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First published online February 18, 2004
doi: 10.1242/10.1242/dev.00986
1 Evolutionary Morphology Research Team, Center for Developmental Biology (CDB),
RIKEN, Kobe, Japan
2 Institut de Génétique et de Biologie Moléculaire et
Cellulaire, UMR 7104, CNRS/INSERM/ULP, BP 10142-67404 Illkirch Cedex, CU de
Strasbourg, France
3 Laboratori di Biologia Cellulare e dello Sviluppo, Università di Pisa,
Via G. Carducci 13, Pisa, Italy
4 Department of Medical Technology, School of Health Sciences, Faculty of
Medicine, Niigata University, Niigata 951-8518, Japan
* Author for correspondence (e-mail: bothrops{at}cdb.riken.go.jp)
Accepted 14 November 2003
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SUMMARY |
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 TOP SUMMARY IntroductionMaterials and methodsResultsDiscussionREFERENCES |
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Key words: Evolution, Hindbrain, Rhombomere, Reticulospinal neuron, Branchiomotor neuron, Lamprey, Hox genes
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Introduction |
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TOPSUMMARYIntroductionMaterials and methodsResultsDiscussionREFERENCES |
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;
Bergquist and Källén,
1953; Vaage,
1969; Fraser et al.,
1990; Figdor and Stern,
1993; Puelles and Rubenstein,
1993). These neuromeres serve as centers for cell proliferation,
migration and neuronal differentiation. The neuromeres in the hindbrain, or
rhombomeres (r) (Orr, 1887;
Vaage, 1969), have attracted
the attention of developmental biologists for their topographical
relationships with peripheral nerves and segmental boundary-related gene
expression patterns.
Rhombomeres appear as repetitive units of neuronal development and as a
prepattern for the spatial deployment of the neural crest-derived
ectomesenchyme in the craniofacial region
(Lumsden and Keynes, 1989;
Clarke and Lumsden, 1993;
Kuratani and Eichele, 1993;
Köntges and Lumsden,
1996) (reviewed by Santagati
and Rijli, 2003). The homeobox-containing transcription factors of
the Hox gene family display nested, segmentally restricted, expression
patterns with sharp anterior boundaries mapping to the rhombomeric borders,
and are involved in position-dependent specification of the rhombomeres and
pharyngeal arch derivatives (e.g. Lufkin et al., 1991;
Chisaka et al., 1992;
Gendron-Maguire et al., 1993;
Mark et al., 1993;
Carpenter et al., 1993;
Rijli et al., 1993;
Studer et al., 1996;
Goddard et al., 1996;
Bell et al., 1999;
Gavalas et al., 1997;
Gavalas et al., 1998;
Rossel and Capecchi, 1999;
Gaufo et al., 2000;
Davenne et al., 1999;
Barrow et al., 2000;
Grammatopoulos et al., 2000;
Pasqualetti et al., 2000;
Hunter and Prince, 2002). Such
a coordinated pattern of development suggests that a primitive metameric
developmental program was acquired in vertebrate ancestors. However,
amphioxus, the sister group of the vertebrates, lacks neuromere organization
while possessing anteroposterior specification along the neuraxis, which
evident in the anatomical architecture
(Lacalli et al., 1994) and
partly in the neuronal repertoire
(Fritzsch and Northcutt, 1993;
Fritzsch, 1996;
Knight et al., 2000;
Lacalli, 2001), as well as in
the expression patterns of developmental genes, including the Hox genes
(Holland et al., 1992;
Wada et al., 1999;
Knight et al., 2000;
Murakami et al., 2001;
Jackman and Kimmel, 2002).
Together, these studies indicate that amphioxus embryos may possess a region
homologous to the vertebrate hindbrain, but that the segmental organization
appeared after the divergence of the vertebrate lineage.
Therefore, the evolutionary origin of rhombomeres, and its relationship to
a metameric developmental program of neuronal patterning, poses an intriguing
question for evolutionary developmental biology. In vertebrates, our knowledge
about rhombomeres is restricted to the gnathostomes. Branchiomotor nuclei are
generated in association with specific pairs of rhombomeres and innervate a
single branchial arch (Tello,
1923; Neal, 1896;
Lumsden and Keynes, 1989;
Noden, 1991;
Gilland and Baker, 1993). The
existence of this 2:1 rhombomere:pharyngeal arch relationship has been
considered the basic developmental unit for patterning of the vertebrate head
(reviewed by Kuratani, 2003).
