Hensen's Node Induces Neural Tissue in Xenopus Ectoderm. Implications for the Action of the Organizer in Neural Induction

Development 113, 1495-1505 (1991) 1495 Printed in Great Britain © The Company of Biologists Limited 1991

Hensen's node induces neural tissue in Xenopus ectoderm. Implications for the action of the organizer in neural induction

C. R. KINTNER1 and J. DODD2

^Molecular Neurobiology Laboratory, The Salk Institute, San Diego, CA 92186, USA 2Department of Physiology and Cellular Biophysics, Center for Neurobiology and Behavior, Columbia University, New York, NY 10032, USA

Summary

The development of the vertebrate nervous system is ectoderm to respond to Hensen's node extends much initiated in amphibia by inductive interactions between later in development than that to activin-A or to ectoderm and a region of the embryo called the induction by vegetal cells, and parallels the extended organizer. The organizer tissue in the dorsal lip of the competence to neural induction by axial mesoderm. The blastopore of Xenopus and Hensen's node in chick actions of activin-A and Hensen's node are further embryos have similar neural inducing properties when distinguished by their effects on lithium-treated ecto- transplanted into ectopic sites in then" respective em- derm. These results suggest that neural induction can bryos. To begin to determine the nature of the inducing occur efficiently in response to inducing signals from signals of the organizer and whether they are conserved organizer tissue arrested at a stage prior to gastrulation, across species we have examined the ability of Hensen's and that such early interactions in the blastula may be an node to induce neural tissue in Xenopus ectoderm. We important component of neural induction in vertebrate show that Hensen's node induces large amounts of embryos. neural tissue in Xenopus ectoderm. Neural induction proceeds in the absence of mesodermal differentiation and is accompanied by tissue movements which may Key words: neural induction, organizer, activin-A, reflect notoplate induction. The competence of the Hensen's node, notoplate.

Introduction tissue when transplanted to ectopic sites in chick or amphibian embryos, respectively. In addition, both Vertebrate neural development begins when ectoderm tissues pattern the induced neural tissue along the on the dorsal side of the embryo is induced to form anterior-posterior axis (Spemann, 1931; Tsung et al., neural tissue. The neural inducer is a region of tissue, 1965; see Hara, 1978). The similarities in the properties known as the organizer, whose properties have been of the organizer in different vertebrate embryos defined by transplantation studies in several vertebrate suggests that the inducing signals underlying neural species. When the organizer is transplanted from one induction may be conserved. To examine this idea, we blastula to the ventral side of another, the host embryo have asked whether Xenopus ectoderm responds to the forms a second dorsal axis (Spemann and Mangold, organizer taken from the chick embryo as it does to its 1924). The nervous system in this second axis is derived own organizer. Chick Hensen's node was isolated and from host ectoderm which, in the absence of the graft, cultured with Xenopus ectoderm in a standard animal would have formed ventral epidermis. This result cap assay. The assay was carried out at room indicates that ectoderm can be induced to form neural temperature (approximately 22°C) such that the ecto- tissue upon appropriate interaction with the organizer, derm can respond to inducing signals while the chick an event that normally occurs only on the dorsal side of tissue is developmentally static. Thus tissue differen- the embryo. In amphibia, the organizer maps to the tiation and morphogenetic movements observed rep- dorsal lip of the blastopore (DLB), a region of the resent the response of Xenopus ectoderm, while the marginal zone of the blastula just above the first site of inducing signals detected in the assay can be attributed invagination at gastrulation. In birds, the organizer to Hensen's node tissue as it existed at the stage of maps to the anterior end of the primitive streak, a isolation from the embryo. region called Hensen's node (Waddington and Schmidt, We show that Xenopus ectoderm responds to 1933; see Hara, 1978). Hensen's node by forming large amounts of neural Both Hensen's node and the DLB induce neural tissue without detectably forming dorsal mesodermal 1496 C. R. Kintner and J. Dodd derivatives. The inducing activity of Hensen's node is embryos. In every assay the size of undigested probes for each different from the potent embryonic inducer, activin-A transcript used was measured on the same gel as the (Asashima et al., 1990; Eijnden-Van Raaij et al., 1990; experimental samples. This permitted us to determine Smith et al., 1990; Thompsen et al., 1990). In addition, whether probe escaped RNAase protection in any experimen- Hensen's node induces ectoderm to undergo morpho- tal samples. logical movements resulting in axial elongation in the Histology and immunohistochemistry absence of mesoderm. These results are discussed in the Recombinants were fixed in 3% trichloracetic acid at 4°C for light of the model in which an early interaction between 30 min. After extensive washing, they were dehydrated ectoderm and organizer tissue in the blastula is an through an ethanol series and embedded in paraffin using important component of neural induction in vertebrate standard procedures (Kintner, 1988). Tissue was sectioned at embryos. 