Download Expression of the zebrafish gene hlx

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71
Development 120,71-81 (1994)
Printed in Great Britain  The Company of Biologists Limited 1994
Expression of the zebrafish gene hlx-1 in the prechordal plate and during
CNS development
Anders Fjose1,*, Juan-Carlos Izpisúa-Belmonte2,†, Catherine Fromental-Ramain3 and Denis Duboule2,‡
1Department of Biochemistry and Molecular Biology, University of Bergen, Årstadveien 19, N-5009 Bergen, Norway
2European Molecular Biology Laboratory, Meyerhofstrasse 1, D-6900 Heidelberg, Germany
3LGME du CNRS, Unité INSERM 184, Faculté de Médecine, 11 rue Humann, 67085 Strasbourg Cédex, France
*Author
for correspondence
Present addresses: †Department of Biological Chemistry, University of California, Los Angeles, CA 90024-1737, USA
‡Departement de Zoologie et Biologie Animale, Sciences III, Universite de Genève, 30 Quai Ernest Ansermet, 1204, Genève, Switzerland
SUMMARY
The zebrafish hlx-1 gene belongs to the H2.0 subfamily of
homeobox genes and is closely related to the mouse Dbx
gene with respect to both homeodomain homology (96.7%)
and neural expression during embryogenesis. Analysis of
hlx-1 expression by in situ hybridization reveals several
particularly interesting features. In late gastrula embryos,
hlx-1 transcripts are detected within a circular area in the
region of the presumptive rostral brain. Subsequently, the
expression domain becomes restricted to the hypoblast and
undergoes dynamic changes involving conversion into a
longitudinal stripe which elongates and retracts following
a temporal sequence. The site of transient hlx-1 expression
along the ventral midline of the rostral neurectoderm,
which in part corresponds to the prechordal plate, suggests
a role in the determination of head mesoderm as well as in
patterning of the rostral brain. As the midline stripe
gradually disappears, the hlx-1 gene becomes regionally
expressed within the diencephalon and at a specific
dorsoventral level along the hindbrain and spinal cord. In
the hindbrain, expression is initiated in dorsoventrally
restricted transversal stripes which correlate with the
segmental pattern of rhombomeres. The stripes fuse into
bilateral columns that are later converted to two series of
paired transversal stripes at the rhombomere borders. This
pattern is consistent with the proposed subdivision of
hindbrain segments into rhombomere centers separated by
border regions.
INTRODUCTION
neural tube (Bastian and Gruss, 1990; Davis et al., 1988; Fjose
et al., 1992; Gruss and Walther, 1992; Krauss et al., 1991a;
Price et al., 1991). Several members of these groups are
expressed within defined domains in the rostral brain during
early stages of embryonic development, when regional identities are established. The majority of these genes are also
expressed at specific dorsoventral levels in the epichordal
division of the neural tube. In the case of murine Pax genes,
expression has been observed in both dividing cells, in the ventricular zone, and in specific postmitotic cells (Gruss and
Walther, 1992). Similar observations have been made in
zebrafish where transcripts and proteins derived from the Pax2
and Pax6 homologues are present in identified interneurons
and the basal plate ventricular zone, respectively (Krauss et al.,
1991b,c; Mikkola et al., 1992; Püschel et al., 1992).
The establishment of Pax gene expression domains in the
hindbrain and spinal cord is probably controlled by the
notochord-floor plate signal cascade which is thought to
determine the dorsoventral organization of the neural tube
(Goulding et al., 1993; Placzek et al., 1991). A different regulatory system is likely to operate in the rostral brain, where
both notochord and floor plate are lacking. However, studies
of the zebrafish cyclops-1 (cyc-1) mutant in which tissues along
A wide range of different transcription factors are expressed
during development of the vertebrate central nervous system
(CNS). One important class consists of the homeodomain
proteins encoded by genes in the Hox complexes, which
probably specify anteroposterior positions of cells in the
nervous system and other tissues (McGinnis and Krumlauf,
1992). Similar to the homologous genes of the Drosophila
homeotic gene complexes (HOM-C), the various Hox genes
have expression domains that extend from specific anterior
boundaries in the embryonic CNS. However, the expression of
individual Hox genes does not seem to be restricted to specific
cell types within the spinal cord region and/or hindbrain rhombomeres. As each hindbrain segment can be tentatively subdivided into a rhombomere center and two border regions
(Trevarrow et al., 1990), it will be of interest to identify the
genes potentially responsible for generating these subdivisions.
