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Differential expression of C-protein isoforms in the developing heart of normal and cardiac lethal mutant axolotls (Ambystoma mexicanum)

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DEVELOPMENTAL DYNAMICS 2 0 5 1 8 g 1 9 5 (1996)
Secondary Axis Induction by Heterospecific Organizers
in Zebrafish
Institute of Neuroscience, University of Oregon, Eugene, Oregon 97403-1254
To investigate the inductive acABSTRACT
tivities of the vertebrate organizer, we transplanted the chicken organizer (Hensen’s node)
into zebrafish gastrula and analyzed resulting secondary axes. Grafted Hensen’s node did not differentiate or participate in the secondary axis. It
also did not induce a secondary notochord or expression of the genes normally expressed by the
fish organizer including IU) tail, axial, goosecoid.
Nevertheless, it recruited fish cells to organize a
variety of tissues: the dorsal portion of the central
nervous system including Rohon-Beard sensory
neurons, otic vesicles, dorsal pigment stripe, dorsal fin, somites, heart, and pronephric ducts. Enlarged neural plate induced by the organizer was
shown by the expression pattern of dlx3 and msxB
genes, which demarcates the early presumptive
neural tissue. In addition, Henscn’s node of an
earlier stage chicken embryo displayed differential movement in zebrafish from that of a later
stage. This might reflect unknown differences in
properties between the organizer at two different
developmental stages related to its normal organizer activity. To create a model system to study
the molecular mechanisms of the organizer, we
next transplanted genetically modified mouse
cells into zebrafish embryos. We found that
Wnt3A-transfected NIH3T3 cells are much more
potent in inducing a secondary axis than NIH3T3
cells alone. These results suggest that formation of
a variety of tissues are controlled by signalling
from the organizer itself with no requirement of
participation of the organizer-derived tissues. Additionally, the activities of the organizer may involve a function of Wnt-family genes.
derm lip of the amphibian embryo was transplanted
into the ventral region of a host embryo (reviewed in
Spemann, 1938). Waddington also showed that the
most anterior tip of the primitive streak in the avian
embryo, Hensen’s node (HN), has similar activities in
inducing a secondary axis (Waddington, 1933; Waddington and Schmidt, 1933). Since the transplant recruited host cells to “organize” a second embryonic
axis, it was designated the “organizer.” Since then, a
number of experiments have been performed to characterize the role of the organizer. Most of these embryological studies were performed with homospecific
transplantation (or between closely related species). In
such a homospecific combination, not only do host-derived cells contribute to a n induced axis, but also the
grafted organizer itself differentiates to produce tissues, such as notochord, that also contribute to the secondary axis. Thus, secondary axis formation is the consequence of combined inductive activities of the
organizer and its derivatives, and it is difficult to dissect out the specific and transient signalling roles of
the organizer itself. Molecular analyses of early inductive events in Xenopus clearly show that patterning of
early embryos is perturbed by ectopic expression of
specific molecules. However, such manipulated blastomeres in Xenopus embryos also undergo morphogenesis, so that it is difficult to assess signalling mechanisms operating at a particular time and embryonic
location during development.
Recently Kintner and Dodd (1991) found that the
chicken Hensen’s node induced neural tissue when it
was combined with a Xenopus animal cap at a temperature (235°C) permissive for Xenopus development. In
this case, the chicken tissue did not differentiate, and it
was concluded that the organizer alone is able to in0 1996 Wiley-Liss, Inc.
duce neural tissues. In other heterospecific organizer
experiments between zebrafish and salamander (OpKey words: Hensen’s node, Wnt3A, Neural induc- penheimer, 1936), and between mouse and Xenopus
tion, Morphogenesis, Pigment pat- (Blum et al., 1992), axial structures were ectopically
terning, Sensory neurons, Rohon- induced. These studies suggest that the signalling
mechanisms by which the organizer recruits its surBeard cells, Otocyst, Heart, Somite
Vertebrate development involves a series of inductive interactions between embryonic cells th a t generate new tissues. The most striking example of embryonic induction was discovered more than 70 years ago
by Spemann and Mangold, who showed th a t a secondary embryonic axis was formed when a dorsal blasto0 1996 WILEY-LISS, INC.
Received July 18, 1995; accepted September 26, 1995.
Yoshiko Takahashi is now a t Department of Biology, Faculty of
Science, Kitasato University, 1-15-1, Kitasato, Sagamihara, Kanagawa, 228, Japan. Address reprint requests/correspondence there.
Kohei Hatta is now a t Institut Pasteur, c/o Dr. Henri Korn, INSERM U261, Laboratoire de Neurobiologie Celldlaire, Department de
Biologie Moleculaire, 25 Rue du Docteur Roux, Paris 75015, France.
