Download Control of Cell Pattern in the Neural Tube: Motor Neuron Induction

Cell, Vol. 73, 673-666,
May 21, 1993, Copyright
0 1993 by Cell Press
Control of Cell Pattern in the Neural Tube:
Motor Neuron Induction by Diffusible Factors
from Notochord and Floor Plate
Toshiya Yamada,’ Samuel L. Pfaff,’ Thomas Edlund,t
and Thomas M. Jessell’
*Howard Hughes Medical Institute
Department of Biochemistry and Molecular Biophysics
Center for Neurobiology and Behavior
Columbia University
New York, New York 10032
tDepartment of Microbiology
Umea University
Umea S-901 87
Sweden
Summary
The identity of cell types generated along the dorsoventral axis of the neural tube depends on inductive
signals that derive from both mesodermal
and neural
cells. To define the nature of these signals, we have
analyzed the differentiation
of cells in neural plate explants. Motor neurons and neural crest cells differentiate in vitro from appropriate
regions of the neural
plate, indicating that the specification
of cell fate along
the dorsoventral
axis of the neural tube begins at the
neural plate stage. Motor neuron differentiation
can
be induced by a diffusible factor that derives initially
from the notochord and later from floor plate cells. By
contrast, floor plate induction
requires contact with
the notochord.
Thus, the identity and patterning
of
neural cell types appear to involve distinct contactmediated and diffusible
signals from the notochord
and floor plate.
Introduction
During the early development of the vertebrate nervous
system, distinct cell types appear at specific locations,
establishing a primitive pattern within the neural tube. In
the caudal region of the neural tube that gives rise to the
spinal cord, the first differentiated cell types are found at
distinct dorsoventral positions. For example, floor plate
cells occupy the ventral midline of the neural tube, and
motor neurons appear in a ventrolateral position. Cells in
the dorsal neural tube give rise to sensory relay neurons,
to neural crest cells, and (at the dorsal midline) to roof
plate cells. Thus, the embryonic spinal cord contains two
midline cell groups and several neuronal cell types that
are distributed in a bilaterally symmetric manner with respect to the midline.
The organization of cell types along the dorsoventral
axis of the neural tube appears to be controlled by signals
from the ventral midline. In chick embryos, a localized
inductive signal from axial mesodermal cells of the notochord is responsible for the differentiation of floor plate
cells at the ventral midline of the neural tube (van Straaten
et al., 1988; Smith and Schoenwolf, 1989; Placzek et al.,
1990b, 1993; Yamada et al., 1991). The identity of neu-
ronal types generated ventrally also appears to depend
on signals from the ventral midline, since elimination of
the notochord and floor plate prevents the differentiation
of motor neurons and other ventral neuronal types (Yamada et al., 1991; Ericson et al., 1992; van Straaten and
Hekking, 1991). Inversely, grafting the notochord or the
floor plate to the dorsal midline of the neural tube induces
motor neurons in ectopic dorsal positions and suppresses
the expression of dorsal markers (Yamada et al., 1991;
Placzek et al., 1991; Ericson et al., 1992). These findings,
together with studies in other vertebrates (Bovolenta and
Dodd, 1991; Hatta et al., 1991; Clarke et al., 1991; Ruiz
i Altaba, 1992) suggest that the notochord and floor plate
have a critical role in defining the identity of cell types and
their position within the ventral neural tube (Jesse11 and
Dodd, 1992).
The nature and mechanism of action of signals that control the patterning of cell types along the dorsoventral axis
of the neural tube have, however, not been defined. In
particular, the time at which inductive signals specify the
fate of neural cells and establish dorsoventral pattern is
not known. Moreover, it remains unclear whether a single
midline signal is involved in the induction of all ventral cell
types. Floor plate induction by the notochord requires a
contact-dependent
signal (Placzek et al., 1993); however,
motor neurons appear at a distance from the ventral midline (Ericson et al., 1992). This raises the possibility that
signals that determine neuronal identity are distinct from
those that control floor plate differentiation.
In addition,
the relative contributions of the notochord and floor plate
to the control of ventral cell fate have not been resolved.
To define the nature of signals that regulate cell fate in
the neural tube, we have analyzed cell differentiation
in
neural plate explants grown alone and in the presence of
the notochord and floor plate. Our results provide evidence
that the fate of cells found along the dorsoventral axis
of the neural tube is specified at the neural plate stage.
Moreover, motor neuron differentiation can be induced by
a diffusible signal from the notochord and floor plate. The
combined action of contact-dependent
and diffusible signals that derive initially from the notochord and later from
the floor plate appears to be sufficient to define the identity
of distinct neural cell types and the positions that they
occupy in the ventral neural tube.
Results
Neural Cells Acquire Distinct Dorsoventral
Fates
within the Neural Plate
The patterning of cell types along the dorsoventral axis
of the nervous system becomes apparent with the differentiation of distinct cell types at defined positions within the
neural tube. Ventral cell fates appear to be controlled by
signals that originate from axial mesodermal cells of the
notochord. Since the neural plate is contacted by the notochord from the time of its formation, it seemed possible that
Cdl
674
Astage
i
10
ii stage 10
iii stages 15-25
rostra,
iv
Figure
”
1. In Vitro induction
vi
Assay
and Expression
of Markers
Used to Detect
Motor
Neuron
Differentiation
(A) (Diagram i) A dorsal view of the neural plate and neural tube of a stage 10 chick embryo showing the position and rostrocaudal
extent (300350 pm) of the region of neural plate isolated for induction assays. (Diagram ii) Transverse
section showing the neural plate and the dorsal (d),
intermediate
(i), and ventral (v) regions used in in vitro assays. The fate of cells in the ventral midline region (9 was not examined.
(Diagram iii)
Inducing tissue was isolated from stage IO-17 notochord (n) and from stage lo-25 and stage lo-26 floor plate (9. (Diagram iv) To determine the
degree of commitment
of cells in the neural plate, dorsal (d), intermediate
(i), and ventral neural plate (v) explants were grown separately
in collagen
gels. (Diagrams
v and vi) For induction assays, intermediate
neural plate explants (i) were grown in contact with notochord
(n) or floor plate (9
explants (diagram v) or in the presence of notochord- or floor plate-conditioned
medium (cm) (diagram vi). For details see Experimental
Procedures.
(6) Coexpression
of SC1 and Islet-1 by embryonic
spinal motor neurons in a transverse
section of stage 16-17 chick spinal cord. SC1 (green
label) is expressed
by motor neurons and also by floor plate cells and the notochord.
Islet-l expression
(red label) is restricted
to motor neurons
at this stage of spinal cord development.
(C) Dark-field micrograph
showing the localization
of ChAT mRNA by in situ hybridization
histochemistry
in a section of a stage 26 chick embryo.
Hybridization
is detected over cells in the motor column but not over other cells in the spinal cord.
Scale bar: (B), 60 urn; (C), 250 urn.
neural cells are exposed to inductive signals and acquire
specific fates prior to neural tube closure.
To determine the time at which neural cells are committed to distinct fates, we monitored cell differentiation
in
explants isolated from the caudal region of the neural plate
of stage 10 chick embryos (Figure 1A). A segment (300350 pm long) of the neural plate was divided along the
futuredorsoventralaxisintoventral,
intermediate, anddorsal regions, discarding theventral midline region that gives
rise to the floor plate (Figure 1 A). Each region was placed
in a collagen gel and maintained in vitro for up to 96 hr
(Figure 1A). In these experiments we monitored the differentiation of two well-characterized
cell types that appear
at different dorsoventral positions: motor neurons that appear ventrally and neural crest cells that migrate from the
dorsal neural tube.
