Development 121, 1755-1768 (1995)
Printed in Great Britain © The Company of Biologists Limited 1995
1755
Expression of zebrafish nk2.2 is influenced by sonic hedgehog/vertebrate
hedgehog-1 and demarcates a zone of neuronal differentiation in the
embryonic forebrain
Katrin Anukampa Barth and Stephen W. Wilson
Developmental Biology Research Centre, Randall Institute, King’s College London, 26-29 Drury Lane, London WC2B 5RL, UK
SUMMARY
We have isolated zebrafish nk2.2, a member of the Nk-2
family of homeobox genes. nk2.2 is expressed in a continuous narrow band of cells along a boundary zone demarcating the location at which two of the earliest nuclei in the
brain differentiate. This band of cells is located within a
few cell diameters of cells expressing the signalling
molecule sonic hedgehog/vertebrate hedgehog-1 (shh/vhh1). Injection of shh/vhh-1 RNA results in ectopic expression
of nk2.2 and concomitant abnormalities in the forebrain
and eyes. Moreover, cyclops mutant embryos, which
initially lack neurectodermal expression of shh/vhh-1, show
a concomitant lack of nk2.2 expression. Together, these
results suggest a requirement of shh/vhh-1 protein for the
spatial regulation of nk2.2 expression.
INTRODUCTION
families, the Nk-2 family is characterised by an additional
conserved motif, the Nk-2 domain (Price et al., 1992). This
motif, the prototype of which is found in the Drosophila NK2 gene (Kim and Nirenberg, 1989), consists of at least 17
amino acids located carboxyterminal to the homeobox. Since
the isolation of the first vertebrate family member Nkx2.1/TTF-1 (Guazzi et al., 1990; Lazzaro et al., 1991; Price et
al., 1992), five other family members have been isolated in
mice. Nkx-2.1 to Nkx-2.4 are all closely related (Price et al.,
1992; Price, 1993), while Nkx-2.5 and Nkx-2.6 represent more
divergent members of the family (Lints et al., 1993). In
addition to being expressed in the thyroid and lung, Nkx2.1/TTF-1 is also transcribed in restricted regions of the
forebrain, as is Nkx-2.2 (Lazzaro et al., 1991; Price et al.,
1992). Nkx-2.5 is thought to be involved in heart development
(Lints et al., 1993), while no detailed expression data have been
reported for Nkx-2.3, Nkx-2.4 and Nkx-2.6. Zebrafish nk2.2 is
most closely related to Nkx-2.2 and the Xenopus gene XeNk2
(Saha et al., 1993).
In this study, we suggest that the signalling molecule
shh/vhh-1 and the transcription factor axial are involved in the
spatial regulation of expression of nk2.2. shh/vhh-1 is the
zebrafish homologue of the Drosophila hedgehog gene (Krauss
et al., 1993; Roelink et al., 1994), and axial (Strähle et al.,
1993) is the homologue of mouse HNF-3β, a member of the
winged-helix family of transcription factors (Pani et al., 1992;
Lai et al., 1993). shh/vhh-1 and axial/HNF-3β are both
involved in regulating the patterning of midline structures in
mesoderm and in the ventral CNS (Ang and Rossant, 1994;
Smith, 1994; Strähle and Blader, 1994; Weinstein et al., 1994).
The notion that axial/HNF-3β is a key regulator of floorplate
The last few years have seen significant advances in our understanding of the mechanisms underlying anteroposterior and
dorsoventral patterning of the hindbrain and spinal cord
(Krumlauf et al., 1993; Smith, 1994). However, the morphogenesis of more rostral brain regions is less well understood,
and there is still debate over such basic issues as defining the
neural axes in the forebrain. It has been demonstrated that,
similar to the hindbrain, distinct neuromeres are present in the
developing forebrain (Puelles et al., 1987). However, controversy remains as to the number and exact positions of the
forebrain neuromeres and whether they correspond to true
segmental subdivisions (Figdor and Stern, 1993; Puelles and
Rubenstein, 1993; Macdonald et al., 1994).
Because of its relative simplicity, the embryonic zebrafish
CNS is well suited for studies of early forebrain development.
By 24 hours of development (h), a simple scaffold of axon
tracts has been established by a small number of neurons at
invariant locations within the brain (Chitnis and Kuwada,
1990; Wilson et al., 1990). We have recently shown that
neurons that pioneer this scaffold differentiate at boundaries of
gene expression domains (Macdonald et al., 1994). For
example, the nuclei of the tract of the postoptic commissure
(nTPOC) and of the medial longitudinal fasciculus (nMLF)
both develop and extend axons along the ventral boundary of
pax6 and rtk1 expression.
In this study, we report the isolation of a member of the Nk2 family of homeobox genes, termed nk2.2, which is expressed
along the boundary zone at which the nTPOC and nMLF differentiate. Like several other homeobox-containing gene
Key words: boundary, Nk2, hedgehog, axial, zebrafish, forebrain,
neuronal differentiation
1756 K. A. Barth and S. W. Wilson
development is supported by the finding that ectopic
expression of HNF-3β results in the ectopic appearance of
floorplate markers (Sasaki and Hogan, 1994; see also Ruiz i
Altaba and Jessel, 1992). It seems likely that shh/vhh-1 is
responsible for the induction of axial/HNF-3β in the presumptive floorplate since ectopic expression of shh/vhh-1
results in ectopic axial/HNF-3β expression (Echelard et al.,
1993; Krauss et al., 1993; Roelink et al., 1994), while COS
cells secreting shh/vhh-1 induce floorplate differentiation in
adjacent neuroectoderm (Roelink et al., 1994).
In zebrafish, analysis of embryos carrying the cyclops
mutation has also provided results consistent with the possibility that shh/vhh-1 and axial/HNF3β regulate floorplate
development. The cyclops mutation affects specification of the
ventral midline of the CNS such that homozygous mutant
embryos lack a floorplate and exhibit fusion of the eyes (Hatta
et al., 1991). The cyclops gene may be involved in the signalling pathway between mesoderm and neuroectoderm that
specifies ventral CNS cell types (Hatta et al., 1991, 1994). In
agreement with this interpretation, neither shh/vhh-1 nor axial
are initially expressed in the neurectoderm, while mesodermal
expression of these genes is present (Krauss et al., 1993;
Strähle et al., 1993).