TABLE 1. Induced Secondary Tissues by Various Transplants
Grafted embrvos
Analvsed embrvos"
% of analyzed embryosb
RB neurons >10 (1-10)
100 (0)
0 (33)
0 (0)
84 (0)
0 (61)
"In some operated zebrafish embryos, a grafted Hensen's Node (HN) was found dorsal in the vicinity of the host original axis
and did not demonstrate its characteristic inductive activity. Therefore, we analyzed operated embryos in which the graft
remained in the lateral to ventral region. Some embryos were fixed at earlier than 26 h. 3T3W3Aproduced duplication of
notochord and paired somites in three cases, which were also fixed early. These are not included in this table.
bThe secondary axes containing more than ten neurons, which were labelled with 21-1-12 and had the characteristic morphology
of Rohon-Beard cells, were scored. The secondary axes containing one to ten RB cells were also scored and are shown in
parentheses. Heart was identified by its beat. Other tissues were identified morphologically.
st 4
St 8
26 h
.. .
Chicken embryos
cDNA of
a signalling molecule
cell line
Fig. 1. Schematic of heterospecific organizer experiment. A small
piece of avian Hensen's node (HN) or a clump of cultured cells genetically
engineered to express a candidate signalling molecule is manually trans-
planted using a tungsten needle into the ventral region of an early zebrafish gastrula to observe the resultant secondary axis. An: animal pole;
D: dorsal; LB: lateral blastoderm; Veg: vegital pole.
rounding cells to organize a basic body pattern is con- the zebrafish embryo, the chicken tissue showed no sign
of self-differentiation, presumably due to the difference
served during vertebrate evolution.
In the present study, we exploited a heterospecific in optimum temperature between the fish (28.5"C) and
combination between chicken or mouse donor tissues the chicken (385°C). Nevertheless, chicken HN could
and the zebrafish embryo as a host. The zebrafish em- induce a secondary axis, containing a variety of tissues,
bryo has advantages for studying vertebrate develop- which were solely derived from fish cells. We studied
ment because of its transparent body and fast growth, early influences of the transplants by examining the
as well as availability of genetics (Kimmel et al., 1991; host expression patterns of tissue specific genes.
Rossant and Hopkins, 1992; Driever et al., 1994). When
In order to analyze the molecular mechanisms by
chicken HN was transplanted into the ventral region of which the organizer induces a n axis, we next trans-
planted genetically engineered mouse cells into zebrafish embryos. As a candidate signalling molecule
for inductive events, we chose a Wnt family gene. The
Wnt genes are vertebrate homologs of wingless (wg) in
Drosophila and encode secretory glycoproteins. Some of
the Wnt gene products including Wnt3A have been
shown to induce secondary axes when their mRNAs are
injected into early frog embryos (reviewed in Moon,
1993). We found here that Wnt3A-transfected mouse
cells partially mimicked the activities of the HN, when
grafted into zebrafish embryos. Thus, this experimental system seems to be powerful for genetic manipulation of zebrafish embryos with a signalling molecule of
interest at a particular region and at a particular stage
of a n embryo.
In addition, we followed the movements of the transplants and found that consistent differences occurred
between grafts from different sources.
HN Induces a Secondary Axis in Zebrafish
The stage 4 HN, grafted into the ventral marginal
zone of zebrafish embryos, induced a secondary axis in
the host, which was visible within 6 h r after operation
as a thick structure containing somites. The induced
axis typically extended from the graft towards the primary host axis where the secondary axis diagonally
joined the dorsal portion of the host embryo (see Figs.
2,3F,9A). Characteristic features of the induced tissues
at 28 h r in a secondary axis are schematically shown in
Figure 2, and the data are summarized in Table 1. As
a control, transplantation of lateral blastoderm including the epiblast and underlying endoderm (see Fig. 1)
at Stage 4 or Stage 8 did not induce any detectable
axial structure (e.g., see Fig. 9C).
Neural tissue. The monoclonal antibody zn-12 intensively stains Rohon-Beard (RB) sensory neurons
(Metcalfe et al., 19901, which are normally arranged in
bilateral rows in the dorsal portion of the spinal cord.
In the secondary axis induced by the chicken HN at
Stage 4,similar neurons were often found to be labeled
along the axis although they usually did not consist of
regular rows as in the original axis. In all cases, the
row of these zn-12 positive neurons joined the RB cells
in the spinal cord of the primary axis, showing a
Y-branching pattern (see Fig. 3F). These neurons in
the secondary axis had neuronal processes which displayed intensive branching over the skin as well as
central axons extending along the axis in the neural
tube. Since these antigenic and morphological features
were characteristic of RB cells, we suggest they were
ectopically induced RB cells. In histological sections,
these neurons were sometimes located on the ventral
floor of the induced neural tissue (not shown). Neither
notochord nor floor plate labeled with mz15 was found
in the secondary axes (see Fig. 3C,D). These results
suggest that the induced axis contained the dorsal but
not the ventral portion of the central nervous system.