As a general rule, the trigeminal motor nucleus is generated in r2 and r3, and
that of the facial nerve in r4 and r5. Hox gene expression patterns
(Hox code) appear to be conserved in the gnathostome hindbrain (Hunt
et al., 1991a; Hunt et al.,
1991b; Lumsden and Krumlauf,
1996), with minor modifications in teleosts, which have
experienced additional Hox cluster duplication
(Amores et al., 1998;
Naruse et al., 2000;
Schilling and Knight, 2001;
McClintock et al., 2002;
Prince, 2002). Loss- and
gain-of-function experiments indicate that Hox genes are involved in providing
branchiomotor subtype positional identity along the anteroposterior axis
(Studer et al., 1996;
Goddard et al., 1996;
Gavalas et al., 1997;
Gavalas et al., 1998;
Davenne et al., 1999;
Jungbluth et al., 1999;
Gaufo et al., 2000; Dominguez
del Toro et al., 2001; McClintock et al.,
2002; Pattyn et al.,
2003).
Unlike branchiomotor neurons, the spatial patterns of interneurons are less
conserved among gnathostomes. In avians and teleosts, rhombomeres initially
generate similar repeated sets of neurons belonging to distinct classes
(Metcalfe et al., 1986;
Mendelson, 1986a;
Mendelson, 1986b;
Kimmel et al., 1988;
Lee et al., 1993; Hanneman et
al., 1988; Kimmel, 1993;
Clarke and Lumsden, 1993).
Thus, each rhombomere seems to represent in its composition a serial homologue
of a repeated repertoire of reticular neurons. In another divergent group, the
amniotes, such segmental repetition is less obvious, and reticular neurons
assume a rather columnar distribution with modulations or interruptions, so
that each rhombomere is instead identified in terms of the particular
reticular neuron class it produces (e.g. reticulospinal or vestibulospinal)
(Auclair et al., 1999).
These observations raise the question of whether hindbrain segmentation and metameric neuronal patterning are intimately linked or independently established. To address this question, the developmental patterns of regulatory genes and neuronal specification, as well as neuroepithelial compartmentalization, should be systematically compared along the phylogenetic tree of the chordates. In particular, the lamprey, an agnathan (jawless) vertebrate, may provide useful insights to partly fill the gaps in our knowledge, because it appears to have diverged early in the vertebrate lineage.
The extant agnathan animals, including the lamprey and hagfish, are thought
to form a monophyletic group (Mallatt and
Sullivan, 1998; Kuraku et al.,
1999; Kuratani et al.,
2003) as a sister group of the gnathostomes. Segmentation in the
hindbrain has been identified in agnathan embryos (Kuratani et al., 1998;
Horigome et al., 1999;
Pombal et al., 2001;
Kuratani et al., 2001). In a
series of detailed embryological observations of a Japanese lamprey,
Lethenteron japonicum, conserved topographical relationships between
rhombomeres, cephalic crest cell populations and cranial nerve roots were
identified (Kuratani et al.,
1997; Horigome et al.,
1999). The lamprey also has reticulospinal neurons, and their
activity appears to be involved in swimming behavior
(Nieuwenhuys and Nicholson,
1998). The neuronal patterning sequence of this animal, however,
has not yet been elucidated. In this study, we analyzed the patterns of motor
and reticular neuron development and of regulatory gene expression in relation
to the rhombomere pattern in the lamprey hindbrain. Our data suggest that
registering of rhombomeric segmentation, neuronal patterning, and the Hox code
may have occurred through successive independent evolutionary changes in the
vertebrate lineage.
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Materials and methods |
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TOPSUMMARYIntroductionMaterials and methodsResultsDiscussionREFERENCES |
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) at
20°C. Embryonic stages were assessed morphologically according to the
sequence established by Tahara (Tahara,
1988) for L. reissneri, a brook lamprey species closely
related to L. japonicum.
Retrograde labeling of hindbrain neurons
Rhodamine- or fluorescein-conjugated dextrans (Sigma, St Louis, MO) were
injected into the spinal cord or pharyngeal arches of the embryo to label
reticulospinal or branchial motoneurons, according to the method described by
Glover (Glover, 1995). The
injected embryos were incubated at room temperature for 30 minutes to allow
the dextran to label neurons retrogradely. Embryos were then washed with 10%
Steinberg solution, and fixed in 4% paraformaldehyde and 1% methanol in 0.1 M
phosphate-buffered saline (PFAM/PBS). The fixed specimens were dehydrated, and
clarified with a 1:2 mixture of benzyl alcohol and benzyl benzoate (BABB).