Other groups of transcription factors are encoded by Pax
genes and several members of the vertebrate homeobox subfamilies related to the Drosophila genes engrailed, evenskipped and Distal-less. These factors appear to have important
roles in regionalization and/or dorsoventral patterning of the
Key words: neurulation, prechordal plate, rostral brain, spinal cord,
hindbrain segmentation
72
A. Fjose and others
the entire ventral neuraxis do not form, have provided evidence
that, also in the rostral brain, neural tissues located at the
ventral midline have organizational functions (Hatta et al.,
1991; Hatta, 1992). Moreover, analyses of genetic mosaics of
cyc-1 indicate that mutant cells of the ventral midline, in the
regions of the rostral brain and hindbrain/spinal cord, are not
able to respond to inductive signals from the prechordal plate
and notochord, respectively (Hatta et al., 1991). In this context,
identification of transcription factors whose expression
patterns are restricted to the prechordal plate and ventral
midline tissues of the rostral brain would be of interest.
We have isolated a zebrafish gene (hlx-1) related to the
Drosophila homeobox gene H2.0 (Barad et al., 1988). In early
stages of neurulation, the gene displays a highly dynamic
expression pattern in the hypoblast of the head region. The
expression domain is rapidly transformed from a circular area
into a longitudinal stripe, which is transiently detected in the
prechordal plate underneath the rostral brain. At later stages,
the hlx-1 gene is expressed at multiple sites in all major subdivisions of the CNS. A particularly interesting expression
pattern is observed within the developing hindbrain. The individual rhombomere primordia appears to control the temporal
activation of hlx-1 and, at later developmental stages,
expression is detected near the interrhombomeric boundaries.
MATERIALS AND METHODS
Embryos
Zebrafish (Brachydanio rerio) were maintained and bred essentially
as described in Stuart et al. (1988). Developmental age is given as
hours postfertilization (hpf) at 28.5°C, which was the temperature of
incubation.
Isolation and sequence analysis of the cDNA
A cDNA library was made from poly(A)+ RNA isolated from 12-48
hpf zebrafish embryos. Total RNA was isolated by the guanidinium
thiocyanate-CsCl method (MacDonald et al., 1987) and poly(A)+
RNA was purified on an oligo(dT)-cellulose column (Pharmacia). The
cDNA was synthesized by random priming according to standard procedures (Sambrook et al., 1989) and 1.5×106 recombinants were
obtained by cloning in the phage vector λZAPII (Stratagene). The
library was screened at low stringency with a probe mixture of
homeobox sequences from class I Hox genes.
Immunohistochemistry and double staining of in situ
hybridized embryos
Zebrafish embryos were manually dechorionated and fixed for 12
hours at 4°C as described by Krauss et al. (1991b,c). After fixation,
embryos were rinsed 3×5 minutes in 0.1 M PO4 (pH 7.3), 0.5% Triton
X-100 and incubated for 30 minutes in a solution containing 2% goat
serum, 1% bovine serum albumin (BSA), 1% dimethylsulfoxide
(DMSO) and 1×PBS (pH 7.3). The embryos were subsequently
incubated for 3 hours with a 1:50 diluted zn-12 antibody (Metcalfe et
al., 1990; Trevarrow et al., 1990) in 0.1 M PO4 (pH 7.3), 1% goat
serum, 0.2% BSA and 1% DMSO at 37°C. The embryos were then
washed 6×10 minutes in the same buffer followed by incubation
overnight at 4°C with a 1:200 diluted horseradish peroxidase (HRP)conjugated secondary antibody in the same buffer as used for the
primary antibody. Embryos were rinsed again 6×10 minutes in the
same buffer and incubated for 20 minutes with 1 ml of 0.5 mg/ml 3,3′diaminobenzidine in 0.05 M PO4 (pH 7.3). The embryos were stained
for about 10 minutes after adding 2 µl of a 3% H2O2 solution. After
washing 3× in cold 0.1 M PO4 (pH 7.3) buffer and dehydration in
100% methanol, the stained embryos were cleared in a 1:2 solution
of benzyl alcohol and benzyl benzoate before mounting in Permount.
For double labelling, the hlx-1-stained embryos were washed 4×5
minutes in 0.1 M PO4 (pH 7.3) followed by incubation with the zn12 antibody and immunohistochemical detection as described above.
RESULTS
Cloning and characterization of the hlx-1 gene
The hlx-1 (H2.0-like homeobox; Allen et al., 1991) cDNA was
cloned as a part of an effort to isolate homeobox genes with
developmental functions (see Materials and Methods). When
compared to the homeodomain sequences of other genes (Fig.
1) it is clear that hlx-1 belongs to a vertebrate group related to
the H2.0 gene of Drosophila (Barad et al., 1988). Moreover,
the level of sequence identity between hlx-1 and the murine
Dbx homeodomain (96.7%) is considerably higher than for any
of the other known vertebrate genes of this group (Fig. 1). This
suggests that hlx-1 and Dbx may be direct homologues.
However, to resolve this issue, it will be necessary to obtain
the complete DNA sequences of both genes. In addition, a
more extensive analysis of the embryonic Dbx expression is
required for direct comparison to hlx-1 (see Discussion).
