Dev Genes Evol (1997) 206:515–531
© Springer-Verlag 1997
O R I G I NA L A RT I C L E
&roles:Julie E. Cooke · Qiling Xu · Stephen W. Wilson
Nigel Holder
Characterisation of five novel zebrafish Eph-related receptor tyrosine
kinases suggests roles in patterning the neural plate
&misc:Received: 22 November 1996 / Accepted: 3 January 1997
&p.1:Abstract Eph-related receptor tyrosine kinases (RTKs)
are the largest known subfamily of RTKs, comprising at
least a dozen members. Expression studies suggest roles
for these genes in patterning and differentiation of the
nervous system, the neural crest, developing limbs and
somites. Some of the recently isolated family of ligands
for Eph-related RTKs have been shown to function as
positional identifiers in the retinotectal system. We have
previously characterised three Eph-related RTKs in the
zebrafish (rtk1-3). Here we report the identification of
five new zebrafish Eph-related RTKs (rtk4, rtk5, rtk6,
rtk7 and rtk8) and describe their dynamic expression patterns. Based on these expression patterns, we propose
that rtk4-8 play various roles in establishing territories
within the developing central nervous system (CNS) and
in the subsequent differentiation of defined neuronal
populations.
&kwd:Key words Eph receptors · Receptor tyrosine kinases ·
Zebrafish · Neural plate · Neural patterning&bdy:
Introduction
Receptor tyrosine kinases (RTKs) perform a central role
in transducing signals for growth and differentiation
throughout embryonic development and in the adult. Intercellular communication via RTKs can result in the acquisition of specific cell fates and in the depletion, expansion or maintenance of defined cell populations.
RTKs, therefore, make an important contribution to embryonic pattern formation. For example, in Drosophila,
the sevenless RTK is required for the adoption of the R7
photoreceptor cell fate in the developing eye and, in
mammals, the c-kit RTK plays a key role in the mainteEdited by J. Campos-Ortega
J.E. Cooke · Q. Xu · S.W. Wilson · N. Holder ( ✉)
Developmental Biology Research Centre, Randall Institute,
Division of Biomedical Science, King’s College London,
26–29 Drury Lane, London, WC2B 5RL, UK&/fn-block:
nance of melanocyte, haematopoietic stem cell and primordial germ cell populations (see review by Pawson
and Bernstein 1990).
The Eph-related subfamily is currently the largest
group of RTKs, with at least 12 members. The subfamily is characterised by shared sequence motifs including
an immunoglobulin-like domain, 19 conserved cysteine
residues, 2 fibronectin type III repeats and an uninterrupted catalytic domain whose sequence is preferentially conserved amongst Eph-related RTK subfamily
members (Tuzi and Gullick 1994; Van der Geer et al.
1994). Eph, the prototype member of the subfamily, was
first identified as an oncogene from an erythropoietinproducing hepatoma cell line (Hirai et al. 1987; Maru et
al. 1990). Eph-related RTKs have been identified in a
variety of vertebrates including mammalian species,
chick, Xenopus and zebrafish (Van der Geer et al.
1994).
In situ hybridisation and protein localisation analyses
have provided evidence to support potential roles for
Eph-related RTKs in early patterning events in several
developing systems, including the nervous system, developing limb, somites and neural crest (Lai and Lemke
1991; Nieto et al. 1992; Becker et al. 1994; Xu et al.
1994). Such studies have also implicated these receptors
in later, differentiation-associated events in the nervous
system such as axon pathfinding and fasciculation
(Cheng and Flanagan 1994; Cheng et al. 1995; Drescher
et al. 1995; Winslow et al. 1995). More recently, functional studies using a dominant negative strategy have revealed specific roles for the Sek-1 Eph-related RTK in
establishing segmental patterns of gene expression during hindbrain development in Xenopus and in zebrafish
(Xu et al. 1995), and in the formation of the zebrafish
eye (Xu et al. 1996).
The recent isolation and characterisation of a family
of ligands for the Eph-related RTKs (reviewed by Pandey et al. 1995a) has enabled the functions of these receptors to be further analysed using in vivo and in vitro
techniques. Eph family ligands are membrane anchored,
either via a glucosyl phosphatidylinositol (GPI) linkage
516
A
Fig. 1A–C Comparison of the deduced amino acid sequences of
rtk4, rtk5, rtk6, rtk7 and rtk8 with other Eph-related receptor tyrosine kinase (RTK) genes. A Schematic diagram of RTK structure
showing the extent of partial cDNA clones used for sequencing
and in situ hybridisation analysis. Bar represents RTK coding region, numbers represent approximate amino acid positions. Lines
represent cDNA clones, arrows at the 3′ end indicate that these
clones extend beyond the coding region. For rtk6 and rtk8, the longer of the two clones illustrated was used for sequence analysis.
Asterisks denote clones from which antisense RNA probes for in
situ hybridisation analysis were synthesised. B Alignment of deduced amino acid sequences from partial cDNAs representing
rtk4-8 and the complete coding sequences of htk (Bennett et al.
1994) and Eph (Hirai et al. 1987). Sequences were aligned and
displayed using the Genetics Computer Group Pileup and Pretty
programs. Dots represent gaps inserted in order to optimise sequence alignment. Probable transmembrane domains are underlined, arrows mark the extent of the kinase domain. Shaded boxes
indicate amino acid identity in more than 50% of the sequences
shown at that point. C Dendrogram generated using the Genetics
Computer Group Pileup program, illustrating the relatedness of
rtk4, rtk5, rtk6, rtk7 and rtk8 (shown underlined) with other members of the Eph family from human (eck, erk, htk, hek7, hek, hek8,
hek11 and eph), mouse (Sek-2, Sek-4, Sek-3, mdk2, mek4, sek and
ebk), rat (elk, ehk3, ehk2 and eek), chick (cek4, cek5, cek6, cek7,
cek8 and cek9) and Xenopus (tck, Xelk and Xsek). All sequences
were obtained from GenBank or EMBL databases&ig.c:/f
or an intrinsic transmembrane domain, a feature necessary for their activity. Considerable cross-specificity of
binding exists between the Eph-related RTKs and their
ligands in vitro (Brambilla et al. 1995). Expression pattern analyses and in vitro experiments have implicated
Eph family ligands in establishing the correct topographical relationship between retinal ganglion cell axons and the optic tectum (see review by Tessier-Lavigne
1995).
We have previously described the isolation of three
zebrafish Eph-related RTKs and reported on their spatially regulated expression during gastrulation (Xu et al.
1994). In this paper, we describe the identification of five
novel zebrafish Eph-related RTKs (rtk4-8) and their dynamic expression patterns throughout embryonic development and propose possible roles for these receptors.
Materials and methods
Maintenance of fish
Breeding fish were maintained at 28.5°C on a 14-h light/10-h dark
cycle. Embryos were collected by natural spawning, raised in 10%
Hank’s saline and staged according to Kimmel et al. (1995).
