein, 1985).
Northern blotting analyses. Total RNA was isolated by the
AGPC method (Chomczynski and Sacchi, 1987), and poly(A) ⫹ RNA
was purified with Oligotex dT30 beads. Northern blot hybridization was carried out using standard procedures (Sambrook et al.,
1989), and filters were washed under high-stringency conditions
(hybridization, 6⫻ SSPE, 0.1% SDS, 1⫻ Denhardt’s solution, 50%
formamide at 42°C for 16 h; washing, 2⫻ SSC– 0.1% SDS at 60°C
for 30 min, 1⫻ SSC– 0.1% SDS at 60°C for 30 min, 0.1⫻ SSC– 0.1%
SDS at 60°C for 30 min). Entire regions of cDNAs were labeled with
[ 32P]dCTP using a random prime labeling kit (Boehringer Mannheim, Indianapolis, IN) for hybridization probes.
Genomic Southern analyses. High-molecular weight genomic
DNA of L. japonica was extracted from a single adult by the
standard procedure (Sambrook et al., 1989). After exhaustive digestion with EcoRI, HincII, SalI, and BglII, and 1% agarose gel
electrophoresis, the DNA fragments were blotted onto
Hybond-N⫹ nylon membranes (Amersham). The blots were hybridized with random-primed 32P-labeled DNA probes at 42°C for
16 h and washed under high-stringency conditions.
In situ hybridization. L. japonica embryos were fixed in 4%
PFA in 0.1 M PBS, dehydrated, and stored in 100% methanol at
⫺20°C. The digoxigenin-labeled antisense riboprobe was prepared
according to the manufacturer’s instructions (Boehringer Mannheim). After H 2O 2 treatment, specimens were rehydrated and
digested with proteinase K (Merck, Darmstadt, Germany). This
digestion could be replaced by treatment with a detergent mix (1%
IGEPAL, 1% SDS, 0.5% deoxycholate, 50 mM Tris–HCl buffer, pH
Copyright © 2000 by Academic Press. All rights of reproduction in any form reserved.
Pax1/Pax9-Related Genes in the Lamprey
FIG. 1. Comparison of the nucleotide and predicted amino acid sequences of PCR fragments of L. japonica Pax1/Pax9-related genes.
Identical nucleotides are indicated by asterisks. The remarkable triplet nucleotide difference is indicated by the shaded box, and
corresponding amino acid changes, D (asparagine in LjP1-PCRF) and T (threonine), are indicated by arrowheads. The HincII site that is
present only in LjP9-PCR is underlined.
8.0, 1 mM EDTA, 150 mM NaCl). The samples were then postfixed
and incubated in hybridization buffer [50% formamide, 5⫻ SSC,
1% SDS, 50 ␮g/ml yeast tRNA, 50 ␮g/ml heparin sulfate, 0.1%
Chaps, 5 mM EDTA, 1% blocking solution (Boehringer Mannheim)]. Hybridization was performed in freshly prepared hybridization buffer containing 2.0 ␮g/ml probe for overnight (O/N) at 70°C.
After hybridization, the specimens were washed in solution 1 (50%
formamide, 5⫻ SSC, 1% SDS) for 30 min at 70°C and the solution
was substituted gradually by solution 2 (0.5 M NaCl, 10 mM
Tris–HCl, pH 7.5, 0.1% Tween 20). RNase A was added at a final
concentration of 50 ␮g/ml in solution 2 and incubated for 30 min
at room temperature. The embryos were then washed twice for 30
min each in 50% formamide, 2⫻ SSC at 70°C, twice for 30 min
each in 2⫻ SSC/0.3% Chaps at 70°C, and once in 0.2⫻ SSC/0.3%
Chaps for 30 min at 70°C. For immunological detection of digoxigenin epitopes, the embryos were blocked with blocking solution
for 60 min before incubation with 1:4000-diluted antidigoxigenin–AP Fab fragments (Boehringer Mannheim) at 4°C for
O/N. The specimens were then washed five times for 1–2 h each
time and O/N in washing buffer (Boehringer Mannheim) at room
temperature. Alkaline phosphatase activity was detected with
NBT/BCIP in detection buffer (Boehringer Mannheim). The stained
specimens were postfixed in PFA/PBS and photographed. Stage 27
embryos were embedded in Technovit 8100 (Heraeus Kulzer,
Friedrichsdorf, Germany) and sectioned at a thickness of 7 ␮m.
