sign in sign up
<<
Home , Cranial neural crest, Neural crest, Neural fold, Neural plate, Neurula

Development 121, 767-777 (1995) 767 Printed in Great Britain © The Company of Biologists Limited 1995

Induction of the prospective of

Roberto Mayor2, Richard Morgan1 and Michael G. Sargent1,* 1Laboratory of , National Institute for Medical Research, The Ridgeway, Mill Hill, London, NW7 1AA, UK 2Departemento de Biologia, Universidad de Chile, Casilla, 653, Santiago, Chile *Author for correspondence

SUMMARY

The earliest sign of the prospective neural crest of Xenopus with another signal that is present throughout the ventral is the expression of the ectodermal component of Xsna (the side of the and (b) that Xslu is unable to express in Xenopus homologue of snail) in a low arc on the dorsal the either because of the absence of a co- aspect of stage 11 , which subsequently assumes inducer or by a positive prohibition of expression. The the horseshoe shape characteristic of the neural folds as the ventral co-inducer, in the presence of overexpressed convergence-extension movements shape the neural plate. , seems to generate an anterior/posterior pattern in A related zinc-finger gene called Slug (Xslu) is expressed the ventral part of the embryo comparable to that seen in specifically in this (i.e. the prospective crest) when neural crest of normal embryos. We suggest that the the convergence extension movements are completed. Sub- prospective neural crest is induced in normal embryos in sequently, Xslu is found in pre- and post-migratory cranial the that overlies the junction of the domains that and and also in lateral plate express noggin and Xwnt-8. In support of this, we show after stage 17. Both Xslu and Xsna are induced by animal cap explants from blastulae and gastrulae, treated mesoderm from the dorsal or lateral marginal zone but not with bFGF and noggin express Xslu but not NCAM from the ventral marginal zone. From stage 10.5, explants although the mesoderm marker Xbra is also expressed. of the prospective neural crest, which is underlain with Explants treated with noggin alone express NCAM only. tissue, are able to express Xslu. However expression of Xsna An indication that induction of the neural plate border is is not apparently specified until stage 12 and further regulated independently of the neural plate is obtained contact with the inducer is required to raise the level of from experiments using ultraviolet irradiation in the pre- expression to that seen later in development. Xslu is period. At certain doses, the cranial crest domains specified at a later time. Embryos injected with noggin are not separated into lateral masses and there is a mRNA at the 1- stage or with plasmids driving noggin reduction in the size of the neural plate. expression after the start of zygotic transcription express Xslu in a ring surrounding the embryo on the ventroposte- Key words: neural crest, neural folds, neural plate border, bFGF, rior side. We suggest this indicates (a) that noggin interacts noggin, NCAM, Xbra, Xslu, Xsna, Xwnt-8, Xenopus, ultraviolet

INTRODUCTION (Schroeder, 1970). The premigratory cephalic crest is typically massive compared with the trunk crest and never comes to lie The emergence of neural crest in the evolution of on top of the (Sadaghiani and Thiebaud, 1987). In is of special importance because of its supposed role in facili- Anurans, unlike Urodeles and all other vertebrates, there is a tating cephalisation (Gans and Northcott, 1983). Substantial two-layered neurepithelium. The prospective neural crest is differences distinguish the organisation of neural crest in the derived from the deep and superficial layers of the ectoderm major classes of vertebrates although the presumptive neural flanking the neural plate although only the latter contributes to crest is always derived from a tissue at the edge of the neural the roof of the neural tube (Schroeder, 1970; Essex et al., plate. Thus in the mouse, neural crest cells are found delami- 1993). nating from the neuroepithelium before the neural tube is We have recently shown that Xsna is expressed ectodermally closed and are only briefly part of an (Nieto et al., in the band of cells that surrounds the prospective neural plate 1992), whereas in the chick a premigratory form of neural crest that includes the prospective neural crest and the anterior exists in the most dorsal part of the closed neural tube. Dorsal (transverse) that we have termed the neural plate neural tube cells can become neurones in the peripheral or border (Essex et al., 1993; Mayor et al., 1993). Changes in the central and furthermore, if this tissue is expression pattern of Xsna reflect the dynamics of ablated, the adjoining tissue is able to reconstitute the neural in a striking way. Xsna expression in the neural plate border crest (Scherson et al., 1993). In amphibia, the neural folds mark first appears as a low arc on the dorsal aspect of the embryo at the edge of the neural plate and contain the premigratory crest stage 11, when the convergence-extension movements that