10 fsm on a rotary microtome and mounted on gelatin-subbed slides. For histology, sections were stained with haematoxylin and eosin or Giemsa's stain. For immunohistochemistry, the Methods sections were dewaxed, labelled with monoclonal antibodies, using a fluorescein-labelled second antibody and counter- Animals and reagents stained with Hoechst dye. Monoclonal antibodies used were Embryos were obtained from Xenopus laevis adult frogs as follows: anti-NCAM (gift of Drs K. Sakaguchi and W. (NASCO and Xenopus 1) by hormone-induced egg-laying Harris, UCSD), 12/101 (Kintner and Brockes, 1985) and Not- and in vitro fertilization using standard protocols. Animal 1 (Placzek et al., 1990; Yamada et al., 1991). caps were dissected from appropriately staged (Nieuwkoop and Faber, 1967) embryos in 0.5x MMR containing penicillin and streptomycin, as previously described (Dixon and Results Kintner, 1989). For lithium treatment, embryos were incubated at the 64 cell stage with 0.25 M LiCl for 10 minutes and Hensen's node induces neural tissue then washed extensively with 0.5 x MMR. To measure Fate mapping and transplantation studies have led to competence, ectodermal caps were isolated from embryos at the idea that Hensen's node in the chick is functionally appropriate times after the start of gastrulation by removing equivalent to the dorsal lip of the blastopore (DLB) in ectoderm that was not yet contacted by involuting tissue. amphibia (see Hara, 1978). We therefore examined Hensen's node tissue was dissected from chick embryos whether Hensen's node could substitute for DLB tissue (White Leghorns from Spafas, CT, Truslow Farms, VA, and Mclntyre Poultry, CA) at stages 3-10 (Hamburger and in a Xenopus animal cap assay by combining Hensen's Hamilton, 1951) in ice cold L15 medium. The chick tissue was nodes, dissected from stage 3.5 and stage 4 chick placed between two pieces of animal cap tissue as shown in embryos, with pairs of ectodermal caps dissected from Fig. 1, and the resulting recombinants maintained on agarose- stage 9 Xenopus laevis embryos (Fig. 1). The recombi- coated dishes in 0.5 x MMR with penicillin and streptomycin nants were allowed to develop for 18-24 hours at room at room temperature (approximately 22°C). Recombinants temperature and then assayed for the expression of were harvested for RNA analysis after 20 hours or were tissue-specific RNA transcripts. maintained for 2.5 days with frequent changes of the culture medium for observation and histological analysis. The recombinants were examined for the presence of A highly purified preparation of porcine activin-A was neural tissue by measuring the expression of two kindly provided by Drs W. Vale and J. Vaughan in the Peptide neural-specific transcripts, NCAM and NF-3. NCAM is Biology Laboratory at the Salk Institute for Biological expressed in Xenopus ectoderm soon after neural Studies. induction and is restricted to neural tissue (Jacobson and Rutishauser, 1986; Kintner and Melton, 1987) RNA analysis providing an early general marker of neural differen- RNA was isolated from embryos or explants and assayed for tiation. NP-3 encodes a neuronal intermediate filament the expression of specific RNA transcripts using an RNAase protein (Charnas et al., 1987) that is expressed in protection assay described previously (Melton et al., 1984; neurons beginning about 4 hours after neural tube Kintner and Melton, 1987). The hybridization probes used to closure (Dixon and Kintner, 1989). All transcripts were detect NCAM, NF3, muscle-specific actin and EF-1 alpha detected with an RNAase protection assay in which the RNA are described elsewhere (Kintner and Melton, 1987; Dixon and Kintner, 1989). The hybridization probe for Xenopus tissue but not the chick tissue generated full- XlHbox6 RNA is a fragment of the XlHbox6 cDNA (Sharpe length protected fragments. et al., 1987) kindly provided by Drs C. Wright and E. Recombinants formed between Xenopus ectoderm DeRobertis. The hybridization probe for en-2 RNA is a and Hensen's node expressed both NCAM and NF-3 portion of the Xenopus engrailed-2 cDNA that was isolated transcripts (Fig. 2, lane 4). The levels of the two and provided for us by Drs R. Harland and A. Hemmati- transcripts expressed in the ectodermal caps after Brivanlou (Hemmati-Brivanlou et al., 1991). The amounts of induction by Hensen's node were approximately equiv- RNA in each sample were normalized by monitoring the alent to the levels observed in stage-matched control levels of EF-1 alpha RNA (Krieg et al., 1989). RNA samples prepared from 4 recombinants or embryos were assayed embryos normalized for the expression of EF-1 alpha simultaneously with all probes except for the muscle-specific RNA (Fig. 2, lane 6). Control explants of ectoderm actin probe, in which case 10% of the RNA sample was cultured alone expressed neither NCAM nor NF-3 (Fig. assayed separately. The Xenopus hybridization probes were 2, lane 2). Of the tissue in the chick blastula, only fully protected by transcripts isolated from frog tissue but Hensen's node was able to induce large amounts of were not protected by transcripts isolated from chick neural tissue in Xenopus ectoderm, although very low Neural induction in Xenopus laevis 1497