In situ hybridization and sectioning of stained embryos
Dynamic changes of hlx-1 expression in the rostral
In situ hybridization to whole-mount embryos was performed essenhypoblast
tially as described by Krauss et al. (1991b,c). Stained embryos were
To investigate the expression of the hlx-1 gene during embryodehydrated in 100% methanol and cleared for 30 minutes in a 1:2
genesis, the cDNA was labelled with digoxigenin and used for
solution of benzyl alcohol and benzyl benzoate. Subsequently, the
in situ hybridization to whole embryos. Initially, at 8-9 hpf,
embryos were mounted in Permount and analysed with DIC optics in
expression was seen in dispersed cells within a small circular
a Nikon Microphot-FXA microscope.
To make tissue sections, the stained embryos
were dehydrated in ethanol and treated with
methyl salicylate for 10 minutes, followed by
infiltration with a solution containing one part
methyl salicylate and one part resin (Epon 812Araldite 502) overnight at room temperature. The
embryos were then placed in pure resin (2
changes of solution, 2 hours each) and embedded
in fresh resin. The blocks were polymerized
Fig. 1. Comparison of the amino acid sequence of the Hlx-1 homeodomain with those of
overnight at 60°C. A tungsten carbide-tipped
other homeodomains. Dashes indicate positions where amino acids are identical with
knife was used to cut 10 µm sections on a 2055
those in the Hlx-1 sequence. The homeodomains listed are murine Dbx (Lu et al., 1992),
Autocut Rotary Microtome. The sections were
chicken CHoxE (Rangini et al., 1991), murine Hlx (Allen et al., 1991), Drosophila H2.0
dried onto glass slides and mounted in Permount.
(Barad et al., 1988) and Antp (McGinnis et al., 1984).
Expression of zebrafish hlx-1 gene
area in the region of the prospective
midbrain and forebrain (not shown).
Subsequently, this domain became
enlarged and stronger hybridization
signals were detected in the individual cells (Fig. 2A). During the 9 hour
stage (Figs 2A-C, 8A), the
expression
domain
became
elongated, probably due to migration
of lateral cells towards the midline.
Also, a higher and more uniform
staining intensity was observed.
As judged from tissue sections of
stained 9 hpf embryos (Fig. 3A,B),
the positive cells were located in the
hypoblast that underlies the rudiment
of the rostral brain. Cross-sections in
the posterior region of the stripe
demonstrated that expression was
confined to a flat triangular area that
spans about 10 cell diameters
ventrally (Fig. 3A). Interestingly,
hlx-1 expression was not detected in
the most ventral cell layer of the
hypoblast which includes prospective endodermal cells. Dorsally, the
expression domain seemed also to
include some cells of the epiblast.
However, at a more rostral position
where a weak ventral bending was
first observed in whole mounts of
this developmental stage (Fig.
2C,D), cross-sections demonstrated
restriction of the expression domain
within the hypoblast (Fig. 3B). At
this level of the body axis, the width
and thickness of the stripe were
about six and three cell diameters,
respectively.
Further elongation and ventral
bending was observed at later stages
(Fig. 2E-H) and, in 11 hpf embryos,
the hlx-1 expression domain
appeared as a narrow stripe (about 6
cells wide) located at the ventral
midline of the rostral brain (Fig. 4A).
The stripe extended up to the anterior
end of the forebrain by 12 hpf but the
posterior border of the expression
domain simultaneously retracted
(Fig. 4B). Soon after this stage,
further shortening of the hlx-1 stripe
was observed (Fig. 4C,D) and, in 22
hpf embryos, it was no longer
detected (Fig. 7A).
To estimate precisely the location
of the hlx-1 domain during early
development at a time when morphological markers are not yet
visible, embryos were hybridized
simultaneously with probes derived
73
Fig. 2. Expression of hlx-1 in the rostral region of zebrafish embryos during early stages of
neurulation. Transcripts were localized by in situ hybridization on a series of whole-mount
embryos at 9-10 hpf (A-H). Dorsal views of the rostral region (anterior to the left) of subsequent
stages are shown in A,B,E and G. Side views of the same embryos as in C,E and G are shown in
D,F and H, respectively. Some embryos were hybridized simultaneously with digoxigenin-labelled
probes of both hlx-1 and the pax[b] gene (Krauss et al., 1991b). The double stained embryos
shown in I and J are comparable to the stages presented in C and G, respectively. Arrows indicate
the location of the presumptive midbrain-hindbrain boundary. Bar, 60 µm. Abbreviations: dc,
diencephalon; hb, hindbrain; mb, midbrain; y, yolk.
74
A. Fjose and others
from hlx-1 and the pax[b] gene. The latter is expressed within
two transversal stripes located in the posterior midbrain region
which fuse at the dorsal midline in 10 hpf embryos (Krauss et
al., 1991b). At later stages, the posterior boundary of the
pax[b] expression domain coincides with the furrow separating the midbrain and hindbrain. In double labelled 9 hpf
embryos (Fig. 2I), the posterior border of the hlx-1 domain
coincided with the site at the dorsal midline where the pax[b]
stripes will eventually fuse. This also showed that hlx-1expressing cells migrate much faster towards the dorsal
midline than the neurectodermal, pax[b]-positive cells. At the
10 hour stage, the hlx-1 expression domain showed its
maximum posterior extension (Fig. 2J). In these embryos, the
posterior border of the hlx-1 stripe was located at the same
anteroposterior level as the fusing pax[b] stripes.
