1 Centre for Developmental Genetics, Department of Biomedical Science,
University of Sheffield, Sheffield S10 2TN, UK
2 Beckman Institute 139-74, California Institute of Technology, Pasadena, CA
91125, USA
3 Center for Advanced Biotechnology and Medicine, UMDNJ-Robert Wood Johnson
Medical School, 679 Hoes Lane, Piscataway, NJ 08854, USA
Author for correspondence (e-mail:
m.placzek{at}sheffield.ac.uk)
Accepted 19 June 2003
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SUMMARY |
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Key words: Floor plate, Prechordal mesoderm, Chick, Nodal, Sonic hedgehog
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Introduction |
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Although the role of the floor plate in patterning the neural tube is well
accepted, its ontogeny has been a controversial subject
(Le Douarin and Halpern, 2000;
Placzek et al., 2000
). The
prevailing view, for which there is extensive evidence in the chick, is that
axial mesodermal notochord cells are the source of an instructive inducing
signal that mediates floor plate differentiation in medial cells of the
overlying neural plate (Jessell,
2000
; Placzek et al.,
2000
). A large body of evidence, moreover, suggests that the
secreted signalling molecule Sonic hedgehog (Shh) mediates the ability of
notochord to induce floor plate differentiation. Shh is expressed in the node
and the notochord prior to floor-plate differentiation. Gain-of-function
experiments show that Shh can induce the ectopic differentiation of
floor-plate cells in the neural plate in vitro
(Marti et al., 1995
;
Roelink et al., 1994
), while
blockade of Shh in the notochord eliminates its ability to induce floor plate
cells (Ericson et al., 1996
).
In support of studies in the chick, mutations in the Shh gene, and in
components of the Shh signalling pathway, in mouse, block ventral midline
differentiation (Chiang et al.,
1996
; Ding et al.,
1998
; Matise et al.,
1998
; Wijgerde et al.,
2002
).
Several recent studies, however, have questioned this model of floor plate
induction. In particular, analysis of chick-quail chimaeras in which quail
cells from the chordoneural hinge (CNH), a derivative of the Node, are grafted
into chick embryos, have led to the proposal that medial floor plate cells are
derived from a population of precursors that are initially situated in the
Node, can segregate into either notochord or floor plate, and are already
pre-specified within this region (Le
Douarin and Halpern, 2000;
Teillet et al., 1998
). In this
model, floor-plate cells thus derive from pre-specified cells that intercalate
from the node into the neural midline.
A further challenge to the paradigm of notochord/Shh-mediated floor plate
induction arises through observations of zebrafish embryos. Floor-plate cells
persist in embryos in which notochord precursors are surgically ablated,
demonstrating that a normally developed notochord is not a pre-requisite for
floor plate differentiation in this species
(Shih and Fraser, 1995).
Analyses of zebrafish mutant embryos further supports this contention.
Mutations in both no tail (ntl) and floating head
(flh) affect notochord formation
(Amacher and Kimmel, 1998
;
Halpern et al., 1993
;
Schulte Merker et al., 1994
;
Talbot et al., 1995
). Despite
this, in ntl mutant embryos the most medial set of floor plate cells
are present, and even expanded (Halpern et
al., 1997
; Odenthal et al.,
1996
; Odenthal et al.,
2000
). flh mutants likewise contain patches of cells at
the ventral midline that express medial floor-plate markers
(Halpern et al., 1995
;
Halpern et al., 1997
;
Schier et al., 1997
).
Moreover, while there is compelling evidence for a requirement for Shh
signalling in the induction of floor plate character in amniotes, in zebrafish
embryos Hh signalling appears crucial to the induction of lateral floor plate
cells, but is not required for the differentiation of medial floor-plate cells
(Chen et al., 2001
;
Etheridge et al., 2001
;
Odenthal et al., 2000
;
Schauerte et al., 1998
;
Varga et al., 2001
). Instead,
the TGFß superfamily member Nodal appears essential for medial floor
plate induction. Mutations in the zebrafish nodal-related gene
cyclops (ndr2) and one-eyed pinhead (oep),
an obligate co-factor for Nodal signal transduction
(Gritsman et al., 1999
) both
cause loss of the medial floor plate throughout the length of the neural tube
(Hatta, 1992
;
Hatta et al., 1991
;
Krauss et al., 1993
;
Rebagliati et al., 1998
;
Sampath et al., 1998
;
Schier et al., 1997
;
Shinya et al., 1999
;
Strahle et al., 1997
;
Zhang et al., 1998
).
Intriguingly, while analysis of the requirement for Nodal signalling in
zebrafish has suggested that medial floor plate specification occurs early in
development and within the organiser region, evidence from the analysis of
oep mutants suggests that it is nevertheless the result of an
inductive interaction (Gritsman et al.,
1999
; Strahle et al.,
1997
).
These extensive analyses of floor plate development appear to point towards
very disparate models of floor plate formation in distinct species. However, a
caveat, and possible explanation for the varied conclusions of these studies
is their failure to analyse floor plate development at equivalent stages of
embryogenesis. To perform a more direct comparative analysis, we have examined
the differentiation of floor-plate cells in the early chick embryo, over the
period of gastrulation/early neural plate formation. Our studies show that
many medial floor plate cells that form at this time do not derive from
Hensen's node itself. Instead, they derive from a region of the prenodal
epiblast that lies anterior to Hensen's node, previously shown to contribute
to the floor plate, and designated `area a'
(Garcia-Martinez et al., 1993;
Schoenwolf et al., 1989
;
Schoenwolf and Sheard, 1990
).
