Download Cell-autonomous shift from axial to paraxial mesodermal

Development 121, 4257-4264 (1995)
Printed in Great Britain © The Company of Biologists Limited 1995
DEV4610
4257
Cell-autonomous shift from axial to paraxial mesodermal development in
zebrafish floating head mutants
M. E. Halpern1,*, C. Thisse1,†, R. K. Ho1,‡, B. Thisse1,†, B. Riggleman2, B. Trevarrow3, E. S. Weinberg4,
J. H. Postlethwait1 and C. B. Kimmel1
1Institute of Neuroscience, University of Oregon, Eugene, OR 97403-1254, USA
2Department of Genetics and Cell Biology, Washington State University, Pullman, WA99164-4234,
3Department of Biology, California Institute of Technology, Pasadena, CA 91125, USA
4Department of Biology, University of Pennsylvania, Philadelphia, PA 19104-6017, USA
USA
*Author for correspondence at current address: Department of Embryology, Carnegie Institution of Washington, Baltimore, MD 21210, USA (e-mail:
[email protected]. edu)
Current addresses: †IGBMC, BP 163, 67404 Illkirch Cedex, CU de Strasbourg, France
‡Dept. of Molecular Biology, Princeton University, Princeton, NJ 08544
SUMMARY
Zebrafish floating head mutant embryos lack notochord
and develop somitic muscle in its place. This may result
from incorrect specification of the notochord domain at
gastrulation, or from respecification of notochord progenitors to form muscle. In genetic mosaics, floating head acts
cell autonomously. Transplanted wild-type cells differentiate into notochord in mutant hosts; however, cells from
floating head mutant donors produce muscle rather than
notochord in wild-type hosts. Consistent with respecification, markers of axial mesoderm are initially expressed in
floating head mutant gastrulas, but expression does not
persist. Axial cells also inappropriately express markers of
INTRODUCTION
Commitment of cells to a particular pathway of differentiation
is thought to occur through successive specification steps,
involving the interplay of extrinsic signals with a regulated
program of gene expression. Mutational analyses in invertebrates have shown that single gene defects can disrupt appropriate cellular interactions, thereby preventing subsets of cells
from acquiring their correct developmental fates. Two wellcharacterized examples are mutations that perturb the
induction of vulval cell fates in C. elegans (reviewed in
Eisenmann and Kim, 1994), and mutations in the Drosophila
visual system that alter photoreceptor cell fates (reviewed in
Dickson and Hafen, 1993, Zipursky and Rubin, 1994).
While single gene mutations have not been well described
in vertebrates that cause a population of cells to form a
different tissue type, overexpression of regulatory genes can
confer new fates on cells in vivo and in vitro (Weintraub et al.,
1991; Lee et al., 1995). Together, genetic and molecular
studies demonstrate that misexpression of genes involved in
cellular signaling or in transcriptional control can dramatically
alter pathways of cellular differentiation.
In zebrafish, mutations at two genes encoding putative DNA
paraxial mesoderm. Thus, single cells in the mutant midline
transiently co-express genes that are normally specific to
either axial or paraxial mesoderm. Since floating head
mutants produce some floor plate in the ventral neural
tube, midline mesoderm may also retain early signaling
capabilities. Our results suggest that wild-type floating
head provides an essential step in maintaining, rather than
initiating, development of notochord-forming axial
mesoderm.
Key words: notochord, gastrulation, embryonic axis, mesoderm,
zebrafish, floating head
binding proteins disrupt notochord development in different
ways. Embryos mutant for no tail (ntl), the homolog of mouse
Brachyury, fail to form notochords and tails (Halpern et al,
1993; Schulte-Merker et al., 1994); however, cells that express
some notochord-specific genes and that are related by lineage
to notochord, appear to persist beneath the mutant neural tube
(Halpern et al., 1993; A. Melby and C. Kimmel, personal communication). Somites also exhibit morphological defects, but
for the most part are bilaterally paired in the ntl mutant trunk.