At 12 hpf, the hlx-1 gene was expressed within a region of
about three cell layers underneath the diencephalon (Fig. 3C).
The expressing cells were located in a ventral protrusion (Fig.
3C), called the ‘polster’, which according to Kimmel et al.
(1990), may be equivalent to the prechordal plate of amphibians (Meier, 1981; Ballard, 1982). In rostral cross-sections of
16 hpf embryos, hlx-1 transcripts were also detected at the
ventral midline (Fig. 3D), but the expression domain mainly
included ventral parts of the diencephalon.
At 15-16 hpf three transversal stripes of different signal intensities were present in the anterior part of the hindbrain (Figs 4D,
5D,E and 8B). Similar to the stripe in the region of the Mi2
rhombomere, expression was restricted to a specific dorsoventral level (Fig. 4D). To determine further whether the anterior
hindbrain stripes would correlate with the segmental rhombomeric pattern, hlx-1 stained embryos were double labelled
with the monoclonal antibody zn-12. This antibody, which recognizes similar or identical cell surface glycoproteins as HNK1, labels the second hindbrain rhombomere (Ro2) and the
trigeminal ganglia flanking Ro1 at 15-16 hpf (Fig. 5F; Metcalfe
et al., 1990; Mikkola et al., 1992; Trevarrow et al., 1990). In
double-labelled embryos, the zn-12 stripe (Ro2) did coincide
well with the second (and strongest) hlx-1 stripe. As the first
(weak) and third stripes were of similar width, they may correspond to Ro1 and Ro3, respectively. The strong transversal
stripe first detected at 12 hpf was still present at 15-16 hpf and
was separated from the Ro3 stripe by a nonlabelled region which
was about one segment wide. Also, the spatial correlation
between this stripe and the otic placodes suggested that it was
located in the fifth rhombomere (Mi2). However, it is probable
that this rather narrow stripe included only the anterior two thirds
of Mi2. Further posteriorly, some hlx-1 expression was also
present in the anterior part of the sixth rhombomere (Fig. 5D,E).
Following the 16 hpf stage, hlx-1 transcripts appeared at a
similar dorsoventral level in the remaining part of the hindbrain
and the expression level increased. As a consequence, two
bilateral columns of expressing cells were detected along the
entire hindbrain at 19 hpf (Fig. 4E). Cross-sections and hori-
Embryonic expression in the hindbrain and spinal
cord
Neural hlx-1 expression in the hindbrain and spinal cord
regions was first observed at 10-11 hpf (Figs 4A, 5A). At this
developmental stage, two bilateral
clusters of cells expressing the hlx1 gene appeared at a rostrocaudal
level which corresponded to the
future location of the fifth rhombomere (Mi2; Hanneman et al.,
1988; Trevarrow et al., 1990). Two
rows of cells which expressed the
gene at a somewhat lower level,
extended posteriorly from the two
hindbrain clusters. By 12 hpf,
stronger hybridization signals were
detected within a transversal stripe
in the region corresponding to the
position where the two bilateral
clusters were seen earlier (Figs 4B,
5B). Even though this stripe may
correspond to a presumptive rhombomere (Mi2), which will eventually become visible at 15-16 hpf
(Fig. 8B; Trevarrow et al., 1990),
the transcripts were clearly
restricted only to particular cells at
a specific level in the dorsal part of
the basal plate (Fig. 4B). Relatively
strong
hybridization
signals
extended posteriorly from the
Fig. 3. Analysis of the tissue distribution of hlx-1 transcripts in rostral cross-sections of stained
anterior end of the spinal cord in 12 embryos. Cross-sections near the posterior and anterior ends of the hlx-1 stripe in the same 9-10 hpf
hpf embryos (Fig. 4B,C). This embryo are shown in A and B, respectively. Arrows indicate the location of a ventral layer of
expression domain span the neural presumptive endodermal cells where transcripts are not detected. Cross-sections in the diencephalic
keel and included only cells in the region of 12 and 16 hpf embryos are shown in C and D, respectively. Bar, 30 µm. Abbreviations: dc,
dorsal part of the basal plate.
diencephalon; ep, epiblast; hp, hypoblast; ov, optic vesicle; p, polster; y, yolk.