Real-time lineage analyses reveal that `area a'-derived cells exist as a
separate population of floor-plate precursors and do not contribute progeny to
Hensen's node, arguing that the shared origins with notochord cells are not a
requirement for floor-plate development along the length of the neuraxis. Our
studies show that `area a' floor-plate precursors are induced by rapid
signalling events mediated by the early forming prechordal mesendoderm.
Together, our evidence shows that floor-plate cells along the neuraxis are
induced to differentiate and argues against a requirement for
pre-specification within the organiser.
In addition, we observe that Shh and Nr1 are co-expressed in the nascent prechordal mesoderm at the time of `area a' differentiation, suggesting that Nodal signalling may play a role in amniote floor plate induction. In support of this, we find that Nodal and Shh can co-operate to induce floor plate character in `area a' cells in vitro. The data presented in this study thus indicate that different signalling events mediate early and late floor plate induction in the chick and support the development of an integrated model of floor plate differentiation in both amniote and anamniote embryos.
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Materials and methods |
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For time-lapse analysis of cell movement, embryos were then cultured in
plastic culture dishes over thin albumen in a culture dish in which the
central plastic area had been replaced with a thin glass coverslip to
facilitate visualisation. Embryos were visualised using an inverted confocal
microscope as previously described (Kulesa
and Fraser, 1998). The microscope was surrounded with an
insulating chamber maintained at 38°C for the duration of the time-lapse
experiment. Single confocal images were taken at 5 or 10 minute intervals for
the duration of the analysis.
Tissue dissection and explant culture
All embryos were staged and dissected in cold L15 medium (Gibco-BRL). `Area
a' explants were prepared from HH stage 4 embryos by making two parallel cuts
either side of and anterior to Hensen's node, followed by two cuts at right
angles to remove a square of tissue from the region anterior to Hensen's node.
The epiblast layer was then isolated from underlying tissue with dispase (1
mg/ml). Explants of `area a'-derived tissue at HH stages 4+, 5 and 6 were
isolated by taking an equivalent area of tissue just anterior to Hensen's
node. In all cases, explant culture was performed in collagen gels according
to published techniques (Placzek and Dale,
1999).
Nodal protein, produced by transient transfection of 293T cells with pcDNA3-mNodal (containing the coding sequence for mouse Nodal) was concentrated tenfold using Centri-plus columns (Amicon) and then diluted 1:10 in explant culture medium. Human Shh-N protein (Biogen) was added to the tissue culture medium at the concentrations indicated.
For tissue recombination experiments, HH stage 4+ prechordal mesendoderm
was identified by morphology. Explants were prepared by making cuts either
side of Hensen's node and at the anterior and posterior limits of the
prechordal mesendoderm using sharpened tungsten needles prior to separation of
the tissues using 1 mg/ml dispase. Intermediate neural plate tissue from E9.5
rat embryos was isolated as previously described
(Placzek et al., 1993).
Prechordal mesendoderm was placed in contact with either `area a' or rat
neural plate explants in collagen gels and cultured as previously described
(Placzek and Dale, 1999
).
In vivo grafting of notochord and prechordal mesoderm
In vivo grafting experiments were performed as previously described
(Placzek et al., 1990).
Briefly, a small incision was made between the open neural groove and adjacent
presomitic mesoderm in the caudal region of HH stage 10 chick embryos in ovo.
Explants of notochord taken from the caudal region of HH stage 10 embryos or
nascent prechordal mesendoderm from HH stage 4+ embryos were inserted into the
incision adjacent to the neural plate at an intermediate position, in between
basal and alar plates. After operations were performed eggs were resealed and
incubated until HH stage 19-21 prior to fixation and analysis by
immunohistochemistry.
Prechordal mesoderm ablations
HH stage 4, 4+ or 5-embryos were prepared for New culture, leaving the
ventral surface of the embryo exposed. Removal of the prechordal mesendoderm
was performed by making a shallow cut through the endodermal and mesodermal
layers just anterior to Hensen's node and then scraping away the mesendoderm
anterior to it. Following operation, embryos were prepared for New culture as
described (New, 1955;
Stern and Ireland, 1981
) and
allowed to develop prior to fixation and further analysis.
Immunohistochemistry
Embryos and explants were analysed by immunohistochemistry according to
standard techniques (Placzek et al.,
1993). The following antibodies were used (dilutions in
parentheses): 68.5E1, anti-Shh mAb (1:50)
(Ericson et al., 1996
); 4C7,
anti-HNF3ß mAb (1:40) (Ruiz i Altaba
et al., 1995
); anti-Sox2 pAb (1:500)
(Pevny et al., 1998
);
anti-Lim1/2 (1:50); and anti-Not 1 (1:50). Appropriate secondary antibodies
(Jackson Immunoresearch) were conjugated to Cy3.
In situ hybridisation
Embryos and explants were processed for in situ hybridisation as described
previously (Vesque et al.,
2000). The following template DNAs were to generate a digoxigenin
labelled antisense RNA probes: plasmid pCM21 containing a cDNA encoding chick
Netrin 1 was linearised with EcoRI and transcribed with T7
polymerase; plasmid pcvhh containing a cDNA encoding chick sonic hedgehog was
linearised with SalI and transcribed with SP6 polymerase; plasmid
pcp7 containing a cDNA encoding HNF3ß was linearised with
HindIII and transcribed with SP6 polymerase; plasmid pcGsc containing
a cDNA encoding chick Goosecoid was linearised with EcoRI and
transcribed with SP6 polymerase (Vesque et
al., 2000
).