In contrast, floating head (flh) mutants have left and right
trunk somites fused together beneath the neural tube, in place
of notochord. Recent molecular studies (Talbot et al., in press)
demonstrate that flh is the zebrafish homolog of the Xenopus
Xnot gene, first identified by its homeodomain homology (von
Dassow et al., 1993; Gont et al., 1993). During Xenopus gastrulation, Xnot is expressed above the dorsal lip and later in presumptive notochord and floor plate. On the basis of its early
expression pattern and similarity to known transcription factors,
Xnot has been proposed to regulate other notochord and floor
plate-specific genes (von Dassow et al., 1993).
Among vertebrates, flh is the first example of a mutation that
causes cells in the fate map position of one tissue type to make
another tissue. Labeling of single cells has revealed that cells
4258 M. E. Halpern and others
A
A
B
B
*
C
Fig. 1. flh mutants have fused somites and discontinuous floor
plate. (A) Live flh− pharyngula (approximately 24 hours) with
fused somites in the midline. (B) Transverse section through the
trunk region of a 24 hour mutant stained with methylene blue-azure
II and basic fuchsin (Humphrey and Pittman, 1974) shows
differentiating muscle fibers across the midline. The developing
spinal cord (arrowheads), and the position where the notochord
would normally be located (asterisk), are indicated. (C) Scattered
patches of floor plate cells (arrowheads) in the trunk and tail spinal
cord of a 24 hour flh mutant are revealed by in situ hybridization
with a probe for α-collagen II, which normally is expressed in the
floor plate, notochord and hypochord (Yan et al., 1995). Scale bar,
100 µm for B and C.
C
Fig. 2. WT-derived notochord can differentiate in flh mutant hosts.
(A) Side view of a live 24 hour flh mutant that differentiated a short
stretch (8 cells) of morphologically identifiable notochord
following cell transplantation at the blastula stage. (A) Nomarski
optics and (B) the corresponding fluorescent image. (B) Notochord
cells developed from rhodamine-dextran labeled WT donor cells.
Labeled WT cells also gave rise to floor plate above the notochord
(arrow) and hypochord cells (arrowhead) beneath it. (C) A subset
of the transplanted WT cells (arrow) stained with MZ15 (Smith and
Watt, 1985), an anti-keratan sulfate monoclonal antibody specific
for notochord. MZ15 also labels floor plate and the spinal cord
central canal (Hatta, 1992). Scale bar, 20 µm for A and B;
30 µm for C.
Cell autonomy of flh in axial mesoderm 4259
A
B
C
D
E
F
H
Fig. 3. Dorsal axial transplants of flh mutant cells differentiate into floor plate and muscle instead of floor plate and notochord. Sagittal
views of 24 hour WT host embryos with (A, B) WT or (C-F) flh mutant donor cells (dark brown) derived from dorsal axial transplants. A
transplant was defined as a dorsal axial transplant if donor cells contributed to >20 floor plate cells in the ventral spinal cord of the trunk and
tail. (A,B) In the case of control experiments with WT cells, donor cells that gave rise to floor plate (arrowheads), also gave rise to
notochord cells (93% of dorsal axial transplants, n=27/29, present study and 95%, n=56/59, Halpern et al., 1993), and sometimes hypochord
cells (arrow in B). (C) flh− donor cells contributed to floor plate (arrowheads) but not notochord. (D) Instead, mutant cells formed muscle
fibers. A total of 72 blastulas were transplanted. Out of 17 WT embryos receiving flh− cells, 7 had dorsal axial transplants. Of these, mutant
donor cells gave rise to more than 50 muscle fibers in 5 embryos and approximately 35 fibers in 1 embryo. Mutant muscle fibers were often
found in the middle of the myotome, with respect to the dorsoventral axis, at the position of muscle pioneers (Felsenfeld et al., 1991).