Some of the stained embryos were dissected under the microscope
for observation of the mesodermal expressions.
Pax1/Pax9-Related Genes in L. japonica
RT-PCR using larval lamprey cDNA and degenerate
oligonucleotide primers corresponding to the conserved
paired domain sequences yielded two types of clones containing a 296-bp PCR fragment encoding the Pax1/Pax9related paired domain. Comparison of the nucleotide sequences of these PCR fragments revealed that these
sequences were 94.7% identical (Fig. 1). The most remarkable difference was an in-frame triple-nucleotide displacement of GAC to ACG (Fig. 1) causing an amino acid change
from asparagine, characteristic of Pax1, to threonine, characteristic of Pax9 (cf. Fig. 4A). Molecular phylogenetic
analyses by the neighbor-joining method based on the
amino acid sequences encoded by these fragments revealed
that they were Pax1-like and Pax9-like paired domains (not
shown): the clone containing the Pax1-like paired domain
was named pLjP1-PCRF and that containing the Pax9-like
paired domain was named pLjP9-PCRF. One of the most
distinctive differences was the presence of a HincII site in
pLjP9-PCRF, which was absent in pLjP1-PCRF (Fig. 1). The
HincII site in pLjP9-PCRF was confirmed by sequencing of
the Pax9 cDNA clone and also by Southern blotting analyses (see below).
Genomic Southern blotting analyses using the 32P-labeled
pLjP1-PCRF fragment encoding Pax1-like paired domain as
a probe detected two to three bands in the lanes of the EcoRI
(about 5.8 and 4.3 kb), HincII (about 7.2, 2.2, and 1.5 kb),
SalI (about 14.0, 8.6, and 1.8 kb), and BglII (about 13.0 and
4.9 kb) digests (Fig. 2A). When we used the pLjP9-PCRF
fragment as a probe, the results of genomic Southern
blotting were the same as those obtained with the Pax1-like
probe (not shown). These results suggested that the nucleotide sequences of paired domains were very similar, and
the probes cross-reacted and both genes were detected.
Northern blotting analyses using a 32P-labeled PCR fragment containing the paired domain detected a slightly
broad signal of about 3.4 kb (Fig. 2B).
Isolation and Characterization of L. japonica Pax1/
Pax9-Related Gene of cDNA Clones
Using pLjP1-PCRF and pLjP9-PCRF as probes, we
screened a larval lamprey cDNA library and found eight
Copyright © 2000 by Academic Press. All rights of reproduction in any form reserved.
Ogasawara et al.
FIG. 2. (A) Genomic Southern blotting analysis of the L. japonica
Pax1/Pax9-related genes. Aliquots of genomic DNA isolated from
a single adult were digested separately with EcoRI, HincII, SalI, and
BglII. 10 ␮g of digested genomic DNA was loaded per lane, and
blots were hybridized with the 32P-labeled DNA probes of the
paired domain. The numbers indicate sizes (in kb) of the signals. (B)
Northern blots of poly(A) ⫹ RNA prepared from larval lamprey were
hybridized with the 32P-labeled paired domain DNA probes. 10 ␮g
of poly(A) ⫹ RNA was loaded in each lane. The numbers indicate
sizes (in kb) of signals.
cDNA clones. Sequencing of these clones revealed that all
contained the same Pax9-like paired domain that was
included in pLjP9-PCRF. The longest clone included 1977
nucleotides excluding the poly(A) tail and contained a
single open reading frame that encoded a polypeptide of 393
amino acids (Fig. 3). In addition to the paired domain, this
clone also included octapeptides, but not the homeodomain, showing the structural features characteristic of
Pax1/Pax9-related (Group I) genes. Analyses of the amino
acid sequence with other group Pax genes as well as Group
I genes of C. elegans (K07C11.1) and Drosophila (Pox meso)
revealed that the isolated clone belonged to Group I Pax
genes with a bootstrap value of 78% (not shown). In the
same analyses, this clone belonged to Pax9 genes with a
bootstrap value of 53% (not shown). This low bootstrap
value is probably due to the high conservation of paired
domains and the length of available amino acids (see
below); only the paired domain could be used for this
analysis especially since Pax4/6 genes lack octapeptides.