768 R. Mayor, R. Morgan and M. G. Sargent shape the neural plate are at an early stage. The appearance of development without effect on the blue colour, by placing them in ectodermal Xsna in a distinct domain of prospective neural fold hydrogen peroxide, formamide, SSC (1%, 5%, 0.5× respectively) over before neural plate formation is complete, indicates that a fluorescent light for about 1 hour. In some cases, specimens were induction of the border should be considered independently of cleared with tetrahydronaphthol to obtain better photographic images. formation of the neural plate. Some stained embryos were sectioned in wax and counterstained with Pioneering studies on Urodeles suggested that induction of eosin for microscopic examination. In situ hybridisation has been used to demonstrate Xslu in conjugates so that the number of expressing the neural crest could be mediated by underlying tissues, inde- conjugates can be determined. pendently of the neural plate. The roof underlying the neural folds was able to induce neural crest derivatives Embryos and explants principally, whereas the medial part of the archenteron roof Xenopus embryos were obtained as described previously (Mayor et tended to induce neural plate and less neural crest (Raven and al., 1993) and staged according to Nieuwkoop and Faber (1967). Kloos, 1945). More recently, the capacity to induce Animal caps, dissected at stage 8 or stage 10.5, were cultured in 1× melanophores (a derivative of neural crest) without neurulation NAM (Slack, 1984) supplemented with FGF or noggin protein, as by co-culture of ventral ectoderm and lateral mesoderm but not indicated, until the equivalent of stage 17. dorsal mesoderm has been demonstrated (Mitani and Okamoto, Preparation of fate map of stage 10+ embryos 1991). The induction of neural crest Xtwi by mesoderm has Dots of Nile Blue (one per embryo) were made on the surface of stage been demonstrated (Hopwood et al., 1989). In contrast, other 10+ Xenopus embryos by applying the dye for a few seconds, using investigators have argued that the signals passing through the a glass microneedle, under the superficial layer of the ectoderm using plane of the ectoderm induce neural folds and placodes as its a micromanipulator. In most cases, the vitelline membranes were still potency is reduced below a critical level (Albers, 1987) or present but a similar result was obtained using demembranated when qualitative changes occur in the competence of ectoderm embryos. Embryos were orientated in a well to apply the dye. The to respond (Servetnick and Grainger, 1991). Neural fold and positions of these dots were recorded, using a camera lucida, and neural crest, derived from ectodermal or neurectodermal plotted as described by Keller (1975) and their subsequent fate was material, respectively, are induced when neural plate and determined by examining embryos at stages between 12 and 25. ectoderm are juxtaposed experimentally (Moury and Jacobson, To mark the prospective neural plate border for specification exper- 1990). Two recent investigations suggest the position of the iments, the animal caps of the embryos were stained to include the prospective folds only. This was done using a grid of precisely neural plate border may be regulated by the balance of two machined holes, which were filled with Nile Blue. Stage 10 embryos opposing inducing signals that emanate from the neural plate were inverted into these wells so that their caps were stained. The size and ectoderm (Coffman et al., 1993; Zhang and Jacobson, of the holes used were chosen so that about 1/3 of the embryo was 1993). immersed in the dye, to cover the prospective neural folds. The dorsal Neural crest induction has often been assessed using half of these caps was dissected from embryos between stage 10 and melanophores as a marker although these are expressed a con- 12, for the specification experiments described below. Embryos were siderable time after the primary inductive event. In this inves- microinjected as described previously (Mayor et al., 1993). Nile Blue, tigation, we have used molecular markers (Xsna and Xslu) that in Danilchiks buffer (Keller et al., 1985) (5-10 nl), was injected into are expressed early to study induction of the prospective neural the blastocoels of embryos in 5% Ficoll in 0.75 NAM. crest. Ultraviolet irradiation Fertilised eggs to be irradiated were dejellied immediately after rotation and were exposed to 254 nm ultraviolet light in a silica cell MATERIALS AND METHODS for 2-3 minutes at 40 minutes after (Youn and Malacin- ski, 1981). At stage 10, embryos were sorted into fast and slow devel- Isolation of Xslu oping types. As noted by Cooke (1985), the latter class is highly A stage 17 Xenopus cDNA library in lambda GT10 was screened enriched with embryos with reduced dorsoanterior development. (Kintner and Melton, 1987) using a slug clone (Nieto et al., 1994) as a probe. Inserts of these clones were subcloned into pSP72 Expression constructs (Kreig and Melton, 1987) and were sequenced by the double stranded Full-length capped transcripts of Xwnt-8 and noggin were prepared dideoxy procedure. The sequence has been deposited with EMBL by standard methods or provided generously by Dr Vincent Cunliffe. (Accession number X80269). pCSKA-Xwnt-8 and the noggin expression clones were the gift of Dr Richard Harland. Synthetic RNA was injected in 5-10 nl volumes as Whole-mount in situ hybridisation described previously (Mayor et al., 1993). noggin protein was Digoxigenin containing RNA probes were prepared from the Xslu obtained by injecting oocytes with a plasmid (500 pg per oocyte) that cDNA sequence. The sequences from 968 to 1701 were used to transcribes noggin from the TFIIIA (base pairs 40-355, Pfaff prepare sense and antisense probes. This sequence has no similarity et al.,1990). Oocytes were injected in 5% Ficoll in 0.8 NAM and were to the Xsna sequence and would not be expected to hybridise with cultured at 18¡C in 0.8× NAM containing 1 mg/ml of bovine serum Xsna sequences. Sense transcripts consistently gave no staining. Other albumin for 3 days after injection. The concentration of noggin was expression patterns were obtained using antisense transcripts of expressed in arbitrary units. NCAM was induced in stage 8 caps by sequences 1200-1800 and 0-723 of Xsna and NCAM, respectively. 10 u/ml. Specimens were prepared, hybridised and stained by the method of Harland (1991). We found that after a 1 hour fixation, proteinase K Nuclease protection studies and RNAse treatment were unnecessary as neither treatment improved RNAse protection probes were made as described by Kreig and the result and mesodermal signals such as Xsna were not adversely Melton, (1987). Solution hybridisation of five probes to the same affected (Essex et al., 1993). After colour development, embryos were sample (at 50¡C, for 16 hours in 50% formamide and 0.23 M NaCl) washed in methanol for 1 hour, to reduce background and enhance was performed to minimise differences in recovery of the mRNA. The blue colour. Some stained specimens was bleached, after colour probes used were Xslu bp 189-469, Xsna bp 138-369, NCAM bp 523- Induction of prospective neural crest 769