Labeled neurons were then examined using a fluorescence microscope.
Isolation of cDNA clones of lamprey genes
A partial cDNA clone of the lamprey Krox20 gene was isolated from
a L. japonicum stage 24-26 embryo cDNA library, using a previously
described low stringency hybridization protocol
(Pasqualetti et al., 2000). We
used a probe derived from a mouse Krox20 cDNA
polymerase-chain-reaction (PCR) product amplified with the specific mouse
primers: 5'-ATCCGTAATTTTACTCTGGGGGG-3' (sense) and
5'-GTCACAGGCAAAGGGCTTCTC-3' (antisense). Four independent cDNA
clones were isolated, all sharing 100% homology in the regions of overlapping
sequence. We could not distinguish whether these genes are the homologues of
Krox20 or Krox24. However, because the expression of these
genes was restricted to r3 and r5, we designated our clones LjKrox20
(Lethenteron japonicum Krox20). The partial sequences have been
assigned DDBJ/EMBL/GenBank Accession Number AY275715. PCR amplification of a
518 bp LjKrox20 fragment was achieved using the
LjKrox20-specific primers 5'-CTTCTCACGCTCGGACGAGCT-3'
(sense) and 5'-GACGTCACCGACGATGAGGACAT-3' (antisense), for use in
in situ hybridization.
Eph lamprey homologues were isolated by low-temperature PCR using
degenerate primers directed against two conserved regions of the
EphA4 molecule. A sense primer
(5'-ATGATIATCACIGAGTAYATGGARAA-3') and an antisense primer
(5'-TTGATIACITCCTGGTTIIICATRTCCA-3') were used. We isolated
several clones, and these sequences were significantly homologous to lamprey
EphC, which had been cloned previously from the lamprey species
L. reissneri (Suga et al.,
1999). We therefore named our Eph clones LjEphC
(Lethenteron japonicum EphC).
The lamprey Hox3 fragment was PCR amplified from cDNA of L. japonicum using a set of degenerate primers directed against two highly conserved regions of the Hox3 molecule: a sense primer (5'-CGGAATTCYTNGARYTNGARAARGARTT-3') and an antisense primer (5'-CGGGATCCNCKNCKRTTYTNRAACCADATYTT-3'). Underlines indicate restriction enzyme sites. This short Hox3 fragment allowed the design of 3'-RACE primers, as well as appropriate nested primers for marathon cDNA amplification (MarathonTM cDNA Amplification Kit, Clontech, Palo Alto, CA). The primers were: 3'-RACE primer, CCTCTGCCGCCCTCGACGAGTT; and 3'-RACE nested primer, AATGGCCAACCTACTTAACCTC. The PCR led to the isolation of a lamprey Hox3 fragment. This fragment was used as the probe to screen a lamprey cDNA library. Screening was performed under low-stringency conditions and isolated clones were sequenced. The deduced protein sequence was significantly homologous to Hox3, therefore we named our Hox clone LjHox3 (L. japonicum Hox3). The partial sequences have been assigned the DDBJ/EMBL/GenBank Accession Number AB125270.
Neuronal labeling in combination with in situ hybridization
After neuronal labeling, whole-mount in situ hybridizations were performed
as previously described (Murakami et al.,
2001). Stained embryos were fixed in PFAM/PBS, and incubated with
streptavidin-horseradish peroxidase (HRP; Vector) in Tris-buffered saline
(TST; diluted 1:500) at 4°C overnight. The specimens were washed five
times for 60 minutes each in TST at room temperature, then HRP activity was
detected using the peroxidase substrate, 3,3'-diaminobenzidine (DAB, 100
mg/ml), in TST with 0.01% hydrogen peroxide.
Retinoic acid treatment of lamprey embryos
Retinoic acid (RA) treatment was performed according to the protocol
described previously (Kuratani et al.,
1998b). In the present study, embryos from stages 12 through 18
were treated with 0.01 µM, 0.05 µM, or 0.1 µM all-trans RA. As a
negative control, only the dimethyl sulfoxide (DMSO) was applied.
