Dorsal Differentiation of Neural Plate Cells Induced by BMP-Mediated Signals from Epidermal Ectoderm

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Karel F. Liem, Jr., Gabi Tremml, cells) are able to differentiate in the absence of notochord- Henk Roelink, and Thomas M. Jessell derived signals (Yamada et al., 1991 ; Ericson et al., 1992; Howard Hughes Medical Institute G. T. et al., unpublished data). Moreover, in the absence Department of Biochemistry and Molecular Biophysics of the notochord, certain genes that are normally restricted Center for Neurobiology and Behavior to dorsal regions of the neural tube are expressed at all Columbia University dorsoventral levels (Yamada et al., 1991; Basler et al., New York, New York 10032 1993; Goulding et al., 1993). These observations raise the issue of how the dorsal fates of neural plate cells are acquired. One possibility is that neural plate cells are pre- Summary disposed to differentiate into dorsal cell types unless exposed to a ventralizing signal from the notochord. Alterna- The cellular interactions that control the differentia- tively, the acquisition of dorsal fates might require the tion of dorsal cell types from neural progenitors have action of inductive signals that originate from adjacent tis- been examined in neural plate explants. Certain genes sues. Evidence for the existence of dorsalizing signals has that are expressed in the dorsal neural tube are initially derived from the analysis of neural crest cell differentia- expressed uniformly within the neural plate and ap- tion. Epidermal ectoderm cells that flank the neural plate pear to achieve their dorsal restriction through a Sonic and mesodermal cells that underlie the lateral border of hedgehog (SHH)-mediated repressive signal from the the neural plate have each been proposed as sources of notochord. The acquisition of definitive dorsal cell signals that induce neural crest cells (Moury and Jacob- fates, however, requires a contact-dependent signal son, 1989, 1990; Takada et al., 1994; Dickinson et al., from the epidermal ectoderm. BMP4 and BMP7 are 1995; Selleck and Bronner-Fraser, 1995; Mayor et al., expressed in the epidermal ectoderm, and both pro- 1995; de la Torre and Tessier-Lavigne, personal communi- teins mimic its inductive activity. BMP4 and a related cation). Neural crest cells can be induced in vitro by expo- gene, DSL1, are subsequently expressed by cells in sure of neural plate explants to dorsalin-1 (DSL1), a TGF~- the dorsal neural tube. The differentiation of dorsal related factor (Kingsley, 1994) expressed in the dorsal cell types, therefore, appears to be initiated at the neu- region of the neural tube (Basler et al., 1993). DSL1 is, ral plate stage and to involve the opponent activities of however, not expressed in the epidermal ectoderm and a BMP-mediated dorsalizing signal from the epidermal appears in the neural tube only after neural crest cells ectoderm and a SHH-mediated ventralizing signal from have been specified (Basler et al., 1993; Nieto et al., 1994; the notochord. Nakagawa and Takeichi, 1995), indicating that DSL1 is not involved in the initial steps of neural crest cell differentiation. Thus, the cellular interactions that initiate the dorsal Introduction differentiation of neural plate cells and the molecular identity of relevant inducing factors remain uncertain. The diverse neuronal and glial cell types generated during In the present studies, we have analyzed the interac- the development of the vertebrate nervous system derive tions that specify the dorsal fate of neural plate cells by from a simple columnar epithelium, the neural plate. The using an in vitro assay of cell differentiation in neural plate differentiation of distinct cell types from neural plate pro- explants. We first examined whether neural plate cells are genitors is thought to be controlled by the actions of se- predisposed to dorsal fates or whether inductive signals creted inductive factors (Smith, 1994; Johnson and Tabin, from adjacent cells are required. Our results show that 1995). Cell types generated from the medial region of neu- certain genes that characterize the dorsal neural tube are ral plate (notably floor plate cells and motor neurons) popu- initially expressed by all neural plate cells and achieve late the ventral half of the neural tube and are induced by their dorsal restriction through a SHH-mediated repressive Sonic hedgehog (SHH), a secreted glycoprotein that is signal from the notochord. The acquistion of definitive dor- synthesized by axial mesodermal cells of the notochord sal cell fates, however, does not occur by default and in- (Echelard et al., 1993; Krauss et al., 1993; Ericson et al., stead involves a contact-dependent inductive signal from 1995; Marti et al., 1995; Roelink et al., 1994, 1995; Tanabe the epidermal ectoderm. Two members of the TGF~ gene et al., 1995). Elimination of the notochord prevents the family, BMP4 and BMP7 (encoding bone morphogenetic differentiation of floor plate cells and motor neurons (van proteins 4 and 7), are expressed in the epidermal ectoderm Straaten and Hekking, 1991 ; Yamada et al., 1991; Ericson flanking the neural plate, and recombinant BMP4 and et al., 1992), establishing that a signal from the notochord, BMP7 mimic the dorsalizing activity of the epidermal ecto- presumably SHH, is required for the differentiation of ven- derm. The acquisition of dorsal neural fates is, therefore, tral cell types. initiated at the neural plate stage and appears to involve Cell types derived from the lateral region of the neural the opponent activities of a BMP-mediated dorsalizing sig- plate that populate the dorsal half of the neural tube (neural nal from the epidermal ectoderm and a SHH-mediated crest cells, dorsal commissural neurons, and roof plate ventralizing signal from the notochord. Cell 970