To assess motor neuron differentiation we used three
markers: the LIM homeodomain
protein Islet-l (Karlsson
et al., 1990) (Figure 1 B), the SC1 immunoglobulin-like
glycoprotein (Tanaka and Obata, 1984; Tanaka et al., 1991)
(Figure 1 B), and expression of the chick gene-encoding
choline acetyltransferase (ChAT), which is the rate-limiting
enzyme in the synthesis of acetycholine, the neurotransmitter used by motor neurons (Figure 1C). Motor neurons
can be distinguished from other neural cells by their coordinate expression of Islet-l, SCl, and ChAT (Table 1).
Table 1. Biochemical
Markers and Other Properties
in Combination,
Distinguish
Cell Types Derived
from the Caudal Neural Plate
Cell Type
Biochemical
Motor neurons
Dorsal neurons
Dorsal root ganglion
neurons
Neural crest cells
Islet-l+,
islet-l+,
Islet-l+,
Floor plate cells
Xl+,
SCl-,
SCl’,
That,
Markers
ChAT+
ChATChAT-
Migration, HNK-l+, 5, integrin’,
generation of melanocytes
Islet-l-, SCl+, 3AiO-
p75+,
In the ventral region of the embryonic
chick spinal cord, Islet-l is expressed selectively
by motor neurons soon after their final mitotic division (Figure 1 B) (Ericson et al., 1992). At later stages, Islet-l is also
expressed
by a small group of cells in the dorsal spinal cord and by
neurons in the dorsal root and sympathetic
ganglia (Thor et al., 1991;
Ericson et al., 1992). In the spinal cord, the SC1 glycoprotein
is restricted to floor plate cells and motor neurons (Figure IB) (Tanaka
and Obata, 1964). ChAT is expressed
selectively
by motor neurons
in embryonic
chick spinal cord (Figure 1C).
SC1 is a 100 kd immunoglobulin-like
glycoprotein
(Tanaka et al.,
1991); Islet-l is a LIM homeodomain
protein (Karlsson
et al., 1990);
MAb 3AlO recognizes
an uncharacterized
filament-associated
protein
(Furley et al., 1990): ChAT is the rate-limiting enzyme in acetylcholine
biosynthesis;
the HNK-1 epitope is a sulfated glucuronyl
lactosamine
(Chou et al., 1965); MAb JO22 recognizes
the chick 6, integrin subunit
(Greve and Gottlieb, 1962).
For details see Experimental
Procedures,
Motor
675
Neuron
Induction
by Diffusible
Factors
Neural crest cells were defined by their migratory properties, the surface expression of the HNK-1, PI integrin, and
p75 antigens (Maxwell et al., 1988; Delannet and Duband,
1992; Bernd, 1985; Stemple and Anderson, 1992), and
their ability to differentiate into neurons and melanocytes.
Ventral neural plate explants did not express Islet-l+,
SCl+, or ChAT messenger RNA (mRNA) at the time of
isolation, but when maintained in vitro for 48 hr, they were
found to contain both Islet-l + (Figure 2A) and SC1 + (data
not shown) cells and to express ChAT mRNA (Figure 28).
These results provide evidence that the specification of
motor neurons occurs at the neural plate stage (Table 2).
In contrast, intermediate neural plate explants contained
few (usually <5) Islet-l+ (Figure 2A) and SCl+ (data not
shown) cells and did not express detectable levels of ChAT
mRNA when grown alone for up to 72 hr in vitro (Figure
2B). Although motor neuron markers were not detected,
labeling with the general neuronal marker 3AlO (Figure
2C) indicated that many neurons differentiated over the
48 hr period that intermediate neural plate explants were
maintained in vitro.
Cells derived from dorsal neural plate explants maintained in vitro for 48 hr also expressed Islet-l (Figures 2A,
2G, and 2H) and SC1 (data not shown) but did not express
ChAT mRNA at detectable levels (Figure 28; Table 2),
suggesting that these cells are not motor neurons. Moreover, extensive cell migration was observed from dorsal
but not ventral or intermediate neural plate explants (Figures 2C and 2D-2F). Over 90% of the cells that migrated
from dorsal neural plate explants expressed the HNK-1
epitope (Figure 21) and the PI integrin subunit (Delannet
and Duband, 1992; data not shown), and 30%-50% expressed the low affinity neurotrophin receptor p75 (Figures
2J and 2K). These results suggest that the cells that migrate from dorsal neural plate explants are neural crest
cells. To determine further the identity of these migratory
cells, we examined their ability to generate neurons and
melanocytes. About 20% of the cells that had migrated
from dorsal neural plate explants expressed Islet-l and
SC1 (Figures 2G and 2H), exhibited neuronal morphology,
and expressed the 155 kd neurofilament subunit (data not
shown). These cells are likely to be neural crest-derived
sensory or sympathetic neurons, which have been shown
to express Islet-l (Ericson et al., 1992). In addition, when
dorsal neural plate explants isolated from quail embryos
were grown in the presence of chick embryo extract and
fetal calf serum, lo%-20%
of cells exhibited the intense
pigmentation and dendritic morphology characteristic of
melanocytes (Figure 2L). These results show that cells
derived from prospective dorsal and ventral regions of the
neural plate adopt distinct and appropriate fates in vitro
and that the specification of motor neurons, neural crest
cells, and (by inference) dorsoventral patterning begins at
the neural plate stage.
The lack of expression of motor neuron markers and the
absence of neural crest cell differentiation in intermediate
neural plate explants grown alone in vitro provides the
basis for assays to assess the nature of signals that direct
the dorsal or ventral fates of neural plate cells. The role
-‘Z-----^
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r,dl
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et al. (1993 [this issue of Ce/fl). In the studies described
below, we have focused on the differentiation
of motor
neurons to provide information on the factors that control
neuronal identity in the ventral neural tube.
Induction of Islet-l + Ceils in Neural Plate Explants
To determine whether signals from the notochord and floor
plate can induce motor neuron markers in vitro, intermediate neural plate explants were placed in contact with segments of stage 1 O-20 notochord or stage 16-26 chick floor
plate (Table 3) and grown in vitro for 12-48 hr. Islet-l+
cells were not detected after 12 hr, but by 18 hr there was
a significant increase in the number of Islet-l+ cells over
controls (see Figure 4A), and by48 hr the numberof Islet-l +
neurons was 50- to lOO-fold greater than in control explants (Figures 3A-31; Figure 4A). The maximum number
of Islet-l+ cells induced by the notochord or floor plate
( - 400 cells per explant) represents 25%-30% of the total
number of cells present in intermediate neural plate explants after 48 hr (see Experimental Procedures). In addition to inducing Islet-l+ cells, exposure of intermediate
neural plate explants to signals from the notochord and
floor plate resulted also in a 2.3-fold increase in cell number when compared with explants grown alone (see Experimental Procedures). Although the extent of proliferation
is low, from these studies we cannot resolve whether the
increase in Islet-l+ cells involves the rapid proliferation of
asmall population of committed motor neuron progenitors
present in intermediate neural plate explants or the commitment of unspecified neural plate cells to a motor neuron
fate.
Three lines of evidence indicate that the Islet-l’ cells
detected in the presence of notochord or floor plate appear
to derive from cells in the intermediate neural plate explants. First, Islet-l+ cells were not observed in notochord
or stage 16-26 floor plate explants grown alone in vitro
(see Figures 3J-3L; data not shown). Second, labeling of
conjugate explants with notochord-specific
(monoclonal
antibody [MAb] Not-l) and floor plate-specific (MAb FPl)
markers (Yamada et al., 1991) showed that Islet-l+ cells
were present in the intermediate neural plate explant (Figure 5A). Third, in some experiments, conjugates of quail
notochord and chick intermediate neural plate explants
were labeled with a quail-specific perinuclear marker (recognized by MAb QCPN), revealing that all Islet-l+ cells
are derived from the chick tissue (data not shown).