Thus, we named this clone LjPax9 (Lampetra japonica
Pax9). This cDNA was shorter than that of the native
transcript detected by Northern blotting analyses (Fig. 2B).
Since this clone contained the initial methionine, an inframe stop codon, and 371 bp of 5⬘ untranslated region
(UTR), it was thought to lack part of the 3⬘ UTR.
Figure 4 shows the comparison of the paired domain and
octapeptide amino acid sequences of Group I, Pax1/Pax9-
related proteins. The LjPax9 paired domain amino acid
sequence was highly homologous to those of human Pax9
(96.1%), mouse Pax9 (96.1%), chick Pax9 (96.1%), fish Pax9
(96.9%), human Pax1 (94.5%), mouse Pax1 (94.5%), chick
Pax1 (94.5%), amphioxus Pax1 (89.8%), ascidian HrPax1/9
(89.8%), ascidian CiPax1/9 (90.6%), acorn worm PfPax1/9
(93.8%), Drosophila Pox meso (86.7%), and C. elegans
K07C11.1 (68.8%). LjPax9 contained some characteristic
amino acid residues that are typical of Pax9: phenylalanine
at position 2, threonine at position 86, and arginine at
position 90 in the paired domain (Fig. 4A). Furthermore,
aspartate at position 5 in the octapeptides was also typical
of Pax9 (Fig. 4B). To determine the evolutionary relationship of Pax1/Pax9 genes, a molecular phylogenetic tree was
constructed by the neighbor-joining method based on the
amino acid sequences of paired domains and octapeptides.
Since Drosophila Pox meso and C. elegans K07C11.1 have
no octapeptides, they were not included in the tree, and
human Pax2 was used as an outgroup. As shown in Fig. 4C,
the tree clearly indicates that LjPax9 belongs to the Pax9
Expression Pattern of LjPax9 in Developing L.
japonica Embryos
The earliest expression of LjPax9 was observed around
stage 23.5 when the cheek process had already clearly
formed on the surface of the embryonic head (Fig. 5A). The
transcripts were found in the rostral endoderm surrounding
the first and future second pharyngeal pouches. The expression of LjPax9, therefore, seemed to take place after
endodermal protrusion. As embryos developed, the order of
pouch expression followed that of the appearance of the
pouches, which proceeded from anterior to posterior (Figs.
5A–5G). The last pouch to express LjPax9 was pouch 8, the
caudalmost permanent pharyngeal pouch of the lamprey
(Fig. 5G, stage 26). The morphology of the first pouch differs
from that of the others in that it never perforates and lies in
an oblique plane; the posterior pouches protrude laterally
and expand along dorsoventral axis (Figs. 5E and 5F). No
other overt expression domains were found in the endodermal region, including the endostyle, indicating that LjPax9
serves as a specific marker of laterally protruding pharyngeal pouches.
From early stages, the lateral wall of the pouches ceased
expressing LjPax9, possibly corresponding to the perforating part of the endoderm; this loss of expression did not
occur in the first pouch that does not open externally. In
later stages, LjPax9 expression was restricted to the medial
lining of the branchial arch and not in the epithelium
covering the lateral lamella of the arch (Figs. 5E–5J).
From stage 25.5 onward, the pouch expression began to be
restricted to the medial portion of the posterior wall (or the
rostromedial wall) of the third and more caudal pharyngeal
(or real branchial) arches (Figs. 6A and 6B). This part of the
endoderm covers a part of the branchial arch called the
ciliary band (Figs. 6A and 6B; Kuratani et al., 1997; “Wim-
Copyright © 2000 by Academic Press. All rights of reproduction in any form reserved.
Pax1/Pax9-Related Genes in the Lamprey
FIG. 3. Nucleotide and predicted amino acid sequences of L. japonica Pax9 gene cDNA clone. The single ORF encodes a polypeptide of
393 amino acids, the molecular mass of which was estimated to be 40.9 kDa. The paired domain is boxed and the octapeptide is double
underlined. The asterisk indicates the termination codon. The sequence is in the DDBJ/EMBL/GenBank database under Accession No.
perschnur” of von Kupffer, 1896). In the first and second
arches, no such changes occurred in this pouch (Fig. 5G,
also see Fig. 7). Around this stage, a new expression domain
appeared in the ectodermal nasohypophysial plate (Fig. 5E).
At stage 28, LjPax9 expression was found in the velum (Fig.