[ pax-3 ]1 msx-1 II msx 1/2 II sluq I m np~ HHm /1/ p Figure 1. Expressionof PAX3 and MSXl and SLUG in the Neural Plate and NeuralTube Panels show the distribution of PAX3 (A-D), MSXI (E-H), MSX proteins(I-L), and SLUG protein (M-P) in the neural plate and neural tube of stage 10 chick embryos. (A, E, I, and M) Sectionsthrough the neural plate rostral to Hensen's node. Expressionof PAX3 (A), MSXl (E), and MSX proteins (I) in neural plate cells. SLUG is not expressed in the neural plate at this level (M). (B, F, J, and N) Sectionsthrough a more rostral level of the neural plate. Expressionof PAX3 (B), MSXl (F), and MSX proteins(J) is not detectable in cells at the midlineof the neural plate. SLUG is expressed by cells in the lateral region of the neural plate (N). (C, G, K, and O) Sectionsthrough the neural fold. Expressionof PAX3 (C), MSXl (G), and MSX proteins(K) is restricted to the dorsal regionof the neural folds. SLUG (O) is expressedby dorsal cells. (D, H, L, and P) Sectionsthrough the neural tube. Expressionof PAX3 (D), MSXl (H), MSX proteins(L), and SLUG (P) is restricted to the dorsal neural tube. Scale bar, 100 pro.

Results the differentiation of neural plate cells, irrespective of their eventual dorsoventral fate. Molecular Markers of Dorsal Cell Differentiation The early extinction in expression of these two genes We determined how dorsal fates are acquired by neural from the midline of the neural plate suggests that signals plate cells by analyzing the expression in situ and then in from the notochord are responsible for their repression. vitro of four genes expressed by cells in the dorsal half In support of this, notochord grafts repress the dorsal ex- of the neural tube: MSXl, PAX3, DSL1, and SLUG. pression of PAX3 in the neural tube in vivo (Goulding et We examined the pattern of neural expression of PAX3 al., 1993) and repress PAX3 and MSX expression in vitro and MSXl at caudal levels of stage 10 chick embryos. In (see below). Conversely, notochord removal results in ex- the newly formed neural plate, cells at all mediolateral pression of PAX3 in the ventral neural tube (Goulding et al., positions expressed PAX3 mRNA, MSXl mRNA, and 1993). Thus, the expression of PAX3 and MSX by dorsal MSXI/MSX2 proteins (termed MSX)(Figures 1A, 1E, neural tube cells appears to be acquired by default, in the and 11). At more rostral levels at which the neural plate sense that these genes are initially expressed uniformly had begun to fold, PAX3 and MSX were not expressed within the neural plate and are subsequently repressed medially (Figures 1B, 1F, and 1J). At a level just caudal from prospective ventral regions by notochord-derived to the point of neural tube closure, the expression of PAX3 signals. and MSX was restricted to the most lateral, prospective In contrast with PAX3 and MSX, DSL1 expression was dorsal, region of the neural folds (Figures 1C, 1G, and not detected in neural plate cells (Figure 2A), appearing 1K). Consistent with previous observations (Goulding et dorsally only after neural tube closure (Basler et al., 1993). al., 1993; Robert et al., 1991 ; Takahashi et al., 1992), PAX3 Thus, DSL1 expression is associated with the differentia- and MSX were restricted to dorsal regions of the closed tion of cells in the dorsal neural tube but does not appear neural tube (Figures 1 D, 1 H, and 1L). Thus, the expression to define a specific dorsal cell type. of PAX3 and MSX appears to delineate an early stage in The SLUG protein was not expressed by early neural BMPs Dorsalize Neural Plate Cells 971

A Expression of pax3 and dsl B Induction by ectoderm C Induction by BMPs D Repression by notochord and SHH

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Figure 2. RT-PCR Analysis of PAX3, DSL1, and $17 Expression in Neural Plate Explants In all lanes, upper bands (C) indicate competitive RNA templates; lower bands, tissue-derived transcripts. Grouped lanes are from the same experiment. The chick $17 gene was used as an internal control for the amount of tissue. In (C), BMP-4 and BMP7 were added in the form of COS cell conditioned medium diluted 1:2 in F12 medium. DSL1 was added at 3 x 10 " M. In (D), conditioned medium containing amino-terminal SHH was added at a concentration of -10 ~ M. Abbreviations: v, ventral neural plate explant; d, dorsal neural plate explant; SHH, Sonic hedgehog; n, notochord; ect, epidermal ectoderm; DSL1, dorsalin-l. Numbers above lanes indicate culture time, in hours. Each lane is representative of at least three different experiments. plate cells (Figure 1M; Nieto et al., 1994) and appears in tral, intermediate, and dorsal regions of the neural plate cells in the extreme lateral region of the neural plate only isolated from the caudal region of stage 10 chick embryos after it has begun to fold (Figures 1N and 10). After neural (a level similar to that shown in Figures 1 B, 1F, and 1J). tube closure, SLUG + cells were found at the most dorsal Ventral neural plate explants'examined at the time of extreme of the neural tube (Figure 1P) and define a single isolation (data not shown) and after 18 hr in culture ex- dorsal cell type, premigratory neural crest cells (Nieto et pressed few, if any, MSX ÷ cells (Figure 3A) but did express al., 1994). low levels of PAX3 (see Figure 2A). The absence of expression of MSX suggested that cells in ventral neural plate Dorsal Fates of Neural Plate Cells Grown In Vitro explants have been exposed to a notochord-derived signal With these four genes as markers, we examined the differ- at the time of isolation. Consistent with this, cells in ventral entiation of cells in explants derived from prospective ven- neural plate explants give rise to motor neurons when