To examine the specificity of induction of Islet-l+ cells,
intermediate neural plate explants were grown in contact
with explants of dorsal spinal cord obtained from stage
16-26 embryos or with somites from stage lo-17 embryos. The number of Islet-l’ cells in intermediate neural
plate explants grown in contact with these two tissues was
similar to that in explants grown alone (see Figure 48).
Thus, of a limited number of relevant cell types examined,
the ability to induce Islet-l’ cells is confined to the notochord and floor plate.
We also determined the temporal expression of Islet-linducing activity in the notochord and floor plate. Islet-linducing
activity was detected in the caudal notochord at
ctann
1 D. whereas
the overlvina
midline
reaion
of the neu-
B
r
l-
k
ii
‘;i65 30 =
8
+, 20 ZG
9
10 -
4 Int Std
3
Q 100 2
2
@J8
4 ChAT
,F
5
.F
j_
60 40 20 -
O-
WInp
Figure
2. Variation
[il w
WI v
[VI “P
in the Fate of Cells Derived
from
Different
Dorsoventral
Regions
of the Neural
[iI v
[VI v
Plate
(A) Quantitation
of Islet-l+ cells present in dorsal (d), intermediate
(i), and ventral (v) neural plate (np) explants grown in isolation for 46 hr. Each
column shows the mean f SEM of 9-16 explants.
(8) Detection of mRNA encoding ChAT in dorsal (d), intermediate
(i), and ventral (v) neural plate (np) explants grown in vitro for 72 hr. Upper band
indicates internal standard (lnt Std). Lower band shows endogenous
ChAT mRNA levels (ChAT). Quantitation
of the ChAT mRNA band indicates
that ventral neural plate explants contain at least 50-fold higher levels of ChAT mRNA than dorsal and intermediate
neural plate explants.
(C) Quantiation
of migratory
cells derived from dorsal (d), intermediate
(i), and ventral (v) neural plate (np) explants.
Each column shows the
mean f SEM of 14-25 different explants.
(D) Phase-contrast
micrograph
showing a dorsal neural plate grown in vitro for 46 hr. Numerous cells have migrated from the explant.
(E and F) Phase-contrast
micrographs
showing intermediate
neural (E) and ventral neural (F) plate explants grown for 46 hr in vitro. Few, if any,
migrating cells are observed.
(G and f-l) lmmunofluorescence
and phase-contrast
micrographs
of the same field showing Islet-l expression
in a subset of cells that have migrated
from dorsal neural plate explants. The Islet-l+ cells exhibit neuronal morphology
and express neurofilament
(data not shown). Approximately
20%
of migratory cells express Islet-l. Arrowheads
in (H) point to the Islet-l+ cells shown in (G).
(I) lmmunofluorescence
micrograph
showing HNK-1 expression
by cells that have migrated from dorsal neural plate explants.
This field is the
sama ne ,hP, dlnurn in IC, .“A ,YI
Motor
677
Neuron
Induction
Table 2. Characteristics
Plate Explants In Vitro
Region
of Neural
Plate
Dorsal neural plate
Intermediate
neural plate
Ventral neural plate
by Diffusible
of Cell Types
Migratory
Cells
+++
-
Factors
That Derive
from Neural
Islet-l
SC1
ChAT
mRNA
++
-
+a
-
+
+
++
Neural plate explants were derived from the caudal neural plate of
stage IO embryos and biochemical
markers and migratory properties
assayed after 46 hr in vitro or 72 hr for ChAT expression.
Islet-l, SCI,
and ChAT mRNA were not expressed
at significant levels at the time
of isolation of explants. The number of migratory cells was assessed
by phase-contrast
microscopy,
and quantitation
is provided in Figure
2C. Islet-l+cellsweredetermined
by immunocytochemistry,
with quantitation shown in Figure 2A. SC1 expression
was determined by immunocytochemistry.
ChAT mRNA was determined
by a PCR-based
assay, and the results are shown in Figure 28.
a Most of the SCl’ cells that derived from dorsal neural plate explants
had migrated from the explant and had neuronal morphology.
ral plate (see region f in Figure 1A) did not exhibit significant activity (Table 3). By stage 16/l 7, the ventral midline
of the neural tube had acquired strong inducing activity
that persisted up to stage 25/26, whereas inducing activity
was absent from the notochord after stage 20 (Table 3).
These results show that the notochord acquires inducing
activity before the overlying region of neural plate, but that
activity persists for a longer period in the floor plate than
in the notochord.
Islet-l Expression Is Indicative of Motor
Neuron Differentiation
Since Islet-l is expressed by several classes of neurons
(Thor et al., 1991; Ericson et al., 1992) the presence of
Islet-l+ cells in intermediate neural plate explants does
not establish that motor neurons have been induced. To
determine whether the Islet-l + cells induced in intermediate neural plate explants have other properties of motor
neurons, we examined the expression of the SC1 glycoprotein and of ChAT mRNA. About 60% of Islet-l+ cells
detected in intermediate neural plate explants grown with
notochord or floor plate for 46 hr coexpressed SC1 (Figure
56). Although motor neurons are the only cells in the embryonic spinal cord that coexpress SC1 and Islet-l (Ericson et al., 1992) it is conceivable that the induced Islet-l’
and SCl+ cells represent sensory or sympathetic neurons
that derive from the neural crest. This seems unlikely since
neural crest cells do not appear to differentiate in intermediate neural plate explants grown alone (see Figure 2).
Nevertheless, to provide evidence that the Islet-l+ and
SCl+ cells are motor neurons, we monitored the expression of ChAT mRNA. Intermediate neural plate explants
Table 3. Temporal
Hamburger-Hamilton
Stage
10
16117
20
25126
35136
Expression
of Motor
Notochord
+++
+++
+++
-
Neuron-Inducing
Midline
Tissue
Activity
Neural
+++
+++
+++
-
Notochord and floor plate tissue was dissected from the brachial level
of chick embryos
at different stages and assayed for their ability to
induce Islet-l’ cells in intermediate
neural plate explants. The minus
sign indicates fewer than IO Islet-l + cells per explant. The triple plus
signs indicate greater than 250 Islet-l+ cells per explant. The table
summarizes
results from 5 to 20 explants per stage.
grown in isolation for 72 hr expressed low or undetectable
levels of ChAT mRNA (Figure 6, lane l), whereas explants
grown in contact with the notochord or floor plate expressed -50-fold higher levels of ChAT mRNA (Figure 6,
lanes 2 and 3).
The coexpression of Islet-l and SC1 and the induction of
ChAT mRNA in intermediate neural plate explants indicate
that the notochord and floor plate can induce the differentiation of motor neurons from intermediate neural plate cells
and suggest that most or all of the Islet-l+ cells in such
explants are motor neurons. The ability of notochord or
floor plate tissue to induce motor neuron differentiation in
intermediate neural plate explants in vitro also establishes
that signals from each of these two cell types are sufficient
to induce motor neuron differentiation and that these signals can act directly on neural plate cells.
Spatial Organization
of Induced Islet-l + Cells
In the embryonic chick spinal cord, Islet-l+ motor neurons
are separated from the notochord and floor plate by a
region of intervening neural epithelium that contains other
defined neuronal types (Yamada et al., 1991; Lo et al.,
1991; Ericson et al., 1992). We therefore examined
whether the spatial relationship between Islet-l+ cells and
the notochord or floor plate can be reconstituted within
intermediate neural plate explants. To assess this, the
position of Islet-l+ cells was determined with respect to
the junction of the inducing tissue and the intermediate
neural plate explant.