5I), the pumping apparatus derived from the mandibular
arch of the lamprey, where the expression was mainly seen
in the mesenchyme. On histological sections, this expression was seen to be heavily accumulated in the cells
surrounding the muscle (Figs. 6C and 6D), implying that the
positive cells were of neural crest origin (see below). Mesenchymal expression was also seen in the second pharyngeal arch (Figs. 6C and 6D). In the trunk region of the same
histological specimen, LjPax9 expression was not observed
in the somitic mesoderm, nor was it found in any part of the
mesoderm when whole-mount stained embryos ranging
from stage 23 to 29 were dissected under the microscope
and carefully observed.
Group I Pax Genes of L. japonica
Pax1/Pax9-related genes encode polypeptides with the
paired domain and octapeptides but without a homeodomain. In higher vertebrates, two Pax1/Pax9-related genes
named Pax1 and Pax9 have been reported in human, mouse,
chick, and fish. On the other hand, only one Pax1/Pax9related gene has been isolated from invertebrates: amphioxus, ascidians, acorn worms, sea urchins, flies, and
nematodes. Therefore, one ancestral Pax1/Pax9-related
gene may have duplicated and evolved to Pax1 and Pax9
during the evolution of vertebrates.
In the present study, we isolated two different clones
containing lamprey Pax1/Pax9-related PCR fragments. The
nucleotide sequences of these fragments closely resembled
each other. One of the clones (pLjP1-PCRF), however,
Copyright © 2000 by Academic Press. All rights of reproduction in any form reserved.
Ogasawara et al.
FIG. 4. Relationship of Pax1/Pax9-related gene products. Amino acid sequences of paired domain (A) and octapeptides (B) of the lamprey LjPax9
compared to corresponding sequences encoded by related genes (with accession numbers): human Pax9 (L09745), mouse Pax9 (X84000), chick
Pax9 (X82443), zebrafish Pax9 (U40931), human Pax1 (AL035562), mouse Pax1 (NM_008780), chick Pax1 (U22046), amphioxus pax1 (U20167),
ascidian HrPax1/9 (AB021138), ascidian CiPax1/9 (AB020762), acorn worm PfPax1/9 (AB020763), Drosophila Pox meso (X16992), and C. elegans
K0C11.1 (U53336). Identical amino acids are indicated by dots. Characteristic amino acids of Pax9 are indicated by arrowheads. (C) A
neighbor-joining tree based on comparison of the amino acid sequences of the paired domains. Human Pax2 (L09747) was used as an outgroup.
Branch length is proportional to the number of amino acid substitutions; the scale bar indicates 0.1 amino acid substitution in the sequence. The
numbers indicate the relative robustness of each node as assessed by boot strap analysis (100 replications).
encoded an aspartate residue at position 86 in the paired
domain, which is typical for Pax1 (Figs. 1 and 4A). In
contrast, the other clone pLjP9-PCRF encoded a threonine
residue characteristic of Pax9 at this position (Figs. 1 and
4A). Since this amino acid change arose from triplet nucleotide displacement, it was not due to PCR-induced mutation. Furthermore, genomic Southern blotting analyses using these paired domains as probes alluded to the possibility
that there are two closely related Pax1/Pax9-related genes
in the genome (Fig. 2A). The two signals in the EcoRI and
BglII digests might have been derived from the loci of both
genes. In the HincII digest, two of the three signals may
have been derived from the Pax9-like gene and one from the
Pax1-like gene, because only the Pax9-like paired domain
has the HincII site. These observations favor the hypothesis
that L. japonica possesses two Pax1/Pax9-related genes,
Pax1 and Pax9, in single copies per haploid genome, though
the definite conclusion waits for the isolation of the hypothetical LjPax1, which has not been successful so far.
On Northern blotting analyses using the paired domains
as probes, we detected only a single slightly broad band in
the larval lamprey (Fig. 2B). On screening of a larval cDNA
library, we isolated only the same type of cDNA clone
containing the paired domain and octapeptides without
homeodomain. These structural profiles support strongly
the idea that the isolated clone encodes a Group I, Pax1/
Pax9-related protein (all the other group members possess
the homeodomain). Amino acid residues in this paired
domain (phenylalanine 2, threonine 86, and arginine 90) and
octapeptides (asparagine 5) are characteristic of Pax9 (Figs.