Figure 3. MSX, SLUG, and HNK1 Expression I (vj II m II rdl I in Neural Plate Explants MSX and SLUG expression was assayed after 18 hr and migratory HNK1 ÷ cells after 40 hr. (A-C) Ventral neural plate explants express few if any MSX÷ cells (2 _+ 1 cells per section, mean _+ SEM, n = 6) (A). MSX is expressed by over 90% of cells in intermediate neural plate explants (61 _+ 3 cells per section, mean _ SEM, n = 6) (B) and dorsal neural plate explants (68 _+ 5 cells per section, n = 6) (C). (D-F) SLUG + cells are absent from ventral (D) and intermediate (E) but present in dorsal (F) neural plate explants. Sections of dorsal neural plate explants contained 39 4- 4 SLUG + cells per section (n = 10). (G-I) Migratory HNK1 ÷ cells are absent from ventral (G) and intermediate (H) but present in dorsal (I) neural plate explants (56 _+ 6 migratory HNK1 -~ cells per explant; n = 24). SLUG expression was not detected in migratory HNK1 + cells (data not shown). I [i]+notochord II II [i] + as SHH I (J) Neural cells in a conjugate of intermediate neural plate explant and notochord (n) do not express MSX. The notochord explant is detected with MAb Not-l. (K) No MSX ÷ cells are detected in intermediate neural plate explants grown in vitro on COS cells transfected with sense rat Shh. (L) MSX is expressed by most cells in intermediate neural plate explants grown in vitro on COS cells transfected with antisense rat Shh. Similar results were obtained in at least 12 explants. Scale bar in (A)-(F) and (J)-(L), 80 ~m; in (G)-(I), 100 pm. Cell 972

grown alone in vitro (Yamada et al., 1993). Although ven- plants, however, did contain SLUG + cells (Figure 3F) and tral neural plate explants appear to have been exposed to gave rise to migratory HNK1 + cells (Figure 31), suggesting notochord-derived signals, DSL 1 expression was detected that cells in dorsal neural plate explants have been ex- at low levels in these explants after 18 hr in vitro (see Figure posed to dorsalizing signals at the time of their isolation. 2A). This finding, together with the ventral expression of Taken together, this analysis of cell differentiation in DSL1 after elimination of the notochord (Basler et al., neural plate explants suggests that certain genes charac- 1993), suggests that the continued presence of notochord- teristic of dorsal neural tube cells (PAX3, MSX) are ac- derived signals is necessary to repress DSL expression quired by default but that the acquisition of distinct dorsal in ventral regions of the neural tube. Thus, DSL1 may also cell fates requires additional inductive signals. achieve its dorsally restricted expression in the neural tube through inhibition of its expression ventrally. No SLUG + Epidermal Ectoderm Is the Source cells were detected in ventral neural plate explants (Figure of a Dorsalizing Signal 3D), and after 40 hr, migratory neural crest cells, as de- To define the source of dorsalizing signals, we examined fined by HNK1 + expression, were not detected (Figure 3G). whether the dorsal differentiation of neural plate cells Thus, cells in ventral neural plate explants that have been could be induced by tissues adjacent to the neural plate. exposed to notochord-derived signals do not give rise to We focused on epidermal ectoderm and paraxial meso- definitive dorsal cell types. derm, since these tissues have been implicated in the In intermediate neural plate explants examined at the differentiation of neural crest cells. Ventral neural plate time of isolation (data not shown) and after 18 hr, virtually explants were grown in contact with epidermal ectoderm all cells expressed MSX (Figure 3B). PAX3 expression was or paraxial rnesoderm derived from stage 10 quail or chick also detected (see Figure 2A). The detection of MSX sug- embryos and assayed for the expression of SLUG and gests that cells in intermediate neural plate explants have HN K1 + migratory cells. In addition, we assayed expression not been exposed to notochord-derived signals at the time of MSX (and DSL1 and PAX3) to determine whether induc- of isolation. To examine whether MSX expression does tive signals could also overcome an earlier repressive in- indeed provide a sensitive indicator of the exposure of fluence of the notochord. neural plate cells to notochord-derived signals, intermedi- Ventral or intermediate neural plate explants grown for ate neural plate explants were grown for 18 hr in contact 18 hr in contact with epidermal ectoderm contained nu- with notochord. The expression of MSX by neural cells was merous SLUG + cells (Figures 4C and 4D; data not shown), repressed over a distance of >100 I~m from the junction of and by 40 hr, many HNK1 + neural crest cells had migrated the explants (Figure 3J; data not shown). MSX expression from the explants (Figures 4G and 4J). MSX + cells were was also repressed when neural plate explants were grown detected in the region of the explant closest to the ecto- transfilter to a notochord explant (data not shown), provid- derm (Figures 4A and 4B), and high levels of PAX3 and ing evidence that the repressive effect of the notochord DSL1 were also induced (see Figure 2B). In contrast, the is mediated by a diffusible factor. We tested whether SHH induction of dorsal markers was not detected when neural mimics the notochord-derived factor by growing intermedi- plate explants were grown at a distance from epidermal ate neural plate explants on COS cells transfected with ectoderm (data not shown). Moreover, no induction of dor- Shh. MSX expression was repressed under these condi- sal markers was obtained in ventral neural plate explants tions (Figures 3K and 3L), suggesting that SHH mediates grown in contact with paraxial mesoderm (data not shown). the long-range notochord-derived repression of MSX de- These results show that the epidermal ectoderm is the tected in vitro and inferred in vivo. The detection of MSX source of a contact-dependent signal that can induce the in intermediate neural plate explants therefore supports differentiation of neural crest cells in vitro, consistent with the idea that, in vivo, cells in these explants have not been other studies (Dickinson et al., 1995; Selleck and Bronner- exposed to notochord-derived signals at the time of iso- Fraser, 1995). They also show that epidermal ectoderm lation. can overcome an earlier repressive influence of the noto- Although DSL1 was not detected in intermediate neural chord. plate explants at the time of their isolation, the gene was expressed after 18 hr in vitro (see Figure 2A). More infor- BMPs as Mediators of Epidermal matively, intermediate neural plate explants did not con- Ectoderm-Derived Signals tain SLUG + cells or give rise to migratory HNK1 ÷ cells The ability of epidermal ectoderm to induce dorsal cell (Figures 3E and 3H). Similarly, the differentiation of a sub- differentiation in ventral neural plate explants sewed as set of dorsal commissural neurons defined by expression the basis of an assay to identify ectodermally derived fac- of the LIM homeodomain protein LH-2 did not occur (G. T. tors that dorsalize ventral neural plate cells. Although not et al., unpublished data). These results provide evidence expressed in the epidermal ectoderm, DSL1 induces neu- that definitive dorsal cell types do not differentiate simply ral crest cells (Basler et al., 1993). We therefore examined as a consequence of isolating neural plate cells from the whether members of the TGF~ family that are related influence of notochord-derived signals. structurally to DSL1 are expressed in the epidermal ecto- Dorsal neural plate explants examined after 18 hr in derm at the time that dorsalization of the neural plate is culture contained MSX + cells (Figure 3C) and expressed thought to occur. high levels of PAX3 and DSL1 (see Figure 2A). These ex- Degenerate polymerase chain reaction (PCR) primers BMPs Dorsalize Neural Plate Cells 973