The timing and spatially restricted expression of nk2.2
suggests that this gene may play a role in the regulation of a
zone of neuronal differentiation within the embryonic zebrafish
forebrain. Furthermore, we present evidence suggesting that
shh/vhh-1 may be involved in the spatial regulation of nk2.2
expression. We show that nk2.2 expression is initially absent
from the neuroectoderm of cyclops mutant embryos which concomitantly lack shh/vhh-1 expression and that overexpression
of shh/vhh-1 results in ectopic expression of nk2.2.
MATERIALS AND METHODS
Fish stocks
Breeding fish were maintained at 28.5°C and embryos were collected
by natural spawning and staged up to 24h (30 somites) according to
Westerfield (1993); beyond this time, embryonic stage is given as
hours post fertilisation. Cyclops (cycb16) mutant carrier fish were
obtained from C. Kimmel and C. Nüsslein-Volhard.
Isolation of the nk2.2 cDNA clone
To isolate zebrafish NK-2 homologues, primers to the region flanking
the homeobox and Nk-2 domain of the Xenopus XeNk-2 gene (Saha
et al., 1993) were used to amplify a 370 bp cDNA fragment from
Xenopus cDNA (stage 17). The PCR fragment obtained was cloned
and used to screen 1.2×106 recombinant clones of a zebrafish neurula
stage cDNA library at low stringency (50% formamide, 6× SSC at
37°C). A single clone containing 1.5 kb of cDNA was obtained,
subcloned and sequenced using internal primers and the Sequenase
Version 2.1 sequencing kit (USB). Sequence data were analysed using
the Genetics Computer Group Sequence Analysis Software Package,
Version 7.0 (Devereux et al., 1984).
In situ hybridisation and immunohistochemistry
Antisense digoxigenin-labelled RNA probes were synthesized using
the digoxigenin (DIG) RNA labelling kit (Boehringer Mannheim).
For nk.2.2, probes either comprising the entire 1.5 kb cDNA clone,
or comprising a 630 bp 3′ region starting immediately downstream of
the homeobox and including the Nk-2 domain gave best results. A
710 bp probe derived from the 5′ region upstream of the homeobox
resulted in a spatially identical signal, but gave higher background
staining. For axial, shh/vhh-1 and hlx-1, full-length cDNA probes
were synthesized. To decrease background staining, probes were fractionated over a G-50 (Sigma) drip column to remove unincorporated
DIG-UTP. Whole-mount in situ hybridisations were carried out as
described (Xu et al., 1994). After staining with NBT/X-phosphate
(Boehringer Mannheim), embryos were refixed overnight in 4%
paraformaldehyde/PBS, washed in PBS and cleared in 70% glycerol.
Embryos were dissected from the underlying yolk and mounted in
70% glycerol for photography. Immunohistochemistry was carried
out according to standard procedures (Wilson et al., 1990).
RNA injections
shh/vhh-1 RNA for injections was derived from the pSP64T-shh
plasmid kindly provided by J.-P. Concordet and P. Ingham; see Krauss
et al., 1993. RNA for injections was transcribed in vitro and several
picoliters were injected at a concentration of 0.1 mg/ml into blastomeres
of 1- to 4-cell stage embryos using a pressure-pulsed Picospritzer II
(General Valve Corp.). To assess the extent of chimerism, lacZ RNA
was co-injected at a lower concentration of 20 µg/ml. For control injections, RNA encoding β-galactosidase was injected at the same concentration (0.1 mg/ml). Analysis of β-galactosidase activity was performed
on embryos that had been fixed for 10 minutes in 4% paraformaldehyde, 0.5% glutaraldehyde at stages between 12 and 24 hours. After
several washes in PBS, 0.1% Tween 20 (Sigma), embryos were rinsed
in buffer A (1 mM MgCl2, 15 mM K3Fe(CN)6, 12 mM K4Fe(CN)6)
and incubated at 37°C in buffer A containing X-gal (Stratagene) to a
final concentration of 800 µg/ml. After staining, embryos were washed
several times, refixed and processed for in situ hybridisation.
RESULTS
nk2.2 is homologous to mouse Nkx-2.2 and
Xenopus XeNk-2
Through screening a zebrafish neurula stage cDNA library with
a fragment of XeNk-2, a single clone containing 1.5 kb of
cDNA was isolated and termed nk2.2 based on its homology
to murine Nkx-2.2 (Price et al., 1992) and Xenopus XeNk-2
(Saha et al., 1993). The sequence of the 1470 bp nk2.2 clone
shows a single open reading frame with a coding potential of
269 amino acids (Fig. 1A).
To define the extent of homology between nk2.2 and other
Nk-2 gene family members, we compared the nk2.2 translation
product to other sequences. Within the homeobox and Nk-2
domain, nk2.2 is 100% identical to mouse Nkx-2.2 and
Xenopus XeNk-2 proteins, and 93% identical to the same
domains of the Drosophila NK-2 protein (Kim and Nirenberg,
1989; Fig. 1B,C). Comparison of nk2.2 to the published 472
bp genomic fragment of Nkx-2.2 shows that, allowing for a
possible frameshift in the published Nkx-2.2 sequence, nk2.2
and Nkx-2.2 are 93% identical over the entire published Nkx2.2 sequence.
The region of high identity (94%) between nk2.2 and
Xenopus XeNK-2 comprises amino acid sequences both
aminoterminal (20 aa) and carboxyterminal (30 aa) to the
domain containing the homeobox and Nk-2 domain. To either
side of this core region, the sequence diverges and the putative
proteins differ in size, with nk2.2 being 64 amino acids longer
than the published XeNk-2 protein. However, 58 amino acids
upstream from the reported translational start site of XeNk-2,
there is another in frame ATG, which is identical to the
proposed translational start site for nk2.2.