4A and 4B). Finally, molecular phylogenetic analyses indicated that the isolated cDNA clone encoded Pax9 of lamprey. These results suggested that the signal observed on
Northern blotting was the Pax9 transcript, and the expression level of the gene which contained the Pax1-like paired
domain appeared to be very low in the larval lamprey.
Alternatively, the lengths of the putative Pax1 and Pax9
transcripts may be similar, resulting in apparently single
Copyright © 2000 by Academic Press. All rights of reproduction in any form reserved.
Pax1/Pax9-Related Genes in the Lamprey
FIG. 5. Expression pattern LjPax9 in L. japonica embryos. Expression patterns of LjPax9 are shown successively in staged embryos (stages
23.5 to stage 29). Embryos are shown as lateral views except for F, which is a ventral view. In some of the embryos, the latest pharyngeal
pouches to express LjPax9 are indicated by arrows. Note that expression of the gene followed the sequence of pharyngeal pouch
development, which proceeds in a rostral-to-caudal direction. Asterisks in A and C indicate nonspecific staining of the embryo. The
morphology of the first pharyngeal pouch (p1) differs from that of the others in that it develops in an oblique plane (E and F). This pouch
is never perforated during development. From stage 27 onward, a de novo and temporary expression domain appears in the velum (double
arrows in I). Abbreviations: cp, cheek process; llp, lower lip; nhp, nasohypophysial plate; p1– 8, pharyngeal pouches (numbered); ulp, upper
lip. Bars, 100 ␮m.
Copyright © 2000 by Academic Press. All rights of reproduction in any form reserved.
Ogasawara et al.
FIG. 6. Histological observations of LjPax9 expression. Whole-mount specimens were sectioned after in situ hybridization with LjPax9
probe and viewed under Nomarski optics. (A and B) Horizontal sections of the pharyngeal region of a stage 27 embryo in which velar
expression of this gene was first observed. LjPax9 expression is seen as blue precipitate. Note that LjPax9 was expressed more intensely
on the posteromedial wall of the endodermal pouch and no expression is seen in the inside of branchial arches (arrowheads in A and B). This
part of the endodermal wall covers the ciliary band of the branchial arch. (C and D) Rostral region of the same specimen as in A and B,
showing the mesenchymal expression (double arrows) of LjPax9 in the velum (vel) and second arch (a2). The fibrous structure in the velum
(arrows in D) is the velar muscle that may also express LjPax9. Abbreviations: a2, the second pharyngeal arch; cb, ciliary band; ph, pharynx;
ps, pharyngeal slits; vel, velum. Bar in A, 50 ␮m.
band. Further studies are required for isolation and characterization of the clone for Pax1-like paired domaincontaining gene.
Expression Pattern of LjPax9 in the Lamprey and
Evolution of the Pharynx: Pharyngeal Pouches—
Plesiomorphic Expression
In the present study, expression of a Pax9 gene cognate
was first observed in the developing agnathan species. In
addition to the paired domain, the probe for in situ hybridization included the 3⬘ UTR of the cDNA that would not be
highly conserved with the putative paralogue of LjPax9, and
furthermore, our preliminary in situ hybridization using
only the paired domain did not give specific signals in the
whole-mount embryos (not shown). The expression data
presented in this study, therefore, are most likely to reflect
the LjPax9 expression specifically. It was expressed in the
endodermal pharyngeal pouch, nasohypophysial plate, and
Copyright © 2000 by Academic Press. All rights of reproduction in any form reserved.
Pax1/Pax9-Related Genes in the Lamprey
FIG. 7. Summary of LjPax9 expression in the developing pharynx of the lamprey. Horizontal sections of early (left) and late (right) L.
japonica embryos are shown schematically. LjPax9 was expressed in a series of endodermal pouches, of which the indentation lateral to
the oropharyngeal membrane (opm) corresponds to pharyngeal pouch 1 (p1). The velum develops rostral to this pouch. In later development,
LjPax9 becomes mesenchymally expressed also in the first and second pharyngeal arches. The endodermal expression of LjPax9 was
restricted to the rostral surface of ciliary bands (medial portions) in caudal branchial arches. No such expression was seen in arch 1 or 2,
which do not differentiate into ciliary bands. This mesenchymal expression was absent from velar muscle and is probably associated with
neural crest-derived ectomesenchyme.
velar epithelium and mesenchyme. Similar expression patterns have been detected not only in vertebrates, but also in
other chordates and even a hemichordate, P. flava (Ogasawara et al., 1999). This latter group expresses an ancestral
form of Pax1/9 in the endodermal pharyngeal pouches.