[ [v] ]1 [v] * ectoderm I I BMP.4 II BMP-7 I II A ~i;i !~':~ ~ ~ ~~ ~•~!:

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Figure 4. Induction of MSX, SLUG, and HNK1 ÷ Cells by Epidermal Ectoderm (A) Section through a ventral neural plate explant grown in culture for 18 hr. No MSX- cells are detected• (B) Section through a ventral neural plate explant grown for 18 hr in contact with epidermal ectoderm isolated from a stage 10 quail embryo. The section was labeled with antibodies directed against MSX (nuclear) and the quail-specific perinuclear marker QCPN. The border between the quail ectoderm (ect) and chick neural plate tissue is marked with arrowheads. MSX + cells (55 ± 8 cells per section; n = Figure 5. Expression of BMP4 and BMP7 in Epidermal Ectoderm and 3) are detected in the chick neural plate explant close to the border Dorsal Neural Tube with the quail ectoderm. The distribution of BMP4 and BMP7 was determined by in situ hybrid- (C) Ventral neural plate explant grown alone. No SLUG + cells are de- ization analysis of stage 10 chick embryos. tected. (A and B) Sections of the neural plate at a level rostral to Hensen's (D) Ventral neural plate explant grown in contact with stage 10 quail node. BMP4 (A) and BMP7 (B) are expressed in the epidermal ecto- epidermal ectoderm, labeled with anti-MSX and QCPN antibodies. derm adjacent to the neural plate. No expression is detected in the SLUG + cells (16 ± 3 cells per section, n = 8) are induced. The junction neural plate. of the neural and ectodermal (ect) explants is shown by arrowheads. (C and D) Sections of the neural plate at a more rostral level, showing Ectodermal tissue is located close to the region of SLUG + cells. a restriction in BMP4 expression (C) to the ectoderm flanking the neural (E and H) Ventral neural plate explant grown alone in culture for 40 plate and to the dorsal folds of the neural plate. BMP7 expression (D) hr. Cells in the explant express HNK1, but there are no migratory is maintained in the epidermal ectoderm. HNK1 + cells• (E and F) Sections through the neural tube showing high levels of (F and I) Chick epidermal ectodermal explant grown alone in culture BMP4 expression (E) in the dorsal midline of the neural tube and in for 40 hr. No HNK1 expression is detected. overlying midline ectoderm. BMP7 expression (F) has disappeared (G and J) Conjugate of ventral neural plate and epidermal ectoderm from the epidermal ectoderm but is expressed at low levels in the explants, grown in culture for 40 hr. HNK1 ÷ cells (38 ± 5 cells per dorsal neural tube. explant; n = 10) have migrated from the neural plate explant. (G and H) Sections through the neural tube at prospective forebrain Images are representative of at least ten explants. level, showing BMP4 expression (G) by cells at the dorsal midline of Scale bar in (A)-(D), 30 pm; in (E)-(J), 100 ~m. the neural tube but not in the epidermal ectoderm. BMP7 is expressed (H) at high levels in the epidermal ectoderm but not in the neural tube. Scale bar in (A)-(D), 80 p.m; in (E)-(H), 100 pro. were used to isolate DSLl-related genes expressed in the region of the epidermal ectoderm that flanks the neural plate in stage 10 chick embryos. Of thirteen PCR products genes were not detected in the epidermal ectoderm by cloned, three encoded BMP4 (Francis et al., 1994), one reverse transcription-PCR (RT-PCR). BMP5 (Kingsley, 1994), and nine BMP7 (Houston et al., Epidermal ectodermal cells flanking the caudal neural 1994). cDNAs encoding chick BMP4, BMP5, and BMP7 plate expressed both BMP4 and BMP7 but not BMP2, were then used to determine the patterns of expression BMP5, or BMP6 (Figures 5A and 5B; data not shown). of those genes in stage 10 chick embryos. We also ana- BMP4 and BMP7 expression was lost from the epidermal lyzed the expression of BMP2 and BMP6, although these ectoderm at the level of neural tube closure (Figures 5C Cell 974