Zebrafish nk2.2 gene 1757
A
nk2.2 is expressed from late gastrula in the
presumptive forebrain adjacent to cells expressing
shh/vhh-1 and axial
nk2.2 expression is first detected around 95% epiboly (9.5h) as
a small patch of cells at the animal pole in the presumptive
brain (Fig. 2A). Between bud/1 somite (10h) and 3 somites
(11h), the domain of expression is a narrow column of cells at
the midline of the condensing neural keel (Fig. 2B,C). At later
stages, cavitation of the neural keel bisects this column of cells
to generate bilaterally symmetrical stripes of expression. Preliminary data indicated that expression of axial (Strähle et al.,
1993), shh/vhh-1 (Krauss et al., 1993) and the homeobox-containing gene, hlx-1 (Fjose et al., 1994) may be localized to
regions neighbouring nk2.2 expression and so the evolving
pattern of nk2.2 expression was examined with respect to the
expression domains of these genes.
At 5 somites (11.7h), the nk2.2 expression domain can be
divided into two components. The rostral domain of expression
extends from the anterior end of the neural keel to the mid-diencephalon (Fig. 2E) and lies adjacent and dorsal to cells that
express both shh/vhh-1 and hlx-1 (Fig. 2D,F), but anterior to the
Fig. 1. Sequence, gene structure and deduced amino acid sequence of
the zebrafish nk2.2 gene. (A) The nucleotide sequence of nk2.2 is
shown along with the conceptual translation of the open reading
frame. The putative translational initiation site (ATG) is indicated,
the homeodomain is boxed, and the Nk-2 motif underlined (thick
line). The 3′ polyadenylation consensus sequence is marked by a thin
line. (B) Gene structure and partial restriction map of nk2.2. Thick
bars at the 5′ and 3′ ends, representing 280 bp and 368 bp
respectively, indicate untranslated regions, while the open box
represents the protein coding region. The homeobox (light striped
box) and Nk-2 domain (dark striped box) are highlighted.
(C) Amino acid sequence comparison of the nk2.2 homeobox and
Nk-2 domain to other members of the Nk-2 family in mouse,
Xenopus and Drosophila as well as to a more distantly related mouse
gene, Dlx-1. Percentage of amino acid identity is given for the core
17 amino acids of the Nk-2 domain and to the slightly larger region
of 21 amino acid that is identical between nk2.2, Nkx-2.2 and
XeNK-2. Adapted from Price et al. (1992) and references therein and
Saha et al. (1993).
1758 K. A. Barth and S. W. Wilson
Fig. 2. Comparison of the developmental time course of nk2.2 expression in the rostral brain with that of shh/vhh-1 and axial. Whole-mount
embryos hybridised with antisense RNA to nk2.2, shh/vhh-1, axial or hlx-1. Lateral views (except A,B) are shown with rostral to the left. In DY, the skin, yolk and eyes have been removed. (A,B) Frontal views (with dorsal up) showing nk2.2 expression (arrowheads) at 95% epiboly
(9.5h) (A) and bud/1s (10h) stage (B). Dots outline the yolk plug in A. (C) Lateral view of nk2.2 expression at 3 somites (11h) and 5 somites
(11.7h). (D) hlx-1 expression in the forebrain of a 5 somites (11.7h) embryo. (E-Y) Comparison of rostral brain expression domains of nk2.2
(E,H,K,N,Q,T,W), shh/vhh-1 (F,I,L,O,R,U,X) and axial (G,J,M,P,S,V,Y) from 5 somites (11.5h) to 44-48h. The arrowheads in E-G indicate a
small groove in the mid-diencephalon at which the cephalic flexure will later form. Arrowhead in Q indicates the gap between rostral and
caudal nk2.2 expression domains. In V, the embryo was also labelled with an antisense RNA probe to wnt1 which is expressed in cells beneath
the epiphysis (Macdonald et al., 1994). Abbreviations: cb, cerebellum; cf, cephalic flexure; e, epiphysis; fp, floorplate; hy, hypothalamus; mb,
midbrain; mdb, mid-diencephalic boundary; or, optic recess; p; anlage of the anterior pituitary; rd and cd, rostral and caudal domains of nk2.2
expression; t, telencephalon; te, tegmentum; III, third ventricle. Scale bar=100 µm
Zebrafish nk2.2 gene 1759
domain of axial expression (Fig. 2G). The weaker caudal
domain of forebrain expression of nk2.2 (Fig. 2E,H) is directly
dorsal to cells expressing both shh/vhh-1 and axial in the caudal
diencephalon and midbrain. The junction between the rostral
and caudal domains overlies a small transverse groove in the
ventral neuroepithelium (Fig. 2E-G) at which the cephalic
flexure will later form (see Fig. 2P), and corresponds to the
anterior boundary of both axial expression and the presumptive
floorplate. We have previously described this position along the
rostrocaudal axis as the mid-diencephalic boundary (MDB,
Macdonald et al., 1994), which may, at later stages, correspond
to the zona limitans interthalamica described in other species
(Puelles and Rubenstein, 1993; Rubenstein et al., 1994).
By 15 somites (16.5h), a dorsally directed deflection in the
band of nk2.2-expressing cells at the MDB becomes apparent
(Fig. 2H). By this stage, shh/vhh-1 is no longer detectable in
the ventralmost cells of the rostral forebrain (Fig. 2I), and the
rostralmost domains of shh/vhh-1 and nk2.2 expression
partially overlap. However, within the caudal forebrain, nk2.2
continues to be restricted to cells dorsal to the domains of both
shh/vhh-1 and axial (compare Fig. 2H to Fig. 2I and J). From
this stage onwards, the pattern of hlx-1 expression becomes
complex and highly dynamic (Fjose et al., 1994) and shows no
obvious correlation with nk2.2 expression (not shown).
Between 22 (20h) and 28 somites (23h), the dorsal deflection of nk2.2 expression at the MDB becomes more pronounced (Fig. 2K-N). Concurrent with this change, the
domains of shh/vhh-1 and axial expression extend further
dorsally at the MDB (axial expression expands dorsally several
hours before shh/vhh-1), with the dorsal tip of expression
coming to underlie the anterior epiphysis (Fig. 2L,M,O,P).
During these stages, cavitation of the neural keel begins to
generate the ventricular system of the CNS and it becomes
apparent that the anterior domain of nk2.2 and shh/vhh-1
expression is located directly ventral to the optic recess/third
ventricle (Fig. 2K,L). The rostral domain of nk2.2 expression
thus overlaps the ventralmost cells within the diencephalic
expression domains of both pax6 and rtk1 (see Macdonald et
al., 1994).