In vertebrates, Pax1 and Pax9 genes have been suggested
to play roles in the development of the pharyngeal
endoderm and it derivatives; Pax9 gene disruption leads to
the loss of pharyngeal pouch derivatives such as the thymus, parathyroid, and ultimobranchial body (Peters et al.,
1998). The presence of Pax1/Pax9 genes in the lamprey,
which does not possess a thymus, implies roles of these
genes upstream of pharyngeal endodermal differentiation.
In Pax9-deficient murine embryos, the defect is already
seen in the early stage of pouch development (Peters et al.,
1998). The Pax9 gene seems, therefore, to function in the
primary stage of pharyngeal segmentation, both in ontogeny and in phylogeny. Moreover, these genes are associated
with paired pharyngeal pouches and not the medial deriva-
tives such as the thyroid, development of which is regulated
by another Pax group gene (Pax8) (Plachov et al., 1990). It
would be of interest to determine whether Pax2/5/8 homologues are associated with the endostyle in ammocoetes or
in other chordate species.
Vertebrate-Specific Expression
Some Pax9 expression domains are found only in vertebrates; the expression of LjPax9 in the velar mesenchyme
(Figs. 6C and 6D) resembles Pax9 expression in the mandibular arch ectomesenchyme of gnathostome embryos
(Ten Berge et al., 1998), which is homologous to the
lamprey velum. Similar conservation was again encountered in the nasal– hypophysial complex (Fig. 5G). These
expression domains are missing in invertebrates and are
probably synapomorphic to vertebrates.
In the mouse, Pax9 expression in the mandibular mesenchyme is associated with tooth development (Peters et al.,
Copyright © 2000 by Academic Press. All rights of reproduction in any form reserved.
Ogasawara et al.
1998; Amano et al., 1999). From the expression profile of
this gene, the domain apparently corresponds to the crestderived ectomesenchyme (Ten Berge et al., 1998; Peters et
al., 1998; Lu et al., 1999). In amniotes, the neural crestderived ectomesenchyme has been shown to play fundamental roles in patterning of head muscles and for differentiation of major groups of connective tissues in the
craniofacial region (reviewed by Noden, 1988). In the lamprey, neural crest-derived cells have also been observed to
migrate into the pharynx, including the mandibular arch,
and populate the outer layer of the mesenchyme similar to
that in gnathostome embryos (Horigome et al., 1999). These
cells are also thought to be skeletogenic as in gnathostomes
(Langill and Hall, 1988). Although more detailed observations are necessary, it seems likely that in the lamprey
LjPax9 is also expressed in the mandibular arch ectomesenchyme, although its expression in the mandibular mesoderm (Kuratani et al., 1999) could not be excluded (Fig. 6D).
Moreover, the developmental role of LjPax9 in the mandibular mesenchyme of lamprey larvae is unclear, since mammalian Pax9 has been shown to be prerequisite for tooth
development and the lamprey does not possess mineralized
teeth (Peters et al., 1998). Further experiments are necessary to address this question.
Evolution of the jaw has been an intriguing topic in
comparative embryology. Traditionally, the cheek process
of the lamprey embryo represents the mandibular arch and
the first pharyngeal pouch. The velum, which develops
from the cheek process, has been compared to the mandibular arch of gnathostomes (reviewed by Mallat, 1996; Janvier, 1996). However, the endodermal pocket found caudal
to the velum (named the first pouch in the present study)
differs markedly from the corresponding pouch in gnathostomes; the shape of the pocket is also different and it never
perforates during development, while the first pouch of
gnathostomes always opens as spiracles. In the embryonic
pharynx also, this pocket appears different from ordinary
pouches (Figs. 5E, 5F, and 7). The present study revealed
that this pharyngeal pouch marker was also expressed in
this structure, confirming the serial homology of this
endodermal structure as a pharyngeal pouch. This was also
consistent with the close relationship between the velum
and the mandibular arch (reviewed by Mallat, 1984; Janvier,
Gnathostomes and Lampreys
The lamprey embryos did not seem to express LjPax9 in
some other tissues in which gnathostome Pax9 genes are
reported to be expressed, the synapomorphic domains associated only with gnathostome or more restricted groups
within gnathostomes. In mammalian and avian development, Pax1/Pax9 genes are expressed in somites or their
derivatives and are involved in somitic differentiation in
response to sonic hedgehog signal from the notochord
(Spörle and Schughart, 1997; Borycki et al., 1998; Hacker
and Guthrie, 1998; Watanabe et al., 1998; Peters, 1999;
FIG. 8. Deuterostome evolution and Pax9 gene expression. The
cladogram shows the generally accepted splitting sequence of
major deuterostome animal groups. Toward the branch of amniotes, the Pax9 cognates appear to have gained additional series of
organ systems along phylogeny as shown by the nested pattern of
green bars. Thus, Pax9 gene regulation has evolved concomitant
with the body plan. In addition to the pharyngeal slits, LjPax9 was
also expressed in the pharyngeal ectomesenchyme of arches 1 and
2, but not in somites as in gnathostomes. This pattern of expression
was consistent with the phylogenetic position of the lamprey. The
acquisition of the neural crest in vertebrates was the basis for
mesenchymal expression of Pax9.