723, Xbra 278-377, EF1a bp 122-186; accession numbers, X80269, the deep layer (Fig. 2B) as we noted previously with Xsna X53450, M25696, M77243 and M25504, respectively. UTP was used expression (Essex et al., 1993). By stage 16 (Fig. 2C), Xslu − − at a specific activity of 800 µCi nmole 1 (or160 µCi nmole 1 for EF1a). expression was clearly organised into the characteristic prem- Unhybridised RNA was digested with RNAse A and RNAse T1. igratory aggregates reported by Sadaghiani and Thiebaud (1987). At stage 18 (Fig. 2D), the neural tube was closed in the trunk and the cranial crest was in condensed masses. Xslu- RESULTS expressing cells were dispersed over the neural plate in a pattern surrounding the . At stage 22 (Fig. 2E), Isolation of Xslu, a cDNA related to Xsna that is neural crest derivatives such as the branchial arches and the expressed specifically in premigratory neural crest tissue surrounding the eyes and forebrain expressed Xslu. A stage 17 Xenopus cDNA library of Kintner and Melton Further to the posterior, migrating cells expressing Xslu (1987) was screened using a chicken Slug cDNA as a probe demarcated rhombomeres. After stage 17, there was weak (Nieto et al., 1994). Isolates were screened for sequences that expression in which increased at stage differed from Xsna, by a PCR sequencing protocol using DNA 26 but was down-regulated in the pronephros region (Fig. 2F) from purified lambda clones and a primer within the zinc finger at stage 26. region. A novel sequence was found in a number of separate Expression of Xslu in the deep and superficial layer of the isolates. The deduced amino acid sequence of this protein (267 ectoderm was seen in transverse sections through the cephalic amino-acids) has high similarity to Xsna over the first 40 crest of stage 18 embryos (Fig. 2G). In the trunk, Xslu amino-acids (90%) and the last 123 aminoacids (94%) which expression was in the deep layer of the ectoderm, on the top contain five zinc fingers (Fig. 1). The long 3′ untranslated of the neural folds (Fig. 2H). A parasagittal section of a stage sequence, which has no similarity to that of Xsna, has been 20 embryo shows Xslu in three premigratory masses in the used as an in situ hybridisation probe and the middle of the deep layer of the ectoderm (Fig. 2I). No mesodermal staining coding sequence has been used to prepare RNAse protection was evident in sections. probes. Both Xsna and Xslu are significantly more similar to escargot, a recently discovered family member from The origin of the prospective neural fold, Drosophila (Whitely et al., 1992) (79 and 80% respectively) relationships with underlying tissues and in the zinc finger region than to Drosophila snail (66 and 69% expression of Xslu in dorsal marginal zone explants respectively) but there are no significant similarities between The neural plate border originates from a low arc of tissue just the non-finger regions of the and the invertebrate above the marginal zone of early gastrulae, which develops members of the family. The amino-acid sequence of Slug in into the characteristically keyhole shaped neural folds as a Xenopus and chicken is very well conserved (92% identical) result of convergence-extension movements within the neural compared to conservation of snail between the two species plate. We have determined from where the neural plate border (data not shown). is derived in a stage 10.25 embryo, using dye marking (Fig. Whole-mount in situ hybridisation showed Xslu was first 3A). This information facilitates dissection of ectodermal expressed on the lateral aspect of stage 12 embryos (Fig. 2A), regions that will always contain prospective neural folds and in a region in which ectodermal Xsna was expressed (Essex et which can be cultured and tested for their capacity to express al., 1993; Mayor et al., 1993). RNAse protection assays of Xslu neural fold markers. The prospective folds at stage 10.25 lie indicated that there was very little, or no mRNA for Xslu on an arc on the dorsal marginal zone, midway between that present before stage 11.5 (data not shown). At stage 14 (Fig. described by Keller (1975) and Suzuki and Harada (1988). The 2B), the lateral neural folds were strongly labelled and the angle subtended by the dorsal blastopore lip and the point on transverse fold unlabelled while the trunk folds were distinctly the dorsal midline that passes through the neural plate border but weakly labelled. The less strongly labelled superficial was about 70-90¡. ectoderm contribution to the cranial folds was seen medial to As the prospective neural plate border was derived from a narrow strip of the dorsal marginal zone, it was of interest to know if the prospective folds were underlain by tissue at this time and if this tissue was the inducer. The first question was investigated by injecting the with Nile Blue at various stages between stage 8 and stage 11 and determining whether stage 16 folds were stained. The prospective neural folds were stained blue by the contents of the blastocoel until stage 10.5, but thereafter the neural folds were unstained. This indicates that after stage 10.5 the prospective folds were underlain with tissue. To determine whether this underlying tissue was the inducer, we dissected the marginal zone (including tissue underlying the ectoderm) into equal-sized upper and lower strips as shown in Fig. 3B at stage 10.25. These explants were cultured until stage 13 and were examined by in situ hybridisation for Xslu. The upper dorsal marginal zone contains that part of the ectoderm Fig. 1. Sequence alignment of Xslu and Xsna. Start of the five zinc that can form the neural folds and express Xslu (Fig. 3C), and fingers shown by bold numbers. appears almost the same as that found in entire dorsal marginal 770 R. Mayor, R. Morgan and M. G. Sargent

Fig. 2. Expression pattern of Xslu in Xenopus embryos obtained using whole mount in situ hybridisation. (A) Stage 12, Xslu on lateral aspect of embryo (c), (b) blastopore. (B) Stage 14, Xslu in prospective (d, deep layer of ectoderm; s, superficial). Weak expression in trunk crest. (C) Stage 16, Xslu in cranial premigratory crest (Sadaghiani and Thiebaud, 1987). d, deep layer of ectoderm; s, superficial layer Xslu expression extending over the neural plate. (D) Stage 18. Neural tube now closed with cranial crest masses condensed (m, h and b, mandibular, hyoid and branchial aggregates. Xslu expression over neural plate demarcating rhombomeres. t, trunk neural crest. (E) Stage 22, Xslu in trunk crest (t), branchial arches and, in surrounding the eyes (e) and forebrain, Xslu seems to demarcate rhombomeres. (F) Stage 25, Xslu in branchial arches (m, mandibular; h, hyoid and b, branchial), in lateral plate mesoderm (lp) with pronephros not expressing (p). (G) Transverse section through cephalic crest of stage 18 embryo. Xslu expression in deep layer (d) and superficial layer (s). np, neural plate; n, ; so, ; a, archenteron. (H) Transverse section through trunk of stage 18 embryo. Xslu expression on top of neural folds in deep layer (d); nt, neural tube. (I) Parasagittal section of stage 20 embryo showing Xslu in three premigratory masses (m, mandibular; h, hyoid and b, branchial). zone explants (Fig. 3E). The lower part (principally the invo- could only be cleanly separated from mesoderm up to stage 12. luting marginal zone) did not express Xslu (Fig. 3D) and Explants prepared at stage 10 and 11 were unable to express neither did the remainder of the embryo (Fig. 3F). Xsna when cultured to the equivalent of stage 16, whereas explants prepared at stage 12 did express Xsna (Fig. 4A-C). Time of commitment to expression of Xsna and Xslu However, this level of expression was evidently established at We next investigated when ectodermal Xsna and Xslu could be stage 12 because, if these explants were fixed at stage 12, the expressed autonomously, by dissecting explants containing the level of Xsna expression was the same (Fig. 4D); this indicates prospective neural folds at stage 10-12 and culturing them until that further contact with the inducer is required to raise the stage 16 and scoring the appearance of Xsna and Xslu using in level of expression to that seen later in development. Similar situ hybridisation (Fig. 4). To mark the prospective neural plate experiments were also carried out for Xslu expression. border, the animal caps of the embryos were stained with Nile Prospective folds dissected at stages 10, 11 and 12, cultured to Blue so that the same region was used at each time point. This stage 16 did not express Xslu (Fig. 4E-G). However, prospec- was done by staining the top 1/3 of each embryo, as described tive folds, dissected at stage 12 with a small amount of the above, at stage 10. At each time point, the labelled cap was adjoining section of mesoderm, expressed Xslu strongly at dissected from the embryo, freed of mesoderm and the dorsal stage 16 (Fig. 4H). This suggests that, although the prospec- half was used for the specification experiments. Ectoderm tive crest ectoderm is in contact with its inducer from stage