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Results |
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To confirm that `area a' cells colonise the midline, we examined whether `area a'-derived cells express the ventral midline cell marker Shh. DiI was injected into `area a' cells in vivo at HH stage 4, and embryos developed in ovo until HH stage 8. Examination of sections confirmed that DiI-labelled cells were confined to the ventral midline floor plate and revealed that they populated medial-most floor plate cells (Fig. 1K-M). `Area a'-labelled cells were never detected within axial mesoderm. These analyses also indicated that `area a' cells contributed largely to anterior ventral midline regions extending from the diencephalon, through the midbrain and hindbrain, with only sporadic labelling detected more posteriorly. Labelled cells were never detected anteriorly within the telencephalon (n=10).
These results demonstrate that cells from the midline, prenodal region of the chick epiblast rapidly populate the midline of the developing neural tube during gastrulation, extending long cellular processes as they migrate. In addition, they demonstrate that `area a'-derived cells largely populate medial-most ventral midline cells that form in the anterior neural tube.
`Area a'- and Hensen's node-derived cells do not mix during early
floor plate formation
We next addressed whether, in addition to populating the ventral midline,
`area a' cells contribute to Hensen's node to form a population of floor plate
precursors within this structure.
Focal injections of DiI and DiD were made into the epiblast layer at HH stage 4 (n=5), and the embryos were followed for a 4 hour period, until they reached the equivalent of HH stage 6 (Fig. 2; see Movie 2 at http://dev.biologists.org/supplemental/). DiI was used to label cells in `area a' (red labelling in Fig. 2). DiD was used to label cells in the superficial, epiblast layer of Hensen's node (blue labelling in Fig. 2); this also served to mark the position of Hensen's node. During time-lapse confocal imaging analyses, DiI-labelled cells from `area a' were again observed to rapidly move both anteriorly and posteriorly to populate the midline of the embryo (Fig. 2B-H). However, at no point in this analysis were cells from `area a' observed to colonise Hensen's node itself.
|
Together, these analyses indicate firstly that `area a'-derived cells remain separate from Hensen's node as neurulation proceeds and do not contribute at this time to any population of floor plate precursors residing in Hensen's node. Second, it suggests that the principal contributor to the earliest forming, and most anterior ventral midline is `area a' and not Hensen's node, with Node-derived cells only contributing to the later forming floor plate.
`Area a' cells become progressively specified as floor plate
following axial mesoderm formation
The majority of studies in amniotes showing the induction of floor plate by
notochord have examined the differentiation of floor-plate cells that form in
thoracic regions of the neuraxis (Artinger
and Bronner-Fraser, 1993;
Placzek et al., 2000
;
van Straaten and Hekking,
1991
; Yamada et al.,
1991
) and have not examined the induction of the anterior, `area
a'-derived population. We therefore next addressed the mechanisms by which
`area a'-derived cells differentiate into floor plate and examined whether
these differ from those which generate floor plate in the posterior neural
tube.
We first addressed the state of specification of `area a'-derived cells as
development proceeds. Explants of `area a'
(Fig. 3A), or its derivatives,
were cultured and examined for expression of markers of floor plate and neural
character. Despite being adjacent to Shh-expressing cells in Hensen's node
(Fig. 3A, arrowhead), cells
explanted from `area a' at HH stage 4 do not express the floor-plate markers
Shh, Hnf3b/Foxa2 or Netrin1
(Fig. 3B-D), indicating that at
this stage they are not specified to become floor plate. Expression of floor
plate markers was not observed at any time point analysed (20, 24, 36, 40
hours; n>50). Analysis of Sox2, a marker of undifferentiated
neuroepithelial cells (Streit et al.,
1997) indicates that `area a' cells are specified as neural at the
time of isolation (Fig. 3E).
Thus, prior to the overt formation of axial mesoderm, floor plate precursors
in `area a' exist as committed neural precursors.
|
Emerging mesendoderm rapidly induces `area a' cells to a floor-plate
fate
The specification of `area a' cells to a floor-plate identity coincides
with the onset of axial mesoderm formation, raising the possibility that the
first emerging axial mesoderm cells are responsible for inducing `area a'
cells to a floor-plate fate. To test this, we removed the earliest forming
axial mesendoderm, together with the deep layers of Hensen's node at HH stage
4 (Fig. 4A,B; n=8).
Previous studies have indicated that these layers contribute to axial mesoderm
and not to neural tissue (Selleck and
Stern, 1991).
|
We next determined whether `area a'-derived floor plate cells require a prolonged period of contact with early emerging mesendoderm for their differentiation, by performing ablations at HH stage 4+. Embryos could be staged precisely, as when endoderm was removed at HH stage 4+, a fan of axial mesendoderm could be seen extending from Hensen's node (Fig. 4C, white arrowhead). After removal of this mesendoderm, and culture for 18 hours, embryos again appeared morphologically normal, although development was retarded to HH stage 7-8 (Fig. 4F; n=5). In situ analysis revealed that Shh was expressed within anterior ventral midline cells (Fig. 4F,I), although far fewer Shh-expressing cells were detected in the neural midline than were present in control embryo, with expression of Shh on these cells much weaker than on control floor-plate cells (compare Fig. 4F,I with Fig. 4E,H).
Together these analyses suggest that the early emerging mesendoderm is crucial for the normal differentiation of the anterior floor plate. In addition, they suggest that a short exposure to this mesendodermal population is sufficient to begin to induce the differentiation of `area a' cells to a floor-plate identity.