ectoderm

Fig. 1. Pieces of chick epiblast with underlying hypoblast were dissected as shown from Hensen's node (HN), the primitive streak (PS) and other regions of the embryo (e). These were sandwiched between two animal Chicken Xenopus ectodermal caps dissected from embryo blastula stage 9-10 Xenopus blastulae. levels of NCAM RNA were induced by primitive streak chick tissue and immediately posterior to the node (Fig. 2, lane 5). frog ectoderm Equal-sized pieces of tissue dissected from lateral or UNPROTECTED PROTECTED anterior epiblast (see Fig. 1) or caudal primitive streak PROBES ^ 8 PROBES did not induce neural transcripts in isolated frog 8 « 1a z muscle O Q f) I 8 ectoderm (Fig. 2, lane 3). Thus, neural inducing 2 w to muscle capacity was restricted to the region of the chick that is actin Q. actin thought to be equivalent to the amphibian DLB (Hara, 1978). Hensen's node does not appear to differentiate within NF-3 NF-3 the recombinates An important aspect of these experiments is that the recombinants were maintained at room temperature (approximately 22°C) in 0.5x MMR, conditions in which development of Hensen's node should be NCAM arrested. The inducing signals detected in this assay are NCAM therefore likely to be present in the node at the time of explantation and unlikely to be generated by tissues arising from the node during the course of the assay. To determine whether Hensen's node arrests within the EF-1A recombinant, or alone at 22°C in 0.5x MMR, we EF-1A examined sections of the node for the expression of the differentiation antigen, Not-1. Not-1 is expressed by chick notochord, beginning at stage 5, as Hensen's node 12 3 4 6 7 begins to regress and notochord is laid down (Placzek et al., 1990; Yamada et al., 1991). Hensen's node Fig. 2. RNA transcripts expressed in Xenopus ectoderm in incubated in the presence or absence of ectodermal response to Hensen's node. Total RNA samples prepared caps in 0.5 x MMR overnight at room temperature did from 4 recombinants or control tissues were assayed not express Not-1. The antigen was expressed by stage 5 simultaneously for the presence of NF-3, NCAM and EF1- or 6 chick notochord before and after incubation of the alpha transcripts by RNAase protection. 10% of each tissue in 0.5x MMR overnight, indicating that the sample was assayed separately for the presence of muscle- antigen itself is not destroyed by incubation under these specific actin transcripts. Lane 1 shows the position of the conditions (not shown). Not-1 was expressed in stage 4 unprotected probes, indicated by arrows. Arrows on the Hensen's node that had been incubated at 37°C in extreme right show the position of the probes after isotonic medium overnight. These results suggest that protection with the appropriate RNA transcripts. Ectoderm Hensen's node is arrested developmentally at room cultured with stage 4 Hensen's node expresses large amounts of NCAM and NF-3 transcripts (lane 4) at levels temperature and that its effects on Xenopus ectodermal found in stage-matched control embryos (lane 6). tissue do not depend on differentiation into its Ectoderm cultured with anterior primitive streak tissue mesodermal derivatives. expressed NCAM RNA at just detectable levels (lane 5). Note that ectoderm cultured alone (lane 2) or combined The inducing properties of Hensen's node change with with chick epiblast (lane 3) expresses EFl-alpha but not developmental stage NCAM RNA. None of the probes are protected by tRNA (lane 7). Transplantation of the amphibian DLB taken from different developmental stages results in the induction 1498 C. R. Kintner and J. Dodd HENSEN'S of tissues with different regional characteristics (Spe- mann, 1931). Dorsal lip tissue taken from an early NODE c gastrula stage induces anterior structures while dorsal lip taken from later-staged embryos induces posterior in o 8 CO tissues such as spinal cord. A similar phenomenon has CO CO c/D x been observed in the chick embryo in that secondary en-2 neural tubes induced by a subepiblast transplant of Hensen's node became progressively more posterior in character as donor nodes were taken from later staged embryos (Tsung et al., 1965; but see Dias and Schoenwolf, 1990). These observations suggest that the inducing properties of Hensen's node and the DLB change similarly with developmental age. We therefore tested the ability of Hensen's nodes, dissected from NCAM chick over a range of ages, to induce neural tissues of anterior and posterior character in Xenopus ectoderm. To do this we measured the expression of two r homeobox-containing genes, en-2 and XlHbox6, that are expressed at different levels of the A-P axis in Xenopus ectoderm. En-2 is expressed in the neural plate at the level of the midbrain (Hemmati-Brivanlou and Harland, 1989; Hemmati-Brivanlou et al., 1991). EF-1A XlHbox6 transcripts are expressed in the posterior region of the neural plate (Sharpe et al., 1987) and in lateral plate mesoderm (Wright et al., 1990). •HI? In animal caps induced with stage 4 Hensen's nodes the en-2 transcript was observed at levels comparable to those observed in stage-matched control embryos (Fig. 3, lanes 1,4). However, when Hensen's nodes from late XIHBOX 