Induction of prospective neural crest 771

10.5, the tissue is unable to express Xsna and Xslu not significantly in the mesoderm before stage 19, it is possible autonomously for a surprisingly long time. to devise a simple assay for induction of Xslu using a conjugate containing any tissue under test and the blastocoel roof of stage Location of Xslu-inducing activity within the embryo 10.25 embryos as the responding tissue. Conjugates prepared at stage 10.5 with these pieces were cultured to stage 17 in 0.75× NAM, by As Xslu is expressed only in a specific part of the ectoderm and which time expression would have occurred in control

Fig. 3. Fate map of stage 10.25 embryos and expression of Xslu in dorsal marginal zone explants. (A) Fate map of stage 10.25 embryos. Each spot represents dye marks applied to embryo at this stage. Fate of each spot is ᭹, neural folds; ᭺, ; ᭪, eye lens; x, neural plate. a, V, D and b = Animal pole, vegetal pole, dorsal and blastopore respectively. (114 embryos analysed). (B) Dissection of UDMZ (A) and LDMZ (B). Both explants were cut as strips extending round 180¡ of the embryo as shown; m indicates height of the floor of the blastocoel. C-F were hybridised with Xslu (C) UDMZ (A) (D) LDMZ (B) (E) Complete DMZ (A)+(B) (F) Remainder of embryo (C). 772 R. Mayor, R. Morgan and M. G. Sargent XSna XSlu

Fig. 4. Time of commitment to Xslu and Xsna expression. Animal cap explants containing the prospective neural crest, dissected at stages shown and cultured until the equivalent of stage 16. A-D stained with Xsna (122 caps analysed), E-H stained with Xslu (245 caps analysed). (Arrows show expression). (A-C) Prospective folds dissected at stages 10, 11 and 12, cultured to equivalent of stage 16. (D) The prospective folds dissected at stage 12 and fixed immediately. (E-G) Prospective folds dissected at stages 10, 11 and 12, cultured to equivalent of stage 16. (H) Prospective folds dissected at stage 12 with a small amount of mesoderm (m) attached cultured to equivalent of stage 16. embryos. The conjugates were then fixed and examined by the ventral explants did not (Fig. 5F). One of the ventral whole-mount in situ hybridisation using an Xslu antisense explant conjugates expressed Xslu (Fig. 5F arrow) (32 probe. The responding tissue alone (animal caps) (Fig. 5A) did analyzed), but was evidently incorrectly dissected as it had not express Xslu but in a conjugate with a very small piece of elongated. These experiments therefore indicate that the dorsal marginal zone containing the organizer, Xslu expression capacity to induce Xslu resides in dorsal and lateral mesoderm was observed (Fig. 5B) while the organiser alone gave no Xslu but not ventral mesoderm. expression (Fig. 5C). In a similar experiment, the mesoderm of the marginal zone of stage 10.5 embryos was dissected as Effect of noggin and Xwnt-8 mRNA on Xslu one piece and then divided into four, the dorsal, ventral and expression two lateral pieces which were tested in conjugates. Dorsal and noggin and Xwnt-8 have reciprocal expression patterns in lateral mesoderm fragments induced Xslu (Fig. 5D,E) while mesoderm at stage 10, with noggin (Smith and Harland, 1992) ventral mesoderm gave a low response (Fig. 5F). The appear- in the dorsal quadrant and Xwnt-8 (Christian and Moon, 1993) ance of the conjugates at stage 17 indicates that the dorsal and in the remainder. As the earliest expression of ectodermal Xsna ventral regions were correctly dissected as, conjugates con- seemed to overlie the junction of these two domains (Essex et taining dorsal and lateral explants (Fig. 5D,E) elongated while al., 1993), we decided to examine the effect of overexpression

Fig. 5. Induction of Xslu in stage 10.25 animal caps by mesoderm measured by in situ hybridisation. (A) Animal caps alone (43 caps analysed). (B) Organiser in conjugate with stage 10.25 ectoderm (32 conjugates analysed). (C) Organiser alone (12). (D-F) Dorsal, lateral and ventral marginal zone respectively in conjugate with stage 10.25 ectoderm (41 conjugates analysed). (Arrows show expression.) Induction of prospective neural crest 773