I msx II slug II .NK-1 I Figure 6. Induction of MSX, SLUG, and HNK1÷ Cells by BMPs (A-C) Ventral neural plate explants exposed to medium from COS cells transfected with a truncated DSL1 construct neither contain MSX÷ cells (A) or SLUG + cells (B) nor give rise to migratory HNK1 + cells (C). (D-F) Ventral neural plate explants grown in medium derived from BMP4-transfected COS cells contain MSX+ cells (71 _+ 5 cells per section, n = 6) (D) and SLUG+ cells (26 _ 2 cells per section, n = 6) (E) and give rise to migratory HNK1÷ cells (49 _ 5 cells per explant; n = 10) (F). (G-I) Ventral neural plate explants grown in medium derived from BMP7-transfected COS cells contain MSX- cells (100 _+ 6 cells per section; n = 6) (G) and SLUG + cells (18 _+ 3 cells per section; n = 10) (H) and give rise to migratory HNK1 + cells (88 _.+ 15 cells per explant; n = 4) (I). (J-L) Ventral neural plate explants grown in the presence of 3 x 10 11 M DSL1 contain MSX÷ cells (86 _+ 3 cells per section; n = 5) (J) and SLUG + cells (33 - 2 cells per section; n = 7) (K) and give rise to migratory HNK1÷ cells (65 _ 20 cells per explant; n = 5) (L). Each image is representative of at least four explants. Scale bar in (A)-(K), 80 #m; in (L), 130 p.m.

and 5F), with the exception that BMP4 expression per- bility that neurally expressed BMPs are induced by 8MP- sisted in epidermal ectodermal cells above the dorsal mid- mediated signals from the epidermal ectoderm. To test line of the caudal neural tube (Figures 5C and 5E). At this, we examined whether DSL1 is induced in ventral neu- prospective midbrain and forebrain levels of the neural ral plate explants by BMP4 or BMP7. Explants exposed tube, BMP7 expression was maintained at high levels in to BMP4 or BMP7 were induced to express high levels of the epidermal ectoderm (Figure 5H). BMP4 was also ex- DSL1 (see Figure 2C). Moreover, DSL1 (3 x 10 -11 M) mim- pressed by cells in the dorsal folds of the neural plate and ics the ability of BMP4 and BMP7 to enhance PAX3 and subsequently at high levels by cells at the dorsal midline DSL1 expression and to induce MSX ÷ cells, SLUG + cells, of the neural tube (Figures 5C, 5E, and 5G). BMP7 was and the emigration of HNK1 + cells in ventral neural plate also expressed, albeit at much lower levels, by cells in the explants (see Figure 2C; Figure 6, lanes J-L). Thus, BMPs dorsal region of the caudal neural tube (Figure 5F; data expressed by dorsal neural cells appear to provide a sec- not shown). ondary source of dorsalizing signals that might operate at The pattern of expression of BMP4 and BMP7 raised atime when the epidermal ectoderm is no longer in contact the possibility that these two proteins mediate the ability with the neural epithelium. of the epidermal ectoderm to initiate dorsal cell differentiation in neural plate cells. To test this, cDNA-derived Opponent Actions of Dorsalizing expression vectors encoding BMP4 and BMP7 were and Ventralizing Signals transfected into COS cells. Medium from BMP4- or BMP7- The long-range repression of MSX expression detected transfected COS cells enhanced the expression of PAX3 in vitro and inferred in vivo suggests that a ventralizing and DSL1 and induced MSX ÷ cells, SLUG + cells, and mi- signal from the notochord might normally block the spread gratory HNK1 ÷ cells in ventral neural plate explants (see of BMP-mediated dorsalizing signals within the neural Figure 2C; Figures 6D-61). Medium derived from un- tube. We therefore examined whether the acquisition of transfected COS cells or cells that had been transfected dorsal cell fates in dorsal neural plate explants could be with a truncated DSL1 cDNA did not induce any of these repressed by the notochord. Dorsal neural plate explants markers (see Figure 2C; Figures 6A-6C). Thus, BMP4 grown in contact with notochord expressed few, if any, and BMP7 mimicked the ability of epidermal ectoderm to MSX + cells (Figure 7A) and exhibited markedly reduced induce or elevate the expression of markers of dorsal neu- levels of PAX3 and DSL1 (see Figure 2D). In addition, the ral tube cells and to promote the differentiation of neural number of SLUG + cells was reduced by 90% (Figure 7B), crest cells. and the number of migratory HNK1 + cells was reduced by The expression of BMP4, BMP7, and DSL1 by cells in 80% (Figure 7C). Similarly, exposure of dorsal neural plate the dorsal region of the neural tube and the induction of explants to SHH almost completely eliminated MSX, DSL 1 expression by epidermal ectoderm raised the possi- PAX& and DSL7 expression (see Figures 2D and 7D) and BMPs Dorsalize Neural Plate Cells 975