Rescue of anterior floor plate differentiation by early exposure to
prechordal mesoderm
The ablation of emerging mesendoderm at HH stage 4 and 4+ removes both
prechordal mesendoderm and notochord progenitor cells. To distinguish which of
these two cell types might be responsible for the induction of `area a' cells
to a floor plate fate, we performed ablations on slightly older, HH stage
5 embryos, and ablated only the early notochord, leaving anterior-most
prechordal mesendoderm cells intact (Fig.
5A-D).
|
These analyses show that early exposure to the prechordal mesoderm can rescue anterior floor-plate cells and reveal that the notochord is not required for their differentiation.
HH stage 4+ prechordal mesoderm is a potent inducer of floor-plate
character
Our studies indicate that prechordal mesoderm is required to rapidly induce
`area a' cells to a floor-plate fate. We next determined whether it is
sufficient to induce their differentiation, by performing in vitro
recombinations of HH stage 4+ prechordal mesendoderm with intermediate neural
tissue from E9.5 rat embryos or with `area a' from HH stage 4 chick embryos
(Fig. 6A). Prechordal mesoderm
induced mature floor-plate cells within rat neural plate, as assessed by the
expression of the floor plate marker, FP3
(Fig. 6B)
(Placzek et al., 1993).
Similarly, when HH stage 4+ quail prechordal mesoderm was recombined with
`area a', expression of HNF3ß and Shh, but not markers of axial mesoderm,
were induced (Fig. 6C,D and not
shown). Thus, HH stage 4+ prechordal mesendoderm is able to induce floor-plate
cells in vitro.
|
Co-operation between Nodal and Shh signalling promotes floor plate
differentiation in `area a'-derived cells
Given the requirement for prechordal mesoderm in anterior floor plate
induction, we assessed the early axial mesodermal expression of Shh
and Nodal, the factors most strongly implicated in floor plate
induction in amniote and anamniote embryos respectively. In situ hybridisation
at HH stage 4 reveals that neither Shh or Nr1 is expressed
in tissues underlying `area a' at this stage of development
(Fig. 7A,B). Coincident with
the appearance of the nascent prechordal mesoderm at HH stage 4+, however,
both Shh and Nr1 are expressed in prechordal mesoderm cells
as they pass beneath `area a' (Fig.
7C,D). In notochord cells that follow immediately behind,
expression of Nr1 is completely absent while Shh is
expressed only very weakly in a subset of cells
(Fig. 7E,F). Thus, when `area
a' cells are being specified to a floor-plate fate, co-expression of
Shh and Nodal is detected in the prechordal mesoderm cells
lying directly underneath them. Subsequent to their transient exposure to
Shh/Nr1-expressing prechordal mesoderm, `area a'-derived cells
themselves begin to express Shh, while underlying notochord cells
express Shh at barely detectable levels. Given our observation that
prechordal mesoderm can rapidly specify `area a' cells to a floor-plate fate
we therefore tested the ability of both Shh and Nodal to specify `area a'
cells to a floor plate fate in vitro.
|
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Discussion |
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`Area a' and Hensen's node-derived floor plate cells: discrete
populations of ventral midline cells
Previous fate-mapping studies have shown that in the HH stage 4 chick
embryo, epiblast cells in `area a' contribute to the floor plate
(Schoenwolf and Sheard, 1990).
However, these studies did not analyse whether, prior to populating the
midline, `area a' cells might transiently populate Hensen's node. Our in vivo
time lapse analyses reveal no evidence for this possibility: we do not observe
that `area a' cells enter the node. Previous lineage analyses have shown that,
prior to HH stage 4, Hensen's node cells do not give rise to floor plate,
suggesting in turn that `area a'-derived cells do not themselves migrate out
of Hensen's node (Selleck and Stern,
1991
; Lopez-Sanchez et al.,
2001
). Together, these results suggest that `area a' and Hensen's
node cells are distinct populations. Importantly, this separation demonstrates
that a shared lineage between notochord and floor plate cells is not a
prerequisite for floor plate differentiation.
Our real-time analysis of cell movement also reveals that, although floor
plate precursors are actually present in the node at HH stage 4, they do not
migrate out until HH stage 6 (see also
Selleck and Stern, 1991;
Lopez-Sanchez et al., 2001
).
Together these studies suggest the existence of two early populations of
floor-plate precursors in the chick, one in the prenodal epiblast (`area a'),
which gives rise exclusively to cells of the neural midline, principally in
anterior regions, and one in the epiblast layer of Hensen's node, the
descendants of which leave the Node only after HH stage 6 and are later found
in the more posterior ventral midline. Our observation that cells in `area a'
form an earlier floor plate population than cells in the Node is supported by
previous studies in the chick
(Lopez-Sanchez et al., 2001
)
and raises the possibility that `area a' cells, or their progenitors, exist as
a specialised population prior to formation of the organiser. In support of
this, even before primitive streak formation, the midline of the epiblast
exhibits specialised properties, and itself undergoes powerful anterior
extension movements (Kelly et al.,
2002
; Lawson and Schoenwolf,
2001
).