Fig. 6. Effect of overexpression of Xwnt-8 and noggin on Xslu. (A) Embryo viewed from ventral aspect at equivalent of stage 13 injected with noggin RNA (100 pg) at 1 cell stage. (52 embryos). (B) Embryo viewed from posterior side, at equivalent of stage 17 injected with noggin RNA (5 ng) at 1 cell stage. Normal cephalic Xslu expression on lateral aspect extended in three banded pattern to ventral side (v). Microscopic examination of these embryos indicates a three-banded structure unambiguously (64 embryos). (C) Embryo viewed from lateral aspect at equivalent of stage 16 injected with pCSKA-noggin DNA (100 pg) at 1 cell stage (16 embryos). (D) Group of embryos at equivalent of stage 13 injected with Xwnt-8 RNA at 1 cell stage. Note greatly reduced expression (32 embryos). of synthetic RNA of these two genes on the expression of Xslu by a pair of factors derived from the dorsal and ventral side of using whole-mount in situ hybridisation, as this marker is the embryo, was investigated by examining the effect of specific for prospective neural crest. Embryos obtained from noggin and bFGF proteins on expression of Xslu and markers eggs injected in the animal cap with noggin RNA, gastrulated of neural tissue (NCAM) and mesoderm (Xbra) on a cultured slightly abnormally but with no indication of a double axis. At explant of naive ectoderm using RNAse protection assays (Fig. stage 13, we observed Xslu expressed in a continuous band 7). Animal caps from stage 8 and stage 10.5 embryos were around the ventral side of the embryo but not on the develop- cultured in NAM containing these factors at various concen- ing neural plate (Fig. 6A). At the equivalent of stage 17, this trations and in combination. Xslu expression was induced in pattern was evidently composed of three parallel bands (Fig. gastrula animal caps by a combination of noggin and FGF 6B). Injection of large amounts of other mRNAs (such as β- galactosidase and other sequences, data not shown) have no effect on the pattern observed. These bands appear to corre- spond to the three aggregates of cranial crest (mandibular, hyoid and branchial), which are extended in a ring around the ventral side of the embryo. We reasoned that this pattern indicates that injected noggin interacts with a signal on the ventral side and jointly causes ectopic Xslu expression on the ventral side while in the neural plate this effect was prohibited. As it was interesting to know if noggin was acting after the start of zygotic transcription, we injected embryos with a plasmid construct containing the Xenopus borealis promoter to drive overexpression of noggin starting at stage 10 (Christian and Moon, 1993). This created a pattern of expression of Xslu at stage 16 (Fig. 6C) that in the best cases was similar to that obtained with mRNA injections with a band circling the ventral side of the embryo but not on the neural plate. This indicates that the interaction between the ventral signal and noggin occurs after zygotic transcription starts. In contrast, Xwnt-8 injected embryos did not express Xslu significantly although there were a very small number of embryos with weak expression in the normal position (Fig. Fig. 7. RNAse protection study of the effect of soluble noggin and 6D). When Xwnt-8 and noggin RNA were injected together, bFGF on ectodermal explants. Animal caps of stage 8 (Fig. A) and Xwnt-8 suppressed almost all Xslu expression (data not stage 10.5 (Fig. B) embryos were cultured 1× NAM containing (a-c) shown). The effect of Xwnt-8 expression after the start of noggin 100, 30 and 10 u/ml, respectively; (d-f) bFGF 90, 30 10 u/ml zygotic transcription using the Xenopus borealis actin respectively; (g) noggin (30 u/ml) plus bFGF 10 u/ml; (h) noggin (30 promoter has not been investigated. u/ml) plus bFGF (30 u/ml); (i) noggin (10 u/ml) plus bFGF (10 u/ml); (j) no additions. At the equivalent of stage 17, the relative Xslu can be induced by a combination of noggin proportions of Xslu, Xsna, NCAM, Xbra and EF1a in the RNA and bFGF in naive ectoderm prepared from the explants was determined by RNAse protection. Each track contains RNA from 10 explants. Individual probes The hypothesis, that Xslu may be induced directly or indirectly hybridised with whole embryo RNA are shown in Fig. 7C. 774 R. Mayor, R. Morgan and M. G. Sargent

Fig. 8. Effect of UV irradiation on Xslu and Xsna. (A) Effect of UV on Xslu distribution at stage 12 Embryos are viewed from dorsal side. (a) Control; (b) regions of cephalic crest slightly closer; (c) cephalic crest regions joined; (d) cephalic fold fused in single dorsal spot; (e) no expression perfectly radially symmetrical embryo. (B) Effect of UV on Xsna distribution at stage 12.5. Note presence of labelled mesoderm underlying ectodermal pattern. Embryos are viewed from dorsal side, arrows indicate ectodermal expression. (a) Control; (b) Xsna in cephalic crest with slightly reduced transverse fold; (c) transverse fold diminished further; (d) cephalic fold fused in single dorsal spot; (e) no expression perfectly radially symmetrical embryo. c, cranial crest; m, mesoderm; y, yolk. (C) Effect of UV on NCAM distribution at stage 17 in a population of selected slow developing embryos. N indicates a normal control. All of this group have shortened axes at the equivalent stage. Most extreme NCAM patterns (x) are extremely short and extend around the blastopore. protein (10 u/ml) at modest concentrations (Fig. 7B track i) concentrations of noggin used (Fig. 7A g), NCAM and Xslu while the factors alone at the same concentration do not induce are both found in the explants. We have also examined the Xslu (Fig. 7A and B, c and f). effect of FGF concentration on Xslu induction in stage 10.5 The presence of FGF with noggin also prevents the caps in the presence of a constant amount of noggin (data not induction of NCAM that normally occurs when noggin is shown). Xslu and Xbra are both induced while NCAM is not administered alone (Fig. 7A and B, a-c) (Lamb et al., 1993). expressed at 3 u/ml of FGF but at 1 u/ml and below, NCAM Although Xslu is entirely ectodermal in the embryo, the con- is expressed and Xslu and Xbra are not expressed. We are ditions that induce Xslu also induce the mesodermal marker therefore unable to dissociate induction of the ectodermal Xslu Xbra in blastula and gastrula caps. Stage 10.5 caps have greatly from induction of the Xbra-expressing mesoderm. reduced competence for formation (Green et Xsna, which is expressed in both mesoderm and ectoderm al., 1990) and, indeed, we have not observed shape changes in (Sargent and Bennett, 1990), is induced by FGF alone but not the explants. In blastula animal caps, the pattern of expression by noggin alone. In blastulae, it is induced at a lower concen- of the four markers, with respect to factor concentration, is tration of FGF than Xbra. more complex. At the lower FGF concentrations (Fig. 7A i), noggin and FGF administered alone at very high concentra- Xslu and Xbra are not induced, while Xsna is and, at the highest tions can induce Xslu in gastrula caps. This probably indicates Induction of prospective neural crest 775