Expression of zebrafish hlx-1 gene
75
zontal views of the hindbrain of 22 hpf embryos (Fig. 6A,B)
mitotic and postmitotic cells were included. Clearly, hlx-1 transhowed that the columns (2-3 cell layers), which were located
scripts were present only within a subpopulation of the cells
in the dorsal region of the basal plate (Fig. 6B), spanned the
localized in this column (Figs 3F, 6H). Double labelling experwalls of the neural tube. At later stages, each longitudinal
iments with hlx-1 and the zn-12 antibody showed that the zncolumn started to transform into a series of transversal stripes,
12-positive Rohon Beard neurons, in the alar plate (Metcalfe
generating a repeated pattern of paired stripes by 30 hpf (Figs
et al., 1990), were localized about two cell diameters dorsal to
6C,E, 8B). The individual stripes were 2-3 cell diameters wide
the hlx-1-expressing cells (Fig. 5G).
and extended from the luminal side
into the marginal zone. A similar
segmental expression pattern was
observed in the hindbrain at 45 hpf
(Fig. 6D). However, at this developmental stage, the stripes had narrowed
up to a width of 1-2 cell diameters.
Interestingly, the spacing between
each pair of hlx-1 stripes correlated
well with the width of hindbrain rhombomeres, and the position of the first
pair seemed to coincide with the
boundary between the two anteriormost rhombomeres (Hanneman et al.,
1988; Trevarrow et al., 1990). Consistent with this, the proposed location of
Mi2 was directly adjacent to the otic
vesicle (Fig. 6E). It is also noteworthy
that only a single transversal stripe of
lower staining intensity was located at
the presumptive boundary between the
first rhombomere (Ro1) and the cerebellar anlagen (Fig. 6E). Therefore,
also with respect to the hlx-1 gene, this
border is distinguishable from the
interrhombomeric boundaries.
We further confirmed the correlation between the double stripes and
rhombomere borders by visualizing
the segmental arrangement of reticular
neurons in 30 hpf embryos with hlx1/zn-12 double labelling. The
clustered location of the zn-12 labelled
neurons in the rhombomere centers
(Hatta, 1992; Trevarrow et al., 1990)
were consistent with the proposed
pattern of paired hlx-1 stripes in the
border regions (Fig. 6G).
Similar to earlier developmental
stages (Fig. 4C,D), expression of hlx1 in the spinal cord of 19 hpf embryos
was restricted to dispersed cells within
a column in the dorsal part of the basal
plate (Fig. 4E,F). Moreover, this
expression domain, which extended to
the tip of the tail, was directly contiguous with the hlx-1 staining
localized in the hindbrain. CrossFig. 4. Whole-mount in situ hybridization analysis of hlx-1 expression of different developmental
sections of the spinal cord of both 16 stages. Side views (anterior to the left) of 11, 12, 13, 16, 19 and 30 hpf are shown in A, B, C, D, E
and 22 hpf embryos (Figs 5H, 6I) and G, respectively. The spinal cord of the 19 hpf embryo is shown at high magnification in F.
revealed the presence of hlx-1- Arrows indicate the location of the midbrain-hindbrain boundary and open triangles mark the
expressing cells within a layer of 1-2 position of rhombomere-restricted sites of expression in the hindbrain. Bar, 60 µm. Abbreviations:
cells extending through the wall of the dc, diencephalon; e, eye; fb, forebrain; fp, floor plate; hb, hindbrain; mb, midbrain; nc, notochord;
neural tube, indicating that both sc, spinal cord; tc, telencephalon; y, yolk.
76
A. Fjose and others
Fig. 5. Analysis of hlx-1
expression during early
stages of hindbrain and
spinal cord
development. Dorsal
views of the posterior
hindbrain region at 1011 hpf and 12 hpf are
shown in A and B,
repectively. Dorsal
views of the anterior
spinal cord region at 12
hpf (C) and 15 hpf (D).
(E) Dorsal view of the
hindbrain in a 15-16 hpf
embryo double labelled
with hlx-1 and the zn-12
antibody. (F) Dorsal
view of the hindbrain of
a 16 hpf embryo after
immunohistochemical
staining with the zn-12
antibody. The
arrowheads in D, E and
F indicate the
approximate location of
rhombomere borders.
The open triangles in D
and E mark the position
of the anterior border of
the first rhombomere
(Ro1). (G) Side view of
the spinal cord region of
a 16 hpf embryo double
labelled with hlx-1 and
zn-12. Arrowheads and
open triangles indicate
the location of Rohon
Beard neurons and hlx1-labelled cells,
respectively. (H) Crosssection of the spinal cord at 16 hpf after whole-mount hlx-1 staining. Arrows indicate the dorsoventral location of columns of hlx-1-expressing
cells. Bar, 30 µm. Abbreviations: hb, hindbrain; nc, notochord; op, otic placode; s, somite; sc, spinal cord; tg, trigeminal ganglia; y, yolk.
Multiple sites of hlx-1 expression in the rostral brain
In the rostral brain, expression of the hlx-1 gene was initiated
later (13-14 hpf) than in the epichordal division of the CNS
(not shown). By the 16 hpf stage, sites of expression were
present in both the midbrain and the ventral part of the diencephalon (Fig. 4D). A somewhat longitudinal expression
domain was seen in the presumptive tegmental region of the
midbrain and this extended further into a column by 19 hpf
(Fig. 4E). Thus, at this stage, longitudinal expression domains
with similar dorsoventral restrictions were present in both the
hindbrain and midbrain. Additional hybridization signals were
observed at a site in the rostral midbrain which coincided well
with a particular group of cells associated with the posterior
commissure (see Discussion).