Although flh− cells did not contribute to WT hypochord, due to the lower frequency of donor cells forming hypochord in control transplants,
more flh genetic mosaics are needed to confirm that flh− cells are unable to form hypochord. (E,F) Mutant donor cells produced muscle
fibers in newly formed somites, however, undifferentiated mesodermal cells in more posterior, and hence, developmentally younger, regions
were situated in the midline at the level of the WT notochord (arrow). (C,D, and E,F) Different focal planes of the same embryos. Scale bar
= 50 µm for A, C, D, E and F; 75 µm for B.
G
4260 M. E. Halpern and others
located within the notochord domain of the gastrula fate map
differentiate instead as muscle fibers in flh mutants (A. Melby
and C. Kimmel, personal communication). This may result
from either incorrect specification of cells within the notochord
territory, or from a failure in subsequent steps in notochord
development by cells that were initially developing correctly.
We now present evidence in favor of the latter hypothesis,
suggesting that flh axial cells autonomously respecify during
gastrulation to a paraxial mesodermal fate, that of somitic
muscle. Evidence also suggests that this shift in cell fate
involves an abnormal state, in which midline cells simultaneously express markers of both axial and paraxial mesoderm.
Thus, flh+ function is essential for early gastrula cells, initially
developing on an axial mesodermal pathway, to progress
correctly along that pathway.
MATERIALS AND METHODS
Fish
Zebrafish, Danio rerio, were maintained at 28.5°C as described
(Westerfield, 1993). All experiments were performed using the spontaneous flhn1 allele which is most likely a null mutation (Talbot et al.,
in press). Embryos were obtained following natural matings of flh/+
heterozygous fish (25% flh−; n=373/1,523). Alternatively, embryos
were obtained by early pressure treatment of eggs that had been gently
squeezed from flh/+ mothers and fertilized in vitro with u.v.-inactivated sperm (Streisinger et al., 1986). This method produces gynogenetic progeny consisting of close to 50% flh homozygous mutant
embryos (n=243/492), due to the close proximity of flh to the centromere. Embryos were sorted, staged and maintained in embryo
medium (EM; Westerfield, 1993), until they reached the desired
developmental stage. Staging criteria were followed according to
Kimmel et al. (1995).
Cell transplantation
During the blastula period, 10-20 cells were transplanted from dyelabeled (a mixture of tetramethylrhodamine dextran and lysine fixable
biotinylated-dextran, Molecular Probes) donor embryos to unlabeled
hosts as described (Ho and Kane, 1990; Halpern et al., 1993), with
one modification. As explained in the Results section, we performed
heterochronic rather than isochronic transplants. Cells were taken at
random positions from donor blastulas that were approximately 2-3
cell divisions ahead of host embryos (about 1 hour older), and transplanted to the host marginal region. Transplants were routinely carried
out between sphere stage donor embryos (4 hours postfertilization)
and 1k-cell stage (3 hours) host embryos, and always prior to doming
(4D hours) of donor embryos. At these stages, embryos can not be
sorted by phenotype; therefore, transplantations were performed with
unidentified embryos. Donors were kept alive in EM supplemented
with penicillin-streptomycin (Sigma) and their genotypes were
inferred from their phenotypes at early somite stages and confirmed
the following day.
At 24 hours, transplanted cells were visualized using a low light
level silicon-intensified camera (Videoscope) and images were
obtained using AxoVideo imaging software (Myers and Bastiani,
1991). Host embryos were fixed in 4% paraformaldehyde and
processed for detection of donor cells containing biotinylated-dextran
using a Vectastain kit (Vector Laboratories, Inc.) or for notochordspecific labeling using the monoclonal antibody MZ15 (Smith and
Watt, 1985) and standard immunocytochemical techniques
(Trevarrow et al., 1990).