Fig. 9. Model of induction of the neural plate border. (A) The neural plate border is induced in ectoderm when it receives two signals from the underlying mesoderm one from the dorsal mesoderm region (expressing noggin; //////) and another from the ventral mesoderm region (expressing Xwnt-8; \\\\\\). M,H,S, mandibular, hyoid and branchial aggregates of the cranial crest in ectoderm. Note expression of the neural plate border occurs above the region of mesoderm expression. (B) Effect of noggin on distribution of cranial folds. M,H and S in three-banded Xslu expressing region encircling the ventral part of embryo. (C) Distribution of the three influences that determine expression in the neural plate border. (a) noggin expression (//////); (b) Ubiquitous ‘ventral’ signal (\\\\\\); (c) prohibitory signal (ϻϻ). Expression occurs only in the presence of a and b but without c. that there is a low level of a co-inducer present in stage 10.5 neural folds (15%) and (c) those with apparently normal folds ectoderm. (50%). The most severely affected embryos have greatly reduced NCAM expression but none-the-less slightly dor- Effect of ultraviolet treatment of fertilised eggs on salised but no Xslu expression. Xslu and Xsna expression To investigate the relationship between induction of the neural plate border and the neural plate, we examined expression of DISCUSSION Xslu in embryos treated with ultraviolet light. Treatment of Xenopus embryos with UV during the first cell cycle reduces Markers of the prospective neural crest and the the dorsal axis-forming capacity of the embryo producing a neural plate border range of phenotypes from the extreme radially symmetrical The fate map reported above shows the neural plate border embryo with no neural folds to less severe phenotypes with originates in a narrow arc above the marginal zone at stage reduced folds (Malacinski et al., 1977). Embryos cultured to 10+, that extends laterally to about 90¥ from the dorsal midline. the equivalent of stage 16 after 2-3 minutes of UV irradiation Xsna is first expressed at stage 11 in an arc slightly higher than during the first cell cycle showed the range of phenotypes the stage 10+ prospective crest which also ends about 90¡ from described by Malacinski et al. (1977). Using Xslu or Xsna as the dorsal midline (Mayor et al., 1993; Essex et al., 1993). Xsna a probe, we identified a continuous spectrum of modified folds expression on the midline of this arc is weaker than in the ranging from normal to almost radially symmetrical embryos lateral parts and is down regulated before stage 12. Xslu is with no ectodermal Xslu or Xsna expression (Fig. 8A,B). There never expressed on the transverse neural fold but is first was also mesodermal Xsna expression of Xsna that tends to detected on the lateral part of the neural plate border demar- obscure the ectodermal pattern (Essex et al., 1993). Between cated by Xsna at about stage 12. It is a specific marker for all the extreme phenotypes, we found embryos in which the neural parts of the cranial and trunk prospective neural crest until folds were not separated into lateral masses but were fused on stage 17 when expression in lateral plate is observed. In the the dorsal midline. Less affected specimens had the character- chicken, there is, in addition to expression in the neural crest, istic pair of cranial crest masses separated by varying a low level transient expression of Slug in the migratory mes- distances. The frequency of this effect and related effects on enchyme of the (Nieto et al., 1994). No com- NCAM distribution were compared in a group of embryos parable tissue exists in Xenopus as involves the selected as showing significant retardation of blastopore involution of a continuous sheet of tissue rather than indepen- formation. Cooke (1985) has shown that such a population is dently migrating cells. In contrast to Xenopus there is no enriched for embryos with increased radial symmetry. A pop- expression in later stages of the chick comparable to that seen ulation of 90 slow developing embryos was divided into two in the lateral plate. groups and stained with DIG in situ hybridisation probes for Xslu at stage 12.5 or NCAM at Stage 17 (Fig. 8C). Almost all Induction of Xsna and Xslu by dorsal mesoderm of the slow developing group had a shorter neural plate and in The specificity of Xslu for prospective neural crest provides an the most extreme cases was reduced to a small arc of assay for induction. The capacity to induce Xslu is present in expression above the blastopore (Fig. 8C) but some NCAM- dorsal and lateral but not ventral mesoderm, at stage 10.5 when staining material was always present on the dorsal side. The the prospective crest becomes underlain by mesoderm appearance of Xslu-stained embryos fell into three groups (a) although ectodermal Xsna and Xslu are not expressed until those with no Xslu expression (35%), (b) those with fused stage 11 and 12 respectively. Even a very small piece of dorsal 776 R. Mayor, R. Morgan and M. G. Sargent mesoderm (less than 60¡ arc) has the capacity to induce Xslu sequences of overexpression of Xwnt-8 after the start of zygotic and ectodermal Xsna (Mayor and Sargent, 1993). The transcription. mesoderm present in the upper dorsal marginal zone at stage Independent evidence that two factors jointly induce 10.25 is sufficient to induce the overlying ectoderm in explants prospective crest comes from experiments in which we show to produce a neural fold pattern comparable to intact embryos. that soluble noggin protein and bFGF administered to blastula Although these explants are able to express Xslu, the ectoder- or gastrula blastocoel roof induces Xslu expression while the mal component is unable to express Xslu or ectodermal Xsna separate factors have no effect. The two factors induce Xslu autonomously until after stage 12. but not NCAM, seemingly reflecting the clear, binary differ- Xtwi (another marker of the neural plate border; Essex et al., entiation seen in