Two ectopic otic vesicles were recognized by their char-
beating heart
central nervous system
Fig. 2. Schematic summary of secondary tissues induced by HN
Stage 4 at 26 h.
acteristic morphology with one or two reflective otoliths (normally two), which were also labeled with
mz15 Ab (see Fig. 3D). The induced otic vesicles were
associated with the formation of the third and fifth
segments of the hindbrain in the secondary axis, indicated by the expression of krox20 mRNA (see Fig. 3B).
Eyes were never formed, although the induced CNS
had more anterior structure than krox20-positive hindbrain. The HN taken from Stage 8 also induced a secondary axis when transplanted in zebrafish embryos.
However, the induced axes contained only a small
number of sensory neurons detected by zn-12 staining
and no other tissues were morphologically recognized
(see Fig. 3G, Table 1).
Somites. The somites in the induced axis were morphologically and immunohistologically recognized with
the anti-myosin antibody a t 26 hr, suggesting myotoma1 differentiation. The induced somites were, however, not paired and constituted a single row underneath the induced neural tissue (see Fig. 3E). The
muscle differentiation in the secondary somites was
delayed, although the somite formation occurs at the
normal schedule. At 14 hr, when muscle development
had begun in cells in the most medial portion of the
somite jaxtaposed to the notochord (Felsenfeld et al.,
1991), no myosin-positive cells could be detected in the
secondary axis (data not shown). The delay of the initiation of the muscle differentiation may be due to the
lack of the notochord in the secondary axis.
Heart and kidney. When the heart started to beat
in the original axis of the host embryo about 20 hr after
operation, beating heart cells were also observed in the
secondary axis (Fig. 2). In addition, histological sections demonstrated that the secondary axis contained a
pair of pronephric ducts (Fig. 3E, triangles).
Neural crest and pigment pattern. At 18 hr, neural crest cells expressing krox20 were found near the
induced hindbrain regions which were also positive for
krox20 (Oxtoby and Jowett, 1993; Fig. 3B, arrows). At
later stages, two neural crest-derivatives, pigment
cells, and the dorsal fin were also observed in the secondary axes (Fig. 4A,C,D; see also Fig. 9A). Secondary
dorsal fins initially were fused to the original dorsal
Fig. 3. Secondary axes induced by transplantation of chicken Hensen's nodes. A-F: Secondary axes induced by HN Stage 4. A: A secondary axis at 17 h. The dark shadow in the anterior region of the induced
axis (arrowhead) indicates the position of the transplanted tissue. B:
Induced hindbrain rhombomeres 3 and 5 labeled with kroxZU probe at 18
h. The neural crest cells migrating from these rhombomeres are also
labeled (arrows).The position of the secondary otocysts is indicated by
the triangle. C: m z l 5 staining at 18 h indicates no secondary notochord
induced (arrows). D: Induced otocycts (arrows) labeled with mz15 at
26 h. E: Induced somites labeled with anti-myosin antibody at 26 h. The
fins and showed a Y-branching pattern a s schematically drawn in Figure 2.
We further analyzed the effects on pigment pattern
formation a t later stages. By 3 weeks, the normal fish
starts to develop a golden pigment stripe along the dorsal midline of the body. In the fish with a secondary
axis, a golden pigment stripe was found to stretch over
the operated side of the body. This golden line bifur-
transverse section demonstrates the fused somites (arrow) under the
neural tissue (star) of the secondary axis, in contrast to the paired
somites in the original axis located at the opposite side of the yolk. The
triangles indicate secondary pronephric ducts. F: Induced Rohon-Beard
sensory neurons labeled with zn-12 antibody. G: Secondary axis induced
by the HN Stage 8 is less prominent. zn-12 labelling reveals induction of
a few neurons. The transplants are indicated with an arrowhead in F and
G. Bar = 50 pm.
Fig. 4. Induction of secondary pigment stripes and fins. The secondary axis creates a new dorsal pigment stripe which interferes with the
original pigment stripes. A: Three weeks after formation of the secondary
axis in response to transplantation of 3T3W3A.A dorsal golden stripe
bifurcates from the original dorsal stripe, runs across and interrupts other
original lateral stripes. The bilaterally associated melanocyte stripes
along the dorsal golden stripe also follow the secondary dorsal stripe. B:
Pigment pattern in a normal fish for comparison. C : Five months after
transplantation of HN, Stage 4. The secondary dorsal dark golden stripe
remains, and parts of the original stripes near the secondary stripe were
further interrupted to form dots. The stripe pattern in the opposite side of
this fish was normal. D: Secondary dorsal fin fully developed at 5 months
after transplantation of HN, Stage 4 (arrowhead). E: Skeletal pattern of
induced pectoral fin by 3T3W3Aat 3 week stage (arrowhead). Red: bones;
blue: cartilages. Bar = 1 mm.