By 26-27h, a small gap between the rostral and caudal
domains of nk2.2 expression is visible (Fig. 2Q and see Fig.
5G). This gap overlies the narrow dorsally directed finger-like
projection of shh/vhh-1- and axial-expressing cells at the MDB
(Fig. 2R,S, and see Fig. 5H). Throughout later developmental
stages, the expression domains of the three genes maintain
similar spatial relationships as the forebrain undergoes further
morphogenesis (Fig. 2T-Y). Finally, nk2.2 transcripts are
detected in the anlage of the anterior pituitary (Fig. 2Q) though
expression is transient and decreases during further development (Fig. 2T).
Low levels of nk2.2 transcripts are present in the
hindbrain and in cells ventral to the notochord
Although the most prominent site of nk2.2 expression lies within
1760 K. A. Barth and S. W. Wilson
Fig. 3. nk2.2 expression in the hindbrain and in a group of cells ventral to the notochord. Lateral views (A,B,E) with rostral to the left and
transverse sections (C,D) of 20-22 somites (19-20h) embryos from which the eyes and yolk have been removed. The alkaline phosphatase
colour reaction was developed 5-6 times longer than usual to reveal weak expression. The approximate levels of the sections shown in C and D
are indicated in A. (A) Low magnification view of the entire embryo. The arrow indicates very faint staining in the caudal spinal cord, and the
arrowhead points to the group of cells shown in D and E. (B) nk2.2 expression in the brain. The arrow indicates the discontinuity in the band of
nk2.2-expressing cells. (C) Transverse section through the caudal hindbrain revealing expression in cells adjacent to the floorplate. (D)
Transverse section near the hindbrain/spinal cord junction showing nk2.2 expression in cells beneath the notochord and hypochord (arrow).
(E) Lateral view of the same group of cells as (D). Abbreviations: fb, forebrain; fp, floorplate; h, hypochord; hb, hindbrain; hy, hypothalamus;
mb, midbrain; n, notochord; s, somite; sc, spinal cord; t, telencephalon. Scale bar: A,B=100 µm, C-E=20 µm.
the forebrain, lower levels of mRNA were also detected in more
caudal parts of the CNS, as well as in a cluster of cells ventral
to the notochord (Fig. 3). Within the CNS, the column of nk2.2expressing cells extends from the ventral optic stalk to the caudal
spinal cord with highest expression rostrally, lower transcript
levels in the hindbrain and only barely detectable expression in
the spinal cord (Fig. 3A). There is one small gap in this column
of expression at the boundary between midbrain and hindbrain
(Fig. 3B). Within the hindbrain, nk2.2 is expressed in several
cells to either side of the floorplate (Fig. 3C).
The only site of nk2.2 expression outside the neuroepithelium is a patch of cells ventral to the hypochord at the
hindbrain/spinal cord boundary (Fig. 3A). Expression is first
detected in this location around 15 somites (16.5h), peaks at
Fig. 4. Gene expression boundaries of nk2.2, shh/vhh-1 and axial
demarcate sites of neuronal differentiation and axogenesis in the
forebrain and midbrain. Embryos are hybridised with nk2.2
(A,B,E,F,L,M), shh/vhh-1 (C,G-I), axial (D,J,K,N) antisense RNA
and HNK-1 antibody (brown labelling of neurons and axons).
(A-D) Lateral views of sagittal hemisections with rostral to the left
and eyes removed. (A) nk2.2 expression with respect to the nTPOC
and nMLF. The dark blue alkaline phosphatase reaction product
masks the nTPOC in A and C. (B) High magnification of nk2.2
expression with respect to the nTPOC. The arrowheads indicate the
course of the axons in the TPOC, and the white arrow indicates
HNK1 labelling within the nk2.2 expression domain. (C,D)
Correlation of shh/vhh-1 (C) and axial (D) expression domains with
the locations of the nTPOC and nMLF. The arrows in D indicate a
few axial-expressing cells dorsal and ventral to axons in the TPOC
and the positions of the sections shown in H and I are indicated in C.
(E-N) Transverse sections. (E-G) nk2.2 (E,F) and shh/vhh-1 (G)
expression at the level of the nTPOC. The arrowheads in F indicate
immunoreactive processes within the nk2.2 expression domain.
(H-J) shh/vhh-1 (H,I), axial (J,K,N) and nk2.2 (L,M) expression at
the level of the nMLF (H and J are through the rostral part of the
nucleus and I,K and L are through the caudal part of the nucleus).
The arrowheads in M and N indicate immunoreactive processes
connecting to the ventricle. The section shown in M is from a
slightly older embryo than in L. Abbreviations: cf, cephalic flexure;
hy, hypothalamus; mb, midbrain; mdb, mid-diencephalic boundary;
nMLF and MLF, the nucleus of the medial longitudinal fasciculus
and its associated tract; nTPOC and TPOC, the nucleus of the tract of
the postoptic commissure and its associated tract; or, optic recess; t,
telencephalon. Scale bars for A,C,D,E, G-L and B,F,M,N=100 µm.
Zebrafish nk2.2 gene 1761
1762 K. A. Barth and S. W. Wilson
Table 1. Alterations in gene expression following injection
of RNA encoding shh/vhh-1
% Embryos affected
n*
Total
nk2.2
shh/vhh-1
Axial
hlx-1
114
59
71
12
63
34
64
67
MDB MDB+eyes Mb
37
34
64
67
26
Total n=
256
145
101
30
20
14
Severely
abnormal
% WT embryos**
37
66
36
33
7
11
2
107
20
In total, 256 shh/vhh-1-injected embryos were analysed for changes in the
pattern of gene expression.
‘total n=’ represents the number of embryos in each category.
n* are the numbers of embryos examined for each gene.