Islet-l+ cells were found in a band 30-50 urn away from
the junction of the inducing tissue and the intermediate
neural plate explant with few, if any, Islet-l+ cells in the
intervening region (see Figure 5A). The position of this
band of Islet-l+ cells with respect to the inducing tissue
was independent of the original orientation of the neural
plate tissue (data not shown). In vitro studies of floor plate
(J and K) lmmunofluorescence
and phase-contrast
micrographs
showing expression
of p75 by a subset of cells that have migrated from dorsal
neural plate explants.
(L) Bright-field
micrograph
showing pigmented cells with dendritic morphology,
presumably
melanocytes,
which differentiate
after 120 hr in vitro
from cells that have migrated from a dorsal neural plate explant isolated from somite stage 10 quail neural plate. About 20% of cells are pigmented.
Scale bar: (D-F), 50 pm; (G-K), 25 pm; (L), 20 urn.
Cdl
676
Figure
3. Induction
of Islet-l
Expression
in Neural
Plate Explants
Intermediate
neural plate explants were placed in collagen gels, grown for 46 hr in vitro, and labeled with rabbit anti-islet-l
antibodies (middle
panels) and MAb 3A10 (right-hand
panels). Nomarski
images of explants are shown in left-hand panels.
(A-C) Cells in an intermediate
neural plate explant grown alone in vitro do not express Islet-l (B), but numerous 3AlO’ neuronal cell bodies and
axons are present (C).
(D-F) Numerous
Islet-l+ cells are present in an intermediate
neural plate (i np) explant grown in contact with notochord (nc). There are no obvious
changes in the density of 3AiO’ cells and axons (F).
(G-l) Numerous
Islet-l’ cells are detected in an intermediate
neural plate (i np) explant grown in contact with floor plate (fp), with no obvious
change in 3AlO’ cells (I) compared
with controls.
(J-L) A floor plate explant isolated from stage 17 chick spinal cord and grown alone in vitro does not contain Islet-l+ (K) or 3AlO+ (L) cells.
(M-O). Many Islet-l+ cells are present in intermediate
neural plate explants grown for 48 hr in the presence
of floor plate-conditioned
medium
(1 x concentration).
All micrographs
show representative
explants from IO-60 similar experiments.
Scale bar: (A-C), 60 pm; (D-L), 50 pm; (M-O), 60 nm.
induction by the notochord have shown that induced floor
plate cells occupy the region of the neural plate explant
immediately adjacent to the inducing tissue (Placzek et al.,
1993; our unpublisheddata).
Thus, the region of unlabeled
neural plate cells interposed between the junction of the
explants and the cluster of Islet-l+ cells is likely to be occupied, in part, by floor plate cells. The spatial relationship
of ventral cell types induced in intermediate neural plate
Motor
679
Neuron
induction
700
by Diffusible
Factors
LL!!
A lil v
0 +nc
600
.
500
400
300
200
100
0
0
10
[II
Figure 4. Time Course
chord and Floor Plate
+fp
nP
20
+"C
and Specificity
30
+fP
40
tdsc
of Islet-l
50h
+som
induction
by Noto-
(A) Graph showing the number of Islet-l+ cells detected in intermediate
neural plate ([i] np) explants grown alone (open triangle) or in contact
with stage 10 caudal notochord (nc) (open circle) or stage 25/26 floor
plate (fp) (closed circle) for varying
lengths of time. Points indicate
mean f SEM of four to seven different explants.
(6) Histogram showing the number of Islet-l+ cells detected in intermediate neural plate ([i] np) explants grown in contact with other mesodermal and neural tissues for 48 hr. In this experiment,
caudal notochord
(nc) was obtained from stage IO, floor plate (fp) from stage 25, dorsal
spinal cord (dsc) from stage 16, and somites (som) from stage 10
embryos.
Dorsal spinal cord tissue up to stage 26 and somites up to
stage 17 lacked Islet-l-inducing
activity. Histogram
shows mean f
SEM for 4-23 separate explants.
explants therefore appears similar to that detected in the
ventral spinal cord at an equivalent developmental stage.
Motor Neuron Induction by Diffusible Factors
Islet-l+ motor neurons in chick spinal cord first appear at
adistancefrom
the notochord(Ericsonet
al., 1992), raising
the possibility that motor neuron induction may not be
dependent on contact with inducing tissues. To examine
this, intermediate neural plate explants were grown alone
in vitro for 48 hr in the presence of medium conditioned
by the notochord or floor plate. Medium conditioned by
notochord and floor plate produced a concentrationdependent induction of Islet-l+ cells (Figures 7A and 78;
Table 4). Medium conditioned by an equivalent mass of
dorsal spinal cord tissue isolated from stages 18-28 did
not induce a significant increase in the number of Islet-l’
cells (Figure 7B). The number of Islet-l* cells induced by
concentrated floor plate-conditioned
medium was similar
to that induced by contact with a single floor plate or notochord explant (Figure 7A). However, when notochord or
floor plate explants were placed at a distance of 100-200
pm from the intermediate neural plate, no significant induction of Islet-l+ cells was observed (data not shown;
see Experimental Procedures for quantitation of inducing
Figure 5. Spatial Analysis
ral Plate Explants
and Identity of Islet-l + Cells Induced
in Neu-
(A) Double-label
immunofluorescence
micrograph
of a conjugate of
notochord
and intermediate
neural plate explant labeled with rabbit
anti-Islet-1
antibodies
(red) and the notochord-specific
MAb Not-l
(green). The highest density of Islet-l’ cells is located about 30-50
pm away from the border of the two explants, defined on the basis of
Not-l expression.
Similar results were observed in many other intermediate neural plate explants in which the boundary between notochord
or floor plate was clearly delineated.
(B) Confocal micrograph
of an intermediate
neural plate explant cultured in contact with stage 26 quail floor plate for 48 hr and labeled
with rabbit anti-Islet-1 (red) and a chick-specific
anti-SC1 (green) MAb.
About 80% of Islet-l+ cells also express
SC1 The coexpression
of
the chick-specific
SC1 epitope and Islet-l by cells establishes
that
induced Islet-l+ cells derived from intermediate
neural plate tissue.
Similar results were obtained with quail notochord as inducing tissue.
Scale bar: (A), 80 pm; (B), 35 Wm.
activity). These results establish that factors from the notochord and floor plate can induce motor neurons in the
absence of direct contact. Contact between the inducing
tissue and intermediate neural plate explant, however, appears to enhance significantly the inductive effect of the
diffusible signal.
To determine whether diffusible factors derived from the
notochord and floor plate can induce motor neuron markers other than Islet-l, we examined the expression of SC1
and ChAT mRNA in intermediate neural plate explants
exposed to floor plate-conditioned
medium. Of the Islet-l+
cells induced by floor plate-conditioned
medium, 25%30% coexpressed SC1 (data not shown). Although SC1
also labels floor plate cells, floor plate-conditioned
medium does not induce floor plate differentiation (Placzek
et al., 1993). Thus, the expression of SC1 in intermediate
Cell
680
Competence
of Neural Plate Cells to Generate
Motor Neurons
The midline of the neural plate is not effective at inducing
A
motor
4 In1Sld
320)
. ChAT
1
2
3
4
5
6
Figure 6. Induction of ChAT mRNA Expression
by Notochord
and
Floor Plate
PCR analysis of ChAT mRNA expression
in intermediate
neural plate
(Ii] np) explants grown for 72 hr.