Whole-mount in situ hybridization
Embryos were fixed in 4% paraformaldehyde and processed for
whole-mount in situ hybridization as described (Thisse et al., 1993).
The snail1 (Thisse et al., 1993), twist (B. Riggleman, unpublished
data), and myoD (Weinberg et al., unpublished data) RNA antisense
probes were all synthesized by T7 polymerase (Boehringer
Mannheim) from XbaI linearized templates. Approximately 50 staged
embryos were used for each hybridization and several experiments
were performed with each probe. For double-labeling, frozen sections
were prepared from embryos already probed by in situ hybridization.
Sections were incubated with anti-ZfT antiserum (Schulte-Merker et
al., 1992) at 1:20,000 in phosphate-buffered saline, pH 7.2 with 1%
BSA, 1% DMSO and 0.1% tween 20. After washing, sections were
incubated in goat anti-rabbit secondary antibody and rabbit peroxidase-anti-peroxidase (Jackson Immunoresearch Laboratories, Inc.),
washed again and stained in diaminobenzidine (Sigma).
RESULTS
Cell-autonomous action of flh in notochord
development
The original floating head allele, flhn1, is a recessive, zygotic
lethal mutation that produces embryos completely lacking
notochords (Fig. 1A). Sectioning of mutant embryos during the
pharyngula period (24-48 hours, see Kimmel et al., 1995)
confirms the absence of notochord and the presence of differentiating muscle fibers under the presumptive spinal cord, in
the region where the notochord would normally be located
(Fig. 1B). Despite their lack of notochord, flh mutants develop
floor plate in the brain and spinal cord. Floor plate is continuous from the midbrain through the hindbrain but posteriorly it
becomes discontinuous, composed of small islands of cells in
the mutant trunk and tail spinal cord (Fig. 1C).
Since flh encodes a homeodomain protein that probably
functions as a transcriptional regulator (van Dassow et al.
1993; Talbot et al., in press), flh mutations are presumed to act
cell autonomously in notochord development. To test this
hypothesis and to determine the fate of flh− axial cells, we
produced genetic mosaic embryos. Groups of cells obtained
from vital dye-labeled embryos were transplanted during the
blastula period, and later assayed for their ability to differentiate into notochord.
Cells derived from wild-type (WT) donors, transplanted into
flh mutant hosts, showed the characteristic morphology and
biochemical properties of notochord (n=6 hosts; Fig. 2). This
demonstrates that WT notochord cells can differentiate in the
flh mutant environment and supports the proposal for a cellautonomous action of flh in notochord development.
flh− donor cells form muscle rather than notochord
To assess whether flh mutant cells could contribute to
notochord in wild-type hosts, only transplants that gave rise to
axial structures were useful for the analysis. It was observed
(these studies and R. Ho, unpublished observations) that transplanting cells from donor blastulas that were approximately 23 cell divisions older than host embryos increased by four-fold
the incidence of donor cells contributing to trunk axial structures (floor plate cells, and in WT controls, notochord and
hypochord). Therefore, heterochronic transplants were
routinely performed to enrich for donor cell contribution to
axial structures. Although their position relative to the
dorsoventral axis could not be determined at the time of transplantation, we refer to these as dorsal axial transplants on the
Cell autonomy of flh in axial mesoderm 4261
basis of the derivatives that the donor cells produced (>20 floor
plate cells).
In control dorsal axial transplants (WT into WT, n=29, Fig.
3A,B), donor blastula cells that produced long stretches of floor
plate almost always contributed to notochord (93%, n=27/29).
Often such transplants also contributed to hypochord, the row
of cells beneath the notochord; however, they rarely gave rise
to somitic muscle fibers (n=5/29 with on average 4 muscle
fibers per embryo).