6 stage 5 to stage 9 were used, the expression of en-2 ceased (Fig. 3, lanes 2,3). Conversely, stage 4 Hensen's node did not induce expression of XlHbox6 (Fig. 3, lane 1), whereas later-staged Hensen's nodes did induce 1 2 3 4 5 XlHbox6 (Fig. 3, lanes 2,3). Fig. 3. Expression of RNA transcripts in Xenopus The results described above indicate that Hensen's ectoderm in response to Hensen's node from embryos at node tissue mimics at least two characteristics of tissue different developmental stages. Hensen's node was isolated in the DLB. First, Hensen's node induces large from chick embryos of different stages and combined with amounts of neural tissue in Xenopus ectoderm. This Xenopus ectoderm from stage 10 embryos. After 24 hours effect is observed with both stage 3 and stage 4 in culture, total RNA samples were prepared and assayed Hensen's nodes, indicating that Hensen's node contains simultaneously for en-2, NCAM, XlHbox6 and EFl-alpha neural inducing signals before gastrulation. Second, the RNA, using RNAase protection. The position of the neural tissue induced has different regional character- protected probe for each transcript is indicated by arrows istics that depend on the age of the embryos from which on the left. Stage 30 embryos expressed all four transcripts Hensen's node is isolated. These results suggest that (lane 4) while ectoderm cultured alone expressed only EF- Hensen's node can be used as a model to study the 1 alpha RNA (lane 5). Ectoderm combined with stage 4 Hensen's node expressed NCAM and en-2 but not properties of the signals that are present in the DLB. XlHbox6 transcripts (lane 1). Ectoderm combined with late We have therefore analysed the response of ectoderm stage 5 (lane 2) or later (lane 3) expressed NCAM and to Hensen's node and compared the responses of XlHbox6 but not en-2 RNA. Xenopus ectoderm to Hensen's node and another potent embryonic inducer, activin-A. induce the expression of the muscle-specific actin Hensen's node does not induce axial mesoderm transcript (Fig. 2, lane 4), indicating that the respond- Ectoderm treated with embryonic inducers such as ing ectoderm did not form somitic mesoderm. In activin-A and FGF forms mesodermal derivatives such contrast, whenever neural tissue was induced by as notochord and muscle (Slack et al., 1987; Smith, activin-A, it was accompanied by the induction of large 1987). In order to determine whether ectoderm also amounts of somitic mesoderm (Fig. 5, lanes 2,3). As an responds to Hensen's node by forming axial mesoderm, alternative assay for somitic muscle, recombinants were we examined recombinants for the expression of examined for the presence of the muscle specific somitic mesoderm using two assays. We first measured antigen, 12/101, by immunohistochemical labelling of the expression of muscle-specific actin RNA in an tissue sections. While patches of muscle were brilliantly RNAase protection assay. Hensen's node did not labelled with this antibody in control explants contain- Neural induction in Xenopus laevis 1499

Fig. 4. Histological analysis of recombinants. (A,B) Haematoxylin and eosin-stained paraffin sections of recombinants formed between, Xenopus ectoderm and Xenopus organizer (A) and Xenopus ectoderm and chick Hensen's node (B). Notochord cells (n) are present in A but not in B. (C) Immunofluorescence micrograph showing labelling of neural tissue with anti-NCAM antibodies. The neural tissue (large arrowhead) is found adjacent to the chick tissue (small arrowheads) which can be identified by Hoechst staining in serial sections (D) (small arrowheads). ing Xenopus DLB tissue, no labelling with the 12/101 transcripts. NCAM labelling was primarily associated antibody was observed in recombinants between with tissue in direct contact with Hensen's node (Fig. ectoderm and Hensen's node (not shown). 4C,D), suggesting that the ectoderm formed neural Although muscle-specific actin and 12/101-reactive tissue in the absence of intervening cells or tissues. tissue were not induced by Hensen's node, it is possible Together, these results indicate that Hensen's node that notochord tissue was induced in the absence of can induce large amounts of neural tissue to form from somitic mesoderm. To determine whether chordameso- Xenopus ectoderm and that this induction occurs in the derm was present we examined sections of recombi- absence of axial mesodermal derivatives such as nants for the presence of notochord and other notochord and somitic muscle. In addition, since mesodermal derivatives. Notochordal tissue, recogniz- Hensen's node induces neural tissue in the absence of able by the presence of large vacuolated cells in axial mesoderm, its properties appear to be different recombinants containing DLB tissue (Fig. 4A), could from those of factors identified as embryonic mesoder- not be detected in recombinates containing Hensen's mal inducers, such as activin-A and FGF. node (Fig. 4B). The lack of detectable mesoderm was independent of the stage of embryo (3-10) from which Hensen's node inducing activity is distinct from Hensen's node was taken (not shown). While this activin-A activity analysis does not exclude the presence of a few isolated Several studies indicate that different parameters can notochordal cells, it provides strong evidence that the alter the responsiveness of ectoderm to