(A) Lane 1, intermediate neural plate (np) explants grown alone. Lane
2, ChAT mRNA is induced in intermediate neural plate explants grown
in contact with Hamburger-Hamilton
stage 10 (Hamburger
and Hamilton, 1951) notochord (nc). Lane 3, ChAT mRNA induction in intermediate neural plate explants grown in contact with stage 25-28 floor plate
(fp). Each sample in (A) derives from two explants.
(B) Lane 4, absence of ChAT in intermediate
neural plate ([i] np) explants grown alone. Lane 5, induction of ChAT mRNA in intermediate
neural plate explants exposed to floor plate-conditioned
medium
(fpcm). Lane 6, stage 10 notochord explant (nc) grown alone. ChAT
mRNA is also not detected in floor plate explants. Each sample in (Et)
derives from eight explants.
(A) and (6) are separate experiments.
Upper band shows the 520 nt
internal standard (Int Std); lower band indicates the 320 nt ChAT mRNA
(ChAT).
neuron
differentiation
in intermediate
neural
plate
explants and acquires significant inducing activity only
after neural tube closure (see Table 3). A contribution of
floor plate-derived
signals to motor neuron induction in
vivo would therefore require that neural cells are capable
of responding to these signals at the time that floor plate
cells have acquired this activity. To address this issue, we
examined the period of time over which cells in intermediate neural plate explants are competent to respond to motor neuron-inducing
signals from the floor plate. Isolated
intermediate neural plate explants were matured in vitro
for varying times and then exposed to floor plate-conditioned medium for a further 48 hr. The number of Islet-l+
cells detected in response to floor plate-conditioned
medium decreased by 50% after maturation of intermediate
neural plate explants for -20 hr in vitro (Figure 7D). After
48 hr of maturation, virtually no Islet-l+ cells could be induced (Figure 7D). Intermediate neural plate explants remained viable for the 96 hr duration of these experiments
as assayed by morphology and expression of the neuronal
marker 3AlO (data not shown). These results suggest that
although the floor plate acquires motor neuron-inducing
activity after the notochord, many neural plate cells are
still competent to generate motor neurons at the time that
floor plate cells have acquired this activity.
Discussion
plate explants is likely to reflect the presence of
motor neurons. Floor plate-conditioned
medium also induced significant levels of ChAT mRNA in intermediate
neural plate explants grown for 72 hr in vitro (see Figure
6, lanes 4-6), indicating that conditioned medium induces
all motor neuron markers examined.
The ability to induce motor neurons with conditioned
medium permitted us to determine the minimum period
of time that cells in intermediate neural plate explants need
to be exposed to the inducing factor to specify motor neurons. Intermediate neural plate explants were exposed to
floor plate-conditioned
medium for 6, 12, 24, or 36 hr,
after which the conditioned medium was replaced with
basal culture medium and explants were grown for a total
of 46 hr before determining the number of Islet-l+ cells.
Intermediate neural plate explants exposed to floor plateconditioned medium for up to 12 hr did not result in the
presence of Islet-l+ neurons assayed at 46 hr (Figure 7C).
These results, taken together with the detection of Islet-l +
cells after 16 hr of exposure to floor plate (see Figure 4A),
indicate that the specification of Islet-l+ cells in intermediate neural plate explants requires between 12 hr and 18
hr exposure to floor plate-derived
signals. The induction
of floor plate differentiation
in vitro by contact-mediated
signals from the notochord requires 12-18 hr of contact
with the inducing tissue (Placzek et al., 1993). Thus, the
time required for the induction of floor plate cells and motor
neurons in vitro is similar.
neural
Grafting studies in chick embryos have provided evidence
that the fate of neural cell types located in the ventral spinal
cord is dependent on inductive signals that derive from
two midline cell groups, the notochord and floor plate.
Such in vivo studies, however, have not resolved the time
at which midline signals act to control neural cell fate, the
mechanism of action of these signals, or the respective
contributions of the notochord and floor plate to neural
patterning. The present in vitro studies and those of Placzek et al. (1993) indicate that the specification of cell fate
and the dorsoventral patterning of cell types begin at the
neural plate stage. Our studies also provide evidence that
two distinct signals, one contact-mediated
and the other
diffusible, are responsible for the differentiation of distinct
ventral cell types. In addition, they suggest that the notochord
provides
the
initial
source
of signals
responsible
both for floor plate and motor neuron differentiation.
Analysis of the temporal expression of the contactmediated and diffusible signals by the notochord and floor
plate and the response of intermediate neural plate cells
to these signals has generated a working model of the
sequential steps involved in the induction and patterning
of ventral cell types within the neural tube (Figure 8). Ventral cell patterning appears to be initiated by a contactmediated signal from the notochord that induces overlying
midline neural plate cells to acquire floor plate properties.
Over the same period, a diffusible signal from the notochord may act on cells in more lateral regions of the neural
Motor Neuron
661
Induction
by Diffusible
Factors
A
500
r
s
400
-
G
2
%
300
-
E
B
250
r
+
;1;
3
200-
100
dsc
I
001
r\
1
10
01
Concentration
of fpcm
Oi
100
D
5
::
s
=
fi
+
7
z
”
20
Exposure
30
40
300
r
250
-
200
-
~
__~_
---.-
0 01
01
C0ncentrat10n
1
01 cm
150100
-
0
50
20
40
Maturation
tome (h)
60
time (h)
Figure 7. Induction of Islet-l’ Cells by Floor Plate-Conditioned
Medium
(A) Islet-l induction by floor plate-conditioned
medium (fpcm) is concentration
dependent.
Medium conditioned
by stage 25-26 floor plate at
different concentrations
was added to intermediate
neural plate explants for 46 hr. Each point represents the mean f SEM for 20-36 explants
from seven separate experiments.
Similar activity was detected in medium conditioned
by stage 1617 floor plate.
(B) Comparison
of Islet-l +-inducing activity in conditioned
medium (cm) derived from stage 25-26 floor plate (fp) (open circle) and stage 25-26
dorsal spinal cord (dsc) (closed circle) explants of similar mass. Each point represents the mean f SEM of four explants.
(C)Time of exposure required for induction of Islet-l+ cells by floor plate-conditioned
medium. Conditioned medium (1 x) was added to intermediate
neural plate explants for the lengths of time indicated (in hours) and then replaced with basal medium for a total of 46 hr. The number of Islet-l’
ceils was then determined.
Each point represents
the mean -f SEM of four different explants.
(D) Competence
of intermediate
neural plate explants to respond to floor plate-conditioned
medium. Intermediate
neural plate explants were
matured in vitro for different periods of time (shown in hours) after which floor plate-conditioned
medium (1 x) was added for an additional 46 hr
and the number of islet-l+ cells determined.
Each point represents the mean + SEM for four different explants.
plate to induce
motor
neurons
and other ventral
neuronal
types (Figure
8A). Since cells at the midline
of the neural
plate are likely to be exposed
to both signals,
the diffusible
signal
may also be required
for floor plate differentiation.
The notochord
appears
to lose its floor plate-inducing
ac-
Specification
of Cell Fate along the Dorsoventral
Axis of the Neural Tube Begins in the Neural Plate
Table 4. Induction of Islet-l+ Cells by
Notochord-Conditioned
Medium
Source
of Conditioned
Medium
Control medium
Stage 6-7 rostra1 notochord
Staoe 16-l 7 caudal notochord
tivity at the time that floor plate cells acquire
inducing
activities. Thus, after neural tub6 closure,
the flocrr plate may play
an increasingly
prominent
role in inductive
signaling
in the
ventral
neural tube, inducing
additional
floor plate cells, motor neurons,
and other ventral
cell types (Figure
86).