cated from the dorsal golden stripe of the original axis (3T3P-ga')into zebrafish embryos. A coherent cell sheet
and extended ventrally to the presumed site of the which was scraped off a culture dish was chopped into
small pieces and transplanted into zebrafish embryos
grafted HN (N = 6).
In normal adults, several parallel blue stripes run in the same way as chicken tissues were grafted. Eighfrom the head to the tail (Fig. 4B). In the operated fish teen hours after the operation, the grafted cells were
(5-month-old), however, these stripes were sometimes recognized as a translucent cell mass and their tight
interrupted into small islands near the secondary dor- association was also confirmed by their P-gal activity
sal stripe, like interference between two waves (Fig. (Fig. 6A). When the cells were dissociated and microinjected as a cell suspension, the mouse cells were scat4C; N = 2/31.
tered in 3-day-old zebrafish host. Figure 6B demonInfluences of the HN on Early Gene Expression
strates a transplanted cell, as a cell suspension, located
in Zebrafish Embryos
in the neural tube just above the notochord, next to the
We next studied early responses of host-derived tis- floor plate cells. This cell has extended filopodia and a
sues to inductive activities of HN by examining the has clear nucleus. Thus mouse cells transplanted into
the zebrafish can survive at least for 3 days after graftexpression pattern of genes at gastrula stage.
In the normal embryo, goosecoid, no tail, and axial ing. It should be noted that the NIH3T3 may have a
genes are expressed in the organizer region (embryonic specific affinity to particular regions in the zebrafish
shield) of early gastrula embryos and in some of its embryo. For example, the 3T3P-ga'cells dissociated and
derivatives at later stages. Briefly summarized at 10 transplanted in the animal pole at the blastula stage
hr, no tail is expressed in notochord precursor (Schulte- seemed preferentially located along the optic nerves (N
Merker et al., 1992). axial is expressed in notochord = 3/10, Fig. 6C,D).
and prechordal plate precursors, as well as in the ventral portion of the central nervous system (Strahle et Inductive Activities of Wnt3A Transfected Cells
al., 1993).goosecoid is expressed in head mesoderm and in the Zebrafish
We next investigated how Wnt3A, a developmental
the overlying ventral midline cells of the neuroectoderm (Thisse et al., 1994).snail1 is expressed in medial signalling molecule, is involved in axis formation by
somitic precursors (adaxial cells) which are closely as- transplanting Wnt3A expressing mouse cells into zesociated with the notochord precursors (Thisse et al., brafish embryos. Since two cell lines, W4 and W11,
1993).Four hours after HN grafting, no ectopic expres- which expressed the highest level of Wnt3A mRNA
sion or altered pattern of expression of these genes was among 11independent cell lines (Fig. 71, gave the same
observed (N = 6 for each), suggesting that no host- result when transplanted into zebrafish embryos, we
derived organizer or midline tissue such as notochord, combined the data for W4 and W11 and designated
3T3P-ga'cells were utilized as a confloor plate, prechordal plate was induced, nor the me- these cells 3T3W3A.
dial part of the somites (Fig. 5C-F). We suggest, there- trol for the transplantation since they showed no signal
for Wnt3A mRNA (Fig. 7; p5 lane). When 3T3P-g"' cells
fore, that the HN did not induce a fish organizer.
dlx.3 (Akimenko et al., 1994)and mshB (Ekker et al., were transplanted as a clump, no morphologically dis1992) are expressed strongly by 10 hr, near the bound- tinct secondary axis structures were recognized. Immuary between presumptive skin ectoderm and presump- nohistochemical analysis also demonstrated no formative neural ectoderm, demarcating the early neural tion of axial or paraxial mesoderm. Staining with zn-12,
plate. Their expression patterns in the zebrafish gas- however, sometimes revealed the presence of a row of a
trula was clearly altered by grafted HN a t Stage 4. An few sensory neurons (Fig. 8B). In contrast, when
cells were transplanted, they induced definitive
expanded bud-like neural plate protruding from the 3T3W3A
original neural plate and surrounding the graft was secondary axes, which were morphologically robust and
observed (see Fig. 5A for dlx3; data not shown for contained numerous sensory neurons (Fig. 8A). MoremshB; N = 6 for each). The lateral blastoderm trans- over, in some cases, unpaired somites were observed in
planted a t the same region did not change expression of a similar way to HN transplantation (see Table 1).Thus
any of these genes (Fig. 5B; N = 6 for each). We sug- Wnt3A-transfected cells remarkably enhanced the forgest that the HN induced an expanded neural plate mation of a secondary axis compared to grafts of control
tissue around it which could be detected 4 hr after 3T3 cells. As was observed when chicken HN was transplanted, the secondary axis also resulted in the formatransplantation.