induction by formation of axial mesoderm is not a major response of polypeptide growth factors. In the case of activin-A, the Xenopus ectoderm to stage 4 Hensen's node. ectodermal response can change as a function of the age Recombinants containing stage 4 Hensen's node of the embryo from which the ectoderm is isolated and were also examined immunohistologically for the by treatment of embryos with a dorsalizing agent, formation of neural tissue, using a monoclonal antibody lithium. To characterize the properties of the inducing against NCAM. Recombinants contained large signals in Hensen's node further, we examined the amounts of NCAM-immunoreactive tissue (Fig. 4C), effect of these parameters on responses of ectodermal confirming the results obtained measuring neural caps to Hensen's node. 1500 C. R. Kintner and J. Dodd (i) Effects of ectodermal competence on the The response of ectoderm to Hensen's node also response to Hensen's node declined with developmental age, but the decline The ability of the ectoderm to respond to mesoderm- occurred at later stages than that observed with activin- inducing signals is known to decline at the beginning of A. Ectoderm continued to respond to stage 4 Hensen's gastrulation and to be lost by midgastrula stages. We node by expressing large amounts of NCAM RNA until therefore tested the response of ectoderm, taken from late gastrula stages (Fig. 5B, lanes 1-3), considerably progressively older Xenopus embryos, to activin-A and later than the developmental stages at which activin-A Hensen's node. In accordance with the results of others is still able to induce NCAM expression. Moreover, (Green et al., 1990), ectoderm taken from embryos even when the ability of the ectoderm to respond to before gastrulation (stages 8.5-10) responded to activin- Hensen's node declined, Hensen's node did not induce A (0.07 nM) by expressing both NCAM and muscle- the expression of muscle-specific actin (Fig. 5B, lane 3). specific actin transcripts (Fig. 5A, lanes 1,2). No Thus the competence of ectoderm to form neural response was observed at 7 pM activin-A (not shown). tissue in response to Hensen's node persisted for a By midgastrulation (stage 11), ectoderm no longer longer period after gastrulation than that to activin-A. expressed NCAM RNA in response to activin-A (Fig. At stage 11, the ectoderm has lost competence to form 5A, lanes 3,4) but continued to express muscle-specific neural tissue in response to activin-A despite the fact actin in response to high concentrations of activin-A that muscle-specific actin RNA was expressed. In (1.4 nM) (Fig. 5A, lane 4). Ectoderm taken from contrast, Hensen's node continues to induce neural embryos older than stage 11 did not express NCAM or tissue at late stages of gastrulation. muscle-specific actin in response to activin-A (not shown). Surprisingly, ectoderm continued to respond to (ii) Effects of lithium on the responses of ectoderm activin-A by expressing XlHbox6 even when its ability to Hensen's node to form neural tissue was lost. The XlHbox6 signal in The response to activin-A is enhanced in ectoderm ectoderm treated with activin-A after stage 11 may taken from embryos treated with lithium at the 64 cell reflect the induction of posterior lateral mesoderm that stage. The ratio of dorsal to ventral induced mesoderm is known to express XlHbox6 in embryos (Wright et al., increases and the competence of the ectoderm to 1990). respond is extended (Cooke et al., 1989; Kao and k B Fig. 5. Effects of competence on the ectodermal response to Ect + Act (nM) Ect + HN activin-A and stage 4 Hensen's 8 node. Animal caps were O i- -r^ o isolated from embryos at different ages (stages 9-11.5) muscle muscle actin and exposed either to activin- actin A (A) or to Hensen's node (B). Total RNA samples were NCAM NCAM prepared from 8 animal caps, 4 recombinants or 4 control tissues, and assayed by RNAase protection for the expression of NCAM, en-2, XlHbox6 and EFl-alpha RNA. EF-1A EF-1A 10% of each sample was assayed separately for the I presence of muscle-specific actin transcripts. The position of the protected probe for XIHBOX 6 each transcript is shown to the XIHBOX 6 left of each panel. Stage 30 m embryos (St 30 con) express all en-2 en-2 *" Av -•-• ~~* ^m "" ^* """' ^ five transcripts (lane 5 in A; 1234 5 1234 'ane 4 in B). (A) Ectoderm from stage 10 embryos expressed muscle actin, NCAM and XlHbox6 RNA in response to both 0.07 nM (lane 1) and 1.4 nM (lane 2) activin-A. When ectoderm was isolated from embryos at a later stage (stage 11, early gastrulation), the response to activin-A was greatly diminished. Stage 11 ectoderm did not express NCAM RNA in response to 0.07 nM (lane 3) or 1.4 nM (lane 4) activin-A. Stage 11 ectoderm could be induced to express muscle-specific actin RNA but in response only to high concentrations of activin-A (1.4 nM, lane 4). The low levels of en-2 signal in lane 4 represent undigested probe (see methods). (B) Recombinants of Hensen's node and ectoderm from stage 10 (lane 1), stage 11 (lane 2) and stage 11.5 (lane 3) embryos express NCAM and en-2 RNA but not muscle-specific actin or XlHbox6 RNA. The levels of NCAM and en-2 transcripts are approximately the same in ectoderm from stage 10 and stage 11 embryos, but decrease in ectoderm from stage 11.5. Although the stage 11.5 recombinant has less NCAM RNA expression, it does not express muscle-specific actin or XlHbox6 RNA- Neural induction in Xenopus laevis 1501