Aruga et al., 1999; Furumoto et al., 1999). Similar mesodermal expression has also been reported in zebrafish embryos
(Nornes et al., 1996). In the present study, no such expression was seen in the lamprey somites as observed by
dissecting stage 23 to 29 whole-mount embryos under the
microscope. Given the assumption that the number of
sclerotomal cells is very small in the lamprey embryo
(Maurer, 1906), however, it is not ruled out that there were
a few LjPax9-expressing cells lateral to the notochord,
which were not possible to detect with the available technique.
In summary, the lamprey Pax9 gene is expressed in the
pharyngeal pouch endoderm and mandibular ectomesenchyme as well as in the nasohypophysial plate ectoderm,
while the homologues of hemichordates, tunicates, and
amphioxus are expressed only in the endodermal derivatives. Furthermore, Pax1/Pax9 genes of gnathostomes have
acquired more expression domains (limb bud and probably
somites) than in lampreys. Along the phylogenetic tree, the
position of the lamprey lineage exactly fits the transition of
Pax9 gene expression domains and this animal may repre-
Copyright © 2000 by Academic Press. All rights of reproduction in any form reserved.
Pax1/Pax9-Related Genes in the Lamprey
sent the basal state of vertebrate Pax9 gene expression (Fig.
8). In such an evolutionary context, it is important that
gene duplication itself does not appear to be the basis of
evolution of the body plan; Pax9 and Pax1 genes have not
differentiated into discrete expression domains. Rather,
Group I Pax genes as a whole appear to have expanded their
expression domains into multiple organ systems during
evolution toward gnathostomes (Fig. 8). The evolution of
the neural crest cells is associated with the mandibular
mesenchyme expression of these genes. These cells constitute the essential component of the vertebrate-specific
developmental plan as the “fourth” germ layer (reviewed in
Hall, 1998). With the evolution of the neural crest, the Pax9
gene seems to have acquired new expression domains
simultaneously. Lampreys are now thought to represent a
secondarily acquired limbless condition (Janvier, 1996). It is
not known, therefore, whether limb-bud expression of the
Pax9 gene is synapomorphic to gnathostomes or to vertebrates.
As is often emphasized with Hox genes, the evolution of
the animal body plan may in part rest upon duplication of
regulatory genes and subsequent diversification of their
functions. The apparent evolutionary trend in Pax9 genes
highlights a contrasting pattern of evolutionary strategy, in
which functions of regulatory genes expand into new domains within embryos. In this context, expression patterns
of the putative paralogue, LjPax1, which have not yet been
identified, would be of great interest in evaluation of Pax
gene evolution.
We thank Dr. Chikara Hashimoto for his technical suggestions
and valuable discussion. M.O. and Y.S. are predoctoral fellows of
JSPS with a Monbusho Research Grant. This work has been
supported in part by grants-in-aid from the Ministry of Education,
Science, and Culture of Japan (Grants 11309010, 11NP0201, and
11152225) to S.K. and also by a Grant-in-Aid for Specially Promoted
Research (No. 0712012) from Monbusho, Japan to N.S.
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Received for publication March 1, 2000
Revised April 10, 2000
Accepted April 17, 2000
Copyright © 2000 by Academic Press. All rights of reproduction in any form reserved.