Islet-l+
0.3
Cells
T 0.3
n
16
3
3
46 j: 12
4
39 I
Intermediate neural plateexplants
were grown aloneor in the presence
of notochord-conditioned
medium for 46 hr, and the number of Islet-l+
cells was determined.
The stage 6-7 rostra1 notochord explants used
to generate conditioned
medium had a tissue mass of about l/5 that
of stage 25-26 floor plate explants used to generate floor plate-conditioned medium. The mass of stage 16-17 notochord
is equal to or
greater than that of stage 25-26 floor plate. Comparison
of the number
of Islet-l+ cells induced by stage 6-7 notochord and stage 25-26 floor
plate (see Figures 7A and 78) suggests that these two tissues release
equivalent
Islet-l-inducing
activity.
The pattern
of cell differentiation
tube becomes
evident
after
within
the caudal
neural
neural tube closure with the
appearance of distinct cell types at different dorsoventral
positions. The differentiation of motor neuron and neural
crest cells from distinct and appropriate regions of the
neural plate in vitro indicates that the specification of two
major neural cell types has occurred at the neural plate
stage.
There
are
few
postmitotic
cells
within
the
neural
plate (Sechrfst and Bronner-Fraser,
1991); thus, the ability
of neural
plate cells to generate specific cell types such
as motor neurons
may be acquired
prior to their final cell
division. Moreover, since the cell cycle time in the chick
neural plate is - 8 hr (Langman et al., 1988), several cell
divisions
could
occur
between
the time
that
the
potential
Cdl
662
-
contact-dependent signal
-r. diffusible signal
A early inductive signals
Figure 6. Model of the Early Inductive InteractionsThat
Establish
ral Cell Identity and Pattern in the Ventral Neural Tube
Neu-
Inductive interactions
appear to begin at the neural plate stage, although the eventual pattern of cell types becomes apparent only after
neural tube closure.
(A) Early inductive signals. Cells in the neural plate, which are assumed
to be equivalent, respond to contact-dependent
(solid line) and diffusible (broken line) signals from the notochord
(N). Cells immediately
overlying the neural plate that are destined to give rise to the floor
plate probably receive both contactdependent
and diffusible signals,
whereas cells located in more lateral regions of the neural plate that
give rise to motor neurons and other ventral neurons are exposed only
to diffusible signals. Thus, the floor plate-inducing
signal appears to
dominate over motor neuron-inducing
signals at the midline of the
neural plate.
(B) Early inductive signals have specified the first group of floor plate
cells (F), motor neurons (M) that are shown in a position that corresponds to their eventual dorsoventral
location after neural tube closure. The initial group of floor plate cells acquires the inductive properties of the early notochord
(N) and provides both contactdependent
signals that recruit additional floor plate cells and diffusible signals
that induce later differentiating
motor neurons and other ventral cell
types. By this time, the notochord has lost its contact-dependent
inductive ability but retains its diffusible signaling properties, which may act
in conjunction
with signals from the floor plate. This model is based
on the results presented
in this paper and on those of Placzek et al.
(1993).
for motor neuron generation is acquired and the initial
expression of motor neuron markers such as Islet-l, SC1 ,
and ChAT.
Although our findings indicate that the specification of
motor neurons occurs within the neural plate, retroviral
lineage analysis of cells in the chick neural tube has shown
that the commitment of an individual cell to a motor neuron
fate does not occur until its penultimate or final cell division
and that clonally related cells give rise to other cell types
(Leber et al., 1990). Inductive signals from the notochord
and floor plate may therefore act on dividing neural plate
cells to confer them with the potential to generate motor
neurons, although the selection of motor neuron fate within
the clonal progeny may occur later and be influenced by
other factors.
Floor Plate and Motor Neuron Induction May Be
Mediated by Distinct Factors
The analysis of floor plate differentiation in vitro has shown
that contact is required for the induction of floor plate properties in neural plate explants (Placzeket al., 1990b, 1993).
By contrast, the present studies show that motor neurons
can be induced by a factor secreted from floor plate and
notochord. Although we cannot exclude the possibility that
a single factor is sufficient to induce both floor plate and
motor neuron differentiation, several lines of evidence indicate that the contact-mediated
and diffusible signals are
distinct. First, a concentration of the diffusible factor that
is over 1 OO-fold greater than that required for induction of
motor neurons does not induce floor plate differentiation
(Placzek et al., 1993; unpublished data). In other systems
in which different concentrations
of secreted factors induce distinct cell fates, the concentration range required
to generate a complete range of cell types appears to be
only about lo-fold. For example, in Xenopus embryos, an
- 1 O-fold difference in activin concentration is sufficient
to induce a range of ventral to dorsal mesodermal cell
markers (Green et al., 1992). Similarly, in studies on dorsoventral patterning in Drosophila embryos, an 8-fold difference in the activity of the decapentaplegic
gene is sufficient to specify the pattern of dorsal cell types (Ferguson
and Anderson, 1992). Thus, the inability to detect floor
plate-inducing
activity with a lOO-fold concentrate of conditioned medium suggests that a single factor is not sufficient to induce motor neurons and floor plate cells. It remains possible, however, that different concentrations of
the notochord- and floor plate-derived
diffusible factor induce distinct neuronal fates in the ventral neural tube.
A second lineof evidence that the two inducing activities
are distinct is that the notochord loses floor plate-inducing
activity at stage 10 (Placzek et al., 1993) whereas the
present studies show that motor neuron-inducing
activity
is retained by the notochord for a much longer period.
Third, notochord and floor plate grafting studies in vivo
have shown that neural tube cells lose the competence
for floor plate differentiation before that for motor neuron
differentiation (van Straaten et al., 1985; Yamada et al.,
1991). Finally, zebrafish cyclops embryos exhibit a normal
number of motor neurons, even though the floor plate is
absent (Hatta et al., 1991; Hatta, 1992). Since the defect
in cyclops embryos results from the inability of neural plate
cells to respond to inductive signals from the notochord,
the separation of motor neuron and floor plate induction
is most easily explained by the existence of separate signaling pathways in neural plate cells and, by inference,
theexistence of twodistinct signals. Taken together, these
observations suggest that distinct factors mediate the induction of floor plate cells and motor neurons.
Although diffusible factors can effectively induce motor
neurons, contact between the inducing tissue and intermediate neural plate explant greatly enhances motor neuron
induction. One possible explanation for this is that only a
small fraction of the notochord- and floor plate-derived
factor is free to diffuse with the remainder sequestered
by the explant, perhaps bound to extracellular matrix components. Thus, placing the floor plate or notochord explant
in contact with the intermediate neural plate explant may
present a much higher concentration of the factor. It is also
possible that the inducing factor exists in related forms that
differ in their capacity for diffusion, in a manner similar to
that described for transforming growth factor a, leukemia
inhibitor factor, and other factors (Massague, 1990; Jes-
Motor
663
Neuron
Induction
by Diffusible
Factors
sell and Melton, 1992). In addition, since contact between
the inducing tissue and intermediate neural plate explant
induces floor plate differentiation (Placzek et al., 1990b,
1993) the induced floor plate may provide an additional
source of motor neuron-inducing
activity that augments
the effects of the conditioned medium.
The range of action of the diffusible motor neuron-inducing signal in vivo remains unclear. Our results show that
motor neurons derive from cells in ventral neural plate
explants that are normally located close to the midline of
the neural plate. Thus, a signal from the notochord may
be required to diffuse only a few cell diameters to act on
the neural plate cells that give rise to motor neurons. The
subsequent migration or proliferation of cells after neural
tube closure may magnify the apparent distance required
for diffusion of this factor. Moreover, in preliminary studies
we have found that notochord and floor plate can induce
motor neurons in a transfilter induction assay under conditions in which diffusion of the factor from the inducing
tissue has to occur over - 50 pm. It seems plausible therefore that a factor from the notochord that is secreted and
freely diffusible in vitro is capable of diffusing over short
distance in vivo to influence the fate of neural plate cells.