B Fig. 6. Effect of lithium on the St11 Ect + Act (nM) st 11 Ect st 11.5 Ect c response of ectoderm to o + HN +HN o activin-A and Hensen's node. o Animal caps were isolated co muscle muscle 8 8 from stage 9-11.5 lithium- actin actin treated or control embryos and were treated with activin-A or combined with stage 4 Hensen's nodes. Total RNA NCAM NCAM samples were prepared from 8 animal caps, 4 recombinants or 4 control tissues and assayed by RNAase protection for the expression of NCAM, en-2, EF-1A XlHbox6 and EFl-alpha RNA. EF-1A 10% of each sample was used to assay for the muscle-specific I actin transcript. The position of the protected probe for each transcript is marked to XIHBOX 6 the left of each panel with XIHBOX 6 arrows. Stage 30 embryos expressed all five transcripts en-2 __ „_ (lane 5 in A; lane 5 in B). (A) Ectoderm from stage 11 embryos does not express t -a 3 4 NCAM RNA in response to activin-A and requires high concentrations of activin-A (1.4 nM) to express muscle-specific actin and XlHbox6 transcripts (lane 2). The low levels of en-2 in lane 2 represent unprotected probe. In contrast, stage 11 ectoderm from lithium-treated embryos responds to activin-A at concentrations of 0.07 nM and 1.4 nM by expressing NCAM, muscle-specific actin and XlHbox6 transcripts. Note that en-2 transcripts are not expressed in ectodenn treated with activin-A even when isolated from lithium-treated embryos. (B) The response of stage 11 (lanes 1,2) and stage 11.5 ectoderm (lanes 3,4) to Hensen's node was the same in animal caps isolated from lithium-treated (lanes 2,4) and untreated (lanes 1,3) embryos. The band in the muscle-specific actin assay (lane 4) represents unprotected probe (see methods).

Elinson, 1989). We therefore compared the effects of 6B, lanes 1-4), in contrast to its effect on the activin-A lithium treatment on the responses of ectodenn to response. Hensen's node and activin-A. As described above, ectoderm from control embryos older than stage 11 The effects of competence and lithium treatment on does not express NCAM in response to activin-A and the expression of region specific markers, en-2 and expresses muscle-specific actm transcripts only in XlHbox6 in Xenopus ectoderm in response to response to high concentrations of activin-A (1.4 nM) Hensen's node (Fig. 6A, lanes 1,2). In marked contrast, lithium- Stage 4 Hensen's node induces the expression of en-2 treated stage 11 ectoderm expressed both NCAM and but not of XlHbox6 in stage 10 Xenopus ectoderm, as muscle-specific actin RNA (Fig. 6A, lanes 3,4) even described above. Conversely, activin-A induces when exposed to low concentrations of activin-A (0.07 XlHbox6 but not en-2 in stage 10 ectoderm. The nM). Thus, as previously reported (Cooke et al., 1989), differential expression of en-2 and XlHbox6 RNA in lithium treatment potentiates the response of ectodenn response to activin-A and Hensen's node was unaffec- to activin-A. ted by lithium treatment or by the age of the ectoderm. The generation of neural tissue by ectoderm in As in control ectoderm, en-2 RNA was not induced by response to stage 4 Hensen's node was not altered by activin-A in lithium-treated ectodenn (Fig. 6A, lanes 1- lithium treatment (Fig. 6B, lanes 1-4). Both the levels 4) but was expressed at control levels in response to of NCAM transcripts and the competence of the Hensen's node (Fig. 6B, lanes 1-4). XlHbox6 RNA was ectoderm to respond to Hensen's node were the same in not expressed in response to stage 4 Hensen's node but lithium-treated and control embryos. Because the was expressed in ectoderm that responded to activin-A Hensen's node signal cannot be titred, we do not know (Fig. 6A, lanes 2-4). whether it was already optimized in these experiments. Thus we cannot definitively conclude that lithium is not Extension movements are induced by Hensen's node capable of potentiating the response of early ectoderm In the course of these experiments, we consistently to Hensen's node. However, the results do show that observed that ectodenn underwent extensive morpho- lithium treatment is not able to extend the competence logical movements in response to Hensen's node. In of older ectoderm to respond to Hensen's node (Fig. some cases these movements resulted in extreme axial 1502 C. R. Kintner and J. Dodd neurulation (Fig. 7 B-D). Movements were not A observed in isolated Hensen's node placed alone in 0.5x MMR at room temperature (not shown) or in isolated ectoderm from Xenopus embryos, indicating that they were initiated in the ectoderm in response to Hensen's node. It is unlikely that ectoderm induced the movements in Hensen's node, since in sections of the recombinants the chick cells appeared to have remained in a local cluster. Elongation movements of the magnitude observed in the Hensen's node recombinants are known to occur in Xenopus embryos only in cells of the involuting marginal zone at the DLB as they form the midline of the prospective dorsal mesoderm, or in cells of the noninvoluting marginal zone as they B form the midline of the neural plate (Keller, 1985). Since the recombinants do not form mesodermal derivatives, the observed movements are likely to be associated with the formation of neural tissue.