In contrast to WT cells, flh− donor cells (flh− into WT)
never differentiated into notochord in equivalent dorsal axial
transplants. Rather, mutant cells that contributed to spinal
cord floor plate (>20 cells, n=7/17 embryos, Fig. 3C,E),
always gave rise to muscle fibers (n=7/7; Fig. 3D,F). flh−
muscle fibers were often situated adjacent to host-derived
notochord, in the middle of the myotome with respect to the
dorsoventral axis, but they were also found at other dorsoventral positions in the myotome. The lack of donor cells in the
notochord and the production of a significant number of
muscle fibers were properties unique to flh− cells. Thus,
genetic mosaic analysis indicates that flh mutant cells behave
differently than WT cells in dorsal axial transplants and that
they exhibit a cell-autonomous tendency to form muscle
fibers.
flh+ function is not essential for floor plate
differentiation
In zebrafish embryos, the floor plate is normally present as a
continuous row of cells extending throughout the length of the
midbrain, hindbrain and spinal cord (Hatta, 1992). As shown
above (Fig. 1C), floor plate is discontinuous in the flh mutant
trunk and tail spinal cord. Interestingly, although transplanted
flh− cells never produced notochord, they contributed to spinal
cord floor plate in the trunk and tail of WT hosts (Fig. 3C,E).
This result suggests that the disrupted floor plate found in flh
mutant embryos is not due to the reduced competence of
mutant cells to form floor plate, but probably results from
defects in non-autonomous influences on floor plate development or defects in cell proliferation.
Markers of axial mesoderm are expressed in flh
mutants
The genetic mosaic data, together with results from fate
mapping analyses of flh mutants (A. Melby and C. Kimmel,
personal communication), indicate that flh− mesodermal cells
autonomously form muscle rather than notochord. In flh
mutants, cells in the notochord domain of the fate map may be
incorrectly specified, developing properties of paraxial
mesoderm rather than axial mesoderm. Alternatively, cells
may be correctly specified as axial mesoderm initially, but fail
in subsequent steps in notochord development. The first
hypothesis predicts that cells in the notochord domain of the
fate map will never express notochord-specific genes; rather,
they will express genes appropriate for a muscle fate on the
normal schedule for cells fated to be muscle. The second
hypothesis predicts that cells in the notochord region of the fate
map will initially express genes appropriate for notochord, but
will later switch to muscle-specific genes. To test these
hypotheses, we compared patterns of mesodermal gene
expression between wild-type and mutant sibling embryos.
The zebrafish homolog of Drosophila twist is expressed in
axial mesoderm which forms the presumptive notochord of
WT gastrulas (80% epiboly stage, Fig. 4A), and it is not
expressed in presomitic mesoderm. As the first few somites
develop, twist expression is down-regulated anteriorly but
remains at high levels in the more posterior, and hence developmentally younger, notochord (Fig. 4C). twist transcripts are
also found in more lateral mesodermal cells forming the
pronephric ducts (Fig. 4C).
Cells in the embryonic shield of flh gastrulas (60%
epiboly stage, data not shown), and later in the midline
mesoderm (80% epiboly, Fig. 4B), also express twist and
other markers of developing notochord (axial, sonic
hedgehog and ntl, unpublished observations and Talbot et al.,
in press). In the mutant axis, expression is not entirely
normal, with a reduction in both the number of expressing
cells and the intensity of labeling (Fig. 4B). This suggests that
the flh− defect arises earlier, prior to or at the onset of gastrulation.
By early somitogenesis, twist expression is entirely lost
throughout the mutant midline, although expression in the presumptive pronephric ducts appears normal (Fig. 4D). These
data demonstrate flh mutant midline cells express properties of
axial mesoderm, ruling out the hypothesis that midline cells are
incorrectly specified as paraxial mesoderm from the outset.
However, although twist-expressing cells are initially present
in flh mutant gastrulas, midline expression does not persist as
in WT embryos.