Contributions
of the Notochord and Floor Plate
to Motor Neuron Induction
The present in vitro assays of neural cell differentiation
suggest that the induction of motor neurons in vivo is initiated directly by the notochord. Four related findings support this idea. First, notochord-conditioned
medium can
induce motor neurons under conditions in which floor plate
differentiation does not occur, providing direct evidence
that motor neuron differentiation
does not require prior
induction of a floor plate. Notochord grafting studies in
chick embryos have provided supportive, although indirect, evidence that the notochord is able to induce motor
neurons directly (van Straaten et al., 1985; Yamada et al.,
1991). Second, the present studies and those of Placzek
et al. (1993) show that floor plate and motor neuron induction in vitro require a similar period of exposure to inducing
factors. Thus, by the time that floor plate cells acquire
motor neuron-inducing
activity, many cells in the neural
plate are likely to have committed to a motor neuron fate.
Third, cells in ventral neural plate explants give rise to
motor neurons, whereas the ventral midline region of the
neural plate does not acquire floor plate properties when
placed in vitro (M. Placzek, unpublished data). Thus, the
commitment of neural plate cells to a motor neuron fate
appears to occur before commitment to a floor plate fate.
Finally, the presence of motor neurons in zebrafish cyclaps embryos lacking a floor plate (Hatta, 1992) supports
the idea that the floor plate is not required for motor neuron
induction in vivo.
The acquisition of motor neuron-inducing
signals by
floor plate cells may, however, contribute to the specification of motor neurons and other ventral neurons that are
generated at later times. Intermediate neural plate explants are still competent to respond to motor neuroninducing signals at the time the first floor plate cells have
differentiated and the notochord has become displaced
from the ventral midline of the neural tube. The protracted
period of generation of chick motor neurons (Hollyday and
Hamburger, 1977; Ericson et al., 1992) could therefore
reflect, in part, the sequential action of inducing signals
from the notochord and floor plate. A role for the floor plate
in the induction of some ventral neurons is also suggested
by the absence of y-aminobutyric acid-containing
neurons
from the ventral spinal cord of zebrafish cyclops embryos
(Bernhardt et al., 1992).
Notochord and Floor Plate Signals and
Neural Patterning
Taken together, the present in vitro studies suggest that
contact-mediated
and diffusible signals derived initially
from the notochord and later from the floor plate control
the induction of the distinct cell types that are located in
the ventral region of the spinal cord. The notochord and
floor plate extend rostrally to the midbrain (Kingsbury,
1930); thus, the same contact-mediated
and diffusible signals are likely to regulate the differentiation of ventral cell
types throughout much of the embryonic central nervous
system. However, the rostral-most region of the neural
plate that gives rise to the forebrain is refractory to floor
plate induction (Placzek et al., 1993), suggesting that the
organization of ventral cell types in prospective forebrain
regions is controlled by different signaling mechanisms.
The differentiation of cell types that derive from the dorsal region of the neural tube appears also to be independent of ventral-midline
signals (Yamadaet al., 1991). Nevertheless, the fate of at least one dorsal cell type, neural
crest cells, appears to be established within the neural
plate over the same time period as that of motor neurons.
Dorsal cell fates might depend on the presence of distinct
secreted factors in the dorsal neural tube (Wilkinson et
al., 1987a; Basler et al., 1993). The specification of cell
fate along the dorsoventral axis of the neural tube may
therefore involve secreted factors that derive from the notochord and floor plate and from the dorsal neural tube.
Experimental
Procedures
Isolation
and Culture of Neural Plate Tissue
Fertilized chick or quail eggs were incubated at 36% in a humidified
incubator.
Embryos were collected into L15 medium at 4%, and a
region of the neural plate adjacent to the segmental plate at the caudal
region of Hamburger-Hamilton
stage 10 embryos
(Hamburger
and
Hamilton, 1951) was isolated (Figure IA). The dissected
neural plate
tissue was incubated
in dispase (Boehringer
Mannheim;
1 mg/ml in
L15) at 22OC for 5-10 min to remove any adherent mesodermal
tissue,
transferred
to L15 containing
1% heat-inactivated
fetal calf serum,
and gently washed by triturating
several times with a fire-polished
Pasteur pipette. Neural plate tissue was cut into dorsal, intermediate,
ventral, and ventral midline regions (Figure 1A) with tungsten needles.
The dorsal, intermediate,
and ventral regions, each measuring
50-80
urn along the dorsoventral
axis and 300-350 urn along the rostrocaudal axis, were maintained
in vitro.
The midline of the neural plate (region f) of stage 10 embryos, the
floor plate from stages 16-35 or 16-26, and the caudal notochord from
stages lo-35 or 1 O-36 were dissected in the presence of dispase and
used as inducing tissues (Yamada et al., 1991; Placzek et al., 1993).
In some experiments,
dorsal spinal cord tissue from Hamburger-Hamilton stage 16-26 embryos and somite tissue from Hamburger-Hamilton stages lo-17 (Hamburger
and Hamilton, 1951) were used.
Neural plate explants were cultured in three-dimensional
Collagen
gels (Vitrogen 100, Celtrix Laboratories,
Palo Alto, California; Tessier-
Cdl
664
Lavigne et al., 1966) in 400 Ql of serum-free
F12 medium with NB
supplement
and antibiotics
(Tessier-Lavigne
et al., 1988; Placzek et
al., 1990a) either alone in contact with notochord
or floor plate tissue
or in conditioned
medium.
For analysis of melanocyte
differentiation,
dorsal neural plate explants were isolated from somite stage 10 quail embryos as described
for equivalent chick explants.
Chick embryo extract (10%) and fetal
calf serum (10%) were added to these cultures to permit the differentiation of neural crest cells into melanocytes
(Maxwell et al., 1988; Stocker
et al., 1991).
saint et al., 1992). RNA was purified from stage 26 chick spinal cord
with guanidine thiocynate (Promega), and 1 .O pg was used in a reverse
transcription
reaction with random hexamera and Superscript
reverse
tranacriptase
(GIBCO BRL). This cDNA product was used for PCR
with the primers S-GTN CCN ACN TAY GA and 5’-GGN ACY TGN
SWN GT, and products were subcloned
into Bluescript
II (KS) (Stratagene).
The chick ChAT cDNA clone encodes a protein fragment with 81%
identity to amino acids 440-546 of rat ChAT (Ishii et al., 1990) corresponding to the conserved
region selected for PCR amplification.
Preparation
of Notochord
and Floor
Plate-Conditioned
Medium
Localization
Rostra1 notochord tissue from stage 6-7 and caudal notochord from
stage 16-17 chick or quail embryos and floor plate tissue from stage
16-26 embryos were dissected
and treated with dispase to remove
contaminating
mesodermal
cells. The mass of stage 6-7 notochord
tissue used to prepare conditioned
medium was about I/5 that of floor
plate tissue. The mass of stage 16-17 notochord
was equivalent to
that of floor plate tissue. Floor plate and notochord tissue was plated
overnight
in uncoated
35 mm culture dishes (Nunc) in N,supplemented F12 medium containing 10% heat-inactivated
fetal calf serum
to permit recovery
of the tissue and attachment
to the culture dish.