A small region of hlx-1 expression was detected near the
posterior end of the prechordal stripe at 15-16 hpf (Fig. 4D).
By the 22 hpf stage, this domain had expanded into a transversal stripe of high signal intensity in the diencephalon (Fig.
Expression of zebrafish hlx-1 gene
77
Fig. 6. Analysis of hlx-1
expression during cell
differentiation in the
hindbrain and spinal cord.
A and B show dorsal
views and cross-sections
of the hindbrain in a 22
hpf embryo, respectively.
The arrows in picture B
indicate the location of the
bilateral columns of hlx-1expressing cells. Large
arrowheads in A,C,E and
G indicate the location of
the furrow at the
midbrain-hindbrain
border. C and D are side
views of the hindbrain of
hlx-1-stained 30 and 45
hpf embryos, respectively.
E and F show dorsal view
and cross-section of the
hindbrain in 30 hpf
embryos labelled with the
hlx-1 probe. In E the
location of the individual
rhombomeres are shown
and the predicted segment
borders are indicated by
arrows. The arrows in F
mark the dorsoventral
location of the transversal
hlx-1 stripes. (G) Dorsal
view of the hindbrain of a
30 hpf embryo double
labelled with hlx-1 and
zn-12. Arrowheads mark
the position of
segmentally arranged
clusters of reticular
neurons and arrows
indicate the predicted
rhombomere borders. H
and I show dorsal view
and cross-section of the
anterior spinal cord in a
22 hpf embryo. The two
arrows in I indicate the
dorsoventral location of
the bilateral columns of
hlx-1-expressing cells. Bar, 30 µm. Abbreviations: cb, cerebellum; hb, hindbrain; llf, lateral longitudinal fascicle; m, myotome; mb, midbrain;
nc, notochord; ov, otic vesicle; sc, spinal cord; tg, trigeminal ganglia; v, fourth ventricle; y, yolk; Ro1-Ca1, abbreviations for rhombomeres 1-7.
7A,B). Unlike the other sites of hlx-1 expression in the rostral
brain, this domain was sharply defined and included all the
cells in the wall of the neural tube (Fig. 7B,D).
After the 22 hpf stage, several features of the hlx-1
expression pattern in the rostral brain were changed. In 30 hpf
embryos, expression was still present in the tegmental region
of the midbrain, but additional hlx-1 transcripts were detected
in cells scattered throughout most of the tectum (Fig. 7E,F).
78
A. Fjose and others
Moreover, the rostral part of the
longitudinal midbrain domain
which at 22 hpf also included a
small area of the posterior
forebrain, had become a regionally restricted expression domain
of high signal intensity. The major
part of this domain was located in
the posterior diencephalon (Fig.
7E) and analysis of cross-sections
revealed that this site corresponded to a region in the dorsal
thalamus (Fig. 7G). Another noteworthy feature was the direct correlation between its ventral border
and
the
sulcus
at
the
dorsal/ventral thalamic junction
(Fig. 7G).
During development from 22 to
30 hpf, the regional hlx-1 domain
in the ventral diencephalon had
extended along the original
anteroposterior axis. However,
this extension appeared to be
directed ventrally as a consequence of the ventral bending of
the hypothalamic tissue. Analysis
of cross-sections showed that the
diencephalic domain included a
part of the thalamus and extended
into the dorsal region of the hypothalamus (Fig. 7G,H).
DISCUSSION
We have cloned a gene that is
expressed in all major subdivisions of the embryonic CNS. A
detailed analysis of the spatial distribution of hlx-1 transcripts
during embryogenesis by wholemount in situ hybridization and
double labelling with other
markers revealed a particularly
dynamic expression pattern in the
hindbrain and prechordal plate,
suggesting multiple functions for
this gene during early stages of
CNS development.
Early embryonic expression
in the prechordal plate
In a way similar to the zebrafish
pax[b] and eng-2 genes (Krauss et
al., 1991b; Fjose et al., 1992),
transcripts of the hlx-1 gene are
first detected in the presumptive
rostral region of late gastrula
embryos. However, by contrast to
these genes, which are first
Fig. 7. Analysis of hlx-1 expression in the rostral brain. (A-C) Side view and dorsal views at two
different focal planes of the rostral brain in the same 22 hpf embryo, respectively. Arrowheads mark the
position of the furrow at the midbrain-hindbrain border. (D) Cross-section in the diencephalic region at
22 hpf. (E) Side view of the rostral brain of a 30 hpf embryo. Cross-sections of the midbrain (F) and
diencephalic region (G,H) of a 30 hpf embryo (G is most rostral). The arrows in E and H indicate the
location of the ventral flexure. Arrowheads in G mark the positions of sulci. Bar, 30 µm. Abbreviations:
dc, diencephalon; dt, dorsal thalamus; e, eye; h, hypothalamus; mb, midbrain; t, telencephalon; te,
tectum; th, thalamus; tm, tegmentum; tv, tectal ventricle; v, ventricle; vt, ventral thalamus.