Isolation and characterisation of cDNA clones
Complementary DNA clones for rtk5, rtk6, rtk7 and rtk8 were isolated by screening a λZAPII neurula-stage cDNA library (provided by Dr. David Grunwald) by plaque hybridisation at moderate
stringency. The probe used was a 32P-labelled random-primed
probe derived from a cDNA fragment of rtk1 (Xu et al. 1994),
which included the kinase domain coding sequence. Rtk4 cDNA
was isolated from a 3–15 h random-primed cDNA library (provided by Dr. Jose Campos-Ortega), probed with a fragment of rtk1
cDNA encoding part of the extracellular domain. Standard protocols were followed for prehybridisation, hybridisation and posthybridisation washing (Sambrook et al. 1989). Three rounds of
hybridisation were performed prior to rescue of positive Bluescript
phagemids. Nucleotide sequence of the novel cDNAs was obtained using the Sequenase Version 2.0 kit (Amersham) or the ABI
373 Automated Stretch Upgrade (Applied Biosystems, Perkin-Elmer Corporation) and synthetic oligonucleotide primers. Sequence
comparisons were performed using software from the University
of Wisconsin Genetics Computer Group (Program Manual for the
Wisconsin Package, Version 8, September 1994: Genetics Computer Group, 575 Science Drive, Madison, WI 53711, USA).
Whole-mount in situ hybridisation and immunostaining
Whole-mount in situ hybridisation using digoxygenin-labelled antisense RNA probes was performed essentially as described (Xu et
al. 1994). The probes used spanned the entire length of the partial
cDNAs for each gene (see clones marked with an asterisk in
Fig. 1A) and were hydrolysed to give an expected fragment size of
0.5 kb. Two-colour in situ hybridisations were performed essentially as described (Hauptmann and Gerster 1994). Antibody staining with a 1:20 dilution of the monoclonal antibody HNK-1 was
performed as described (Xu et al. 1994). Manually-sectioned embryos were cut with a hypodermic needle. Embryos were cleared
and mounted in 70% glycerol for photography.
517
B
Continuation of fig. 1 see page 518
518
C
Table 1 Deduced amino acid sequence comparisons between
rtk4-8 and related Eph subfamily receptors. Each zebrafish RTK is
compared with its most closely related database sequence, as
judged using the Genetics Computer Group BlastX program. Percentage similarity and identity were calculated using the Genetics
Computer Group Gap program&/tbl.c:&
rtk4/xsek
rtk5/htk
rtk6/eck
rtk7/cek7
rtk8/mdk2
Overall
Kinase domain
%
%
similarity identity
%
%
similarity identity
88
77
76
81
78
96
96
89
92
95
78
64
60
68
64
91
90
78
85
89
&/tbl.:
Table 2 Deduced amino acid sequence comparisons between
rtk4-8. Percentage identities were calculated using the Genetics
Computer Group Gap program&/tbl.c:&
rtk4
rtk4
rtk5
rtk6
rtk7
rtk8
rtk5
*
75
52
*
52
50
67
57
53
72
Overall
rtk6
rtk7
rtk8
74
70
*
56
50
82
74
72
*
58
77
94
71
75
*
Kinase domain
&/tbl.:
Continuation of fig. 1 (legend see p 516)
Results
Cloning and characterisation of rtk4, rtk5, rtk6, rtk7
and rtk8 - evidence that they are members
of the Eph-related family of receptor tyrosine kinases
In order to isolate novel zebrafish Eph-related RTKs, a
zebrafish neurula-stage cDNA library (kindly provided
by Dr. David Grunwald) was screened using a probe derived from the kinase domain of rtk1 (Xu et al. 1994).
The most strongly hybridising plaques were not selected
in order to avoid re-isolating rtk1. On the basis of restriction maps and preliminary sequence analysis, the isolated cDNA clones were divided into six classes. Two
clones corresponded to rtk2, seven clones corresponded
to rtk3, five clones were classed as rtk5, eight as rtk6,
two as rtk7 and three as rtk8. Rtk4 was cloned by screening a random-primed 3–15 h cDNA library (kindly pro-
vided by Dr. Jose Campos-Ortega) under moderate stringency using a probe derived from the extracellular domain of rtk1.
The nucleotide sequence was obtained for the longest
cDNA representing each of rtk4, rtk5, rtk6, rtk7 and rtk8
(Fig. 1A, B). Deduced amino acid sequence comparisons
with database sequences revealed a high degree of homology primarily to Eph-related RTKs (Table 1, Fig. 1B,
C). Database searches using the Blastx program of the
Genetics Computer Group indicated that the amino acid
sequence of rtk4 is most similar to Xenopus Sek-1, rtk5
is most similar to human htk, rtk6 to human eck, rtk7 to
chicken cek7 and rtk8 to mouse mdk2. Interestingly, htk
and mdk2 are presumed orthologues of each other, so
both rtk5 and rtk8 are most related to Eph-related receptors from the same orthologous group. The implications
of these sequence comparisons are discussed below (see
Discussion). Table 2 shows the percentage identity between rtk4, rtk5, rtk6, rtk7 and rtk8. The putative kinase
domain sequences of these five novel zebrafish genes are
as closely-related to each other as to their presumed closest relatives from other species (see Table 1). However,
the zebrafish sequences share less identity with each other than with their possible orthologues when overall sequence is taken into account (see Discussion).
519
Fig. 2A–D Expression of rtk4. Whole-mounted embryos hybridised with a digoxygenin-labelled antisense RNA probe to rtk4
(blue). Lateral views, anterior is to the left. A Sixteen somites, rostral end. Transcripts are detected in the presumptive telencephalon.
B Sixteen somites, hindbrain region. Low level expression is detected throughout the dorsal half of the hindbrain with higher levels in r3 and r5. Highest level expression is detected specifically in
a group of presumptive neurons in r3. C Twenty-four hours post
fertilisation (hpf), rostral end. Transcripts are detected throughout
the hindbrain with higher levels in r5 and r6. Other sites of expression are the ventral midbrain (tegmentum, arrowhead) and developing branchial arches. D Forty-eight hpf, branchial arch region.
Expression is detected in branchial arches 2, 3, 4 and 5 and in the
ventrocaudal part of the otic vesicle. Numbers refer to branchial
arches (hb hindbrain, ov otic vesicle, r rhombomere, tc presumptive telencephalon, scale bars 50 µm)&ig.c:/f
Expression patterns indicate roles for these genes
in patterning and differentiation of neural
and mesodermal cells
The expression patterns of the five novel Eph-related
RTKs were examined by in situ hybridisation of digoxygenin-labelled antisense RNA probes to whole zebrafish
embryos fixed at regular intervals between the onset of
gastrulation (5 hpf, hours post-fertilisation) and the longpec stage (48 hpf; see Kimmel et al. 1995 for details and
terminology of staging).
Rtk4
Rtk4 transcripts are first detected at the 15-somite stage
(16.5 hpf) in the presumptive telencephalon (Fig. 2A)
and hindbrain (Fig. 2B). There is a low level of expression throughout the dorsal half of the developing hindbrain with a much higher level in rhombomeres (r) 3 and
5 (Fig. 2B). The highest level of expression is detected
specifically in some of the presumptive neurons located
dorsally and medially within r3 (Fig. 2B).
The expression of rtk4 in the presumptive telencephalon is transient and is no longer detectable in embryos at
the 22-somite stage (20 hpf, data not shown). By the
prim-5 stage (24 hpf), expression is detected throughout
the hindbrain with a higher level in r5 and r6 but the high
level neuronal expression has disappeared (Fig. 2C).