Approximately
30 notochord explants were cultured in 1 ml of serumfree medium for 46 hr. The conditioned
medium was removed and
centrifuged
(103 rpm for 5 min at 4OC) to remove cell debris. Conditioned medium was stored at 4OC or -60°C
and concentrated
on a
Centricon
ultrafiltration
membrane
(Amicon) (molecular
size cut off
was 3 kd).
To obtain floor plate-conditioned
medium, 30 floor plates were dissected from stage 25-26 chick embryos and placed in 1 ml of medium.
The mass of the floor plate tissue isolated from each embryo was - 10
times that of the explant used in conjugate experiments.
The medium
was collected after 48 hr of conditioning
and used in induction assays.
Thus, on the assumption
that the rate of secretion of inducing activity
is constant over 48 hr, the floor plate-conditioned
medium is expected
to contain an -240-fold
greater concentration
of inducing activity than
that of medium conditioned
for 24 hr by a single floor plate explant
of the size used in conjugate experiments.
Detectlon
of Neural
Markers
of ChAT
mRNA
Localization
of ChAT mRNA was performed
by in situ hybridization
on stage 20-35 chick embryos
essentially
as described
previously
(Wilkinson et al., 1987b; Klar et al., 1992) using a chick ChAT probe.
A labeled single-stranded
RNA probe complementary
to ChAT mRNA
was transcribed
using T7 RNA polymeraae in the presence of PSJUTP.
Paraffin sectionsof
chick spinal cord were labeled with antisense RNA,
coated with Kodak NTB2 emulsion, and exposed for 2-3 weeks.
ChAT mRNA was first detected in the region of differentiating
motor
neurons at stage 22. Dorsal root ganglion neurons did not express
ChAT mRNA at any embryonic
stage examined
up to stage 35.
Quantltatlon
of ChAT
mRNA
A PCR-based
assay was used to measure ChAT mRNA levels in neural
plate explants grown in vitro for 72 hr. RNA was extracted
from 5-10
collagen-embedded
neural plate explants and purified with 50 jrl of
guanidine thiocynate,
with 5 pg of transfer RNA (tRNA) added as carrier. An internal standard for competitive
PCR analysis of ChAT mRNA
was prepared by subcloning
a 200 bp SaulllAl fragment of Bluescript
into a Bglll site within the chick ChAT sequence. This linearized plasmid was transcribed
in vitro with T3 RNA polymerase
to make sensestrand RNA containing
an insert between ChAT sequences.
Approximately
1 .O fg of internal standard
RNA was added to the
explantderived
RNA before reverse transcription
with random hexamers. This cDNA was amplified by PCR for 18 cycles with heat-stable
Tli DNA polymerase
(Promega) using primers (5’-TCC ATA CGC CGA
TTT GAT GAG GGC and 5%TA TTG CTT GTC AAA TAG GTC TCA)
that flank the insert position of the internal standard
RNA. The PCR
products from chick ChAT mRNA (320 nt) and from the internal standard (520 nt) were resolved on 2% agarose gels and detected
by
Southern hybridization
with a radiolabeled
ChAT probe. Chick genomic DNA did not give a PCR product under these conditions.
Each
experiment
included both a titration to ensure that the amplification
of ChAT sequences
was within the linear range and a negative control
that omitted reverse transcriptase.
Islet-l was detected with rabbit antibodies (Thor et al., 1991; Ericson
et al., 1992) the SC1 glycoprotein
with MAb SC1 (Tanaka and Obata,
1984) and neuronal cell bodies and axons with MAb 3AlO (Furley et
al., 1990; Tanaka et al., 1989). The chick ~75 protein was detected
with MAb 7412. In some experiments,
stage 10-l 7quail notochord and
stage 16-26 floor plate were used as inducing tissues to distinguish
the responding
chick neural tissue. Chick cells were identified by a
chick-specific
SC1 MAb (Yamada et al., 1991; Tanaka et al., 1990)
and quail cells by MAb QCPN (generated by 8. Carlson and J. Carlson;
available from the Developmental
Studies Hybridoma
Bank).
Neural plate explants were fixed with 4% paraformaldehyde
at 4OC
for l-2 hr, washed with phosphate-buffered
saline (pH 7.4) at 4°C for
l-2 hr, and peeled from the bottom of the dish, and the excess collagen
get was trimmed. Explants were incubated with primary antibodies
overnight at 4OC with gentle agitation. After washing with phoaphatebuffered saline for 2 hr at 22OC, neural plate explants were incubated
with fluorescein
iaothiocyanate-conjugated
goat anti-mouse
immunoglobulin G (Boehringer
Mannheim) or Texas red-conjugated
goatantirabbit immunoglobulin
G (Molecular
Probes) for l-2 hr with gentle
agitation at 22°C. The explants were then washed
in phoaphatebuffered saline at 22“C for 2 hr with at least two changes of buffer
and mounted on slides in 50% glycerol containing
paraphenylene
diamine (1 mglml). Explants were examined on a Zeiss Axiophot microscope equipped with epifluorescence
optics. Double labeling with Islet-1 and SC1 antibodies
was analyzed
using a Bio-Rad MRC-500
confocal microscope.
Acknowledgments
laolatlon of the Chick ChAT Gene
A 331 nt fragment of the chick ChAT gene was cloned using degenerate polymerase
chain reaction (PCR) primers directed
against sequences encoding conserved
regions of the ChAT protein, predicted
from cDNA clones isolated from other species (Ishii et al., 1990; Tous-
We thank Monica Ensini for help in cloning of chick ChAT cDNA,
Mark Baldassare
for help with in situ hybridization,
Marysia Placzek
for making available unpublished
data, and John Kuwada for preprints.
David Anderson
provided valuable advice on neural crest cells. We
are also grateful to Richard Axel, Jane Dodd, Monica Ensini, Marysia
Determlnatlon
of Cell Number
In Neural
Plate Explants
To determine cell number, neural plate explants were grown in vitro,
fixed in 4% paraformaldehyde,
and embedded
in paraffin, and serial
6 Qm sections were cut on a microtome.
Sections were stained with
cresyl violet to visualize nuclei, and the number of cells in each explant
was determined
from the number of nuclei. The number of cells in
intermediate
neural plate explants at the time of isolation from stage
10 embryos was 641 + 108 (mean f SEM; n = 3 explants).
After
48 hr in vitro, intermediate
neural plate explants grown alone contained
766 f 101 cells (mean f SEM; n = 6 explants).
Intermediate
neural
plate explants grown for 48 hr in the presence
of 1 x floor plateconditioned
medium contained
1468 rt 281 cells (mean f SEM;
n = 4 explants). An approximately
Bfold increase in cell number was
also detected after 48 hr when intermediate
neural plate explants were
grown in contact with notochord or floor plate explants, although the
estimate of these numbers was complicated
by the difficulty in distinguishing accurately
cells in the intermediate
neural plate explant and
in the inducing tissue.
Motor
685
Neuron
Induction
by Diffusible
Factors
Placzek, and Ariel Ruiz i Altaba for criticism of the manuscript,
to Ira
Schieren for help with figures, to Vicki Leon for typing the manuscript,
and to Eric Hubel for photography.
Antibodies to the chick p75 protein
were kindly provided by Hideaki Tanaka. T. M. J. is an investigator
and S. L. P. a Research
Associate
of the Howard Hughes Medical
Institute. T. Y. is supported
by the Muscular
Dystrophy
Association.
T. E. is supported
by grants from the Swedish National Science Research Council and the Swedish Medical Research
Council.
the control
128.
Received
Kingsbury,
B. F. (1930). The developmental
significance
of the floorplate of the brain and spinal cord. J. Comp. Neural. 50, 177-207.
January
26, 1993;
revised
March
10, 1993
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