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Results |
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TOPSUMMARYIntroductionMaterials and methodsResultsDiscussionREFERENCES |
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). By stage 28, isthmic group (I), B and mir neurons had
increased in number, and all the components of the reticulospinal system
present in the adult animal could be identified
(Fig. 1D)
(Jacobs et al., 1996).
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The expression domain of LjKrox20 corresponded to r3 and r5, as
indicated by the position of the labeled domains relative to the morphological
rhombomere boundaries and the trigeminal and facial nerve roots, which were
stained with anti-acetylated tubulin antibody
(Fig. 2A,B) (see also Kuratani
et al., 1998; Horigome et al.,
1999). The LjEphC domain also seemed to correspond to r3
and r5, because of the relative position of the otic vesicle, which is lateral
to r4 in the lamprey (Kuratani et al., 1998;
Horigome et al., 1999)
(Fig. 4A,B). Based on this
mapping, the cluster of B cells and the Mth neuron could be positioned in r4
(Fig. 3A,B, Fig. 4C-F). The Mth neuron
appeared in the middle of r4 at stage 26 and corresponded to the domain of
lowest LjPax6 expression and to the rostral limit of LjHox3
expression (Fig. 2D, Fig. 3C,D)
(Murakami et al., 2001).
Reticulospinal neurons, including I and B cells, were found lateral to the
LjPax6-expressing domain along the anteroposterior neuraxis, as seen
from the dorsal view of the same stage embryo
(Fig. 2C). The auxiliary
Mauthner neuron (Mth') developed in r5
(Fig. 3A,B). The I4 neuron was
located in r3 (Fig. 3A,B),
whereas the I3 neuron was positioned anterior to the r2/3 boundary
(Fig. 3B). The topographical
relationships between neuron position and gene expression described above did
not change throughout the later stages analyzed.
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;
Gilland and Baker, 1993).
Interestingly, the border between the trigeminal and facial nuclei
corresponded to the anterior limit of LjHox3 expression
(Fig. 6D), with the trigeminal
motor nucleus positioned just rostral to this limit
(Fig. 6D).
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; Lopez et al.,
1995; Marshall et al.,
1992; Morris et al., 1991;
Hill et al., 1995;
Alexandre et al., 1996;
Dupé and Lumsden, 2001;
Maden, 2002;
Gavalas and Krumlauf, 2000).
Interestingly, we could not detect ectopic Mth neurons in r2 at any RA
concentration, although this has been observed in some gnathostomes
(Fig. 7A-C; n=60).
Moreover, the position of the Mth neuron did not change in relation to the
LjKrox20 expression domains (Fig.
7D-F). However, the application of 0.1 µM RA resulted in an
obvious anterior shift of the trigeminal motor nucleus beyond the
mid-hindbrain boundary (compare Fig.
7J,N), as visualized by the expression of Ljfgf8/17
(Fig. 7I,J). Furthermore, the
boundaries of the branchiomotor nuclei became undetectable (compare
Fig. 7K with L). This phenotype
was paralleled by the rostral expansion of the LjHox3 expression
limit, as assessed by its position relative to the Mth neuron
(Fig. 7G,H). Thus, unlike in
gnathostomes, RA signaling did not appear to alter the developmental pattern
of the lamprey reticulospinal neurons. By contrast, it did affect the Hox code
and branchial motoneurons in a coordinated manner, as seen in gnathostome
embryos.
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Discussion |
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TOPSUMMARYIntroductionMaterials and methodsResultsDiscussionREFERENCES |
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Developmental marker genes and rhombomere segmental identities in lamprey
A number of genes have been implicated in the control of hindbrain
segmentation and of odd-even rhombomere segregation in gnathostomes, including
the kleisler/valentino gene
(Frohman et al., 1993;
Moens et al., 1996;
Moens et al., 1998), members
of the ephrin/eph gene family
(Xu et al., 1995;
Wilkinson, 2001) and
Krox20 (Wilkinson et al.,
1989; Schneider-Maunoury et
al., 1997). Of these, Krox20 and EphA4 are
generally regarded as markers for r3 and r5 segmentation in gnathostomes. The
specification of rhombomere segmental identities and of neurons also depends
on the highly organized expression patterns of the Hox genes (Hunt et al.,
1991a; Hunt et al., 1991b;
Krumlauf, 1993;
Rijli et al., 1998;
Schilling and Knight, 2001).