** are the numbers of severely abnormal embryos that were not included in
the analysis (see text). Scored embryos were either wild type (WT), or fell
into one of three classes of altered expression patterns: ‘MDB’ corresponds to
changes in gene expression domains at the mid-diencephalic boundary;
‘MDB+eyes’ indicates ectopic gene expression in the eyes in addition to
altered expression at the MDB, and ‘Mb’ denotes widespread ectopic gene
expression in the midbrain (and sometimes the hindbrain) as well as at the
MDB. In a few cases, embryos that showed ectopic expression of nk2.2 in the
eyes and at the MDB also exhibited a small patch of ectopic expression in the
midbrain (see text). Figures are given as percentages.
about 20-22 somites (19-20h) (Fig 3D,E) and diminishes by
28-30 somites (23-24h). Because expression is transient, we
have not determined the fate of this group of cells.
nk2.2 expression demarcates a zone of neuronal
differentiation in the rostral brain
Boundaries between gene expression domains demarcate the
sites at which the first neurons in the forebrain differentiate and
extend axons (Macdonald et al., 1994). The dorsoventral
position of nk2.2 expression suggested that it may overlie the
boundary at which neurons in the nTPOC and the nMLF differentiate. To test this possibility, we examined the formation
of the nMLF and nTPOC with respect to sites of nk2.2,
shh/vhh-1 and axial expression.
Mature neurons of the TPOC differentiate within the nk2.2
expression domain (Fig. 4A,B,E,F). Indeed, many HNK1immunoreactive radial processes connected to the ventricle lie
within the nk2.2 expression domain; these processes probably
belong to young neurons that still retain ventricular connections (Fig. 4F). There is considerable overlap between the
shh/vhh-1 and nk2.2 expression domains in the rostral
forebrain though shh/vhh-1 expression extends further
ventrally and nk2.2 expression extends more laterally into the
optic stalk (compare Fig. 4E to G). While at least some of the
neurons of the nTPOC appear to differentiate just within the
shh/vhh-1 expression domain (compare Fig. 4F to G), mature
neurons and axons are positioned at the edge of this domain
(Fig. 4G). The axons of the TPOC initially trace a course along
the ventral edge of cells expressing nk2.2 (Fig. 4B) and as they
approach the mid-diencephalon, they extend into a domain of
cells expressing axial (Fig. 4D). A small region not expressing axial (Fig. 4D) was usually observed at the point of entry
of the leading TPOC axons into the domain of axial expression.
The nMLF differentiates along the ventral edge of the caudal
domain of nk2.2 expression (Fig. 4A,L,M). While many
immunoreactive radial processes, probably belonging to young
neurons, lie within the nk2.2 expression domain, most and
perhaps all of the mature nMLF neurons lie just lateral to the
domain and do not express nk2.2 (Fig. 4M). Conversely, many
of the mature neurons lie just within the axial expression
domain and do express this gene (Fig. 4J,K,N) whereas many
of the radial processes lie just dorsal to the axial expression
domain (compare Fig. 4M to N). The shh/vhh-1 expression
domain in the midbrain does not extend quite as far dorsal as
the axial expression domain with the result that there are
usually a few non-expressing cells between the shh/vhh-1
expression domain and the neurons of the nMLF (Fig. 4C,H,I).
Overexpression of shh/vhh-1 RNA results in
elevated and ectopic nk2.2 expression
The observation that all sites of nk2.2 expression are within
several cell diameters of cells expressing shh/vhh-1 raises the
possibility that shh/vhh-1 may be involved in the regulation of
nk2.2 expression. In order to determine if shh/vhh-1 can induce
nk2.2, we analysed embryos that ectopically expressed
shh/vhh-1 after injection of synthetic shh/vhh-1 RNA.
In total, 256 shh-injected embryos were examined for alterations in expression of nk2.2, shh/vhh-1, axial and hlx-1 (see
Table 1). More than half of the injected embryos had specific
alterations in CNS expression domains (see below), a few had
minor deficiencies in the body axis (such as kinked notochords), while the remainder did not show any obvious defects.
A further 20 injected embryos showed severely perturbed
development and were not included in our detailed analysis.
Similarly disturbed development was occasionally seen in
control injections or in the wild-type background.
nk2.2 and axial expression was noticeably altered in about
two thirds, and endogenous shh/vhh-1 expression changed in
one third of injected embryos (Table 1). Because widespread
ectopic expression of shh/vhh-1 was not detected, we assume
that the injected shh/vhh-1 RNA was already degraded by the
stage at which embryos were fixed. Almost invariably, alterations in the expression of all genes examined were apparent
at the MDB. For nk2.2, the expression domain at the MDB was
broader, extended further dorsal and mRNA levels were
elevated as compared to controls (compare Fig. 5A to 2Q). In
some cases, the gap between the rostral and caudal domains of
nk2.2 expression was enlarged (compare Fig. 5D to 2Q and
5G). Ectopic nk2.2 transcripts were also observed in the eyes
(see below), and occasionally in the midbrain (Fig. 5D). No
ectopic nk2.2 expression was observed posterior to the
midbrain or outside the CNS.
Paralleling the changes observed for nk2.2 expression, axial
and shh/vhh-1 expression domains also extended further
dorsal, and mRNA levels were higher and detected in a wider
stripe of cells at the MDB of injected embryos (Fig. 5B,C,E,F).
In severe cases, the width of the band of tissue expressing
shh/vhh-1 and axial at the MDB expanded from the 1-2 cells
normally observed (Fig. 5H) to 10 or more cell diameters (Fig.