Prechordal mesoderm induces `area a' cells
Our analyses show that `area a' cells are induced to a floor-plate identity
between HH stage 4 and 4+, and suggest that the nascent prechordal mesoderm
mediates this induction. Ablation of the mesendoderm as it forms in the deep
layers of Hensen's node leads to the loss of the entire floor plate at early
stages of development, including floor-plate cells that normally arise from
`area a'. This ablation removes both prechordal mesoderm and notochord
precursor cells; thus, in principle, either of these could contribute to the
induction of `area a' cells to a floor plate identity. However, a number of
lines of evidence suggest that prechordal mesoderm, and not notochord cells,
are responsible for `area a' induction. First, anterior floor plate cells form
normally in embryos in which prechordal mesoderm is present, but notochord is
absent. The differentiation of these cells is dependent upon only a very short
exposure to the prechordal mesoderm: in embryos in which prechordal mesoderm
is eliminated after only a short exposure to `area a' cells, early floor-plate
cells still form, albeit fewer in number and expressing lower levels of Shh
than normal. It is likely that homeogenetic lateral induction mediated by
these early specified cells accounts for the complete rescue of the anterior
floor plate in the absence of notochord signals
(Placzek et al., 1993).
Second, in an ectopic situation the prechordal mesendoderm can induce floor
plate in neural tissue with marked potency, supporting the assertion that in
vivo, prechordal mesoderm mediates the rapid induction of `area a' cells.
Finally, we find that the chick nodal homologue Nr1 is
co-expressed with Shh in the nascent prechordal mesoderm at the time
at which this tissue is required for the rapid induction of floor plate
character in `area a' cells. By contrast, nascent notochord cells do not
express Nr1 and barely express Shh.
A role for Nodal signalling in chick floor plate induction: parallels
between amniote and anamniote floor plate differentiation
Many lines of evidence have suggested that in zebrafish, Nodal signalling
is required for medial floor plate formation early in development. Both
cyc and oep mutant phenotypes include a loss of medial floor
plate cells (Hatta, 1992;
Hatta et al., 1991
;
Krauss et al., 1993
;
Rebagliati et al., 1998
;
Sampath et al., 1998
;
Schier et al., 1997
;
Shinya et al., 1999
;
Strahle et al., 1997
;
Zhang et al., 1998
).
Importantly, the cell-autonomous requirement for oep indicates that
the formation of the medial floor plate occurs as the result of an inductive
interaction (Gritsman et al.,
1999
; Strahle et al.,
1997
). In addition to loss of the medial floor plate, both
cyc and oep have defects in prechordal plate formation, in
the case of oep a complete loss of this tissue
(Schier et al., 1997
). This
correlation may suggest that the prechordal plate is in fact the source of a
floor plate-inducing Nodal signal during gastrulation, a possibility supported
by the fact that rescue of the floor-plate phenotype in cyc mutants
requires the presence of wild-type cells within the prechordal plate
(Sampath et al., 1998
).
Our analyses provide a first indication that Nodal may play a role also in
chick floor-plate induction: Nodal can cooperate with low levels of Shh to
induce floor plate character in `area a' cells. Studies of the zebrafish have
suggested that Nodal can induce Shh expression within the neural
tube, providing a potential mechanism of cooperation
(Muller et al., 2000). Whether
such a cooperation does in fact operate in vivo in the chick remains unclear,
but is indicated through studies of mouse embryos: both Shh-null mice
and mice that are conditionally mutant for Smad2, a downstream
effector of Nodal signalling, lose Shh-expressing cells in the
anterior neuraxis (Chiang et al.,
1996
; Heyer et al.,
1999
). Taken together, these data are suggestive of a cooperative
role for Shh and Nodal signalling during floor plate formation in amniote
embryos, potentially via Shh activation.
A dual model for floor-plate induction
Our studies, together with earlier work
(Artinger and Bronner, 1993;
Placzek et al., 2000
;
van Straaten and Hekking,
1991
; Yamada et al.,
1991
) suggest that floor-plate cells are induced by two different
mechanisms along the anteroposterior axis of the chick embryo. Floor-plate
cells that derive from `area a' arise during gastrulation and largely populate
anterior regions of the neuraxis. Our studies show that `area a'-derived floor
plate cells are induced through a rapid interaction mediated by prechordal
mesoderm that may involve a previously unrecognised role for Nodal signalling
in an amniote embryo. By contrast, floor-plate cells that differentiate in the
neurula stage embryo, and occupy posterior regions of the neuraxis, require a
prolonged period of contact with underlying notochord
(Fig. 8). Studies in both mouse
and zebrafish embryos support the idea that distinct mechanisms operate to
specify the floor plate in anterior and posterior regions. Distinct cis-acting
regulatory sequences have been identified within the mouse Shh promoter that
direct Shh expression to specific regions of the neural tube, supporting the
view that multiple genes are involved in activating Shh transcription along
the length of the CNS (Epstein et al.,
1999
). In zebrafish flh and ntl/spt double
mutants, the anterior floor plate develops normally but the posterior floor
plate is severely affected (Amacher et al.,
2002
; Halpern, 1995; Schier et
al., 1997
). Similarly, while ntl acts a partial
suppressor of the oep or cyc phenotypes, rescue is observed
only posteriorly (Halpern et al.,
1997
; Schier et al.,
1997
; Strahle et al.,
1997
).
|
Our observations raise the question of why the floor plate should arise in
this dual manner. A likely explanation is that it occurs because of the
different modes of cellular movements in the gastrula and neurula embryo.
Early in development, the rapid morphogenetic movements associated with
gastrulation and formation of the early neural tube mean that the registration
of floor plate and underlying axial mesoderm is not stable
(Dale et al., 1999;
Woo and Fraser, 1995
). By
contrast, during neurulation, the ventral midline of the caudalmost neural
tube is formed in register with the notochord, so that floor-plate cells arise
through the interaction of two stably apposed tissues. It is likely, then,
that the functional significance of rapid specification of `area a' cells by
the early prechordal mesoderm is to circumvent the requirement for prolonged
exposure to a Shh-expressing notochord until such time as stable tissue
interactions and Shh expression are re-established in more posterior regions
of the embryo following gastrulation.