Rtk4 transcripts are also present in the ventral midbrain
and developing branchial arches (Fig. 2C).
In long-pec stage embryos (48 hpf), rtk4 transcripts
are present in the developing cerebellum and, at a low
level, in the tectum. Expression of rtk4 in the hindbrain
is maintained and expression in the second to fifth developing branchial arches has become more apparent at this
stage (Fig. 2D). In addition, rtk4 transcripts are found in
the ventrocaudal part of the developing otic vesicle
(Fig. 2D) and in a group of cells located in the ventrolateral part of r7.
520
Fig. 3A–F Expression of rtk5. Whole-mounted embryos hybridised with a digoxygenin-labelled antisense RNA probe to rtk5
(blue, A–F) and a fluorescein-labelled antisense RNA probe to
krx20 (red, D). Dorsal views, anterior is to the left (except A
where anterior is to the top). A 70% epiboly. Transcripts are detected in the germ ring and dorsally along the midline (arrowhead). B Five somites, rostral end. Transcripts are detected in
stripes in the presumptive forebrain/midbrain and in presumptive
r2 and r5/6. C, D Ten somites, rostral end C and hindbrain region
D. Stripes of rtk5 expression (blue) are in the caudal diencephalon/rostral midbrain, at the midbrain/hindbrain boundary, in r2 and
in r5/6. Expression of krx20 (red in D) marks r3 and r5. Expression of rtk5 is also detected in the optic stalk region. Arrowheads
in C indicate neural crest; arrow in D indicates the r2/r3 boundary.
E Eighteen somites, rostral end. Stripes of rtk5 expression are still
detected in the caudal diencephalon/rostral midbrain, at the midbrain/hindbrain boundary (arrow) and in r5/6. The expression in
r2 has disappeared by this stage. Additional sites of expression include the optic stalks and ventrolateral cells at the axial level of
r5/6. F Prim-11, trunk region. Rtk5 is expressed in the migrating
primordia of the posterior lateral line (arrowheads; mb midbrain,
os optic stalk, r rhombomere, scale bars 50 µm)&ig.c:/f
Rtk5
Rtk5 transcripts are detected throughout the germ ring at
approximately 50% epiboly (5.25 hpf). By 70–80% epiboly (8 hpf), expression in the germ ring is stronger and
extends along the midline (Fig. 3A). Expression is seen
around the dorsal margin of the yolk plug at 100% epiboly (10 hpf) and in the axial midline and two transverse
stripes near the anterior end of the neural plate. The
more anterior stripe is broader and stronger than the
more caudal stripe. The rostral limit of the neural plate is
also rtk5-positive at this stage.
The two stripes of rtk5 expression in the neural plate
persist and, by the 3 somite stage (11 hpf), groups of
rtk5-positive cells are detected extending anteriorly from
the lateral limits of these stripes. At this stage, an additional stripe of rtk5-expressing cells becomes apparent
more caudally. Initially, the signal is weak but it becomes
stronger and the anterior and posterior boundaries become more defined by the 5-somite stage (11.7 hpf;
Fig. 3B). Regions of rtk5-positive cells are detected lateral to the neural plate, adjacent to the two more caudal
stripes. Their position is consistent with a neural crest
identity. By performing two-colour double in situ hybridisations with probes for rtk5 and krx20 (Oxtoby and
Jowett 1993), and by analysing the positions of the
stripes of rtk5-expressing cells with respect to morphological landmarks in later stage embryos, we concluded
that the most rostral stripe of rtk5 expression corresponds to a region of the presumptive forebrain and midbrain, extending from the presumptive mid-diencephalon
to the midbrain/hindbrain boundary. The middle stripe
521
corresponds to presumptive r2 and the most caudal stripe
corresponds to presumptive r5 then to r5/6 (Fig. 3B-D).
At the 5-somite stage, a lateral view of the embryo
shows rtk5-positive cells surrounding the optic vesicle.
The axial expression of rtk5 is weaker than at earlier
stages and appears to extend as far rostrally as presumptive r6.
By the 10-somite stage (approximately 14 hpf), the
most anterior stripe of rtk5-expressing cells (in the presumptive forebrain/midbrain) has resolved into two
stripes of stronger expression at its previous rostral and
caudal limits, with weaker expression in between
(Fig. 3C). The stripes of rtk5 expression in presumptive
r2 and r5/6 are well defined at their anterior boundaries
but less well defined at their posterior boundaries. The
stripe of rtk5 expression in presumptive r2 partially overlaps with krx20-expressing cells in r3 (Fig. 3D). Rtk5-expressing cells extend anteriorly from the rostral/lateral
limits of the presumptive r2 stripe and from the caudal/lateral limits of the presumptive r5/6 stripe. Their position suggests that these cells are neural crest (Fig. 3C,
D).
A striped pattern of rtk5 expression is apparent until
approximately the prim-5 stage (24 hpf), although the
stripe of rtk5 expression in r2 becomes weaker and is undetectable by the 18-somite stage (18 hpf; Fig. 3E). In
addition to the striped pattern, rtk5 expression is widespread in the telencephalon, is detected at low levels
throughout the hindbrain (Fig. 3E) and is also detected in
the pronephros.
By the 24-somite stage (21 hpf), the two most anterior
stripes of rtk5 expression correspond to the dorsal diencephalon and a narrow band at the midbrain/hindbrain
boundary (see Fig. 7B). The ventrolateral region of expression extending from the r5/6 stripe is very prominent.
By the prim-8 stage (approximately 27 hpf), the stripe
of rtk5 expression in r5/6 is undetectable and instead
there is widespread low-level expression throughout the
hindbrain. Expression of rtk5 in the telencephalon, dorsal diencephalon and at the midbrain/hindbrain boundary
is still apparent (Fig. 8A). The migrating posterior lateral
line primordium is distinctly labelled as an extremely lateral clump of approximately 20 cells at the level of the
myoseptum on either side of the embryo at about the level of the 8th somite. Figure 3F shows posterior lateral
line primordia stained for rtk5 in a prim-11 stage embryo. From the prim-8 stage onwards, rtk5 transcripts are
detected in the primordium of the pectoral fin.
Expression of rtk5 is detected in six posterior lateral
line structures, at approximately 5-somite intervals at the
long-pec stage (48 hpf). The pectoral fin buds and developing branchial arch structures express high levels of
rtk5 at this stage.
Rtk6
Transcripts for rtk6 are detected at approximately 60%
epiboly (6.5 hpf) in a triangular region extending from
the germ ring to a point on the dorsal side of the embryo
(Fig. 4A). This region of expression extends rostrally as
the anteroposterior axis develops.
By 100% epiboly (10 hpf), a high level of rtk6 expression is detected in the developing notochord
(Fig. 4B). In the caudal third of the embryo, cells just
lateral to these midline cells are also rtk6-positive. Close
to the anterior limit of axial expression (approximately
40% of embryo length from the rostral end of the 100%
epiboly stage embryo) a dorsal transverse stripe extends
to the lateral limits of the neural plate (Fig. 4B). The axial level of this stripe is caudal to the future midbrain/hindbrain boundary as shown by double in situ hybridisation experiments with probes for rtk6 and pax2
(Krauss et al. 1991; data not shown). A more rostral
stripe of rtk6-positive cells lies in presumptive forebrain
or midbrain tissue and expression of rtk6 is also detected
around the rostral boundary of the neural plate.
