Control of cell pattern in the neural tube: Motor neuron induction by diffusible factors from notochord and floor plate

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

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vi

Assay

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Motor

Neuron

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(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

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Induction

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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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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

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(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

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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

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(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

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Competence of Neural Plate Cells to Generate Motor Neurons The midline of the neural plate is not effective at inducing

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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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receptors

on

Bernhardt, R. R., Patel, C. K., Wilson, S. W., and Kuwada, J. Y. (1992). Axonal trajectories and distribution of GABAergic spinal neurons in wildtype and mutant zebrafish lacking floor plate cells. J. Comp. Neurol. 326, 263-272. Bovolenta, P., and Dodd, J. (1991). Perturbation of neuronal differentiation and axon guidance in the spinal cord of mouse embryos lacking a floor plate: analysis of Danforth’s short-tail mutation. Development 7 73,625-639. Chou, K. H., Llyas, A. A., Evans, J. E., Quarles, R. H., and Jungalwala, F. B. (1985). Structure of a glycolipid reading with monoclonal IgM in neuropathy and with HNK-1. Biochem. Biophys. Res. Commun. 728, 383. Clarke, J. D. W., Holder, N., Soffe, S. R., and Storm-Mathissen, J. (1991). Neuroanatomical and functional analysis of neural tube formation in notochordless Xenopus embryos: laterality of the ventral spinal cord is lost. Development 172, 499-516. Delannet, M., and Duband, J.-L. (1992). Transforming growth factor-p control of cell-substratum adhesion during avian neural crest cell emigration in vitro. Development 776, 275-287.

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Klar, A., Baldassare, M., and Jessell, T. M. (1992). F-spondin: a gene expressed at high levels in the floor plate encodes a secreted protein that promotes neural cell adhesion and neurite extension. Cell 69,95110. Langman. J.. Guerrant, of neuroepithelial cells Neural. 727, 399.

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Analysis of crest using

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