Finally, the early specification of a population of floor plate cells by signalling from the prechordal mesoderm suggests parallels with anamniote embryos. Our observations that `area a'-derived cells occupy a medial position in the developing anterior floor plate, and that a floor plate is able to develop in the absence of notochord signalling, contingent upon early specification of `area a' cells by the nascent prechordal mesoderm suggest similarities between early floor-plate specification in amniotes and the generation of the medial floor plate in anamniote embryos. In addition, our observation that Nodal signalling may be responsible for mediating this rapid induction of a population of floor-plate cells indicates further parallels with the situation in anamniote embryos. Thus, our studies may go some way towards reconciling models of floor-plate formation in different vertebrate systems.
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ACKNOWLEDGMENTS |
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Footnotes |
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* Present address: Departament de Genetica, Facultat de Biologia, Universitat
de Barcelona, Avenida Diagonal 645, Barcelona 08028, Spain
Present address: Stowers Institute for Medical Research, 1000 East 50th
Street, Kansas City, MO 64110, USA
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Amacher, S. and Kimmel, C. (1998). Promoting
notochord fate and repressing muscle development in zebrafish axial mesoderm.
Development 125,3379
-3388.
Amacher, S., Draper, B., Summers, B. and Kimmel, C. (2002). The zebrafish T-box genes no tail and spadetail are required for development of trunk and tail mesoderm and medial floor plate. Development 129,3311 -3323.[Medline]
Artinger, K. B. and Bronner-Fraser, M. (1993). Delayed formation of the floor plate after ablation of the avian notochord. Neuron 11,1147 -1161.[Medline]
Chen, W., Burgess, S. and Hopkins, N. (2001). Analysis of the zebrafish smoothened mutant reveals conserved and divergent function of hedgehog activity. Development 128,2385 -2396.[Medline]
Chiang, C., Litingtung, Y., Lee, E., Young, K. E., Corden, J. L., Westphal, H. and Beachy, P. A. (1996). Cyclopia and defective axial patterning in mice lacking Sonic hedgehog gene function. Nature 383,407 -413.[CrossRef][Medline]
Corbo, J. C., Erives, A., di Gregorio, A., Chang, A. and Levine,
M. (1997). Dorsoventral patterning of the vertebrate neural
tube is conserved in a protochordate. Development
124,2335
-2344.
Dale, J. K., Sattar, N., Heemskerk, J., Clarke, J. D. W.,
Placzek, M. and Dodd, J. (1999). Differential
patterning of ventral midline cells by axial mesoderm is regulated by BMP7 and
chordin. Development
126,397
-408.
Ding, Q., Motoyama, J., Gasca, S., Mo, R., Sasaki, H., Rossant,
J. and Hui, C. C. (1998). Diminished Sonic hedgehog
signaling and lack of floor plate differentiation in Gli2 mutant mice.
Development 125,2533
-2543.
Epstein, D., McMahon, A. and Joyner, A. (1999).
Regionalization of Sonic hedgehog transcription along the anterioposterior
axis of the mouse central nervous system is regulated by HNF-dependent and
-independent mechanisms. Development
126,281
-292.
Ericson, J., Morton, S., Kawakami, A., Roelink, H. and Jessell, T. M. (1996). Two critical periods of Sonic Hedgehog signaling required for the specification of motor neuron identity. Cell 87,661 -673.[Medline]
Etheridge, L. A., Wu, T., Liang, J. O., Ekker, S. C. and Halpern, M. E. (2001). Floor plate develops upon depletion of Tiggy-winkle and Sonic hedgehog. Genesis 30,164 -169.[CrossRef][Medline]
Garcia-Martinez, V., Alvarez, I. S. and Schoenwolf, G. C. (1993). Location of the ectodermal and nonectodermal subdivisions of the epiblast at stages 3 and 4 of avian gastrulation and neurulation. J. Exp. Zool. 267,431 -446.[Medline]
Giger, R. J. and Kolodkin, A. L. (2001). Silencing the siren: guidance cue hierarchies at the CNS midline. Cell 105,1 -4.[Medline]
Gritsman, K., Zhang, J., Cheng, S., Heckscher, E., Talbot, W. S. and Schier, A. F. (1999). The EGF-CFC protein one-eyed pinhead is essential for nodal signaling. Cell 97,121 -132.[Medline]
Halpern, M. E., Ho, R. K., Walker, C. and Kimmel, C. B. (1993). Induction of muscle pioneers and floor plate is distinguished by the zebrafish no tail mutation. Cell 75, 99-111.[Medline]
Halpern, M. E., Thisse, C., Ho, R., Thisse, B., Riggleman, B.,
Trevarrow, B., Weinberg, E. S., Postlethwait, J. H. and Kimmel, C.
B. (1995). Cell-autonomous shift from axial to paraxial
mesodermal development in zebrafish floating head mutants.
Development 121,4257
-4264.
Halpern, M. E., Hatta, K., Amacher, S. L., Talbot, W. S., Yan, Y. L., Thisse, B., Thisse, C., Postlethwait, J. H. and Kimmel, C. B. (1997). Genetic interactions in zebrafish midline development. Dev. Biol. 187,154 -170.[CrossRef][Medline]
Hamburger, V. and Hamilton, H. L. (1951). A series of normal stages in the development of the chick embryo. J. Morphol. 88,49 -84.