5E,F,I). Embryos examined for changes in axial expression at
earlier stages indicated that cells in the mid-diencephalon
expressed the gene earlier in injected embryos than in controls
(Fig. 5J). About one third of injected embryos examined for
axial expression also exhibited ectopic expression in the
midbrain and/or hindbrain as has been previously observed
(Krauss et al., 1993). hlx-1 was also overexpressed at the MDB
in 8 out of 12 embryos examined (not shown). Embryos that
exhibited changes in gene expression domains also showed
Zebrafish nk2.2 gene 1763
Fig. 5. Injection of shh/vhh-1 RNA results in elevated and ectopic expression of nk2.2, axial and shh/vhh-1. Whole-mount embryos with rostral
to the left. (A-F) Lateral views of 24h shh/vhh-1-injected embryos showing nk2.2 (A,D), axial (B,E) and shh/vhh-1 (C,F) expression. Eyes have
been removed. (D-F) Examples of embryos affected more severely than those in A-C. (G,H) Dorsal views showing the gap in nk2.2 expression
at the MDB of uninjected wild-type embryos (G) and the complementary expression of axial at the same location (H). The eyes are removed in
H. (I) Dorsal view of expanded axial expression domain at the MDB in an shh/vhh-1-injected embryo. (J) Lateral view of axial expression in an
control (left) and shh/vhh-1-injected 12 somite embryos. (K) Detection of β-galactosidase activity (blue) in embryos injected with βgalactosidase-encoding RNA. Some embryos were also examined for nk2.2 expression (eg. dark blue label in the forebrain of embryo at bottom
left). (L) Higher magnification of the tail region of embryo seen bottom right in K. Blue cells are positive for β-galactosidase. Abbreviations:
bl, blood; cf, cephalic flexure; h, hypochord; hy, hypothalamus; mb, midbrain; mdb, mid-diencephalic boundary; n, notochord; or, optic recess;
sc, spinal cord; sk, skin; t, telencephalon; y, yolk; III, third ventricle. Scale bar=100 µm.
1764 K. A. Barth and S. W. Wilson
morphological defects in the anterior brain. In particular, the
cavity of the third ventricle appeared reduced (compare Fig. 5I
and H) and the development of the eyes was abnormal (see
below and Krauss et al., 1993).
Changes in gene expression were usually not apparent at the
sites at which the nTPOC and nMLF differentiate. For
instance, nk2.2 expression never expanded ventrally into the
hypothalamus or floorplate. However, in a few cases, nk2.2
expression was disrupted in the midbrain and in these embryos
we also observed disruption of the nMLF from being a tight
column of neurons to being a much more widely scattered
group of cells (not shown).
To ascertain that changes in gene expression were not due
to the effects of injecting RNA per se, we examined embryos
injected with RNA encoding β-galactosidase for changes in
nk2.2 expression. Of 99 injected control embryos, 94 were
morphologically normal with unchanged expression patterns,
while 5 embryos showed non-specific defects.
We examined the distribution of injected RNA in 42
embryos that had been injected with both RNA encoding
Fig. 6. Ectopic expression of nk2.2 in the optic primordia of shh/vhh-1-injected embryos correlates with impaired eye development.
(A-D) Whole-mount 22-24h shh/vhh-1-injected embryos hybridised with antisense RNA to nk2.2. (A,B) Lateral views showing ectopic nk2.2
expression throughout the optic primordia. (A) Focussed at the level of the eye and (B) focussed through the eye and onto the brain. The white
arrowhead indicates the normal position of the optic stalk and the arrow indicates the dorsocaudal limit of fusion of the optic primordia to the
brain. (C) Ventral view of an shh/vhh-1-injected embryo with ectopic nk2.2 expression in the anterior part of the optic primordia. (D) Frontal
view of an shh/vhh-1-injected embryo with nk2.2 expression throughout the optic primordia. (E,F) Dorsal (E) and frontal (F) views of nk2.2
expression in normal 22-26 somites (20-22h) embryos. (G-H) Eye morphology in living normal (G) and shh/vhh-1-injected (H,I) 30h embryos.
The lens is reduced in H and absent in I. Ventrorostral eye development and pigment formation is affected in both embryos. Abbreviations: cf,
cephalic flexure; ch, choroid fissure; hy, hypothalamus; l, lens; mb, midbrain; mdb, mid-diencephalic boundary; nr, neural retina; op, optic
primodia; or, optic recess; os, optic stalk; pe, pigment epithelium; pnr, presumptive neural retina; ppe, presumptive pigment epithelial layer; se,
surface ectoderm; t, telencephalon. Scale bar: A-F=100 µm, G-I=50 µm.
Zebrafish nk2.2 gene 1765
shh/vhh-1 and β-galactosidase by assaying the distribution of
enzyme activity. In all cases, β-galactosidase-positive cells
were widely distributed throughout the embryo (Fig. 5K) and
detected in all tissue layers (Fig. 5L).
Ectopic expression of nk2.2 in the eyes of shh/vhh1-injected embryos correlates with abnormal eye
development
In normal embryos, nk2.2 is expressed in the proximal, ventral
part of the optic stalk, but not within the eyes (Fig. 6E,F). In
contrast, 41% of shh/vhh-1-injected embryos in which nk2.2
expression was altered, exhibited ectopic nk2.2 expression in
the eyes. The extent of this expression was variable, sometimes
being detected throughout the optic primodia (Fig. 6A,D), in
other cases restricted to more medial and ventral parts of the
developing eyes (Fig. 6C). Although nk2.2 expression spread
laterally into the eyes of injected embryos, it was never
detected in the hypothalamus or within dorsal regions of the
telencephalon (Fig. 6A,C,D).
In embryos that exhibited ectopic nk2.2 expression in the
optic primordia, normal eye development was impaired, and
eyes remained fused to the brain (Fig. 6A,B,D). The area of
fusion extended dorsocaudally from the normal position of the
optic stalk to near the MDB (Fig. 6A,B). In addition, the optic
primordia frequently failed to invaginate to form an optic cup
and showed abnormal development of the presumptive neural
and pigment layers of the retina (compare Fig. 6D with F).
Indeed, the abnormal optic primodia of injected embryos more
closely resembled the undifferentiated optic vesicles of much
younger normal embryos. Possibly as a consequence of
abnormal optic cup formation, the lens was frequently reduced
in size or sometimes even absent from the eyes of injected
embryos (Fig. 6G-I).
cyclops mutant embryos that lack shh/vhh-1 and
axial expression in the neuroectoderm exhibit a
concomitant loss of nk2.2 expression
That overexpression of shh/vhh-1 leads to ectopic induction of
nk2.2 suggests that shh/vhh-1 may be required for the normal
induction of nk2.2 expression. To investigate this possibility,
we examined nk2.2 expression in embryos homozygous for the
cyclops mutation. The cyclops mutation prevents specification
of the ventral midline in the CNS (Hatta et al., 1991) and, at
early stages, mutant embryos do not express shh/vhh-1 within
the CNS (Krauss et al., 1993).
nk2.2 expression was absent from the forebrain of all cyclops
mutant embryos examined between 10 somites (14h) and 24
somites (21h) (n=23; Fig. 7A,B,C). shh/vhh-1 and axial
expression were also absent at comparable stages confirming
previous results (Krauss et al., 1993; Strähle et al., 1993).