At the onset of gastrulation in WT embryos (50% epiboly),
snail1 is first expressed in all cells of the embryonic margin
(Thisse et al., 1993; Hammerschmidt and Nüsslein-Volhard,
1993), but transcripts rapidly clear from the most dorsal region
producing axial mesoderm (75% epiboly, Fig. 5A). Thus,
snail1 comes to be expressed in the adaxial cells, a subset of
paraxial mesodermal cells that flank the dorsal axis, in a pattern
complementary to that of twist. Normally, snail1 continues to
be expressed by paraxial mesodermal cells and excluded from
axial mesoderm (Fig. 5C).
In contrast, flh mutant gastrulas (75% epiboly) express
snail1 in the most dorsal marginal region (Fig. 5B). During
somitogenesis, in addition to those of the paraxial mesoderm,
many cells in the mutant axis produce snail1 transcripts (Fig.
5D).
Zebrafish myoD is also expressed by a subset of the paraxial
mesoderm, commencing at 60% epiboly (E. Weinberg, unpublished data). Unlike snail1, myoD is not expressed throughout
the margin, but its expression is confined to cells flanking the
axial mesoderm (Fig. 5E).
In flh mutants, the dorsal margin is at first largely devoid
of myoD expression, perhaps indicative of its initial axial
identity. Some expression can be found in scattered midline
cells (Fig. 5F), but by early somite stages, when myoD is
solely expressed by paraxial mesodermal cells in WT
embryos (Fig. 5G), many more cells in the flh− axis express
myoD (Fig. 5H). In fact, double labeling of mutant gastrulas
with antibody against Ntl protein, which labels presumptive
notochord (Schulte-Merker et al., 1992), and with snail1 (Fig.
6) or myoD probes (data not shown) demonstrates that single
cells in the flh mutant midline can simultaneously express
markers of both axial and paraxial mesodermal differentiation.
4262 M. E. Halpern and others
A
C
B
A
B
C
D
E
F
G
H
D
Fig. 4. Axial mesodermal differentiation is initiated but not
maintained in flh mutants. twist is expressed by axial mesoderm in
(A) WT and (B) flh mutant gastrulas at 80% epiboly (8.25 hours). At
the 3-somite stage (11 hours) in (C) WT and (D) mutant embryos,
twist is also expressed in the presumptive pronephric ducts. Although
twist transcripts are abundant in newly-forming WT notochord at this
stage (C), expression is entirely absent in the flh− axis (D). In B, flh
mutant embryos were overstained approximately three-fold relative
to WT sibling embryos. Overstaining of flh− embryos at early somite
stages comparable to D, failed to reveal twist transcripts in the axis.
DISCUSSION
We have examined how the zebrafish mutation flh acts at the
cellular level to disrupt notochord development, through the
analysis of genetic mosaics and patterns of mesodermal gene
expression.
flh− midline mesodermal cells autonomously change
their fate
Genetic mosaics were produced by heterochronic transplantation of uncommitted blastula cells (Ho and Kimmel, 1993).
Transplanting slightly older WT or mutant donor cells to the
margins of younger hosts increases the frequency with which
they contribute to dorsal axial structures. The mechanism
underlying this phenomenon is currently under investigation
(R. Ho, unpublished observations).
Mosaic analysis confirms that flh, which encodes a homeodomain protein (Talbot et al., in press), acts autonomously in
axial mesoderm. Because groups of WT cells can develop into
notochord in flh− hosts, the flh mutant environment provides
all extracellular signals necessary for notochord patterning and
differentiation. Reciprocally, flh mutant cells transplanted into
WT hosts fail to form notochord, indicating that flh− cells are
incapable of responding to the same environmental cues.