tion of a golden pigment line on the operated side of the
Transplantation of Mouse Cells Expressing
body after 3 weeks (Fig. 4A). In addition, transplantap-Gal in Zebrafish Embryos
tion of 3T3W3A
cells occasionally resulted in ectopic noMouse cell lines are extensively utilized as vehicles tochord and paired somites (N = 3/47; not included in
which can be engineered with a gene of interest. We the Table 1). Duplication of a host derived notochord
reasoned, therefore, that this system could also be use- was never observed in the embryos transplanted with
ful as a way to introduce cells to make specific gene chicken tissues or 3T3P-ga'.We also observed a duplication of the pectoral fin after transplantation of
products a t precise locations in zebrafish embryos.
cells (Fig. 4E).
We first transplanted NIH3T3 cells expressing P-gal 3T3W3A
Ultimate Location of the Heterospecific Tissue
in Zebrafish Depends on the Properties of
the Graft
During observation of induced secondary axes by
chicken HNs as above, we noticed that even though the
tissues were transplanted into the same position in the
ventral region of zebrafish embryos, their ultimate location in the host depended on the age of the donor HN
(Fig. 9). Twenty hours after the operation many of the
early HNs taken from Stage 4 of chicken embryos (36
out of 43) were found on the anterior yolk region,
whereas HNs from Stage 8 donors were located on the
tail or yolk extension region (9 out of 9). Transplanted
lateral blastoderm taken from either Stage 4 or 8
chicken embryo were located on the yolk extension region (16 out of 22). 3T3W3A
cells and 3T3P-ga’cells were
both located mainly on the yolk extension region (40
out of 43, and 16 out of 18, respectively). These results
suggest that the organizers of different developmental
stages behave differently when transplanted in the
ventral region of the zebrafish embryos. Wnt3A had no
effect on the differential location of the grafts.
Inductive Activities of Chicken Organizer
in Zebrafish
In this paper, we showed that the chicken organizer,
Hensen’s node, which showed no sign of differentiation,
induces a variety of axial structures without creating a
fish organizer. The induced structures include somites,
heart, and dorsal neural tissues. This suggests that the
formation of these tissues does not require a contribution of the organizer-derived midline tissues and may
be the consequence of inductive actions by the organizer itself.
The heterospecific combination of chicken and zebrafish may be useful for studying the inductive activity of developing tissue arrested a t a particular stage.
For example, we observed as a preliminary result that
a chicken segmental plate in the vicinity of the HN a t
Stage 8 also induced neural tissues in a similar way to
the early HN when transplanted into zebrafish (Hatta
and Takahashi, unpublished observations). This may
suggest that not only the organizer but also a somite
precursor plays a role in the formation of the dorsal
portion of the neural plate.
Involvement of Wnt Genes in Inductive Activities
of the Organizer
Transplantation of genetically engineered cells into
the zebrafish embryo seems suitable for investigating
the mechanisms of cell- and tissue-interactions during
development a t the molecular level.
In this report, we showed that 3T3W3Acells have a
higher potency in inducing neural tissues and organizing mesoderm than untransfected control cells. Although efficiency in the mesodermal tissue formation
by 3T3W3Ais lower than that by early HN, the tissues
induced by the graft of 3T3W3A
cells contained the dorsal portion of the CNS and somites in a single row.
Therefore, 3T3W3Apartially mimics a role of the organizer. In mouse, Wnt3A is expressed in embryonic mesoderm, and a null mutant of this gene was shown to
lack caudal somites, have disrupted notochord, and fail
to form a tailbud (Takada et al., 1994). This, together
with our result, suggests that Wnt3A may have a role
in organizing the mesoderm. In Xenopus, Wnt3A is expressed in a specific region of the neural tube and tail
bud but not in the early organizer region (Wolda et al.,
1993). The distribution of Wnt3A in zebrafish remains
to be studied.
In a few cases, 3T3W3A
induced a seconday notochord.
Thus, Wnt3A may have additional properties not expressed by HN under these experimental conditions.