By the 3-somite stage (11 hpf), the domain of rtk6 expression adjacent to the notochord has extended rostrally
and expression in the notochord appears more punctate
(Fig. 4D). A second transverse stripe of rtk6 expression is
detected caudal to the stripe in the presumptive hindbrain
initially detected at 100% epiboly (Fig. 4C, D). Two-colour double in situ hybridisations with probes for rtk6 and
krx20 (Oxtoby and Jowett 1993) show that the rtk6
stripes are in presumptive r3 and r5 (Fig. 4C, D). Unlike
the striped expression domains of rtk5 in the neural plate
(Fig. 3B-E), the stripes of rtk6 expression in presumptive
r3 and r5 do not show sharp boundaries. Cells expressing
rtk6 extend both rostrally and caudally from the extreme
lateral ends of the presumptive r3 stripe. These cells extend the length of the trunk and mark the lateral edges of
the neural plate (Fig. 4D). Rostrally, rtk6-expressing cells
surround the eye primordia (Fig. 4D).
Expression of rtk6 in the notochord is only apparent
at its caudal end by the 10-somite stage (approximately
14 hpf). Rtk6 transcripts are detected in paraxial mesodermal cells along the length of the trunk and in the ventral spinal cord adjacent to the floor plate. A widespread
distribution of rtk6 transcripts is seen throughout the dorsal hindbrain. Ventrally in the hindbrain, regularly
spaced individual cells and doublets, paired across the
midline, express high levels of rtk6 transcripts. Discrete
patches of rtk6-positive cells are detected in the ventral
midbrain, dorsal diencephalon and in the telencephalon.
By the 14-somite stage (16 hpf), expression of rtk6 in
the dorsal hindbrain is stronger in r1-3 and r5-6 and
weaker in r4. The dorsal edge of the otic vesicle is rtk6positive. The ventral region of the hindbrain shows more
widespread expression of rtk6 than at previous stages although sections show that the floor plate is not stained.
Most other elements of the rtk6 expression pattern in 10somite-stage embryos are retained during subsequent development, up to at least the prim-25 stage (36 hpf).
522
Fig. 4 Legend see on page 523
523
▲
At the 18-somite stage (18 hpf), expression of rtk6 is
detected in a clump of approximately 20 cells on either
side of the embryo in a very lateral position (lateral to
the otic vesicle) at the level of r5. In the trunk, rtk6 is expressed in the ventral neural tube adjacent to the floor
plate and in the medial part of the somites. The extreme
caudal end of the notochord still expresses rtk6 and transcripts are also detected around the edge of the developing tail fin and in the cloaca.
At the 24-somite stage (21 hpf), rtk6-positive cells
are still detected in discrete patches in the ventral midbrain, diencephalon and telencephalon (Fig. 4E). The
patch of rtk6-positive cells in the ventral midbrain is
dorsal and slightly caudal to the nucleus of the medial
longitudinal fasciculus (mlf), as shown by HNK-1 immunostaining in this region (Metcalfe et al. 1990;
Fig. 4F). Transverse sections cut through the midbrain,
hindbrain and trunk reveal that rtk6 expression in the
ventral neural tube extends across the entire width of the
neuroepithelium and is dorsal to the floor plate in the
motor region of the neural tube (Fig. 4E-H). In the hindbrain, expression of rtk6 is apparent in the dorsal aspect
of the neural tube as well as ventrally (Fig. 4E, G). Rtk6
expression in a one-cell-wide column in the dorsal spinal cord is apparent from a lateral view of the 24somite-stage embryo. The medial part of the somites are
still rtk6-positive (Fig. 4H).
At the prim-8 stage (approximately 27 hpf), new sites
of strong rtk6 expression include the posterior lateral line
primordium and the pectoral fin bud. Expression domains in the CNS remain similar to previous stages
(Fig. 8B). By the prim-25 stage (36 hpf), expression of
rtk6 in the pectoral fin bud is confined to the fin margin.
Fig. 4A–H Expression of rtk6. Whole-mounted embryos hybridised with a digoxygenin-labelled antisense RNA probe to rtk6
(blue, A–H) and a fluorescein-labelled antisense RNA probe to
krx20 (red, C) or immunostained with the HNK-1 antibody
(brown, F). A–D Dorsal views and E lateral view, anterior is to the
top (A, B) or left (C–E). E–H Manual transverse sections, dorsal
is to the top. A 60–70% epiboly. Rtk6 transcripts are detected dorsally along the extending axial midline. B 100% epiboly. Transcripts are detected in the developing notochord and in a transverse stripe in the presumptive hindbrain. C Three somites, presumptive hindbrain region. High power view showing that transverse stripes of rtk6 expression (blue) coincide with those of krx20
(red) in r3 and r5. D Three somites. Rtk6 transcripts are detected
in the presumptive forebrain, in the axial midline and paraxial mesoderm cells of the trunk, in r3 and r5 and extending along the
edges of the neural plate. E Twenty-four somites. Transcripts are
detected in the forebrain, in discrete regions of the ventral midbrain and in dorsal and ventral longitudinal columns of cells in the
hindbrain and spinal cord. Dashed lines mark the approximate
plane of section in F, G. F Section through midbrain of 24-hpf
embryo. Expression of rtk6 in the neural tube spans the width of
the neuroepithelium and is dorsal to the nucleus of the medial longitudinal fasciculus (nMLF, arrowhead). G Section through hindbrain of 25- to 30-somite embryo. Rtk6 transcripts are apparent in
the motor region (arrow) and a dorsal region of the neural tube. H
Section through trunk of 25- to 30-somite embryo. Transcripts are
detected in the motor region and in the medial part of the somites.
The floor plate does not express rtk6. (f floor plate, fb presumptive
forebrain, n notochord, r rhombomere, s somite, sc spinal cord,
scale bars 50 µm)&ig.c:/f
Rtk6 expression appears to be down-regulated in most
tissues by the long-pec stage (48 hpf).
Rtk7
Rtk7 transcripts are first detected at a later stage than
those of rtk5, rtk6 and rtk8. A low level of expression is
seen at the posterior end of tailbud-stage embryos
(10 hpf), extending anteriorly along the axial midline.
Axial midline expression of rtk7 is still weak at the 3somite stage (11 hpf), but cells showing strong expression of rtk7 are now seen in the presumptive forebrain
region (Fig. 5A). By the 5-somite stage (11.7 hpf), expression of rtk7 in the presumptive forebrain surrounds
the eye primordia and, by the 10-somite stage (approximately 14 hpf), this pattern has resolved into a stronger
signal in the presumptive telencephalon and weaker in
the presumptive diencephalon. At the 10-somite stage,
rtk7 expression is detected in individual cells or small
groups of 2–3 cells paired across the midline in the presumptive hindbrain. The location of these cells and the
timing of this expression suggests that they may be reticulospinal neurons. Transverse stripes of weak rtk7 expression are located in the presumptive midbrain and in
the rostral hindbrain. Rtk7 transcripts are apparent
ventrolaterally in the spinal cord in cells which may be
primary interneurons.