Discussion Both Hensen's node of the chick and the DLB of amphibia induce host ectoderm, in chick and Xenopus respectively, to form neural tissue upon transplantation to an ectopic site (Spemann and Mangold, 1924; Waddington and Schmidt, 1933; Smith and Slack, 1983; Vakaet, 1965; McCallion and Shinde, 1973; Hara, 1978; Hornbruch et al., 1989; Dias and Schoenwolf, 1990). The results reported here show that Xenopus ectoderm also responds in vitro to Hensen's node. The ectoderm forms neural tissue at levels similar to those formed in normal embryos and expresses distinct regional neural markers in response to nodes taken from embryos at different developmental stages. The induction of neural tissue by Hensen's node is not accompanied by the formation of any detectable mesodermal derivatives and occurs under conditions in which development of the node itself is arrested. Thus organizer tissue appears to be able to act as a potent neural inducer before it gastrulates and forms mesodermal derivatives.

The organizer as an early inducer of neural tissue The finding that avian organizer tissue is a potent Fig. 7. Xenopus ectoderm/Hensen's node recombinants inducer before gastrulation and need not develop into show gastrulation-like movements. (A) Several mesodermal derivatives to act as an effective neural recombinants showing extreme elongation and inducer suggests that neural induction in vivo may occur concentration of pigment are shown 18 hours after the in the blastula. This induction may be due to an tissues were placed together. (B-D) A recombinant at 2 interaction between the organizer and ectoderm across (B), 6 (C) and 18 (D) hours after sandwiching of the the boundary they share in the blastula and may be Hensen's node with Xenopus ectoderm. carried by signals travelling in the plane of the ectoderm. This implies that organizer tissue need not elongation of the recombinants (Fig. 7A), during which involute beneath the ectoderm for neural induction to the inner ectodermal cells were extruded. In other occur. This idea has already been suggested from results cases, cell movements were extensive and resulted in a with Xenopus embryos (exogastrulae) and explants complex pigmentation patterns, but seemed not to be (Keller sandwiches) in which DLB tissue does not oriented and the explants did not elongate. The timing involute beneath the prospective neural ectoderm of the movements occurred over the time course of axial during gastrulation. In this situation in Xenopus the elongation in embryos undergoing gastrulation and ectoderm in edgewise contact with the DLB tissue Neural induction in Xenopus laevis 1503 forms large amounts of neural tissue (Kintner and Induction with Hensen's node is distinct from that of Melton, 1987; Keller and Danilchik, 1988; Dixon and growth factors such as activin-A Kintner, 1989). To begin to characterize the inducing signals generated The idea of an early induction between the organizer by organizer tissue, we compared induction by Hen- and ectoderm is also supported by the observation that sen's node to that by another potent embryonic inducer, one of the first responses of ectoderm to Hensen's node activin-A. Three lines of evidence suggest that the is elongation movements. Elongation movements are induction of neural tissue by Hensen's node and activin- known to occur in only two regions of the Xenopus A involve different pathways. First, recognizable embryo. One region corresponds to the cells within the mesodermal derivatives are not observed when neural involuting marginal zone, a region which converges, tissue is induced by Hensen's node. Second, the involutes, comes to lie beneath the neural plate and competence of the ectoderm for neural induction forms notochord. The other region maps to cells in the extends much later than that for induction by activin-A. noninvoluting marginal zone, called notoplate cells, Third, the response of the ectoderm to activin-A is which extend within the ectodermal sheet along the enhanced by lithium treatment while induction by midline of the prospective neural plate, during gastru- Hensen's node is unaffected. lation and neurulation (Jacobson and Gordon, 1976; In Xenopus, neural tissue has been shown to Gordon and Jacobson, 1978; Keller, 1985; Jessell et al., differentiate from animal cap ectoderm exposed to 1989). The notoplate arises from the ectoderm that is activin-A. The induction of neural tissue by these closest to the organizer in the blastula fate map and its factors is always accompanied by the induction of axial movements have been shown to occur in the absence of mesoderm structures, in particular notochordal tissue underlying mesoderm and when dissected free of and muscle. The neural tissue formed in response to prospective mesoderm after stage 11 (Keller and growth factors such as activin-A may therefore be Danilchik, 1988). This suggests that the notoplate is generated in response to signals derived from the specified by an interaction between the organizer and induced mesoderm rather than from direct actions of adjacent ectoderm or by an inductive signal from activin-A on the undifferentiated ectoderm. In support vegetal cells (Jacobson and Sater, 1988) prior to the of this, recent studies have shown that dissociated onset of gastrulation. Our