vivo between prospective neural crest and 1993) is induced in early gastrula ectoderm by gastrula dorsal neural plate (Essex et al., 1993). As Xbra is also expressed in mesoderm (Hopwood et al., 1989). In apparent contradiction these explants, they must also contain mesoderm which may of our results, induction of neural-crest-derived melanophores be the inducer of Xslu. Competence for axial mesoderm has been demonstrated in microconjugates of ventral ectoder- formation in blastocoel roof is lost by stage 10.5 (Green et al., mal cells and stage 10.5 lateral (but not dorsal) marginal zone 1992) and we have not observed shape changes in these mesoderm of Xenopus embryos (Mitani and Okamoto, 1991). explants that might indicate that axial mesoderm is present. However, the experimental situation may not be comparable as Using a microculture system, melanophores were induced in the components of the conjugates were much smaller than ours, animal caps treated with soluble FGF alone from stage 9 (but were cultured for longer and may have autoneuralised to the not at 8), attaining peak competence autonomously at stage extent of providing the dorsal inducer postulated here 10.5 (Kengaku and Okamoto, 1993). As suggested above, (Godsave and Slack, 1991). unknown aspects of microculture may conceal the role of the dorsal inducer. The capacity of FGF as a co-inducer of Xslu in A model for induction of the neural plate border vitro has not been demonstrated in vivo. Furthermore, the The three banded pattern of Xslu expression around the effect of the FGF dominant negative interference posterior, produced after injection of noggin mRNA suggested construct suggests that FGF is not required for expression of that (1) there is probably a factor present throughout the ventral branchial arches or melanophores (Amaya et al., 1991) and side of the embryo that can interact with ectopically expressed may therefore be acting indirectly on an unknown ventral noggin to induce Xslu and (2) that the absence of expression inducer. on the neural plate indicates this interaction is prohibited on The presence of noggin (Smith and Harland, 1992) and FGF the neural plate. These observations suggested to us that (Isaacs et al., 1992) throughout the dorsal region and the lack induction of Xslu requires two elements, one that originates of expression of ectodermal Xsna and Xslu in the neural plate from the dorsal side and another from the ventral side. The suggests a prohibition for expression within the neural plate. dorsal signal could be the secreted protein noggin which is Independent support for this has been adduced from studies in expressed in the dorsal quadrant of the marginal zone (Smith which the overexpression of certain neural plate genes (a and Harland, 1992). The leading edge of this domain, at stage dominant negative form of XOTCH, Coffman et al., 1993; or 11, corresponds with the region in which ectodermal Xsna is Xash-3, Turner and Weintraub, 1994) leads to the suppression first seen (Essex et al., 1993)). The other signal could arise of the prospective neural crest. This suggests a refinement of solely from the ventrolateral mesoderm which is characterised the model by which we propose that one signal is ubiquitous by Xwnt-8 expression (Christian and Moon, 1993) (Fig. 9A) but is unable to interact with noggin in the prospective neural and which is expressed in a reciprocal fashion to noggin. We plate region because of a prohibitory signal (Fig. 9C). postulate the two signals jointly induce a particular type of The effect on dorsal development of precleavage irradiation mesoderm which in turn induces the overlying ectoderm or the of fertilised eggs, which tends to prevent development of the two signals jointly induce the overlying ectoderm directly. In notochord and neural plate (Malacinski, et al., 1977; Youn and a normal embryo, noggin and the co-inducer can only interact Malacinski, 1981) can be explained in terms of the absence of at the boundary of the two domains, but noggin expressed the prohibitory signal. After moderate irradiation, a proportion ectopically throughout the embryo generates a three-banded of the embryos have cranial crest domains that are fused on the ring of Xslu expression that encircles the ventral region of the dorsal midline and the length of the NCAM-expressing domain embryo (Fig. 9B). We suggest that these bands correspond to is reduced. This suggests that, when development the three cranial crest premigratory aggregates in their normal of the neural plate is restricted, the neural plate border can be anterior/posterior orientation but which are now extended to induced across the dorsal midline because the prohibitory the ventral side. As a similar result is obtained from ectopic signal is not propagated far enough towards the anterior. The expression of noggin from the CSKA promoter after the start effect of UV parallels the effect of surgical removal of of zygotic transcription, the interaction between dorsal and precaudal mesoderm which is reported to lead to cyclopia in ventral signals presumably occurs after the start of zygotic other amphibia (Adelmann, 1934) transcription. Recent investigations provide independent evidence for the As Xwnt-8 has a reciprocal expression pattern to noggin, it role of a dorsal and ventral signal in determining the size of is a possible candidate for the ventral signal. Overexpression the neural plate (Moury and Jacobson, 1991; Zhang and of Xwnt-8 by injection of mRNA prevents Xslu expression in Jacobson, 1993). embryos and also suppresses the effect of ectopically expressed noggin when co-injected (data not shown). However Xwnt-8 We gratefully acknowledge the expert assistance of Karen Roberts can down-regulate the Nieuwkoop centre activity (Christian with the library screen and sequencing and of Wendy Hatton who and Moon, 1993). However, we have not investigated the con- prepared the wax sections. We thank Angela Nieto, Jonathan Cooke, Induction of prospective neural crest 777