I m,x II ,lug II HNK-1 I Figure 7. Inductive Activities of Epidermal Ec- toderm and BMPs Oppose Those of Notochord and SHH (A-C) Dorsal neural plate explants grown for 18 hr in contact with notochord (n) contain few MSX÷ cells (A). Dorsal neural plate explants grown in contact with notochord contain few SLUG+ cells (4 ± 2 cells per section; n = 10) (B) and at 40 hr gave rise to few migra- toryHNK1 + cells (11 -,- 6 cells per section; n = 9) (C). (D-F) Dorsal neural plate explants grown in the presence of medium containing the amino- terminal cleavage product of SHH (-10 8 M) for 18 hr did not contain MSX÷ cells (D), contained few SLUG+ cells (1 _ 0.3 cells per section; n = 10)(E), and gave rise at 40 hr to few migratory HNK1+ cells (16 ± 4 cells per explant; n = 10) (F). (G) Intermediate neural plate explants grown for 18 hr in contact with notochord (n) and quail epidermal ectoderm (ect). MSX÷ cells (22 ± 4 cells per section; n = 3) are detected in the neural plate explant close to the epidermal ectoderm. Ectodermal cells are labeled by QCPN. The notochord explant (n) is labeled with Not-l. (H) Dorsal neural plate explants grown for 18 hr in contact with notochord (n) and quail epidermal ectoderm. SLUG+ cells (16 ± 4 cells per section; n = 6) are detected in the neural plate explant close to the ectoderm. The border between the ectodermal and neural explants is shown by arrowheads. (I) Dorsal neural plate explant grown for 40 hr in contact with notochord (n) and chick epidermal ectoderm shows migratory HNK1+ cells (45 ± 11 cells per explant; n = 7). (J) Ventral neural plate explant grown for 18 hr in contact with notochord in the presence of medium from BMP4-transfected COS cells (1:2 dilution). MSX÷ cells (84 ± 10 cells per section; n = 4) are detected in the region of the explant farthest from the notochord. (K) Ventral neural plate explant grown for 18 hr in contact with notochord in the presence of medium from BMP4-transfected COS cells (1:2 dilution). Most SLUG+ cells (34 ± 10 cells per section; n = 4) are detected in the region of the explant farthest from the notochord~ (L) Ventral neural plate explant grown for 40 hr in contact with notochord (n) in the presence of BMP4. Numerous HNK1+ cells (45 ± 11 cells per explant, n = 7) have migrated from the explant on the side farthest from the notochord (n). Images are representative of four to twelve explants. Scale bar, 80 pm.

reduced by 96% the number of SLUG + cells (Figure 7E) mal ectoderm was -4-fold greater that that found in ex- and by 72% the number of migratory HNK1 ÷ cells (Figure plants grown in contact with the notochord alone (Figures 7F). Although some neural crest cells were detected in 7H-71). Moreover, virtually all SLUG + cells were located the presence of ventralizing signals (see also Artinger and close to the ectoderm. The epidermal ectoderm was also Bronner-Fraser, 1992), these results show that the major- able to maintain MSX expression locally in neural plate ity of cells in lateral regions of the neural plate are not explants grown in contact with the notochord (Figure 7G). committed to dorsal fates prior to neural tube closure. The Signals from the epidermal ectoderm may, therefore, nor- repression of an ongoing program of dorsal cell differentia- mally ensure dorsal cell differentiation by counteracting, tion by a SHH-mediated signal from the notochord sup- locally, a long-range ventralizing influence of the noto- ports the idea that an equivalent activity normally restricts chord. the domain of dorsal cell differentiation within the neural Finally, we tested whether the ability of the epidermal tube. ectoderm to maintain dorsal cell fates in the presence of The progressive spread of this long-range ventralizing ventralizing signals from the notochord is mimicked by signal from the notochord could eventually influence the BMPs. Ventral neural plate explants grown in contact with entire neural plate. The expression of dorsal cell properties the notochord but in t~le presence of BMP4 contained in lateral regions of the neural plate might therefore result MSX ÷ cells, SLUG + cells, and migratory HNK1 + cells (Fig- from the ability of signals from the epidermal ectoderm ures 7J-7L; data not shown) and exhibited elevated levels to maintain dorsal markers in cells that are exposed to of PAX3 and DSL1 (data not shown). Under these condi- notochord-derived signals. To address this issue, dorsal tions, the expression of SLUG and MSX was largely re- neural plate explants were grown in vitro, flanked on one stricted to neural plate cells located at a distance of >- 50 side by notochord and on the other byepidermal ectoderm. i~m from the notochord (Figures 7J and 7K), suggesting The number of SLUG + cells and migratory HNK1 + cells that in the vicinity of the notochord, cell fate is still domi- detected in explants grown with the notochord and epider- nated by ventralizing signals. These results suggest that Cell 976