In gnathostomes, the anterior limits of Hox expression domains precisely map
to rhombomere borders, and Hox patterns are generally used as markers
to identify specific rhombomeres.
In lamprey embryos, the expression patterns of both LjKrox20 and
LjEphC and their localization to r3 and r5 appear to be conserved
relative to their gnathostome cognates (Figs
2,
3). By contrast, the rostral
boundary of the expression of LjHox3 is found between the rostral and
caudal expression domains of both LjKrox20 and LjEphC (Figs
3,
4). Therefore, even though the
precise location of the LjHox3 rostral limit could not be defined in
the present study, it definitely mapped within r4 and did not correspond to a
specific rhombomere boundary (Figs
3,
4). This pattern is apparently
not conserved, as the main transcripts of gnathostome Hox paralogue group 3
genes (e.g. Hoxa3 and Hoxb3) have rostral expression
boundaries that map precisely to the r4/r5 border (reviewed by
Carroll et al., 2001). However,
in the larval zebrafish, the Hoxb3 homologue is expressed in r4
(Prince et al., 1998).
Moreover, at least three types of Hoxb3 endogenous transcripts were
described in the mouse, differing in size, splicing patterns, and expression
domains (Sham et al., 1992).
One such transcript displays a rostral expression domain extending into r4 and
even into r3 segments, which is rather a feature of the expression pattern of
the adjacent Hoxb2 than of Hoxb3. Thus, transcriptional
control mechanisms similar to those in gnathostomes may apply to the
regulation of LjHox3 expression in the lamprey hindbrain.
Evolution of the vertebrate reticular neurons
One important question in evolutionary biology concerns the identification
of serial homology, because it implies the presence of metameric developmental
mechanisms (reviewed by Kuratani,
2003). The present analysis allowed us to gain insights into the
evolution and homology of reticular neurons in the vertebrate lineage.
Reticulospinal neurons are arranged corresponding to rhombomeres in several
vertebrate species, including teleosts
(Metcalfe et al., 1986;
Lee et al., 1993;
Hanneman et al., 1998) and the
rat (Auclair et al., 1999). In
particular, in the aquatic vertebrates studied so far, Mth neurons always
develop in r4 (Metcalfe et al.,
1986; Lee et al.,
1993). Based on the r3 and r5 expression of LjKrox20 and
LjEphC (see above), the lamprey Mth neuron was also localized in r4,
suggesting that the developmental program to generate the Mth neuron in r4 has
been conserved through vertebrate evolution.
In teleosts, developing reticulospinal neurons have been classified into
several families based on shared morphological features, and it has been
suggested that each rhombomere develops basically the same set of neurons as
the others. Serial homologies have been recognized in the Mauthner, MiD2 and
MiD3 neurons of zebrafish. The axons of these neurons cross the midline and
descend the spinal cord along the medial longitudinal fascicle (MLF,
Fig. 8) (Metcalfe et al., 1986). In
the lamprey, in addition to the Mth neuron in r4, a similar neuron called the
Mth' neuron develops in r5. The Mth' neuron in the lamprey is
similar to the zebrafish MiD2, as discussed by Kimmel et al.
(Kimmel et al., 1982). In the
adult animal, the axon of this neuron crosses the midline and descends to the
spinal cord, like the Mth neuron (Swain
et al., 1993). Thus, the Mth and Mth' neurons possibly
represent serial homologues.
As for the isthmic reticular group, the I4 neuron is located in r3, and the
I3 neuron in r2, as assessed from the LjKrox20 and LjEphC
expression domains. These two neurons are of similar size, both grow axons
along similar pathways, and occupy the same relative dorsoventral position
within the neural tube (Jacobs et al.,
1996) (Fig. 8).
These neurons thus appear to represent another serial homology group. With
respect to its position and axon projection pattern, the I4 neuron in the
lamprey also seems to be homologous to RoM3 of the zebrafish, and I3 to RoM2
(Figs 7,
8)
(Metcalfe et al., 1986).
From the above discussion, this rhombomere-related serial homology of
selected neuronal subtypes (e.g. the Mauthner neuron) appears to be a shared
feature in the vertebrate lineage. This supports the idea that a metameric
pattern of neuronal development was already present in the `hindbrain region'
of the vertebrate common ancestor. However, some cell types appear to be
present only in the lamprey, as seen in the bulbar neurons with ipsilateral
axons (Jacobs et al., 1996)
(Fig. 8), which are restricted
to r4 and not present in any other vertebrate species. Furthermore, in the
lamprey, a pair of Mth' neurons are observed in r5, whereas in the
zebrafish, two pairs of Mth homologues, MiD2 and MiD3, are located in r5 and
r6, respectively (Fig. 8).