Between the 10- and 18-somite stages (14–18 hpf),
additional presumptive reticulospinal neurons, paired
across the axial midline, become rtk7-positive. By the
18-somite stage (18 hpf), regularly-spaced, labelled cells
are located along the lateral margins of the entire length
of the hindbrain (Fig. 5B). Triangular groups of 10–15
cells, one on either side of the embryo just anterior to the
otic vesicle, show a high level of rtk7 expression
(Fig. 5B). These cells may represent the primordia of
one of the preotic ganglia; rtk2 is expressed in a similar
group of cells (Xu and Holder, unpublished observations). At the 18-somite stage, rtk7-expressing cells are
found in the telencephalon, diencephalon and midbrain
(Fig. 5B). Rtk7 transcripts are also detected in the eye, at
a higher level in the future dorsal/temporal half than in
the future ventral/nasal half (Fig. 5B, C). Low levels of
rtk7 transcripts are detectable in the neuroepithelium of
r2 and r3. Expression of rtk7 ventrolaterally in the spinal
cord remains strong and more cells become rtk7-positive
in the dorsal spinal cord.
By the 24-somite stage (21 hpf), rtk7 is widely expressed in the hindbrain, except for r1 in which only one
or two cells on each side of the midline are rtk7-positive
(Fig. 5C, D). A diffuse staining is apparent in the midbrain, although a group of cells within the ventral midbrain are stained more strongly (Fig. 5C). Rtk7 transcripts are detected at high levels in the telencephalon,
expression in the eye is still apparent and the pattern of
rtk7 expression in the spinal cord remains similar to earlier stages. Transverse sections cut at the level of the
hindbrain or spinal cord show staining in the ventrolater-
524
Fig. 5A–F Expression of rtk7. Whole-mounted embryos hybridised with a digoxygenin-labelled antisense RNA probe to rtk7
(blue) and immunostained with the HNK-1 antibody (brown, F).
A–D Dorsal views, anterior is to the left. E, F Manual transverse
sections, dorsal is to the top. A Three somites, rostral end. Transcripts are detected in the presumptive forebrain. B Eighteen somites. Transcripts are detected in the eye, forebrain, midbrain and
at low levels throughout the hindbrain (except r1). High-level expression is seen in paired, regularly-spaced presumptive hindbrain
reticulospinal neurons and in preotic ganglia (arrows). C Twentyfour somites. Expression is detected in the eye, telencephalon,
midbrain and hindbrain. High-level expression is seen in groups of
cells within the ventral midbrain (see F) and in paired, regularlyspaced presumptive hindbrain reticulospinal neurons. D Twentyfour somites. Region of caudal midbrain/rostral hindbrain showing
high level expression of rtk7 in one or two cells each side of the
midline in rhombomere 1 (arrowheads). E Section through trunk
of 24-somite embryo. Rtk7-positive cells are detected in the ventrolateral aspect of the neural tube (arrow). F Section through
midbrain of 24-hpf embryo. Transcripts of rtk7 are detected in
cells just medial to HNK-1-positive cells in the nucleus of the medial longitudinal fasciculus (nMLF, arrowhead, hb hindbrain, e
eye, fb presumptive forebrain, mb midbrain, n notochord, ov otic
vesicle, r rhombomere, scale bars 50 µm)&ig.c:/f
al regions of the neural tube, primarily in cells at the pial
surface of the neuroepithelium (Fig. 5E). These cells
may be primary interneurons.
Several more cells in r1 express rtk7 by 24 hpf and
more cells in the ventral midbrain appear strongly rtk7positive. These midbrain cells are just medial to cells of
the nucleus of the mlf as shown by HNK-1 immunostaining (Metcalfe et al. 1990; Fig. 5F), so they may contribute to this nucleus once differentiated.
Expression of rtk7 in the spinal cord is down-regulated by the prim-8 stage (approximately 27 hpf). The regions of rtk7 expression in the telencephalon, midbrain
and hindbrain show uniform staining from the prim-8
stage (Fig. 8C) to the long-pec stage of development
(48 hpf) with no individual, strongly-stained cells being
apparent.
Rtk8
Transcripts for rtk8 are first detectable at least as early as
50% epiboly (5.25 hpf), when they are concentrated in
the germ ring. By 70% epiboly (8 hpf), rtk8 is strongly
expressed in the presumptive mesendoderm (Fig. 6A). At
85% epiboly, rtk8 is expressed at higher levels on the future dorsal side (the side of the embryonic shield) than
elsewhere around the germ ring (Fig. 6B). Rtk8-positive
cells spread above and below the germ ring on the dorsal
side (Fig. 6A, B). The rtk8-positive cells on the vegetal
side of the shield correspond to the forerunner cells, a
population of cells which, according to fate mapping
studies, give rise to the epithelial lining of Kupffer’s vesicle (Melby et al. 1996).
At the tailbud stage (10 hpf), the expression of rtk8 is
very strong around the yolk plug, especially at its dorsal
525
Fig. 6A–G Expression of rtk8. Whole-mounted embryos hybridised with a digoxygenin-labelled antisense RNA probe to rtk8
(blue). A, D, E, G Dorsal views, B vegetal view, dorsal to top, C
ventrolateral view and F lateral view; anterior is to the top (A, B,
G) or to the left (C–F). A 70% epiboly. Transcripts are detected in
the germ ring and in the forerunner cells which are just vegetal to
the germ ring on the dorsal side (arrowhead). B 85% epiboly.
Transcripts are at a higher level dorsally (arrowhead). C Five somites, caudal end of embryo. Transcripts are detected in the tail
bud mesenchyme and around the epithelium of Kupffer’s vesicle.
D Five somites. Transcripts are detected in the tailbud region, the
extreme caudal end of the notochord and in a transverse stripe in
the presumptive midbrain. E Fourteen somites. Rtk8 transcripts are
still detected in the tailbud region and presumptive midbrain. In
addition, the optic stalks and otic vesicle express rtk8, and a low
level of expression is detected in the hindbrain. F Prim-5 stage,
caudal end of embryo. Transcripts are detected in the tailbud region and in the ventral part of the most posterior-somites. G Prim8 stage, hindbrain region. In addition to low level expression in the
hindbrain, high level expression of rtk8 is seen in an anteromedial
portion of the otic vesicle (arrowheads; gr germ ring, kv Kupffer’s
vesicle, mb presumptive midbrain, n notochord, os optic stalk, ov
otic vesicle, tb tailbud, scale bars 50 µm)&ig.c:/f
526
Fig. 7A, B Diagrams of the developing zebrafish brain at 6-somites (6s), 18-somites (18s) and the prim-5 stage (24 h) showing
prominent expression domains of rtk4-8. Lateral views, rostral is
to the left. A Prominent domains of expression of rtk4 (violet),
rtk7 (blue) and rtk8 (green). B Prominent domains of expression
of rtk5 (red) and rtk6 (purple). Boundaries and extent of overlap
of expression domains are approximate since data is from in situ
hybridisations performed using only one rtk probe per embryo (fb
presumptive forebrain, mb presumptive midbrain, numbers refer to
hindbrain rhombomeres)&ig.c:/f
margin. Weak axial midline expression extends rostrally
from the yolk plug. A transverse stripe of expression is
apparent in the region of the presumptive midbrain and a
medial patch of expression is found rostral to this stripe.