results support this notion in animal cap ectodermal cells treated with activin-A do that Xenopus ectoderm responds to Hensen's node with not themselves acquire neural characteristics but do striking movements that can be observed within two have neural inducing ability when assayed by the hours of contact. These movements cause the explants response of naive animal cap ectodermal cells (Green to elongate over a time course similar to that of and Smith, 1990). gastrulation and neurulation in whole embryos (Symes In contrast, it is unlikely that Hensen's node induces and Smith, 1987), and occur in the absence of neural tissue indirectly through the induction of detectable mesoderm. The simplest explanation of mesoderm. Axial mesoderm is undetectable in the these movements is that they reflect the movements of recombinants by RNAase protection assays and by notoplate cells which have been induced in the morphological and immunohistochemical analysis. Fur- ectoderm by signals from organizer tissue. thermore, in sections of the recombinants the induced Induction of the notoplate by an interaction between neural tissue is often found to be in contact with the the organizer and ectoderm before gastrulation may be node. It is possible that Hensen's node induces an important component of neural induction. As they prechordal mesoderm, for which there is no reliable converge and extend along the anterior-posterior axis, marker in animal cap conjugates and that this mesoder- notoplate cells may be the source of neural inducing mal cell type is responsible for neural induction. This is signals that act on surrounding dorsal ectoderm to form unlikely for two reasons. First, prechordal mesoderm is lateral regions of the neural plate. This model predicts relatively ineffective in inducing neural tissue in that notoplate extension is necessary for efficient neural Xenopus ectodermal caps (Dixon and Kintner, 1989; induction and that inducing signals travel from the Savage and Phillips, 1989; Sive et al., 1989). Second, the midline of the prospective neural plate as proposed by induction of neural tissue with posterior character by others (Nieuwkoop et al., 1952; Leussink, 1970; late stage Hensen's nodes suggests that prechordal Gordon and Brodland, 1987). This model may also mesoderm is not involved. explain the formation of neural tissue in cases of The different time courses over which ectoderm edgewise induction in Keller sandwiches and exogas- responds to activin-A and Hensen's node provide trulae as a two step process in which the organizer first compelling evidence that the actions of these two induces the notoplate cells in the noninvoluting inducers differ. Hensen's node induced neural tissue in marginal zone. Our results, interpreted in the light of ectoderm taken from late gastrulation stage embryos. this model, suggest that Hensen's node may owe its This time course is similar to the competence of the neural inducing properties to its ability to induce a ectoderm to form neural tissue in response to axial notoplate. We cannot rule out the alternative model, mesoderm (Sharpe and Gurdon, 1990) and extends however, that induction of the notoplate and the rest of much later than that for mesodermal induction. the neural plate are both mediated directly, and in Ectoderm responded to activin-A only until just after parallel, by interactions between the organizer and the onset of gastrulation. These observations support ectoderm. the idea that more than one induction step is required 1504 C. R. Kintner and J. Dodd for the induction of neural tissue by activin-A while, in We thank Cliff Hume, Tom Jesse 11, Nancy Papalopulu, contrast, Hensen's node induces neural tissue more Marysia Placzek and Ariel Ruiz i Altaba for helpful, if heated, directly. discussions about the work and comments on the manuscript. The evidence described above shows that the neural We are also extremely grateful to Drs Ali Hemmati-Brivanlou and Harland for providing us with an en-2 cDNA before inducing signal in Hensen's node cannot be ascribed publication and to Ray Keller for insights into the gastrulation solely to an activin-A-like molecule, suggesting that the movements of neural tissue. Eric Hubel provided excellent Hensen's node-derived signal may be different from photographic assistance and Ira Schieren generated figure 1. activin-A. Alternatively, the neural inducing properties The work was supported by grants from the NIH, to C.K. and of Hensen's node may result from the presence of an to J.D., from The McKnight Fund for Neuroscience, to C.K. activin-A-like molecule together with a second agent and to J.D., and The Esther A. and Joseph Klingenstein that alters the response properties of the ectoderm to Fund, to J.D. the growth factor. In fact, experiments with lithium have previously shown that the response properties of ectodermal cells can be altered (Cooke et al., 1989; Kao References and Elinson, 1989). In addition, a dorso-ventral difference in the response of ectoderm to activin-A has ASASHIMA, M., NAKANO, H., SHIMADA, K., KJNOSHITA, K., ISHn, been observed: activin-A induces anterior dorsal K., SHIBAI, H. AND UENO, N. (1990). 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A homeobox-containing marker of posterior neural (Accepted 19 September 1991)