Instead of forming notochord in a WT host, flh mutant cells
differentiate into muscle fibers. In contrast, dorsal axial trans-
Fig. 5. flh− axial cells express markers of paraxial mesoderm. At
75% epiboly (8 hours) in (A) WT embryos, snail1 is expressed in
the marginal region and in mesodermal cells flanking the dorsal
axis, and in (B) flh mutants, snail1 is also ectopically expressed by
cells in the axis. At the 3-somite stage (11 hours) in (C) WT
embryos, expression persists in paraxial mesoderm and also in (D)
axial cells in flh mutants. Zebrafish myoD is only expressed in
paraxial mesodermal cells flanking the dorsal axis and not
throughout the margin in 60% epiboly (E) WT embryos. Dorsal
expression is mostly absent, however, a small number of cells in
the (F) flh mutant axis (60% epiboly) contain myoD transcripts. By
early somitogenesis (3-5 somites), when myoD is strongly
expressed in (G) WT paraxial mesoderm, many more cells in (H)
the flh mutant axis also express myoD. In other strongly stained
preparations, myo D expression was also found more laterally in
the developing somites (data not shown).
Cell autonomy of flh in axial mesoderm 4263
A
B
C
Fig. 6. Single cells in the flh mutant midline co-express axial and
paraxial mesodermal genes. Transverse sections (7 µm) of 2-somite
stage (10J hours) (A) WT and (B,C) flh mutant embryos double
labeled for Ntl protein and snail1 RNA expression. (A) In the WT
axis, nuclei of presumptive notochord cells express Ntl (arrowheads),
while snail1 expression is confined to the paraxial presomitic
mesoderm. (B,C) Single cells (arrowheads) in the flh mutant axis
express Ntl protein (brown nucleus) and snail1 transcripts (blue
cytoplasm). (B) A more rostral section through the same embryo as
C. Other cells expressing Ntl protein in B and C (arrows), constitute
an epithelium surrounding Kupffer’s vesicle (see Laale, 1985), an
axial structure at the tip of the newly forming notochord in WT
embryos (data not shown). Although axial mesodermal cells express
paraxial mesodermal genes in flh mutants, cells lining Kupffer’s
vesicle only express Ntl protein as normal. Scale bar,
50 µm.
plants of no tail mutant cells into WT embryos do not give rise
to muscle, even though the ntl mutation blocks notochord
development cell autonomously (Halpern et al., 1993), as does
flh. This suggests that flh− (but not ntl−), cells actively
respecify, to produce muscle rather than notochord.
Discontinuous floor plate may result from defective
midline mesoderm
Xnot, the Xenopus homolog of flh is expressed in cellular precursors of the notochord and neural tube floor plate (von
Dassow et al., 1993; Gont et al., 1993). In zebrafish, flh is
expressed in corresponding cells of the axial hypoblast and
epiblast cell layers (Talbot et al., in press). Although flh mutant
embryos exhibit disruptions in spinal cord floor plate, it is
unclear if this is a direct consequence of lack of flh+ function
in floor plate precursors. Disrupted floor plate can not be due
to partial function of flh, since flhn1 is a frameshift mutation
producing a truncated polypeptide, and is most likely a null
allele (Talbot et al., in press). Moreover, embryos homozygous
for flhb327, a deletion encompassing the entire flh region
(Talbot et al., in press), also develop discontinuous spinal cord
floor plate (M. Halpern, unpublished observations).
flh− cells transplanted into WT embryos, form large stretches
of trunk and tail floor plate, typically interspersed with WT
host floor plate cells. This suggests that mutant and WT cells
are equally competent to differentiate into floor plate. It is
possible that, in genetic mosaics, WT notochord (and/or floor
plate) precursors provide non-autonomous signals for specification of flh− floor plate. We propose that similar signals are
also present early in flh mutants, accounting for the partial
formation of floor plate in the spinal cord; however, as midline
mesoderm is altered, floor plate signaling also may be
disrupted. Therefore, a cell-autonomous role for flh+ in floor
plate development remains to be demonstrated.
flh as a regulator of mesodermal cell fate
Our data indicate that axial mesodermal cells lacking flh
function differentiate into muscle rather than notochord. This
shift in cell fate is evident at gastrulation, when dorsal midline
cells express genes normally expressed by paraxial mesoderm
and fail to maintain expression of markers of axial mesoderm.