Expression of zebrafish hlx-1 gene
A
B
Fig. 8. Schematic presentation of the dynamic changes of hlx-1
expression that occur during early stages of development in the
rostral region. (A) Expression domains observed in the prechordal
region of the hypoblast at approximately 9, 9.5, 10 and 11 hpf,
respectively. (B) Expression patterns in the hindbrain of 12 (left), 16
(middle) and 30 (right) hpf embryos. Abbreviations: FB, forebrain;
HB, hindbrain; MB, midbrain; Ro1-Ca1, rhombomeres 1-7.
expressed as two lateral transversal stripes that converge
toward the dorsal midline during neurulation, the hlx-1
expression domain is transformed from a circular area to a longitudinal stripe located in the hypoblast. The formation of the
hlx-1 stripe occurs before fusion of the transversal pax[b]
stripes at the dorsal midline. Several features of the expression
pattern indicate that the hlx-1-positive cells are, at least in part,
located in the prechordal plate. Initially, transcripts are
detected within a circular area near the rostral end of the
embryo which may be the equivalent of the circular prechordal
plate observed in higher vertebrates at the corresponding developmental stages (Meier 1981; Ballard, 1982). Although the
prechordal plate of zebrafish has not been unequivocally identified as a specific group of cells, the hypoblast beneath the
forebrain forms a prominent bulge (‘polster’) at 11-12 hpf,
which probably corresponds to this tissue (Kimmel et al.,
1990). The ventral bending of the hlx-1 stripe observed in the
presumptive midbrain region of 9-10 hpf embryos, seems to
reflect a thickening of the brain rudiment and simultaneous
formation of the underlying polster. In the region posterior to
this bending, the gene appears to be expressed both in the
hypoblast and ventral parts of the epiblast.
The hlx-1 gene, which after the 15 h stage is expressed along
the ventral midline of the diencephalon, may contribute to the
patterning of the rostral brain. In support of this suggestion, it
79
has been shown that proper induction of the forebrain in
Xenopus embryos requires vertical signals from underlying
axial mesoderm (Ruiz i Altaba, 1992). Moreover, analyses of
the zebrafish cyc-1 mutant have revealed a dependence of
ventral midline cells of the rostral brain on signals emanating
from the prechordal plate (Hatta et al., 1991). Interestingly, the
ventral midline cells of the brain seem to have an expanded
organizational function (Hatta et al., 1991; Hatta, 1992) in the
rostral region where hlx-1 expression remains until 20 hpf.
Thus hlx-1 may be involved in the control of both rostrocaudal and dorsoventral differences in the prechordal division of
the brain.
Several genes have recently been identified which are active
both in the prechordal plate and other mesodermal tissues
during early stages of development. Among these, the two
Xenopus transcription factor encoding genes goosecoid and
XFKH-1 are of particular interest as they are also active very
early in the dorsal lip (Cho et al., 1991; Dirksen and Jamrich,
1992) and may thus be located upstream of hlx-1 in a regulatory cascade.
As far as the closely related murine Dbx and chicken CHoxE
genes are concerned, prechordal plate expression has not been
reported (Lu et al., 1992; Rangini et al., 1991). This could be
due to the reduced size of the prechordal plate in mouse
embryos (Tam et al., 1982) and/or that early stages were not
analysed in detail by in situ hybridization.
Dorsoventral restriction of hlx-1 expression in the
hindbrain and spinal cord
The hlx-1 gene is expressed in single cells within longitudinal
columns in the spinal cord at early embryonic stages. During
these early stages of neurogenesis the zebrafish spinal cord is
known to contain a small number of neurons that are organized
in a very simple manner (Bernhardt et al., 1990; Kuwada and
Bernhardt, 1990). The individual hemisegments in 18-20 h
embryos contains approximately 18 postmitotic neurons, and
these are identifiable on the basis of their unique locations,
sizes and axonal outgrowth (Bernhardt et al., 1990).
Within the spinal cord region, hlx-1 expression appears in
dispersed cells at a dorsal level in the basal plate 4-5 hours
before the first postmitotic neurons start to project axons
(Kuwada and Bernhardt, 1990), suggesting that hlx-1 plays a
role in the determination of specific subsets of neurons. A
similar function has been proposed for the zebrafish pax[b]
gene which is known to initiate expression in precursors of
commissural secondary ascending interneurons (CoSA) at 13
hpf (Mikkola et al., 1992). However, expression of hlx-1 is
initiated earlier than pax[b] and the transcripts seem to be
present in both mitotic and postmitotic cells. As judged from
differences in dorsoventral position and cell numbers per
hemisegment, the hlx-1-positive cells do not correspond to any
of the other types of neurons which have been described in 20
hpf embryos (Bernhardt et al., 1990). The identities of the additional postmitotic neurons that have not projected axons at this
early stage are not known but may include three subpopulations of commissural neurons, which are first visible at 22-23
hpf (Bernhardt et al., 1990). The hlx-1 labelled cells could correspond to precursors of one or more of these three types of
interneurons. Alternatively, expression of hlx-1 may be more
directly linked to dorsoventral patterning and possibly the
formation of the border between basal- and alar plate.