Other than Wnt genes, several signalling molecules
have also been reported to have an important role in
mesoderm formation or neural induction. These molecules include Nodal (Zhou et al., 1993; Toyama et al.,
1995), Hepatocyte Growth Factor (Nakamura et al.,
1989; Stern et al., 1990), Follistatin (Nakamura et al.,
1990; Hemmati-Brivanlou et al., 1994), and Noggin
(Lamb et al., 1993). With the technique shown in this
study, the influences of these and other signalling molecules in the formation of a secondary axis in zebrafish
embryos may be compared.
Interference Between Ectopic and Original
Pigment Stripes
The mechanism by which how longitudinal pigment
stripes form is not known, although mutants which
affect this pigment patterning in zebrafish may permit
causal mechanisms to be elucidated (Johnson et al.,
1995). We showed that secondary axis formation leads
to an ectopic stripe formation, which is characteristic of
the dorsal stripe, traversing the operated side of the
fish body. Although the other lateral pigment stripes
were not induced, the original stripes were disrupted in
both 3-week and adult fish (Fig. 4A,C). The secondary
axis may create a positional information field which
interferes with the original field. More research is required to clarify the model and its molecular basis (see
Fig. 10).
Differential Locations of the Heterospecific
Organizers Transplanted Into
Zebrafish Embryos
Ultimate locations of the transplanted HNs are different depending on the stage of an HN. The motive
force might be generated by the fish cells which are
adjacent to the graft. The influences by the HN on
these fish cells to determine the direction they move
may depend on the stage of the grafted organizer. Alternatively, early and late HNs have a differential affinity to fish cells along A-P axis. In this case, for example, the early HN has a high affinity to the cells
which are destined to be located anteriorly whereas the
late HN has an affinity to those destined posteriorly. It
Fig. 5. Altered gene expression in the zebrafish gastrula by the
chicken Hensen's node (HN) grafts. Neural plate but not mesendodermal
derivatives of midline is induced. A,B: Expression pattern of dlx3 which
demarcates the neural plate in the ectoderm of late gastrula (10 h) was
expanded around the transplanted HN, Stage 4 (A), while the chicken
lateral blastoderm (LB), Stage 4 had no effect (6). The dotted line in A
indicates the induced secondary axis. C F : Various markers of midline
tissue precursors, or derivatives of the organizer were not induced by the
HN, Stage 4 transplant. C: The no tail (not) probe labels early mesoderm
and notochord precursors. D: The axial probe marks the zebrafish organizer and axial meso-ectoderm, from tail to the forebrain. E: The
goosecoidgene (gsc) is expressed in the zebrafish organizer early and at
10 h in prechordal plate and overlying midline ectoderm. F: The snail7
(snal) gene is expressed transiently in the early mesoderm and in the
adaxial cells, a medial portion of somites which is associated with the
notochord precursors. X: transplants, a: anterior, p: posterior, d: dorsal, v:
ventral. Bar = 100 urn.
Fig. 6. Transplanted NIH3T3 mouse cells expressing p g a l (3T3P~g”’)
in zebrafish embryos. A: Embryos (26 h) transplanted with a clump of
3T3p-g”’cells at the ventral region of the early gastrula as described in
Figure 1. The cell clusters were often found around the yolk tube. B,C:
Three-day-old a/bb%/bb4 embryos transplanted with 3T3p~9a’
after being
dissociated into single cells with trypsiniEDTA (see Experimental Procedures). Cells were transplanted into the animal pole of blastula at 5 h. B:
One of the transplanted cells was found in the ventral spinal cord, extending filopodia. Lateral view. C : A group of mouse cells were found to
be localized along the optic nerves and tracts. The dissociated 3T3P-gat
cells tend to colonize this particular region (see text). Fish lens tissues at
3 days have intrinsic enzyme activity and turn blue without any transplants. D: Transverse section of the embryo C. Bar = 50 pm.
Fig. 7. Northern blot analysis of RNA isolated from NIH3T3 cell lines
transfected with Wnf3A (W2, W4, W11) or p-galgene (pS),probed with
Wnf3A cDNA. W2, W4, W11 express Wnf3A mRNA (arrowhead), while
p5 shows no detectable expression. The patterns of total RNA are shown
in the lower panel.
has been reported that the goosecoid gene ectopically
expressed in Xenopus changes the morphogenetic
movement and propels the injected cells anteriorly
(Niehrs e t al., 1993), and in chicken goosecoid gene is
expressed in the HN at Stage 4 but not in the later HN.
Such transcriptional regulators may be controlling the
surface molecules.