Finally, whereas teleosts and lampreys have large identifiable reticular
neurons, this is not the case in amniotes, whose small reticular neurons are
clustered in each rhombomere. Thus, various forms of diversifications have
arisen independently in each lineage of animal groups.
Based on these results, we propose an evolutionary scenario in which the vertebrate common ancestor possessed a basic metameric pattern of serially repeated sets of reticular neurons. In the lineage leading to lampreys, specific cell types, such as additional Mauthner neurons, were lost from each metamere, resulting in the anteroposteriorly differentiated neuronal pattern of the present lamprey hindbrain (Figs 8, 9). In amniotes, large identifiable neurons may have been lost secondarily as well, possibly because of the shift to the terrestrial life and related loss of a lateral line-mediated reflex system. An alternative option is that the ancestral hindbrain was already specialized anteroposteriorly as observed in the lamprey. In teleosts, each rhombomere would then have secondarily acquired a similar pattern of interneurons. The latter possibility, however, makes it difficult to explain the origin of such anteroposterior specification in the vertebrate hindbrain. Observation of hindbrain development in the hagfish might therefore provide key information to distinguish among such possibilities.
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), neuronal organization of the rostral hindbrain
is linked to muscle innervation via the trigeminal nerves in vertebrates. This
raises the possibility that the lamprey-specific distribution pattern of
trigeminal motoneurons may be associated with agnathan oral movement and the
specific patterning of the perioral mesenchyme (see
Kuratani et al., 2001;
Shigetani et al., 2002). The
facial motor nucleus of the lamprey, however, is located in r4 and r5. In some
gnathostomes, such as zebrafish
(Chandrasekhar et al., 1997;
Higashijima et al., 2000;
Bingham et al., 2002) and mouse
(Studer, 2001), the
equivalent nucleus migrates caudally to r6 during development. In the lamprey,
the facial nucleus does not show such migration
(Fig. 8), indicating that its
migratory properties were acquired secondarily in gnathostome evolution. It is
noteworthy that the boundary between the trigeminal and facial nuclei did not
correspond to the r3/r4 boundary, which is unlike any gnathostome analyzed so
far (see Fig. 8 for a
comparison).
Together, these observations suggest that variations in motoneuron identity
along the anteroposterior axis of vertebrate embryos are not constrained by
hindbrain segmentation. This is particularly apparent from this analysis of
lamprey branchiomotor neuron development, and from accurate comparisons with
the development of the branchiomotor nuclei in gnathostomes
(Fig. 8). Importantly, the
shift between trigeminal and facial motoneuron identities corresponded well
with the anterior boundary of LjHox3 expression, as inferred from
retrograde labeling of motor axons and in situ hybridization. In gnathostomes,
there is mounting evidence that Hox genes control the identity of motoneurons
along the anteroposterior axis. In the developing hindbrain, Hoxa and
Hoxb genes are involved in the specification of cranial motor
neurons, their projections, and migratory properties
(Studer et al., 1996;
Goddard et al., 1996;
Gavalas et al., 1997;
Bell et al., 1999;
Davenne et al., 1999;
Jungbluth et al., 1999;
Gaufo et al., 2000;
McClintock et al., 2002;
Pattyn et al., 2003;
Guidato et al., 2003;
Gaufo et al., 2003).
Interestingly, Hox genes are also involved in spinal motoneuron positional
specification and innervation, despite the absence of neuromeric compartments
in the developing spinal cord [Hoxa10
(Rijli et al., 1995),
Hoxc genes (Tiret et al.,
1998; Dasen et al.,
2003), Hoxd10 (de la
Cruz et al., 1999; Lance-Jones
et al., 2001)]. Therefore, we speculate that the relationship
between Hox gene expression domains and motoneuron identity may be an
ancestral feature conserved throughout the anteroposterior axis of the central
nervous system (hindbrain and spinal cord), and which is evolutionarily as
well as developmentally independent of neuromeric compartments and of the
segmentation process.