80
A. Fjose and others
Expression in the embryonic hindbrain and spinal cord is
initiated simultaneously at comparable dorsoventral levels in
the neural keel of 10 hpf embryos, indicating a response to the
same signals. Differences are observed later when the
hindbrain becomes subdivided into segmental units. Thus at 12
hpf, a transversal stripe of hlx-1 expression appears at a specific
dorsoventral level in the region of the presumptive Mi2 rhombomere. Similarly, dorsoventrally restricted transversal stripes
correlate with the three most anterior hindbrain segments at 1516 hpf. This pattern shows that hlx-1 expression follows the
maturation of the rhombomeres where it may act as a determinant of the dorsoventral polarity. Several members of other
vertebrate gene families, including Krox20 (Wilkinson et al.,
1989), cytoplasmic retinoic acid binding proteins (Ruberte et
al., 1992), seven-up related nuclear receptors (Fjose et al.,
1993) and some Hox genes (McGinnis and Krumlauf, 1992),
are known to have rhombomere specific expression domains at
equivalent embryonic stages. However, in these cases the
expression domains span most of the rhombomeric units.
Later during embryogenesis, a repeated pattern of paired
hlx-1 stripes appears which seems to indicate a subsegmental
organization of hindbrain rhombomeres, as already proposed
by Trevarrow et al. (1990). According to this proposal, two
regions repeat in an alternating pattern along a series of seven
segments. While the rhombomere centers contain the first basal
plate neurons to develop, the segment boundaries contain the
first neurons to develop in the alar plate. This model is partially
based on the staining pattern of the monoclonal antibody, zn5, which starts to label neurons near the segment borders at 22
hpf (Trevarrow et al., 1990). Interestingly, the zn-5 labelling
observed in the hindbrain of 48 hpf embryos is very similar to
the expression pattern of hlx-1 at a comparable developmental
stage suggesting that the same commissural interneurons are
stained in both cases. Alternatively, the hlx-1 gene may be
expressed in a different subset of neural cells in the rhombomere border regions. In any case, the hlx-1 gene is the first
identified regulatory gene that reveals the segmental subdivisions proposed for the hindbrain rhombomeres. Although the
closely related murine Dbx and chicken CHoxE genes have
similar dorsoventrally restricted expression patterns, correlations with hindbrain segmentation were not observed (Lu et al.,
1992; Rangini et al., 1991).
of the midbrain at 19 hpf and older stages. The position of these
cells correspond to the location of the nuc PC (nucleus of the
posterior commissure) neurons which at 20 hpf project axons
along the posterior commissure located at the forebrainmidbrain border (Chitnis and Kuwada, 1990). The hlx-1 gene
may thus play a role in the ontogenesis of this particular subset
of midbrain neurons.
In 22 hpf embryos, the highest level of hlx-1 expression in
the rostral brain is located within a sharply defined region in
the ventral part of the diencephalon. This domain which is first
detected at 15 hpf, is likely to reflect an early regionalizing
function of the hlx-1 gene. However, partly due to the observed
spatiotemporal changes, it is unclear whether this expression
domain can also be correlated with the segmental subdivisions
which have been proposed for the embryonic chick diencephalon (Figdor and Stern, 1993).
An additional hlx-1 domain with regional features appears
near the forebrain-midbrain border at a later developmental
stage. The ventral boundary of this area, which is mainly
located in the diencephalon, coincides with the morphological
border (sulcus) separating the dorsal and ventral thalamus. This
correlation suggests an involvement of hlx-1 in the formation
of the dorsoventral thalamic border.
On the basis of these features, it is likely that the hlx-1 gene
plays a role in several different aspects of regionalization,
differentiation and border formation in the rostral brain.
Similar functions have been proposed for other homeobox
genes including members of the Pax, Dlx, Emx and Otx subfamilies, which also have regional expression patterns in the
forebrain (Krauss et al., 1991a,b,c; Porteus et al., 1991; Price
et al., 1991; Robinson et al., 1991; Simeone et al., 1992).
Therefore, in the context of rostral brain development, hlx-1 is
likely to be part of a combinatorial system of homeobox genes.
We thank C. B. Kimmel, A. Molven and M. Westerfield for helpful
comments to the manuscript. We are also very grateful to M. Westerfield for providing the zn-12 antibody and S. Nornes for technical
assistance. This work was funded by grants from the Norwegian
Research Council, Norwegian Cancer Society, Nansen Foundation,
HFSPO and EMBL.
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(Accepted 13 October 1993)