Although the 3T3W3Acells partially mimicked the
inductive activities of the early HN, WnBA expression
does not seem to be involved in the mechanism which
differentiates the final localization of heterospecific organizer in zebrafish embryos. This may suggest that
factors which are responsible for this behavior of the
organizer are distinct from those for the inductive activities of the organizer.
Zebrafish Embryos
Zebrafish (Danio rerio) embryos were obtained from
natural spawnings of a colony (AB line) at the University of Oregon. Embryos were staged by hours postfertilization at 285°C (h). Homozygous albino mutants
(albb4/albb4,Streisinger et al., 19861, which lack black
pigment in the body and pigmented retina were also
utilized (e.g., in Fig. 6).
Chicken Embryos
Eggs of White Leghorn (Oregon State University
farm) were incubated at 38.5"C, and staged according
to Hamburger and Hamilton (Hamilton, 1965).
Culture and Transfection of Mouse Cells
Mouse cell lines were cultured at 37°C in Dulbecco's
modified eagle medium, buffered with NaHCO, and 5%
Fig. 8. Secondary axes induced by the NIH3T3 cell line transfected
with Wnt 3A gene (3T3W3A).A: Many sensory neurons were induced by
grafts of 3T3W3A,revealed by labeling with zn-12 at 26 h. B: Only a few
(as shown) or no sensory neurons were induced by grafts of 313@-@.
Arrowheads designate the site of the graft. Bar = 50 pm.
COz, and containing 10% fetal bovine serum and 100
U/ml penicillin-streptomycin. The plasmid, pmiw
WnBA, which contains RSV LTR and the (3-actin promoter was constructed as follow: The CAT-coding region of the pmiw CAT plasmid (Suemori et al., 1990)
was excised with Hind 111 and Hpa I and replaced by the
1.42 Kb cDNA fragment containing the entire coding
region of Wnt3A (kindly provided by Dr. H. Roelink)
(Roelink and Nusse, 1991). Mouse NIH3T3 cells were
transfected withpmiw Wnt3A together with the tkneoB
plasmid (Kato et al., 1987) by a calcium-phosphate
method. After transfection, 11independent G418-resistant clones were obtained. Thepmiw lacZ plasmid (Suemori et al., 1990) was introduced into NIH3T3 cells in
the same way as above to obtain cells expressing p-gal
activity constitutively. For Northern blot hybridization, digoxygenin-labeled Wnt3A DNA probe was prepared and signals were detected by alkaline phosphate
conjugated anti-digoxygenin antibody, followed by
LUMI-Phos detection process (Boehringer, Indianapolis, IN).
Lateral blastoderm
Fig. 9. Differential localizations of the transplanted tissues. (A) Hensen's node (HN), Stage 4, (6)HN, Stage 8 , (C) LB, (D)3T3W3A.The
arrowheads indicate position of the transplants. The embryos are at
24-28 h. Bar = 50 pm. E: Schematic drawing of differential localizations
of the transplanted tissues. Approximate position of each transplanted
tissue at about 26 h was plotted. After transplantation as described in
Figure 1, the HN, Stage 4, tends to localize near the anterior part of the
yolk, whereas the HN, Stage 8 , localizes near the tail. The chicken lateral
blastoderm, 3T3W3A,and 3T3@-ga',
in contrast, are found mostly around
the yolk extension, near the anus.
Development of the chicken embryo is much slower
than that of the zebrafish embryo under normal conditions. For example, i t requires 38 h r of incubation for
chicken embryos to develop from the blastula stage to
the 10 somite stage. In contrast, the zebrafish embryo
requires only 11 h r to do so. When chicken eggs a t H-H
Stage 4 (definitive streak stage) were incubated a t
285°C for 6 hr, they fail to develop further, whereas
Stage 4 embryos which were maintained a t 385°C for
the same duration, developed to reach Stage 6 (head
fold stage) Thus, transplanted chicken tissue remains
within a narrow range of developmental stage, with
virtually no further morphological differentiation.
Transplantation was performed at 285°C. The zebrafish embryos were manually dechorionated with
fine forceps in embryo medium (EM: 10%Hanks' solu-
tion supplemented with Ca2+ at the concentration of
the normal Hanks' solution; Westerfield, 1993). The
zebrafish embryo at early gastrula stage a t about 6 hr,
when the embryonic shield became visible, was placed
on 3% methyl cellulose/EM spread on a depression
slide, overlaid with a drop of Hanks' solution, and oriented with a hair loop so that the ventral side (opposite
to the embryonic shield) faced upwards.