A second transverse stripe of weak rtk8 expression,
caudal to that seen at the tailbud stage, is just detectable
at the 3-somite stage (11 hpf). By the 5-somite stage
(11.7 hpf), the high levels of rtk8 expression at the caudal end of the embryo correspond to the epithelium of
Kuppfer’s vesicle (Kimmel et al. 1995) plus the extreme
caudal tip of the notochord and some of the surrounding
mesenchyme (Fig. 6C, D).
The rostral stripe of rtk8 expression first seen at tailbud stages resolves into two stripes in the presumptive
midbrain by the 10-somite stage (14 hpf). Rtk8-positive
cells are seen just lateral to the neural tube at the level
of the rostral stripe. Transcripts are also detected in a
thin line of cells extending to the anterior limit of the
embryo from this rostral stripe, which delineates the
medial boundary of the developing eye. Weak expression of rtk8 is detected in the hindbrain at the level of r3
and r5, as well as laterally to r5 in the developing otic
vesicle.
At the 14- to 18-somite stages (16–18 hpf), expression of rtk8 at the caudal end of the embryo persists
and expression in the midbrain is similar to the pattern
described for the 10-somite stage embryo (Fig. 6E).
Rtk8 expression is upregulated throughout the hindbrain and appears more uniform than previously. Rtk8
expression continues to be prominent in the otic vesicle, especially along its dorsal margin. The optic stalks
express a high level of rtk8 transcripts and a low level
of expression is detectable throughout most of the forebrain (Fig. 6E).
Regions of the rostral and ventral telencephalon, the
diencephalon, midbrain, optic stalks and lens are
amongst the tissues expressing rtk8 at the 24-somite
stage (21 hpf). Low level expression is detected in the
hindbrain and, in the midbrain, expression surrounds the
ventral edge of the tectal ventricle. Rtk8 is expressed by
the epithelium of the otic vesicle and is still expressed in
the caudal tip of the tail.
The expression pattern of rtk8 between the prim-5
and prim-8 stages (24–27 hpf) is similar to the 24-somite
stage embryo. Notably, CNS expression is still seen in
the telencephalon, diencephalon and ventral midbrain
(Fig. 8D). Additionally, expression is detected in the
pectoral fin buds, the ventral region of the most posterior
somites (Fig. 6F) and, in the otic vesicle, transcripts become restricted to an anteromedial portion of the structure (Fig. 6G).
By the prim-15 stage (30 hpf), expression of rtk8 in
the tail tip is virtually undetectable. The entire midbrain
and the area surrounding the optic recess are rtk8-positive, but the rest of the forebrain is negative or only
weakly rtk8-positive. Transverse stripes of expression,
527
Fig. 8A–D Expression of rtk5, 6, 7 and 8 in the anterior brain at
prim-8 (approx. 27 hpf). Whole-mounted embryos hybridised with
digoxygenin-labelled antisense RNA probe (blue) to rtk5 (A), rtk6
(B), rtk7 (C) or rtk8 (D), and immunostained with anti HNK-1
(brown). Rtk5, 6, 7 and 8 show distinct but overlapping domains of
expression in the anterior brain. Some, but not all, boundaries of
rtk5-8 expression domains lie adjacent to sites of neuronal differentiation (nMLF nucleus of the medial longitudinal fasciculus,
nTPOC nucleus of the tract of the postoptic commissure, Tn telencephalic nucleus, scale bar 50 µm)&ig.c:/f
which appear to correspond to individual rhombomeres,
are apparent in the hindbrain.
Expression of rtk8 in the CNS is weaker between the
prim-25 and long-pec stages (36–48 hpf). At these stages, rtk8 transcripts are present in the developing pectoral
fin, in the hindbrain, midbrain and around the ventral
portion of the otic vesicle, as well as in the branchial
arch region.
The elaborate, dynamic expression patterns of rtk4-8
in the developing brain are schematised in Fig. 7. Relatively simple domains of expression seen at neural plate
stages are superceded by more complex patterns as development proceeds and neuronal differentiation commences. The spatial correlation of rtk5, 6, 7 or 8 expression domains in the anterior brain, with sites of neuronal
differentiation (as shown by HNK-1 immunostaining),
are illustrated in Fig. 8 (prim-8 stage embryos). Rtk5, 6,
7 and 8 show distinct but overlapping domains of expres-
sion in this region. Boundaries of some of the expression
domains shown appear to correspond to regions of neuronal differentiation. For example, the dorsal limit of the
nucleus of the mlf lies adjacent to the ventral boundary
of expression domains of of both rtk6 and rtk7 in the
midbrain; the ventral limit of this nucleus lies alongside
the dorsal boundary of a domain of rtk6 expression
(Fig. 8B, C). However, a low level of rtk8 expression
overlaps with the position of the mlf nucleus (Fig. 8D)
and boundaries of the telencephalic domain of rtk7 expression do not respect the position of the telencephalic
nucleus (Fig. 8C).
Discussion
Spatial and temporal analysis of the expression of rtk4–8
suggests that these receptors may function in several different developmental pathways. Firstly, several of the
genes are expressed in discrete domains or stripes of
neuroepithelial cells during neural plate stages, suggesting roles in establishing pattern in the CNS. The subdivision of the neural plate into tightly restricted domains
with respect to the anterior-posterior axis is probably
necessary for the subsequent elaboration of pattern
which may either be segmental, with reiterated elements
as in the hindbrain, or non-segmental as may be the case
in the forebrain and midbrain. Secondly, some expres-
528
sion appears to be associated with neurogenic domains
of the neuroepithelium (e.g. rtk6 and rtk7). This type of
pattern is reminiscent of the zebrafish achaete/scute
genes ash 1a and b (Allende and Weinberg 1994) which
have been implicated in the control of neuronal fate determination. Thirdly, some of the novel RTKs appear to
be expressed in migratory cells.
The timing of expression of the novel zebrafish genes
is consistent with their involvement in the neuromeric
subdivision of the CNS which occurs at neural plate
stages of development. Within all parts of the developing
brain, the novel RTKs are frequently expressed coincidentally with other regulatory genes believed to function
in establishing regional identity. For example, otx-2 (Li
et al. 1994), pax2 (Krauss et al. 1991) and krx20 (Oxtoby
and Jowett 1993) are expressed in restricted domains of
the neural plate which give rise to forebrain and midbrain tissue, midbrain/hindbrain boundary and rhombomeres 3 and 5, respectively. A role in defining the presumptive forebrain region of the neural plate is suggested by early expression of rtk5, 6 and 7. The recent demonstration of a role for rtk1 in regionalisation of the forebrain supports this possibility (Xu et al. 1996). Like pax6
and rtk1 (Macdonald et al. 1994), rtk6 is broadly expressed in the presumptive forebrain from the end of epiboly and rtk5 and rtk7 show widespread expression in
this region of the neural plate by early-somite stages. At
mid-somite stages all five of the novel RTKs described
here have regionally-restricted expression domains within the forebrain (see Fig. 7).