Jim Smith, and David Wilkinson for their comments on the manu- N., Stahl, N., Yancopolous, G. D. and Harland, R. M. (1993). Neural script. Roberto Mayor was supported by the British Council, the induction by the secreted polypeptide noggin. Science 267, 713-718. Medical Research Council of Great Britain and FONDECYT (grant Malacinski, G. M., Brothers, A. J. and Chung, H-M. (1977) Destruction of 1940231). the neural Induction system of the amphibian egg with ultra-violet irradiation. Devel. Biol. 56, 24-39. Mayor, R., Essex, L. J., Bennett, M. F. and Sargent, M. G. (1993) Distinct elements of the Xsna promoter are required for mesodermal and ectodermal REFERENCES expression. Development 119, 661-671. Mitani, S. and Okamoto, H. (1991). Inductive differentiation of two neural Adelmann, H. B. (1934). A study of cyclopia in Ambystoma punctatum with lineages reconstituted in a microculture system from Xenopus early gastrula special reference to the mesoderm. J. Exp. Zool. 67, 217-227. cells. Development 112, 21-31. Albers, B. (1987). Competence as the main factor determining the size of the Moury, J. D. and Jacobson, A. G. (1990). The origins of neural crest cells in neural plate. Devl. Growth Differ. 29, 535-545. the axolotl. Dev. Biol. 141, 243-253. Amaya, E., Musci, T. J. and Kirschner, M. W. (1991). Expression of a Nieto, M. A., Bennett, M. F., Sargent, M. G. and Wilkinson, D. G. (1992). dominant negative mutant form of the FGF receptor disrupts mesoderm Cloning and developmental expression of Sna, a murine homologue of the formation in Xenopus embryos. Cell 66, 257-270. Drosophila snail gene. Development 116, 227-237. Christian, J. L. and Moon, R. T. (1993). Interactions between Xenopus wnt-8 Nieto, M. A., Sargent, M. G., Wilkinson, D. G. and Cooke, J. (1994). Control and Spemann organiser signalling pathways generate dorsoventral pattern in of cell behaviour during development by Slug a Zinc finger gene. Science the embryonic mesoderm of Xenopus. Genes Dev. 7, 13-28. 264, 835-839. Cooke, J. (1985) Dynamics of the control of body pattern in the development of Nieuwkoop, P. D. and Faber, J. (1967). Normal Table of Xenopus laevis, 2nd Xenopus laevis III Timing and pattern after UV irradiation of the egg and ed. Daudin, Amsterdam, North-Holland. after excision of the presumptive head endo-mesoderm. J. Embryol. Exp. Pfaff, S. L., Tamkun, M. M. and Taylor, W. L. (1990). pOEV: A Xenopus Morph. 88, 135-150. 1985. Oocyte protein expression vector. Anal. Bioch. 188, 192-199. Coffman, C. R, Skogland, P. Harris, W. A. and Kitner, C. (1993). Raven C. R. and Kloos, J. (1945). Induction by medial and lateral pieces of the Extracellular of Xotch diverts cell fate in Xenopus embryos. Cell 73, archenteron roof, with special reference to the determination of the neural 659-671. crest. Acta. Neer. Morph. 5, 348-362 Essex, L. J., Mayor, R. M. and Sargent, M. G. (1993) Expression of xenopus Sadaghiani, B. and Thiebaud, C. H. (1987). Neural crest development in the snail in mesoderm and prospective neural fold ectoderm. Devel. Dynamics. Xenopus laevis embryo, studied by interspecific transplantation and scanning 198, 108-122 electron microscopy. Dev. Biol. 1, 91-110. Gans, C. and Northcott, R. G. (1983). Neural crest and the evolution of Sargent, M. G. and Bennett, M. F. (1990). Identification in Xenopus of a vertebrates: A new head. Science 220, 268-274. structural homologue of the Drosophila gene snail. Development 109, 967- Godsave, S. and Slack, J.M.W. (1991). Single cell analysis of mesoderm 973. formation in the Xenopus embryo. Development 111, 523-530. Scherson, T., Serbedzija, G. Fraser, S. and Bronner-Frazer, M. (1993). Green, J. B. A., Howes, G., Symes, K. Cooke, J. and Smith, J. C. (1990). The Regulative capacity of the cranial neural tube to form neural crest. biological effects of XTC-MIF; quantitative comparison with Xenopus Development 118, 1049-1061. bFGF. Development 108, 173-183. Servetnick, M. and Grainger, R. (1991). Changes in neural and lens Green, J. B. A., New, H. V. and Smith, J. C. (1992). Responses of embryonic competence in Xenopus ectoderm: evidence for a developmental timer. Xenopus cells to activin and FGF are separated by multiple dose thresholds Development 112, 177-188. and correspond to distinct axes of the mesoderm. Cell 71, 731-739. Schroeder, T. E. (1970). Neurulation in Xenopus laevis, an analysis and model Harland, R. M. (1991). In situ hybridisation: an improved whole-mount based upon light and electron microscopy. J. Embryol. Exp. Morph. 23, 427- method for Xenopus embryos. Meth. Cell Biol. 36, 685-695. 462. Hopwood, N. D., Pluck, A. and Gurdon, J. B. (1989). A Xenopus mRNA Slack, J. M. W. (1984). Regional biosynthetic markers in the early amphibian related to Drosophila twist is expressed in response to induction in the embryo. J. Embryol. Exp Morph. 80, 289-319. mesoderm and the neural crest. Cell 59, 893-903. Smith, W. and Harland, R. M. (1992). Expression cloning of noggin, a new Isaacs, H. V., Tannahill, D. and Slack, J. M. W. (1992). Expression of a novel dorsalising factor in the Spemann organizer in Xenopus embryos. Cell 70, FGF in the Xenopus embryo. A new candidate inducing factor for mesoderm 829-840. formation and antero-postior specification. Development 114, 711-720. Suzuki, A. S. and Harada, K. (1988). Prospective neural areas and their Keller, R. (1975). Vital dye mapping of the gastrula and of Xenopus morphogenetic movements during neural plate formation in the Xenopus laevis. 1. Prospective areas and morphogenetic movements of the superficial embryo. 2. Disposition of transplanted pieces of Xenopus borealis animal cap layer. Devl. Biol. 42, 222-241. in prospective neural areas of albino Xenopus laevis gastrulae. Dev. Growth Keller, R., Danilchik, M., Gimlich, R. and Shih, J. (1985). The function and Differ. 30, 391-400. mechanism of convergent extension during gastrulation of Xenopus laevis. J. Turner, D. L. and Weintraub, H. (1994). Expression of Achaete-scute Embryol. Exp. Morph. 89 Supplement, 185-209. homolog 3 in Xenopus embryos converts ectodermal cells to a neural fate. Kengaku, M. and Okamoto, H. (1993). Basic Genes Dev. 8, 1434-1447. induces differentiation of neural tube and neural crest lineages of cultured Whiteley, M., Noguchi, P. D., Sensabaugh, S. M., Odenwald, W. F. and ectoderm cells from Xenopus gastrula. Development 119, 1067-1078. Kassis, J. A. (1992). The Drosophila gene escargot encodes a zinc finger Kintner, C. R. and Melton, D. A. (1987). Expression of Xenopus N-CAM motif found in snail-related genes. Mech. Devl. 36, 117-127. RNA in ectoderm is an early response to neural induction. Development 99, Youn, B. W. and Malacinski, G. M. (1981). Axial structure development in 311-325. ultraviolet irradiated (notochord-defective) amphibian embryos. Dev. Biol. Kreig, P. A. and Melton, D. A. (1987). Synthesis of RNA in vitro using SP6 83, 339-352. RNA polymerase. Methods in Enzymology 155, 397-415. Zhang, J. and Jacobson, A. G. (1993) Evidence that the border of the neural Krieg P. A., Varnum, S. M., Wormington, W. M. and Melton, D. A. (1989). plate may be positioned by the interaction between signals that induce ventral The mRNA encoding elongation factor 1(EF-1a) is a major transcript at the and dorsal mesoderm. Devel. Dynamics. 196, 79-90. midblastula transition in Xenopus. Dev. Biol. 133, 93-100. Lamb, T. M., Knecht, A. K., Smith, W. C., Stachel, S. E., Economides, A. (Accepted 22 November 1994)