Donor tissues taken either from a chicken embryo or
a sheet of mouse cells scraped off a culture dish were
cut with a sharpened tungsten needle into approximately 50-100 pm fragments in Hanks' solution. A
small incision was made with the needle in the enveloping layer (EVL) of a zebrafish embryo near the ventral margin of the blastodisc (see Fig. 1). Donor tissue
was then inserted into the deep cell layer, taking care
not to damage the yolk membrane. The depression
Fig. 10. Alternative models to explain the pigment stripe formation in
zebrafish with a secondary axis. A: The normal adult pigment stripe
pattern. A side of a fish is schematically shown. The bar filled with checkers indicates the dorsal dark golden stripe. The black bars indicate more
laterally distributed melanocyte (blue) stripes. B,C,D: The stripe patterns
with a secondary axis, predicted from three models. The vertical checkered bar indicates the secondary dorsal pigment stripe. In the model B,
the pigment cells are added on top of the original stripes. In the model C,
the dorsal stripe is the center of a diffusible morphogen. The stripes
would follow the contours of certain concentration ranges of the morphogen. In the model D, the stripe formation is based on waves of positional
information. Interferencewould cause pigment cells to localize in discrete
islands. The results shown in Figure 4A, Care partially consistent with the
model D.
slide was then placed into a Petri dish, and immersed
in EM with 100 U/ml penicillin-streptomycin. Several
hours later, after the methyl cellulose had dissipated
and the embryos had detached from the bottom of the
depression slides, they were transferred to another
Petri dish containing EM.
To transplant dissociated mouse cells, the cells on a
dish were rinsed with PBS, then incubated in 0.125%
trypsin, 1 mM ethylenediaminetetraacetic acid
(EDTA),5 mM Hepes-buffered saline (pH 7.5) for 5 min
a t 37.0"C. Digestion was ended by adding culture medium. Cells were washed a few times by centrifugation
a t 1,200 rpm, replacing of the supernate with culture
medium and pipetting, then suspended as single cells
in Hanks' solution. Cells were transplanted near the
animal pole in the zebrafish blastula (4-5 hr) with a
In Situ Hybridization
Whole-mount in situ hybridization was performed
essentially as described by Oxtoby and Jowett (1993).
The cRNA probes for krox20 (Oxtoby and Jowett,
1993), no tail (Schulte-Merker et al., 1992), snail1
(Thisse et al., 1993), axial (Strahle et al., 1993), dlx3,
mshB (Ekker et al., 1992; Akimenko et al., 19941,
goosecoid (Thisse et al., 1994) were as described previously.
Immunohistochemical detection in whole-mounted
and sectioned embryos was performed essentially as
described by Hatta (1992). Mouse monoclonal antibodies used in this study are as follows: zn-12 recognizes a
carbohydrate epitope similar or identical to HNK-1
and labels many neurons and axons at early stages
including Rohon-Beard cells (Metcalfe et al., 1990).
mz15, which recognizes keratan sulfate (Smith and
Watt, 1985) and labels the floor plate, notochord, and
otocysts in zebrafish (see Hatta, 1992). Anti-myosin
was a gift from Dr. D. Stainer.
p-Gal Enzyme Detection
To detect P-gal enzyme activity, manipulated embryos were fixed in 4% paraformaldehyde, 0.2% glutalaldehyde in PBS for 30 min a t room temperature,
washed three times in PBS containing 0.01% NP40 and
transferred to a solution containing 1.2 mM 5-bromo4-chloro-3-indolyl-~-D-galactopyranoside
(X-gal), 0.1%
Triton X-100, 1mM MgCl,, 3 mM K,[Fe(CN),l, 3 mM
K,[Fe(CN),l in PBS and incubated for several hours at
Histological Staining of Bones and Cartilages
After the skin and visceral tissues were removed,
3-week-old fish were placed in a solution of 80% ethanol and 20% acetic acid containing 0.015% Alcian blue
8GX (Sigma,St. Louis, MO) overnight, and then dehydrated in 3 changes of 100% ethanol for 24 hr. Following dehydration, embryos were placed in a solution of
0.5% potassium hydroxide containing 0.01 % alizarin
red S (Sigma) until bone staining became visible (usually for 1 to 2 hr). Embryos were then cleared in 20%
glycerol, and stored in 50% glycerol.
We thank J. Weston and C. Kimmel for their support
and comments on the manuscript. We are also grateful
to T. Jowett, U. Strahle, B. Thisse, C. Thisse, J. Wegner, and M. Westerfield for providing probes for in situ
hybridization, and to R. BreMiller, S. Johnson, M. McDowel1 for technical assistance. This work was supported by NIH grant HD22486. Y.T. was a fellow of
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expressions, developing, mexicanum, heart, norman, mutant, lethal, protein, differential, isoforms, ambystoma, axolotls, cardiaca
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