The spatial relationships between expression domains
of rtk5, 6, 7 and 8 and sites of neuronal differentiation in
the anterior brain were examined in prim-8 stage embryos (Fig. 8). Boundaries of rtk6 and rtk7 expression domains in the forebrain and midbrain corresponded closely to the positions of the developing nuclei of the mlf and
TPOC (tract of the post-optic commisure; Figs. 4F, 5F,
8B, C), in a similar fashion to rtk1 and rtk2 which have
been proposed to play a role in demarcating sites of neuronal differentiation (Macdonald et al. 1994). The
boundaries of domains of rtk5 and rtk8 expression in the
anterior brain appeared to correlate less closely to sites
of HNK-1 immunostaining (Fig. 8A, D). These data suggest that the roles of rtk5 and rtk8 in forebrain patterning
are distinct from those of rtk6 and rtk7 and implicate
rtk6 and rtk7 more strongly as regulators of neuronal differentiation (see below).
Within the hindbrain, it seems very likely that rtk4
and 5 (and, possibly, rtk6 and 7) are involved in establishing or maintaining rhombomeric segmentation. Each
rhombomere appears to express a unique combination of
Eph-family RTKs at around the time segmental subdivisions are first thought to arise. For example, rtk5 is expressed in r2, 5 and 6, rtk1 is expressed in r1, 3 and 5
(Xu et al. 1995) and rtk3 is expressed in r3 and 5 (Xu,
Brennan and Holder, unpublished observations). Furthermore, direct evidence for a role for Eph-related RTKs in
hindbrain segmentation has arisen from experiments in
which the function of rtk1 was analysed by injecting ze-
brafish embryos with RNA encoding a putative dominant-negative form of the receptor (Xu et al. 1995). In
these studies, experimental embryos exhibited disruption
of segmentally-restricted gene expression in the hindbrain. The effects were consistent with an involvement
for rtk1 in the dynamic regulation of cell identity or in
the restriction of cell movement between adjacent hindbrain segments.
The function of rtk4 in the hindbrain is likely to be
different from that proposed for rtk3 and 5, and demonstrated for rtk1, since it is not expressed in this region
until the 15-somite stage. The high level expression of
rtk4 in r3, 5 and, specifically, in a group of presumptive
neurons in r3, suggest that this receptor may be important for the maintenance of segmental identity and/or
may play a role in segment-specific neuronal differentiation. It remains unclear how specificity of Eph-family
RTK function within the hindbrain is achieved. Considerable redundancy of function may operate and it is not
yet known whether these receptors can heterodimerise. If
they do, different heterodimers may show differing ligand preferences/affinities, providing a possible explanation for how alternative combinations of Eph-related
RTKs could give rise to alternative hindbrain segment
identities.
In the developing midbrain, it appears that Eph-related RTKs and their ligands are involved in establishing
the spatially appropriate connections between retinal
ganglion cell axons and the optic tectum which are vital
for visual system function. Positional cues in the tectum
have long been postulated to guide developing nasal and
temporal retinal axons to their topographically correct
target region in the posterior or anterior tectum, respectively. RAGS (repulsive axon guidance signal), an Eph
family ligand, was recently isolated from posterior tectal
membranes of the chick (Drescher et al. 1995). RAGS
exhibits axon-repelling activity and causes growth cone
collapse of temporal retinal axons; it is expressed in a
gradient in the optic tectum with highest levels posteriorly (Drescher et al. 1995). Another Eph family ligand,
ELF-1, has also recently been implicated in retinotectal
patterning (Cheng et al. 1995). Like RAGS, ELF-1 is expressed in a posterior to anterior gradient in the tectum.
Interestingly, Mek4, an ELF-1 receptor, is expressed in
retinal ganglion cells in an opposing gradient to ELF-1
(highest levels in nasal retina) whereas Sek-1, which is
also a receptor for ELF-1, is expressed uniformly across
the retina (Cheng et al. 1995).
Our studies have identified one further Eph family receptor which is expressed in a restricted domain of the
eye. Unlike rtk2, which is expresed in the temporal half
of the eye (Macdonald et al. 1995), rtk7 expression does
not appear to be sharply restricted to a specific domain
of the retina. Instead, transcripts appear highest in the
posterior half of the optic vesicle which later gives rise
to predominantly dorsal and temporal regions of the eye.
The in situ hybridisation analyses of Mek4, rtk2 and rtk7
indicate that Eph-related RTKs show both gradients and
domains of expression in the eye, raising the possibility
529
that combinations of these genes may provide unique
identity labels for retinal ganglion cells. It appears that
retinal ganglion cell identity may be determined by the
combination of Eph-related receptors expressed and that
the combination of Eph family ligands expressed by tectal cells determines their positional identity. The balance
between the repulsive and attractive effects of the ligands
on the different receptors expressed by the axons may be
responsible for accurately establishing appropriate connections, not only along the anteroposterior axis, but also
along the dorsoventral axis.
In addition to broad neuroepithelial domains of expression, some of the zebrafish Eph family RTKs are expressed in neural cells at sites of neurogenesis in the
brain. Thus, both rtk6 and rtk7 are expressed in cells located around the nucleus of the MLF (nMLF). It is interesting that rtk7 is neither expressed in the mature neurons nor in proliferative neuroepithelial cells in this region, raising the possibility that the gene is expressed
transiently in neurons after their final division but before
they express neural antigens. The zebrafish GATA 3 transcription factor orthologue, gta3, is expressed in a similar fashion to rtk7 with respect to the nMLF (Neave et al.
1995). It is thought that gta3 may play a role in specifiying the cell fate of subsets of neurons within the CNS;
perhaps rtk7 is involved in the cascade of molecular interactions required to specify cell fate in presumptive
neurons of the nMLF. Some of the other novel RTKs
presented here are also expressed in domains in neurogenic regions which show little or no neuromeric subdivision. At mid-somite stages, rtk4, 6 and 7 are all expressed at high levels in specific cells of the hindbrain
and/or spinal cord which are often paired across the midline and appear to be segmentally arranged. The position
and size of these cells indicate that they are neurons. At
later stages, rtk4 and 7 become expressed more uniformly throughout the hindbrain and rtk6 is expressed in dorsal and ventral longitudinal columns of neural tissue
throughout the hindbrain and spinal cord. From the position of these columns, it seems likely that they include a
region that gives rise to motor neurons and a region from
which sensory neurons will arise. The expression of rtk4,
6 and 7 initially in specific putative neurons of the hindbrain and then uniformly, suggests roles both in the specification of cell fate within the neuroepithelium and in
later processes associated with differentiated neurons,
for example axon pathfinding and fasciculation. Indeed,
Eph-related RTKs have previously been implicated in
such events. For example, immunolocalisation studies
demonstrated that the mouse receptor, Nuk, is localised
to sites of cell-cell contact often involving migrating
neuronal cells or their extensions and to sites of initial
axon outgrowth (Henkemeyer et al. 1994). In addition,
AL-1, a human homologue of RAGS has been shown to
function in cortical axon fasciculation in vitro, [the receptor implicated in this interaction is Rek7 (Winslow et
al. 1995)], and immunostaining for Cek5 has shown localisation of this receptor to the fasciculated, nonsynaptic portions of parallel fibre axons (Pasquale et al. 1992).