However, in 30 somites (24h) and older mutant embryos, a
small patch of cells expressed nk2.2 (12/16 embryos examined),
Fig. 7. Expression of nk2.2, shh/vhh-1 and axial in homozygous mutant cyclops embryos. Lateral views (A,D,E,F) with rostral to the left, and
transverse sections (B,C) of embryos hybridised with nk2.2, axial or shh/vhh-1 anti-sense RNA. (A-C) nk2.2 expression in 18 somite (18h)
wild-type and cyclops mutant embryos. The transverse sections shown in B and C are at the level of the diencephalon. The small dark patch on
the dorsal surface of the embryo in B is an artefact. (D) 30 somites (24h) cyclops mutant embryos hybridised with antisense RNA to nk2.2,
axial and shh/vhh-1. The arrowheads indicate a small cluster of axial and shh/vhh-1-expressing cells in the forebrain. (E) shh/vhh-1 expression
at the dorsal tip of the mid-diencephalic furrow of a 30 somite (24h) cyclops mutant embryo. (F) nk2.2 expression at the tip of the middiencephalic furrow of a 40-44h cyclops mutant embryo. Abbreviations: cb, cerebellum; e, epiphysis; fe, fused eye; l, lens; mdb, middiencephalic boundary; mdf, mid-diencephalic furrow; ov, optic vesicle; t, telencephalon; te, tectum. Scale bar=100 µm.
1766 K. A. Barth and S. W. Wilson
shh/vhh-1 (14/19) and axial (14/15) at the tip of the furrow that
forms in place of the MDB (see Macdonald et al., 1994; Patel
et al., 1994) in cyclops mutant embryos (Fig. 7D-F).
Low levels of nk2.2 expression were also detected in more
caudal regions of the CNS of cyclops embryos from about 16
somites (14h) (data not shown). Similar observations have
been made for shh/vhh-1 (Krauss et al., 1993) and axial
(Macdonald et al., 1994).
DISCUSSION
We have described the isolation and characterisation of the
zebrafish nk2.2 gene. In common with all members of the Nk2 family of homeobox genes, nk2.2 contains a conserved
sequence characteristic for this family, the Nk-2 domain. The
high conservation of the Nk-2 domain suggests that it is
important for the function of Nk-2 proteins. The nature of this
function is unknown although it has been suggested that the
Nk-2 domain could be involved in mediating protein-protein
interactions (Price et al., 1992).
nk2.2 is most closely related to mouse Nkx-2.2 (Price et al.,
1992) and Xenopus XeNk-2 (Saha et al., 1993). The observed
homology at the amino acid level appears to be paralleled by
the conservation of the expression patterns among nk2.2, Nkx2.2 and XeNk-2, although there are some differences between
our interpretation of expression patterns and others. However,
these differences probably reflect the fact that previous studies
have not analysed expression patterns in such great detail.
Indeed, recent reanalysis of Nkx-2.2 expression in mouse
suggests a very close similarity in expression between this gene
and nk2.2 within the developing forebrain (Rubenstein et al.,
1994; Rubenstein, personal communication).
While expression domains of nk2.2, Nkx-2.2 and XeNk-2
appear to be similar in the CNS, nk2.2 exhibits one additional
site of expression not reported for the mouse and frog homologues. This patch of nk2.2-expressing cells is located in a
region ventral to the hypochord near the hindbrain/spinal cord
boundary. The transient and weak nature of expression at this
site may explain why it has not been described in other species.
nk2.2 expression delineates a zone of neuronal
differentiation in the rostral brain
Many of the early neurons in the rostral zebrafish CNS differentiate at boundaries between regulatory gene expression
domains (Macdonald et al., 1994). For instance, the nTPOC
and nMLF are both positioned at the ventral boundary of
expression of the receptor tyrosine kinase, rtk1, and the paired
box transcription factor, pax6. These observations raised the
possibility that cells at the interface between adjacent
expression domains may have an identity distinct from that of
either of the neighbouring domains (Wilson et al., 1993). nk2.2
is expressed in a band of cells at the interface where both the
nTPOC and nMLF differentiate suggesting that this gene may
be involved in the establishment or maintenance of the identity
of cells at a zone of neuronal differentiation.
From studies performed mainly in Drosophila, at least two
classes of genes have been shown to be important in regulating neurogenesis; proneural genes influence whether ectodermal cells become epidermis or neural tissue while neurogenic
genes influence which of the neural cells differentiate as
neurons (Jimenez and Modolell, 1993). Several members of the
basic helix-loop-helix family of transcription factors act as
proneural genes (Jan and Jan, 1993), while signalling
molecules including Notch and Delta function in the neurogenic pathway (Ghysen et al., 1993). Although nk2.2
expression defines several regions where neurons differentiate,
it is unlikely that it functions as a neurogenic gene. For
instance, nk2.2 is expressed in a continuous longitudinal band
within the rostral brain at which early neuronal differentiation
is only observed in two discrete sites. Therefore, many of the
cells that express nk2.2 do not appear to be in the developmental pathway leading to early neuronal differentiation.
However, it is possible that nk2.2 functions in combination
with other genes to regulate the distribution of the earliest
neurons in the brain (Barth and Wilson, 1994).
Our results indicate that cells at a boundary region are
distinct from adjacent cells in terms of gene expression, though
it remains unknown if this distinction extends to differences in
morphology or cell surface properties, as has been documented
for boundary cells between rhombomeres in the hindbrain
(Heyman et al., 1994). Although there are no published
descriptions of cell surface proteins restricted to boundary cells
in the forebrain, several such proteins are expressed in spatially
restricted domains that respect these boundaries (Allendoerfer
et al., 1994; Redies and Gänzler, 1994.
shh/vhh-1 influences the expression of nk2.2
All sites of nk2.2 expression in the CNS lie within several cell
diameters of cells that express shh/vhh-1. The observations that
all changes in the pattern of shh/vhh-1 expression are accompanied by complementary changes in nk2.2 expression, and
that overexpression of shh/vhh-1 ectopically induces nk2.2,
suggest that secreted shh/vhh-1 may be required for nk2.2
expression. Hence, the spatially restricted domain of nk2.2
expression may arise due to the limited diffusion of shh/vhh1 protein within the neuroectoderm. The Drosophila hedgehog
protein has recently been shown to be cleaved into two active
forms and it is likely that one has short-range, and one has
longer range activities (Lee et al., 1994). Similar cleavage of
zebrafish shh/vhh-1 occurs (Lee et al., 1994) though it remains
unknown over what range the two protein species may signal
and so it is premature to speculate which of the two proteins
may be involved in regulating nk2.2 expression.