BMPs mediate the ability of the epidermal ectoderm to plate cells. Similarly, our studies show that even though maintain dorsal cell fates in the presence of notochord- DSL1 can induce dorsal cell types, its expression by cells derived signals. in intermediate neural plate explants is insufficient to promote their differentiation. This might be because the level Discussion of DSL1 expressed is below the threshold for induction of dorsal cell types. In addition, the competence of neural These studies have examined the cellular interactions that plate cells to respond to inductive signals is lost rapidly control the differentiation of cell types generated in the (Yamada et al., 1991, 1993; Placzek et al., 1993; K. L., dorsal region of the neural tube. Our results provide evi- unpublished data); thus, cells may have lost the compe- dence that neural plate cells acquire dorsal cell fates in tence to respond to DSL1 by the time that it is expressed. part through the maintenance of genes expressed throughout the neural plate at earlier stages and in part as a re- BMPs as Dorsalizing Signals from the sponse to localized inductive signals. They also establish Epidermal Ectoderm three points about the origin and nature of these signals. The present findings, taken together with other studies First, the epidermal ectoderm that flanks the lateral border on neural crest cells (Moury and Jacobson, 1989, 1990; of the neural plate represents a source of signals that dor- Dickinson et al., 1995; Selleck and Bronner-Fraser, 1995) salizes neural plate cells. Second, the TGFI3-1ike mole- provide evidence that the epidermal ectoderm is the cules BMP4 and BMP7 are expressed in the epidermal source of signals that induce dorsal cell differentiation in ectoderm, and both proteins mimic its dorsalizing activity. lateral regions of the neural plate. The local action of these Third, BMP-mediated signals from the epidermal ecto- dorsalizing signals is supported by the early lateral restric- derm can ensure the differentiation of dorsal cell types tion in expression of the dorsal markers SLUG (Figure 1, by opposing the actions of a long-range SHH-mediated Nieto et al., 1994), cadherin 6B (Nakagawa and Takeichi, ventralizing signal from the notochord. These findings sug- 1995), and BMP4 within the neural plate. The action of gest that acquisition of dorsal cell properties by neural signals from the epidermal ectoderm might underlie the plate cells is dependent on the opponent activities of BMPs rapid generation of neural crest cells in the ventral half of from the epidermal ectoderm and SHH from the noto- the neural tube that is observed after excision of dorsal chord. neural tube at cranial levels (Scherson et al., 1993). The notochord has also been implicated in the ventrali- The route by which ectodermal signals are transmitted zation of paraxial mesoderm (Pourquie et al., 1993; Brand- to lateral neural plate cells has not been resolved. The Saberi et al., 1993; Fan and Tessier-Lavigne, 1994)through epidermal ectoderm and neural plate are initially contigu- an activity that appears to be mediated by SHH (Johnson ous; thus, a dorsalizing signal could be transmitted through et al., 1994; Fan and Tessier-Lavigne, 1994; Fan et al., the plane of the epithelium. However, during the folding 1995). Moreover, the epidermal ectoderm is the source of the neural plate, the basal surface of the ectoderm con- of an as yet unidentified signal that dorsalizes the paraxial tacts the lateral, prospective dorsal region of the neural mesoderm (Fan and Tessier-Lavigne, 1994). Thus, the es- plate (Martins-Green, 1988), providing an extended inter- tablishment of dorsoventral pattern within the neural plate face for the transmission of ectodermally derived signals. and paraxial mesoderm appears to be achieved through The major support for the idea that BMPs mediate the a common cellular strategy and, at least in part, through dorsal inductive activity of the epidermal ectoderm derives the same inductive factors. from two observations. First, two members of this family, BMP4 and BMP7, are expressed in the surface ectoderm The Early Character of Neural Plate Cells at the time that the neural plate is formed. BMP4 and BMP7 The possibility that the acquisition of dorsal fates repre- are also expressed in the surface ectoderm in other verte- sents a default state in the differentiation of neural plate brate embryos (Jones et al., 1991 ; Lyons et al., 1995; Fain- cells was raised by the observation that elimination of the sod et al., 1994). Second, BMP4 and BMP7 mimic the notochord not only failed to inhibit the differentiation of ability of the epidermal ectoderm to dorsalize neural plate dorsal cell types but also resulted in the expression of cells. Additional TGFI3-1ike molecules could contribute to certain dorsal markers throughout the entire dorsoventral the inductive activity of the epidermal ectoderm. However, axis of the neural tube (Yamada et al., 1991; Goulding et numerous other factors, including epidermal growth fac- al., 1993; Basler et al., 1993). The present in vitro assays tor, fibroblast growth factors, neurotrophins, and Wntl, provide evidence that cells in neural plate explants that do not mimic the ability of BMPs to induce dorsal markers have not been exposed to ventralizing signals do acquire (K. L. and H. R., unpublished data). Thus, BMPs currently several dorsal characteristics yet fail to differentiate into represent the sole candidates for mediators of ectoder- definitive dorsal cell types. Thus, definitive dorsal fates mally derived dorsalizing signals. Nevertheless, the re- are not acquired by default. quirement for BMPs in the dorsalization of neural plate PAX3 and MSX1 are required for the differentiation of cells remains to be demonstrated. neural crest cells and their derivatives (Stuart et al., 1994; Although the initial dorsalizing influence of the epider- Satokata and Maas, 1994), but the present results suggest mal ectoderm appears to be a local event, BMP4, DSL1, that expression of these two transcription factors is not and low levels of BMP7 appear to be induced in neural sufficient to confer definitive dorsal identities upon neural cells as a component of the program of dorsal cell differen- BMPs DorsalizeNeural Plate Cells 977