Selective effect of retinoic acid on lamprey hindbrain development
It is well established that exogenous RA administration in gnathostomes
causes abnormal development of the brain
(Morriss-Kay et al., 1991;
Conlon and Rossant, 1992;
Marshall et al., 1992;
Durston et al., 1989;
Papalopulu et al., 1991;
Holder and Hill, 1991).
Morphological changes correlate with shifts of Hox gene as well as of other
regulatory gene expression patterns
(Kessel, 1992;
Lopez et al., 1995;
Marshall et al., 1992;
Morris-Kay et al., 1991). In the hindbrain, RA treatment results in complete
changes in the segmental developmental program. For example, the r2 segmental
identity is transformed into that of r4, resulting in both the duplication of
the Mth neuron and the switch in fate from trigeminal to facial branchiomotor
neuron (Hill et al., 1995;
Marshall et al., 1992).
Similar phenotypes occur with the ectopic expression of the paralogue group 1
genes, Hoxa1 or Hoxb1, in r2
(Bell et al., 1999;
Alexandre et al., 1996;
McClintock et al., 2001;
McClintock et al., 2002),
demonstrating that RA-induced repatterning is mediated through Hox gene
function. Thus, in the gnathostomes, rhombomere identity, Hox codes, and
neuronal patterning along the anteroposterior axis are intimately linked and
appear to rely on RA-dependent developmental mechanisms.
In the lamprey, RA treatment also affects axial patterning (Kuratani et al., 1998). In this study, we showed that RA causes a rostral shift in the LjHox3 expression domain, concomitant with the rostral shift of branchiomotor neurons (Fig. 7). As in gnathostomes, the positional specification of lamprey branchiomotor neurons seems to be under the control of RA-dependent and Hox-mediated mechanisms. Interestingly, however, lamprey reticulospinal neurons, including the Mth and bulbar cells, did not change their positions along the neuraxis following RA treatment, nor was the apparent segmental organization of the hindbrain itself altered (Fig. 7). Furthermore, we did not observe duplication of the Mth neurons (Fig. 7).
These data support the idea that neuronal patterning and segmental identity may be independently regulated in the lamprey, and that the dependence of these processes on RA signaling could be distinct in agnathans and gnathostomes.
Evolution of the vertebrate hindbrain: a hypothetical scenario
Based on our findings, we speculate that the gnathostome-like hindbrain
organization may have developed through independent mechanisms of
anteroposterior patterning during evolution
(Fig. 9).
As noted above, an ancestral anteroposterior program of neuronal patterning
would have already been present at the pre-vertebrate, amphioxus-like stage,
dependent upon the position-specific expression of regulatory genes. In
addition to this pattern, rhombomere-like compartments of neuroepithelial
cells giving rise to repeated sets of serially homologous reticular neurons
were also established in the common ancestor of vertebrates. Morphological
segmentation of the rhombomeres would have appeared subsequently in the
vertebrate lineage, perhaps to reinforce such a metameric pattern. Partial
support for this idea comes from studies of amphioxus, which also possesses
interneurons similar to vertebrate reticular neurons
(Fritzsch and Northcutt, 1993;
Fritzsch, 1996) and
anteroposterior molecular regionalization of the neural tube, while lacking a
bona fide segmented hindbrain.
An independent mechanism would have instead involved the anteroposterior
specification of branchiomotor neurons, via a rostrocaudally regulated Hox
code. Indeed, anteroposterior restriction of Hox expression has been
demonstrated in amphioxus (Holland et al.,
1992). Moreover, conservation of cis regulatory mechanisms that
allow Hox expression in the neural tube has been demonstrated between
amphioxus and vertebrates (Manzanares et
al., 2000). Because the Hox code is generally in register with
rhombomere boundaries in gnathostomes, the rhombomere-dependent and Hox
code-dependent neural specification programs have not been distinguished from
one another. Our present work, however, raises the possibility that these two
distinct developmental programs were not acquired simultaneously, but may have
been established as distinct evolutionary events that were secondarily
integrated in the lineage of gnathostomes after the split from agnathans
[Fig. 9; for the concept of
integration, see Hall (Hall,
1998)]. Integration and registering of the two mechanisms in the
gnathostome lineage might have relied partly on the elaboration of cis
regulatory elements at Hox loci. Comparison of Hox regulatory sequences in the
lamprey and gnathostomes may help to test this hypothesis further.
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ACKNOWLEDGMENTS |
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TOPSUMMARYIntroductionMaterials and methodsResultsDiscussionREFERENCES |
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