Hatta, K. (1992). Role of the floor plate in axonal patterning in the zebrafish CNS. Neuron 9, 629-642.[Medline]
Hatta, K., Kimmel, C. B., Ho, R. K. and Walker, C. (1991). The cyclops mutation blocks specification of the floor plate of the zebrafish central nervous system. Nature 350,339 -341.[CrossRef][Medline]
Heyer, J., Escalante-Alcalde, D., Lia, M., Boettinger, E.,
Edelmann, W., Stewart, C. L. and Kucherlapati, R.
(1999). Postgastrulation Smad2-deficient embryos show defects in
embryo turning and anterior morphogenesis. Proc. Natl. Acad. Sci.
USA 96,12595
-12600.
Jessell, T. M. (2000). Neuronal specification in the spinal cord: inductive signals and transcriptional codes. Nat. Rev. Genet. 1,20 -29.[CrossRef][Medline]
Kelly, K. A., Wei, Y. and Mikawa, T. (2002). Cell death along the embryo midline regulates left-right sidedness. Dev. Dyn. 2,238 -244.[CrossRef]
Lawson, A. and Schoenwolf, G. C. (2001). Cell populations and morphogenic movements underlying formation of the avian primitive streak and organiser. Genesis 4, 188-195.
Lopez-Sanchez, C., Garcia-Martinez, V. and Schoenwolf, G. C. (2001). Localization of cells of the prospective neural plate, heart and somite within primitive streak and epiblast of avian embryos at intermediate primitive streak stages. Cells Tissue Organs 4,334 -346
Krauss, S., Concordet, J. P. and Ingham, P. W. (1993). A functionally conserved homolog of the Drosophila segment polarity gene hh is expressed in tissues with polarizing activity in zebrafish embryos. Cell 75,1431 -1444.[Medline]
Kulesa, P. M. and Fraser, S. E. (1998). Neural crest cell dynamics revealed by time-lapse video microscopy of whole embryo chick explant cultures. Dev. Biol. 204,327 -344.[CrossRef][Medline]
Le Douarin, N. M. and Halpern, M. E. (2000). Discussion point. Origin and specification of the neural tube floor plate: insights from the chick and zebrafish. Curr. Opin. Neurobiol. 10,23 -30.[CrossRef][Medline]
Marti, E., Bumcrot, D. A., Takada, R. and McMahon, A. P. (1995). Requirement of 19K form of Sonic hedgehog for induction of distinct ventral cell types in CNS explants. Nature 375,322 -325.[CrossRef][Medline]
Matise, M. P., Epstein, D. J., Park, H. L., Platt, K. A. and
Joyner, A. L. (1998). Gli2 is required for induction of floor
plate and adjacent cells, but not most ventral neurons in the mouse central
nervous system. Development
125,2759
-2770.
Muller, F., Albert, S., Blader, P., Fischer, N., Hallonet, M.
and Strahle, U. (2000). Direct action of the nodal-related
signal cyclops in induction of sonic hedgehog in the ventral midline of the
CNS. Development 127,3889
-3897.
New, D. A. T. (1955). A new technique for the cultivation of the chick embryo in vitro. J. Embryol. Exp. Morphol. 3,326 -331.
Odenthal, J., Haffter, P., Vogelsang, E., Brand, M., van Eeden,
F. J., Furutani Seiki, M., Granato, M., Hammerschmidt, M., Heisenberg,
C. P., Jiang, Y. J. et al. (1996). Mutations affecting the
formation of the notochord in the zebrafish, Danio rerio.
Development 123,103
-115.
Odenthal, J., van Eeden, F., Haffter, P., Ingham, P. and Nusslein Volhard, C. (2000). Two distinct cell populations in the floor plate of the zebrafish are induced by different pathways. Dev. Biol. 219,350 -363.[CrossRef][Medline]
Pevny, L. H., Sockanathan, S., Placzek, M. and Lovell-Badge,
R. (1998). A role for SOX1 in neural determination.
Development 125,1967
-1978.
Placzek, M. and Dale, K. (1999). Tissue recombinations in collagen gels. Methods Mol. Biol. 97,293 -304.[Medline]
Placzek, M., Tessier Lavigne, M., Yamada, T., Jessell, T. and Dodd, J. (1990). Mesodermal control of neural cell identity: floor plate induction by the notochord. Science 250,985 -988.[Medline]
Placzek, M., Jessell, T. M. and Dodd, J.
(1993). Induction of floor plate differentiation by
contact-dependent, homeogenetic signals. Development
117,205
-218.
Placzek, M., Dodd, J. and Jessell, T. M. (2000). Discussion point. The case for floor plate induction by the notochord. Curr. Opin. Neurobiol. 10, 15-22.[CrossRef][Medline]
Rebagliati, M. R., Toyama, R., Haffter, P. and Dawid, I. B.
(1998). cyclops encodes a nodal-related factor involved in
midline signaling. Proc. Natl. Acad. Sci. USA
95,9932
-9937.