Since expression of axial markers is altered at the earliest
detectable times, it is possible that flh mutations affect mesodermal specification prior to or at the onset of gastrulation.
It has been argued previously that thresholds of growth
factors may be important in specifying different types of
mesoderm, with more dorsal derivatives forming at higher concentrations (Green et al., 1992; Gurdon et al., 1994). flh is a
candidate for a gene expressed in response to particular growth
factor levels required for notochord differentiation. Without flh
function, the response may be altered so that cells become
muscle by default. This model is consistent with the behavior
of flh− cells in mosaic embryos; however, it is inconsistent with
their initial entry into the axial mesodermal pathway of development in mutant gastrulas.
Another possible explanation for the mutant phenotype is
that flh regulates the timing of midline expression of axial and
paraxial mesodermal genes. In mutants, snail1 expression
persists in the dorsal midline at an inappropriate stage, while
axial twist expression is lost prior to when it would normally
be down-regulated in WT presumptive notochord. A result of
4264 M. E. Halpern and others
altered temporal regulation of gene expression is the transient
presence of transcripts of both axial and paraxial mesodermal
genes in dorsal midline cells. In some cases, such as with snail1
and ntl, coexpression is reminiscent of that found in all mesodermal cells in the WT margin (Schulte-Merker et al., 1992;
Thisse et al., 1993; Hammerschmidt and Nüsslein-Volhard,
1993), possibly prior to the segregation of distinct notochord
and muscle cell lineages. Although an hypothesis solely based
on temporal control of gene expression can explain some of
our findings, it does not account for the nearly normal early
expression of myoD, or the abnormal early expression of twist,
found in flh mutants. Our data suggest that flh mutations alter
spatial patterns of gene expression in a dynamic manner that
is consistent with the changing fate of notochord progenitors
(A. Melby and C. Kimmel, personal communication).
The demonstration that twist and no tail (Talbot et al., in
press) are first expressed in the flh− axis suggests that mutant
cells not only enter the dorsal mesodermal pathway but that
they also are initially specified as notochord precursors, for in
WT embryos, axial mesodermal cells expressing these genes
go on to develop notochord specifically. However, this does
not imply that the flh gene normally functions to specify the
notochord lineage or that it is sufficient for notochord differentiation. Indeed, expression of notochord-specific markers in
the flh mutant argues against this idea. For this reason, we favor
the hypothesis that flh+ normally functions in axial cells to
preserve their notochordal identity, and also to repress
expression of paraxial mesoderm-specific and muscle-specific
genes. Molecular analyses will determine whether twist, snail1,
myoD or other mesodermally expressed genes are targets of
flh+ regulation.
This work was supported by a MRC of Canada Centennial Fellowship (M. E. H.), a Helen Hay Whitney post-doctoral fellowship
(R. K. H.) an EMBO fellowship (C. T.), a Fogarty Fellowship (B.
Thisse), a Royal Society Visiting Fellowship (B. Trevarrow), and by
the Institut National de la Santé et de la Recherche Médicale, the
CNRS and the Centre Hospitalier Universitaire Régional (C. and B.
Thisse), and by NIH grants HD22486 (C. B. K. and J. H. P.),
1RO1AI26734 and 1RO1RR10715 (J. H. P.). We thank Y.-L. Yan for
generously providing probe prior to publication, S. Schulte-Merker
for antibody, T. Jowett, C. Walker, E. Melancon and S. Amacher for
helpful input, and W. Talbot, A. E. Melby, D. Kimelman and J. Eisen
for valuable comments on the manuscript. R. BreMiller provided
expert technical assistance with histology and C. Norman, W. Kupiec,
and C. Jewell with Fig. preparation. We also thank E. Lawson for help
in maintaining mutant fish lines.
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(Accepted 11 September 1995)