The novel RTKs presented here are expressed in at
least two populations of migrating cells, the neural crest
and the posterior lateral line primordia, suggesting a role
in cell-cell or cell-extracellular matrix interactions. An
involvement in cell migration for Eph family ligand-receptor interactions is not unprecedented, since the chemotropic activity of B61 (a ligand for the Eck receptor)
on endothelial cells in vitro has recently been demonstrated (Pandey et al. 1995b). Furthermore, experimental
evidence supports a role for Eph family molecules in the
movement of growth cones during axon outgrowth in the
retinotectal system (Drescher et al. 1995). The presence
of immunoglobulin-like and fibronectin type III adhesion
motifs in the extracellular domains of Eph-related RTKs
argues for a direct role for these receptors in cell-cell interactions which may be important for cell migration,
since these motifs are also present in neural adhesion
molecules such as L1 and NCAM (Moos et al. 1988;
Cunningham et al. 1987).
Finally, the mesodermal expression of zebrafish Eph
family RTKs suggests that they may be involved in axis
formation and lateral mesodermal development. From
the onset of gastrulation, rtk5 and rtk8 transcripts are
found in the germ ring and this expression is maintained
until gastrulation is complete and the tailbud is formed.
Rtk8 is subsequently expressed in the tailbud mesenchyme and Kupffer’s vesicle, suggesting a role in tail
formation. Rtk6 is expressed early on in the notochord,
paraxial mesoderm and subsequently in the forming-somites. As previously described (Xu et al. 1994), three
other zebrafish Eph-related genes, rtk1-3, are all expressed in the developing mesoderm. Co-expression of a
number of zebrafish Eph family ligands in these mesodermal regions (Brennan, Durbin and Holder, unpublished observations) strongly indicates a role for signalling through these receptors in the formation of the axis
and lateral mesoderm.
Homologies of rtk4-8 with other vertebrate Eph-related
RTKs
The rtk4 sequence presented here shows greatest similarity to Xenopus Sek-1 (Xu et al. 1995), the Xenopus orthologue of mouse Sek-1 (Gilardi-Hebenstreit et al. 1992).
Rtk4 and Xenopus Sek-1 share 91% identity in the kinase
domain and 78% identity overall (Table 1). However,
rtk4 is unlikely to be functionally analogous to Sek-1
since the expression patterns are dissimilar and a zebrafish orthologue of Sek-1, rtk1, has already been identified (Xu et al. 1994). Rtk1 is 94.5% identical to mouse
Sek-1 in the kinase domain and these two genes also
share a high degree of sequence similarity in the C-terminal region; furthermore, their expression patterns are
very closely related (Nieto et al. 1992; Xu et al. 1994,
1995). So, if sequence data alone were taken into account, rtk4 would be assigned as an orthologue of Xenopus Sek-1, suggesting a close evolutionary relationship
between these two genes. The above observations dem-
530
onstrate that an apparent evolutionary relationship does
not necessarily imply conservation of function.
Database searches indicate that rtk5 and rtk8 are most
closely related to human htk (Bennet et al. 1994) and
mouse mdk2 (Ciossek et al. 1995), respectively, presumed orthologues of each other (Table 1, Fig. 1C). Htk
and mdk2 are 96% similar and 92% identical to each other overall, whereas rtk5 and htk are 77% similar and 64%
identical overall, and rtk8 and mdk2 are 78% similar and
64% identical overall (Table 1). These sequence comparisons suggest either that there may not be a direct one-toone relationship between Eph subfamily members in zebrafish and in other vertebrates, or that orthologues for
rtk5 and rtk8 have not yet been discovered in other species (see below). Insufficient data on the embryonic expression patterns of htk and mdk2 prevents parallels from
being drawn with the embryonic expression patterns of
rtk5 or rtk8.
Rtk5 and rtk8 share a greater degree of sequence identity with each other than any other pair of zebrafish
RTKs presented in this paper (Table 2). However, since
their expression patterns are distinct from each other, it
is likely that their regulatory regions differ significantly.
It will be interesting to see whether rtk5 and rtk8 can
compensate for each other functionally.
The sequence of rtk6 presented here is most similar to
human eck (Lindberg and Hunter 1990); the deduced
amino acid sequences of these two receptors are 60%
identical overall, and 78% identical over the kinase domain. The embryonic expression pattern of human eck
has not been described in detail but that of its likely murine orthologue, also termed eck, (90% identical overall
to human eck) has been described in detail at both RNA
and protein levels (Ganju et al. 1994; Ruiz and Robertson 1994). Some elements of the rtk6 expression pattern
are similar to eck, for example the early expression in the
notochord which becomes restricted to caudal regions,
expression in the limb (fin) buds and in the ventral diencephalon. However, one of the most striking features of
eck expression, a tightly-restricted band corresponding to
rhombomere 4, is not seen with rtk6, and many of the expression domains of rtk6, for example in the somites,
spinal cord and forebrain, are not seen with eck.
Sequence comparisons between rtk7 and database sequences indicated the highest degree of similarity to
chick cek7 (Siever and Verderame 1994). The deduced
amino acid sequences are 68% identical overall and 89%
identical in the kinase domain. A lack of detailed description of the embryonic expression pattern of cek7
prevents comparisons from being made with that of rtk7.
No full-length sequence information is currently
available for a zebrafish Eph-related RTK, so it is still
unclear what degree of identity a zebrafish gene might
share with its human, rat, mouse, chick or Xenopus orthologue. However, zebrafish rtk1 and mouse Sek-1 are
94.5% identical to each other over the kinase domain and
the recently characterised zebrafish orthologue of mouse
mdk1, ZDK1, is 96% identical to mdk1 over the kinase
domain (Taneja et al. 1996). If rtk4-8 are as closely relat-
ed to their respective orthologues as rtk1 and ZDK1 are
to theirs, it is probable that many members of this subfamily have not yet been discovered.
In conclusion, caution should be observed when assigning new Eph-related RTKs to orthologous groups
based on sequence data. The reasons for this are several:
firstly, full-length sequence data including untranslated
regions are required and are not always available, even
for published sequences, secondly, it is probable that
there are still members of the Eph-related subfamily of
RTKs yet to be discovered and thirdly, there may not be
a one-to-one relationship between Eph-related RTKs in
different species. Furthermore, implied evolutionary relationships may be of limited value in providing clues
about the function of these receptors. It is clear that, in
addition to full length sequence information, data on developmental expression patterns, receptor-ligand binding
characteristics and in vivo functional analyses are required in order to understand the potentially diverse roles
of vertebrate Eph-related RTKs.
&p.2:Acknowledgements We thank Jose Campos-Ortega, David Grunwald, Robert Riggleman and Kathryn Helde for the cDNA libraries and our colleagues for helpful comments and discussions during the course of this work. We are grateful to Brian Gasking and
Geraldine Millard for maintaining the fish colony. This work is
supported by the MRC and the HFSP. N.H. is a BBSRC Senior
Research Fellow.
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