Roelink, H., Augsburger, A., Heemskerk, J., Korzh, V., Norlin, S., Ruiz i Altaba, A., Tanabe, Y., Placzek, M., Edlund, T., Jessell, T. M. and Dodd, J. (1994). Floor plate and motor neuron induction by vhh-1, a vertebrate homolog of hedgehog expressed by the notochord. Cell 76,761 -775.[Medline]
Ruiz i Altaba, A., Placzek, M., Baldassare, M., Dodd, J. and Jessell, T. M. (1995). Early stages of notochord and floor plate development in the chick embryo defined by normal and induced expression of HNF-3ß. Dev. Biol. 170,299 -313.[CrossRef][Medline]
Sampath, K., Rubinstein, A. L., Cheng, A. M., Liang, J. O., Fekany, K., Solnica-Krezel, L., Korzh, V., Halpern, M. E. and Wright, C. V. (1998). Induction of the zebrafish ventral brain and floorplate requires cyclops/nodal signalling. Nature 395,185 -189.[CrossRef][Medline]
Schauerte, H. E., van Eeden, F. J., Fricke, C., Odenthal, J.,
Strahle, U. and Haffter, P. (1998). Sonic hedgehog is
not required for the induction of medial floor plate.
Development 125,2983
-2993.
Schier, A. F., Neuhauss, S. C., Helde, K. A., Talbot, W. S. and
Driever, W. (1997). The one-eyed pinhead gene
functions in mesoderm and endoderm formation in zebrafish and interacts with
no tail. Development
124,327
-342.
Schoenwolf, G. C. and Sheard, P. (1990). Fate-mapping the avian epiblast with focal injections of a fluorescent-histochemical marker: ectodermal derivatives. J. Exp. Zool. 255,323 -339.[Medline]
Schoenwolf, G. C., Bortier, H. and Vakaet, L. (1989). Fate mapping the avian neural plate with quail/chick chimeras: origin of prospective median wedge cells. J. Exp. Zool. 249,271 -278.[Medline]
Schulte Merker, S., Hammerschmidt, M., Beuchle, D., Cho, K. W.,
de Robertis, E. M. and NussleinVolhard, C. (1994).
Expression of zebrafish goosecoid and no tail gene products in wild-type and
mutant no tail embryos. Development
120,843
-852.
Selleck, M. A. J. and Stern, C. D. (1991). Fate mapping and cell lineage analysis of Hensen's node in the chick embryo. Development 112,615 -626.[Abstract]
Shih, J. and Fraser, S. (1995). Distribution of
tissue progenitors within the shield region of the zebrafish gastrula.
Development 121,2755
-2765.
Shinya, M., Furutanis-Seiki, M., Kuroiwa, A. and Takeda, H. (1999). Mosaic analysis with oep mutant reveals a repressive interaction between floor plate and non-floor plate mutant cells in the zebrafish neural tube. Dev. Growth Differ. 41,135 -142.[CrossRef][Medline]
Stern, C. D. and Ireland, G. W. (1981). An integrated experimental study of endoderm formation in avian embryos. Anat. Embryol. 163,245 -263.[Medline]
Strahle, U., Jesuthasan, S., Blader, P., Garcia-Villalba, P., Hatta, K. and Ingham, P. W. (1997). one-eyed pinhead is required for development of the ventral midline of the zebrafish (Danio rerio) neural tube. Genes Funct. 1,131 -148.[Medline]
Streit, A., Sockanathan, S., Perez, L., Rex, M., Scotting, P.,
Sharpe, P. T., Lovell-Badge, R. and Stern, C. D.
(1997). Preventing the loss of competence for neural induction:
HGF/SF, L5 and Sox 2. Development
124,1191
-1199.
Talbot, W. S., Trevarrow, B., Halpern, M. E., Melby, A. E., Farr, G., Postlethwaite, J. H., Jowett, T., Kimmel, C. B. and Kimelman, D. (1995). A homeobox gene essential for zebrafish notochord development. Nature 378,150 -157.[CrossRef][Medline]
Teillet, M. A., Lapointe, F. and le Douarin, N. M.
(1998). The relationships between notochord and floor plate in
vertebrate development revisited. Proc. Natl. Acad. Sci.
USA 95,11733
-11738.
van Straaten, H. W. M. and Hekking, J. W. M. (1991). Development of a floor plate, neurons and axonal outgrowth pattern in the eraly spinal cord of the notochord-defficient chick embryo. Anat. Embryol. 184, 55-63.[Medline]
Varga, Z. M., Amores, A., Lewis, K. E., Yan, Y.-L., Postlethwait, J. H., Eisen, J. S. and Westerfield, M. (2001). Zebrafish smoothened functions in ventral neural tube specification and axon tract formation. Development 128,3497 -3509.[Medline]
Vesque, C., Ellis, S., Lee, A., Szabo, M., Thomas, P.,
Beddington, R. and Placzek, M. (2000). Development of
chick axial mesoderm: specification of prechordal mesoderm by anterior
endoderm-derived TGFbeta family signalling.
Development 127,2795
-2809.
Wijgerde, M., McMahon, J. A., Rule, M. and McMahon, A. P.
(2002). A direct requirement for Hedgehog signalling for normal
specification of all ventral progenitor domains in the presumtive mammalian
spinal cord. Genes Dev.
16,2849
-2864.
Woo, K. and Fraser, S. E. (1995). Order and
coherence in the fate map of the zebrafish nervous system.
Development 121,2595
-2609.
Yamada, T., Placzek, M., Tanaka, H., Dodd, J. and Jessell, T. M. (1991). Control of cell pattern in the developing nervous system: Polarizing activity of the floor plate and notochord. Cell 64,635 -647.[Medline]
Zhang, J., Talbot, W. S. and Schier, A. F. (1998). Positional cloning identifies zebrafish one-eyed pinhead as a permissive EGF-related ligand required during gastrulation. Cell 92,241 -251.[Medline]