tiation. An initial short-range dorsalizing signal from the range SHH-mediated signal that spreads through the neu- epidermal ectoderm is likely, therefore, to be propagated ral plate over time. within the neural plate and neural tube through the actions Although notochord-derived signals and SHH can sup- of BMP4, BMP7, DSL1, and possibly other BMPs. This press dorsal cell differentiation, DSL1 and BMP4 can, con- secondary source of BMPs may be important in promoting versely, suppress the differentiation of ventral cell types the differentiation of dorsal cell types that are generated (Basler et al., 1993). Thus, the fate of early neural plate at later times, after the epidermal ectoderm loses contact cells is likely to depend on whether they are exposed to with the dorsal neural tube. The transfer of dorsalizing BMPs or to SHH, on the concentration of these factors, signals from the epidermal ectoderm to the dorsal midline and on the time of their exposu re to them. In medial regions of the neural tube is similar in principle to the strategy of the neural plate, SHH-mediated signals appear domi- used to perpetuate ventralizing signals through their trans- nant, whereas in lateral regions, the influence of BMPs fer from the notochord to the floor plate (Yamada et al,, prevails. Cells that differentiate in the intermediate region 1991; Placzek et al., 1993; Marti et al., 1995). of the neural plate exhibit distinct molecular properties The present results, together with studies on dorsal (Rangini et al., 1991; Lu et al., 1992; Zimmerman et al., commissural neurons (G. T. et al., unpublished data), sug- 1993). How such intermediate cell fates are established gest that BMP-mediated signals can induce many or all remains unclear. definitive dorsal cell types. Roof plate cells, neural crest cells, and commissural neurons are generated at distinct Experimental Procedures positions in the dorsal half of the neural tube, raising the issue of whether the concentration of BMP to which a neu- cDNA Clones ral plate cell is exposed defines its specific fate. The ex- PAX3 (Gouldinget el., 1993), MSX2 (Takahashiet al., 1992; Yokouchi et al., 1991), SLUG (Nieto et al., 1994), and s17 (Trueb et al., 1988) pression of several BMPs in the epidermal ectoderm and sequences were isolated by RT-PCR. BMP2, BMP4, BMP5, BMP6, in nested dorsal domains of the neural tube leaves open and BMP7 sequences were isolated by RT-PCR using degenerate the additional possibility that the formation of BMP hetero- primers(Basler et al., 1993). PCR fragmentswere usedto isolatecDNA dimers confers qualitatively or quantitatively distinct induc- clones encoding BMP4, BMP5, and BMP7. Chick BMP2 and BMP4 cDNAs were provided by P. Brickell (Francis et al., 1994), a BMP6 tive activities, through actions on subclasses of BMP re- cDNA by C. Hume, and a PAX3 cDNA by M. Goulding.A BMP4 cDNA ceptors (Massague et al., 1994). was provided by R. Derynck, and a human BMP7cDNA by M. Jones The response of neural cells to BMPs varies at different and A. Furley. rostrocaudal levels of the neural tube. At spinal cord levels, BMPs promote neural crest cell differentiation, whereas Antibodies Monoclonal antibody (MAb) 4G1 recognizesMSXl and MSX2. SLUG in the hindbrain, prospective neural crest cells in odd- proteinwas detectedwith a mouseserum antibody. MAb QCPN (Devel- numbered rhombomeres are induced to undergo apop- opmental Studies Hybridoma Bank) detects quail perinuclear anti- tosis in response to BMP4 (Graham et al., 1994). Thus, gens; MAb Not-1 (Yamadaet al., 1991) detectsnotochord, MAb HNK1 an early restriction in the rostrocaudal identity of neural identifies migrating neural crest cells (Tucker et al., 1984). plate cells appears to define the nature of their response Immunocytochemistry to both dorsalizing and ventralizing (Ericson et al., 1995) Immunocytochemicaldetection of antigens in tissue sectionsand neu- inductive signals. Components of the response of neural ral plate explants was performedas described(Yamada et al., 1993). cells to BMPs may, however, be conserved. Induction of MSX gene expression is observed in response to BMP4 In Situ Hybridization Whole-mount in situ hybridization was performed with digoxigenin- at both spinal cord and hindbrain levels (Graham et al., labeled probes essentially as described (Ericson et al., 1995). 1994). In addition, MSX gene expression can be induced by BMP4 in mesenchymal cells (Vainio et al., 1993). Competitive PCR Analysis PCR analysis was performedas described(Tanabe et al., 1995). De- tails are available on request. Opponent Actions of BMPs and SHH The present results, taken together with studies on ventral COS Cell Transfections cell specification (see Smith, 1994; Johnson and Tabin, COS cells were transfectedwith lipofectamine(GIBCO BRL) (Roelink 1995), suggest that the patterning of the neural plate de- et al., 1994) with BMP4 (in pMT 21), BMP7 (in pcDNA), or DSL1 (in pMT 21). Expressionconstructs encodingthe full-lengthSHH protein pends on the combined actions of a dorsalizing signal from (Roelink et al., 1994) or its amino-terminalcleavage product (Porter the epidermal ectoderm and a ventralizing signal from the et al., 1995) were transfected into COS cells (Roelink et al., 1994, notochord. The ventralizing activity of SHH is likely to rep- 1995). resent a major factor in confering the dorsal restriction in Neural Plate Assays expression of MSX, PAX3, and DSL1 and in limiting the Notochord and ventral, intermediate,or dorsal neural plate explants domain of the neural tube within which the differentiation were dissectedfrom the caudal region of stage 10 (Hamburgerand of definitive dorsal cell types can occur. It is possible, Hamilton, 1951) chick embryos(Yamada et al., 1993). Epidermalecto- therefore, that the induction of ventral cell types by SHH derm tissue was dissected from an area lateral to the neural plate at caudal levels of stage 10 chick embryos, unsegmented paraxial requires the repression of genes such as MSX1 and PAX3. mesodermfrom a region caudalto the first somite. Conjugatesformed In addition, the maintenance of dorsal cell differentiation between notochord, epidermal ectoderm, or paraxial mesodermand in lateral regions of the neural plate might depend upon neural plate were cultured essentially as described (Yarnadaet al., the ability of ectodermally derived BMPs to oppose a long- 1993). Cell 978

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