Further support for a possible requirement of shh/vhh-1 for
the induction of nk2.2 expression is derived from the observed
lack of nk2.2 transcripts in young cyclops mutant embryos,
which lack shh/vhh-1 expression in the CNS. Although recent
results have shown that the mesoderm of cyclopic embryos is
affected (Thisse et al., 1994), the primary consequence of the
mutation appears to be the incorrect specification of ventral
midline cells in the CNS (Hatta et al., 1991, 1994). We suggest
that a secondary consequence of the failure to specify ventral
midline tissue is a failure to induce nk2.2 in more lateral cells.
In older homozygous cyclops mutant embryos, the partial
recovery of shh/vhh-1 and axial expression is accompanied by
late expression of nk2.2. The recovery of ventral midline gene
expression in the neuroectoderm of cyclops mutant embryos is
not understood but suggests that signalling between mesoderm
and ectoderm may not be completely blocked by the mutation.
It also remains unknown if the recovery of gene expression is
accompanied by changes in the phenotype of midline cells.
Zebrafish nk2.2 gene 1767
Although we suggest that shh/vhh-1 influences nk2.2
expression, other molecules may also be involved in the spatial
regulation of expression of this gene since overexpression of
shh/vhh-1 results in spatially restricted ectopic expression of
nk2.2; thus only a subset of neural cells exposed to shh/vhh-1
respond by inducing nk2.2. However, we cannot rule out the
possibility that spatially restricted ectopic nk2.2 expression
may be explained by position-specific differences in
exogenous shh/vhh-1 RNA or protein stability or processing.
The spatial relationship between cells that express nk2.2 and
cells expressing shh/vhh-1 differs slightly between the rostral
and caudal domains of nk2.2 expression in the forebrain. In the
caudal domain, cells expressing nk2.2 and shh/vhh-1 are
discrete populations whereas rostrally the expression domains
of these genes overlap. If shh/vhh-1 is involved in the induction
of nk2.2 transcription, then it is of interest to consider why
nk2.2 expression is not induced within all shh/vhh-1-expressing cells. One possibility is that other gene(s) may repress
nk2.2 expression within many of the cells that express shh/vhh1 and thus it could be the absence of such factors rostrally that
allows overlap of nk2.2 and shh/vhh-1 expression.
Do nk2.2 and shh/vhh-1 expression domains define
the rostro-caudal axis of the brain?
Temporal analysis of shh/vhh-1 and nk2.2 expression indicates
that both genes are transcribed to the anterior tip of the condensing neural keel. As the forebrain differentiates, it becomes
apparent that the anteriormost point of expression lies immediately ventral to the optic recess. These observations support
the hypothesis that the anterior end of the brain is the optic
stalk region and that the telencephalon is a dorsal structure
(Ross et al., 1992; Puelles and Rubenstein, 1993; Hatta et al.,
1994; Rubenstein et al., 1994), not a discrete neuromere rostral
to the diencephalon.
Although nk2.2 is expressed further dorsally in rostral
compared to caudal regions of the brain, we believe that the
position at which this gene is expressed may be equivalent
throughout the CNS in terms of the molecular mechanisms that
regulate dorsoventral patterning. Indeed, in the early neural
keel, there is little difference in the dorsoventral position of
shh/vhh-1 or nk2.2 expression between the rostral brain and
more caudal regions. It is only at later stages that the distinction between more dorsally positioned expression in the diencephalon and ventral expression in more caudal regions
becomes apparent.
Two prominent features of forebrain morphogenesis contribute to the spatial changes in gene expression in the diencephalon. The first is the expansion of the hypothalamus and
the associated development of the cephalic flexure; the second
is the development of the MDB. Fate mapping studies of the
neural plate in Xenopus suggest that the hypothalamus derives
from midline cells (Eagleson and Harris, 1990), and so we
assume that the hypothalamic cells derive from the early pool
of shh/vhh-1-expressing midline cells. The later expansion of
the hypothalamic region is not well understood in any species
and it is unknown if it occurs by recruitment of more dorsal
forebrain cells or by exaggerated proliferation of the most
ventral cells.
The shift in gene expression associated with the development of the MDB takes place after the neural keel has
condensed and continues throughout early forebrain morpho-
genesis. Over time, there is a gradual dorsal extension in axial
expression, followed by a comparable change in shh/vhh-1
expression and complemented by a dorsal deflection of nk2.2
expression either side of the MDB. At least two possibilities
could account for the changes in gene expression at the MDB.
Either cells could migrate from ventral regions to more dorsal
positions at the MDB or, alternatively nk2.2, axial and
shh/vhh-1 expression may be gradually induced in progressively more dorsal cells within the mid-diencephalon. Indeed,
the precocious expression of axial in dorsal cells at the MDB
of shh/vhh-1-injected embryos suggests that dorsal cells may
be responsive to inductive signals at stages before axial is
normally expressed. This would suggest that in normal
embryos, the temporal availability of inductive signals may
contribute to the regulation of gene expression at the MDB.
We thank Phil Ingham, Jean-Paul Concordet, Stefan Krauss, Uwe
Strähle, Anders Fjose, Denis Duboule and Claudio Stern for probes
or antibodies, David Grunwald and R. Riggleman for the cDNA
library and Nigel Holder, Rachel Macdonald and Roger Patient for
comments on the manuscript. This study was initiated with funds from
the Medical Research Council and supported by the Wellcome Trust.
S. W. was a Science and Engineering Research Council Advanced
Research Fellow and is a Wellcome Senior Research Fellow.
The databank accession number for nk2.2 cDNA sequence is
X85977.
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(Accepted 17 February 1995)